JP2009518113A - Flexible device and method for monitoring and delivery - Google Patents

Abstract

The present invention generally relates to systems and methods for co-location in a small system that is flexible and configurable, as well as multi-layered substrate sampling, rapid analysis, biological sample storage, and delivery performed on biological tissue or material obtained from a living body. Regarding functions. Sampling types may include chemical, biochemical, biological, thermal, mechanical, electrical, magnetic, and optical sampling. In general, analysis performed at a sampling point measures a sample taken and records the value. The biological sample storage function encapsulates a small sample of the specimen and stores it for subsequent inspection or analysis in vivo by the system or remotely by an independent analysis system. When stored, the sample can provide a record of the biological state at the correct sampling time. Delivery at the sampling point may include chemical, biochemical, biological, thermal, mechanical, electrical, magnetic, and optical stimuli.

Description

Cross-reference to related applications This application is based on International Patent Application No. PCT / US2005 / 044287, filed Dec. 9, 2005, entitled “Apparatus and Method for Continuous Real-Time Trace Biomolecular Sampling, Analysis”. This claims and claims this priority from US Provisional Patent Application No. 60 / 634,783, entitled “Systems and Methods for Monitoring Health and Delivering Drugs Transdermally”. The contents of which are both incorporated herein by reference).

The present invention relates to the fields of portable biochemical and biomolecular monitoring, remote diagnostics, and connected medicine. More particularly, the present invention relates to a method, apparatus and system for a configurable and flexible personal health monitoring and material delivery system.

Background of the Invention Non-invasive transcutaneous sampling of bodily fluids has long been a goal of medical research. Prior art attempting to achieve this goal is described, for example, by Currie et al., May 3, 2005, entitled "Systems and Methods for Monitoring Health and Delivering Drugs Transdermally," the contents of which are hereby incorporated by reference. It is described in Japanese Patent Application Publication No. JP-A-2001-26853.

  Prior attempts at percutaneous sampling are usually characterized by making relatively large pores in the outermost or stratum corneum of the epidermis. The stratum corneum is effectively the surface of the skin and is mainly composed of dead cells without nuclei. The hole is usually made by thermal or laser ablation or puncture using a fine needle and reaches the underlying living epidermis. Interstitial fluid from the living epidermis, or bodily fluid from the end of the vasculature, is then typically drawn or squeezed from the subcutaneous into the transdermal device, where the transdermal device uses a microfabricated channel and a light-guided system And analyzed spectroscopically.

  Such systems usually have a number of drawbacks, including that the pore size is about tens of microns, sufficient to produce local irritation. This often causes inflammation, which normally prevents the channel from remaining open for hours to days or longer.

  Furthermore, microfabrication of complex systems usually requires the use of a silicon substrate. Silicon substrates are relatively inflexible, thereby making close surface contact difficult and providing lateral movement between the percutaneous detector and the hole through the stratum corneum. Because of the size of the percutaneous hole, which is typically a few tens of microns in diameter, even a slight lateral movement can render such a device inoperable.

It would be desirable to provide a configurable and flexible personal health monitoring and delivery system that may be configured for use in a wider range of applications. It would also be desirable to provide a sampling device that may be used in invasive, minimally invasive, and non-invasive procedures, including transcutaneous and non-transcutaneous sampling.
US Pat. No. 6,887,202

SUMMARY The present invention generally relates to systems and methods for co-installation in a flexible and configurable small system, as well as multilayer substrate sampling, rapid analysis, biological sample storage, performed on biological tissue or material obtained from a living body, And the function of delivery. In several application areas described below, the types of sampling include chemical, biochemical, biological, thermal, mechanical, electrical, magnetic, and optical sampling. Also good. In general, analysis performed at a sampling point measures a sample collected and records the value. The biological sample storage function encapsulates a small sample of the specimen and stores it for subsequent inspection or analysis in vivo by the system or remotely by an independent analysis system. When stored, the sample can provide a record of the biological state at the correct sampling time. Delivery at the sampling point may include chemical, biochemical, biological, thermal, mechanical, electrical, magnetic, and optical stimuli.

  The result of the measurement may provide a quantitative snapshot at the time of sampling and a measure of the health of the tissue being measured or the organism being monitored. If a value is measured that does not fall within the normal biomedical range, the predicted disease state can be reported and a medical practice may be performed to restore the organism to its healthy state.

  Monitoring is broadly defined as a periodic measurement activity of a biological process (eg, physiological, biochemical, or metabolic) and within a healthy area where the entered biological function is normal and functioning. Attempts to define a range so that timely corrective action can be taken against detected defects. Evaluation is the process by which measurement results are analyzed and clearly determined against individual and population standards. Similarly, a person's biological environment (air, food, water, drugs, temperature, etc.), animal tissue or organism can be monitored for its input to a biological process. Examples of the types of monitoring with which the system is configured include biological and chemical monitoring for safety, monitoring to detect and maintain personal health behaviors, and early disease detection This includes monitoring for disease exacerbations, including secondary effects (disease burden), medical interventions including medication, monitoring therapeutic effects, monitoring treatment compliance, and / or monitoring for the likelihood of recurrence.

  Monitoring in these situations can be either short-term or long-term. Only a single measurement may be performed at the desired prepared or controlled time. The time scale for repeated measurements may be variable from minutes to weeks, or from more than one hour to more than tens of hours. The biochemical concentration scale may vary from trace 1 pg / dl to high concentrations 1 g / dl, or over 12 orders of magnitude.

  The disposable sampling component of the system may be mechanically flexible to deform to adhere to and adhere to the tissue to be monitored, multiple thin layers of structural and functional membranes It may be made of. As described below, the resulting contact is minimally invasive, i.e. does not interfere with the survival function of the tissue or does not disrupt the measurement target due to its presence. Similarly, a flexible sampling component can be fitted into a suitable housing for analysis of biological fluids and biological or chemical samples. Sensors may be attached to different sampling sites such as cheeks, gingiva, nose, throat, sinus, ear, and eye tissue. Each location may exhibit unique sampling characteristics. For example, the eyes and inner ear allow access to the central nervous system inside the blood brain barrier.

  The system and its components can be configured depending on the location of the sampling on the organism, the number and type of measurement targets, and the frequency, redundancy, recording and reporting of individual measurements. The types of configuration can be classified as follows. Measurement target identification; measurement target size; sample location; type of sample mechanism, whether non-invasive, minimally invasive or invasive; type of sample collected; sample storage; sample modification; Number of judgments for one measurement target. Furthermore, the details of the configuration may change over time to suit the purpose of health monitoring.

  The system described in more detail below may be used in a wide range of application areas including, but not limited to: Diabetes monitoring and treatment; Resuscitation medicine including hemorrhagic shock, trauma, burns, etc .; Discovery and treatment of pediatric jaundice; Monitoring stamina, physical activity, fatigue, and arousal; Testing tissue viability; Performing tissue biopsy; Early detection of diseases such as cancer and infectious diseases and response to treatment of such diseases; detection and treatment of periodontal diseases such as bacteria, viruses, microbial membranes, inflammation and caries, and normal to assess overall health Hormones, proteins, and metabolites; monitoring obesity, diet, exercise, and weight, and overall body composition management; monitoring cardiac and vascular functions and stroke; drug discovery and development screening; drug pharmacokinetics, individuals Medication, efficacy, safety, toxicity, secondary effects, interference and pharmacological studies, and clinical trials; including influenza, malaria, and dengue Discovery and treatment of non-limiting infections; neural interfaces for short- and long-term interfaces for prosthetic control and therapeutic response, etc .; monitoring of neurological diseases such as depression, anxiety, multiple sclerosis; animals, crops, water, Monitoring of food supply, etc .; Monitoring of the use and abuse of substances and drugs such as smoking, drinking and anesthetics; Hospitals that administer drugs such as anticoagulants. In addition, the system may be used in conjunction with biomedical instruments and may provide direct feedback of therapeutic effects on radiation, chemotherapy, exercise, dialysis, ventilators, tissue and organ support systems. The system may also be used for transplant tissue monitoring for the viability and functionality of the tissue to be removed and the tissue to be transplanted, both pre- and post-operatively.

  It is an aspect of the present invention to provide a method for monitoring a biological state of a subject. In one embodiment, the method includes exposing a sensor of a sampling, analysis and delivery device to a substance containing a target biomolecule and providing an electrical signal to the sensor; Measuring, correlating the measured electrical property with the biological state, determining whether the measured biological state is normal or abnormal, and the measured biological state Generating a signal if is abnormal.

  In certain embodiments, the target biomolecule is selected from the group consisting of glucose, lactate, bilirubin, ethanol, pyruvate, and cytochrome P-450 2A6 enzyme.

  In certain embodiments, the target biomolecules are glycosylated hemoglobin and glycosylated protein, insulin, cholesterol, C-reactive protein, homocysteine, orexin, P. aureus. falciparum histidine-rich protein 2, parasite lactate dehydrogenase, prostate specific antigen, prostate membrane specific antigen, estrogen, epidermal growth factor, insulin growth factor, hemagglutinin, neuroadminidase, cleaved caspase 3 17 kDa subunits, p54-like proteins, immunoglobulins, anesthetics, leptin, ghrelin, vitamins, folic acid, creatine kinase (CK and CK-MB), troponin, C-reactive protein, tumor necrosis factor receptor 1 and 2, creatine phosphokinase (CK and CK-MB), creatinine, troponin, interleukins 1, 2 and 6, interleukin-2 receptor, tumor necrosis factor-alpha, n-nitrosami , Nicotine, cotinine, opiates, chosen cocaine, and from the group consisting of spore metabolites.

  In certain embodiments, the target biomolecule is selected from the group consisting of influenza virus, multiple sclerosis virus, dengue fever, malaria, HIV, and tuberculosis.

  It is another aspect of the present invention to provide a method for biomolecule monitoring. In certain embodiments, the method includes sampling a substance that includes a biochemical substance and analyzing the substance using a pair of electrodes. The pair of electrodes are disposed on a support substrate. The method also includes controlling the sampling and analyzing steps with a controller and communicating information between the controller and the remote device.

  It is an aspect of the present invention to provide a system for sampling, analyzing and / or delivering at least one biochemical. In some embodiments, the system includes a support substrate and a plurality of cells supported by the support substrate and arranged in a plurality of columns and a plurality of rows. Each of the plurality of cells is configured to sample, analyze, and / or deliver biochemicals. The system also includes a controller configured to control the interaction of each of the plurality of cells having materials related to the biochemical material. The controller is connected to the plurality of cells via a plurality of column address conductive paths, a plurality of row address conductive paths, and a plurality of zero-insertion force connectors. The system further includes a communication device configured to communicate with the controller and the external device.

  It is another aspect of the present invention to provide a system for analyzing material properties. In some embodiments, the system includes a support substrate and a plurality of sensors supported on the support substrate. Each sensor includes a pair of electrodes supported by a substrate. The pair of electrodes includes a working electrode and a verification electrode. The working electrode is electrochemically activated and configured to react to the material. When a voltage is applied across the electrode and at least the working electrode is exposed to the material, an electrical property corresponding to the property of the material is generated. The system also includes a controller that controls a series of electrical signals to the electrodes and receives the generated electrical characteristics.

  It is a further aspect of the present invention to provide a sensor that senses the properties of substances including biomolecules. In some embodiments, the sensor includes a pair of electrodes supported by a substrate. The pair of electrodes includes a working electrode and a verification electrode. The working electrode is electrochemically activated and configured to react to the material. When a voltage is applied across the electrode and at least the working electrode is exposed to the material, an electrical property corresponding to the property of the material is generated.

  It is yet another aspect of the present invention to provide a sampling device for sampling materials including biochemicals. In certain embodiments, the sampling device includes a support substrate and a pair of electrodes supported by the substrate. The pair of electrodes are configured to tear a protective film disposed between the support substrate and the substance.

  It is an aspect of the present invention to provide a method for analyzing biochemical substances in a substance. In certain embodiments, the method comprises the steps of exposing at least the working electrode of the sensor to the substance, applying a voltage across the working electrode and the reference electrode, and applying the voltage across the electrode. Measuring the resulting electrical properties and associating the measured electrical properties with the material properties.

  It is a further aspect of the present invention to provide a method for filling cavities in a support layer of a delivery device. In some embodiments, the method includes disposing a sealing layer having a hole on the support layer, filling the cavity with a material, and separating the cavity from the cavity so that the hole is disposed above the cavity. Placing the sealing layer relative to the support layer so as to be covered by the sealing layer, and bonding the sealing layer to the support layer.

  It is an aspect of the present invention to provide a method of manufacturing a device for analyzing material properties. In some embodiments, the method includes connecting between a plurality of connectors and a plurality of electrodes disposed on the support substrate, exposing the plurality of electrodes to a polymer matrix, and a plurality of grounds. A step of applying an electrochemical potential to an electrode selected from the above electrodes, and a step of coating the selected electrode with a polymer matrix.

  These and other aspects of the system will now be described with reference to the accompanying drawings.

  In order to understand the inventive concept of the present invention, it is useful to consider the accompanying drawings. In the drawings, like numbers indicate like elements whenever possible.

DETAILED DESCRIPTION System and Method Overview The present invention relates generally to a method, apparatus, and system for a configurable and flexible personal health monitoring and delivery system. More particularly, the present invention relates to a system and method for selective sampling of materials. The substance includes, but is not limited to, interstitial fluid and biological fluid. Regarding the selective measurement of the target property, it includes, but is not limited to, biomolecules present in the sample. Further, the systems and methods may provide on-command release of stored materials, including but not limited to chemicals, biochemicals, and drugs.

  FIG. 1 is a schematic diagram illustrating an embodiment of a configurable and flexible personal health monitoring and delivery system 10 of the present invention. In the illustrated embodiment, the system 10 includes three cooperating devices. The cooperating device includes a sampling, analysis and delivery device 12, and a control device or controller 14 in communication with the sampling, analysis and delivery device 12, and a remote logger, controller, or any other device. It includes a communication device 16 that enables data transmission. Any other device described above is configured to transmit and / or receive data.

  As will be described in more detail below, the sampling, analysis and delivery device 12 may be used to sample a substance and / or analyze a substance and / or a material such as a chemical, biochemical, or drug. It may be configured to deliver to the sampled and / or analyzed material. As such, the phrase “sampling, analysis and delivery device” should not be taken in the sense that the device needs to provide all three functions in all embodiments. In contrast, in some embodiments, the device 12 may be configured to sample and analyze the substance, or only sample and analyze the substance. Any combination of these three functions is considered to be within the scope of the present invention. As will be described in more detail below, in certain embodiments, device 12 may be a flexible, extensible, sterile, disposable microfluidic electrochemical chip that includes a plurality of electrochemical sensor cells. Good.

  In certain embodiments, the controller 14 may include flexible cables and connectors. The connector connects to the device 12 so that the controller 14 communicates with a plurality of electrochemical sensor cells. The controller 14 also includes a radio control and messaging unit. The messaging unit is configured to communicate with a communication device 16, which is preferably in communication with a wireless remote computer or any other device configured to receive data. May be configured. For example, the remote device may be a cell phone, camera, music player, or any other device including a memory. Controller 14 and communication device 16 are also described in more detail below.

  One skilled in the art will appreciate that embodiments of the system 10 may be used in a wide variety of applications where it is desired to monitor materials for a given condition. In general, system 10 may be used to monitor for certain abnormal biochemical conditions, such as illnesses, and to treat such conditions upon discovery.

  As shown in FIG. 25, in one embodiment, a method 200 for monitoring a biochemical condition, such as a disease of interest, using the system 10 described herein is provided. The subject may be a human, animal, biological tissue, organ, or non-biological material such as food or water. The method 200 of the present embodiment begins at 202. At 204, the sensor of the sampling, analysis and delivery device 12 may be exposed to a substance containing the target biomolecule by placing the device 12 in an appropriate location on the subject. Device 12 may be in communication with the monitoring system via controller 14 and communication device 16. Once the device 12 is in place and the system 10 is functioning, the subject may be monitored during a predetermined time to determine the subject's normal health baseline behavior. For example, biomolecules in a subject's bloodstream that may be discovered by sampling the interstitial fluid of the subject may be analyzed to obtain a “normal” concentration within that particular subject. The sample may be analyzed by providing an electrical signal to the sensor at 206 and measuring an electrical property at the sensor generated in response to the electrical signal at 208. Then, at 210, the measured electrical property may be associated with a biological state. In some embodiments, the subject's normal health baseline behavior may be known and programmed into the system 10. After the criteria are established, the subject uses the same process as 206, 208, and 210 using the system 10 until an abnormality is detected by the system, such as an increase or decrease in the concentration of the target biomolecule at 212. May be continuously monitored.

  If an abnormality is discovered, a signal may be generated at 214 and communicated to the controller 14. At this point, at 216, the controller 14 may determine whether to continue monitoring or at this point the method returns to 206, or signal the device 12 and at 218, the material from the cavity inside the device 12 May decide to deliver the material until then the method may return to 206. Subjects may be continuously monitored, as material effectiveness and compliance may be monitored. In addition, the controller 14 may signal the device 12 to expose another sensor to the substance in order to detect any secondary effects caused by the anomaly. The system 10 may be configured to stop delivery of material if it is determined that the anomaly has disappeared. The subject may then be monitored using the system for possible recurrence of the abnormality. If it is discovered that there is a recurrence, the material may be delivered again to the subject, or another substance may be supplied and the subject may continue to be monitored. If it is determined that monitoring should be stopped at any time, the method ends at 222. In particular, if it is determined at 212 that there is no abnormality, the controller 14 continues to signal and at 220, an electrical signal may be supplied to the same sensor on the device 12 or another sensor, or the controller 14 May stop supplying the signal to the sensor and the method ends at 222.

  The monitoring time interval described above may be variable, or may be set to a predetermined interval in a range from several minutes to several weeks. The sensor inside the cell 18 of the device 12 may be configured to detect the concentration of the target biomolecule over a wide range from about 1 pg / dl (trace amount) to about 1 g / dl (high concentration).

  The device 12 may be configured to be placed at a number of tissue locations on the subject's body, including an internal location and an external location. For example, the external locations may include the subject's arms, legs, neck, hands, feet, and facial skin. Examples of internal locations include the subject's mouth (including cheeks, gingiva, and tongue), nasal and sinus routes, throat, ears, and eye tissue. Information about the central nervous system may be accessed by attaching the device to the part of the eye or inner ear.

  The system 10 may also be configured to monitor an entire chemical panel for individuals, patients, or populations at risk of disease. In some embodiments, the system may be configured for use in resuscitation of patients in need of urgent emergency care, patients in shock, patients with certain trauma, and unconscious patients. Good. By affixing the device 12 to the patient, real-time analysis of the target biomolecule can be performed, and even if the patient cannot speak, further information about the patient can be provided to the health care provider. . In an embodiment, the system 10 may be configured to monitor and treat chronic severe illness to monitor for early detection of disease and / or response to therapeutic therapy.

Sampling, Analysis and Delivery Device FIG. 2 is a schematic diagram showing the functional components of device 12. As shown, device 12 includes a plurality of sampling, analysis and delivery cells 18. The phrase “sampling, analysis and delivery cell” should not be taken to mean that the cell needs to provide all three functions in all embodiments. In contrast, in some embodiments, one cell 18 may be configured to sample a substance and analyze the substance, and one cell 18 only samples the substance and analyzes the substance. However, it may be configured to only deliver material to the substance. Any combination of these three functions in a single cell is considered to be within the scope of the present invention.

  In the illustrated embodiment, the cells 18 are arranged in a plurality of columns and rows. Other arrangements are contemplated and the illustrated embodiment is not intended to be limiting in any way. Device 12 also includes a plurality of electrical contact pads 20. The electrical contact pad is configured to communicate with the controller 14 and the plurality of conductive paths 22. The conductive path connects cell 18 to contact pad 20. The cell 18 is disposed in a region 24 that is disposed in contact with a sample to be analyzed such as tissue. Device 12 may optionally include an electrical device identification area 26. The electrical device identification area allows a particular device 12 to be identified by the controller 14 when the device 12 is connected thereto.

  In one embodiment where biomolecules present in the interstitial fluid will be sampled transcutaneously, the device 12 is placed in close contact with the subject's skin and held in place by pressure or adhesive. . A series of precisely predetermined and controlled electrical pulses may be applied to one or several pairs of various selectable electrodes located within the cell 18 to generate heat and local electric fields. . Thermal and local electric fields rupture dead skin cells in the stratum corneum without damaging living cells beneath them in the living epidermis. This causes the interstitial fluid to flow towards the surface of the point on the device 12 and soak it there for a concentration that is in equilibrium with the underlying tissue concentration for hours or until the stratum corneum is re-formed. It becomes possible to maintain.

  At the exact location where the stratum corneum has been torn, one or more pairs of electrochemical electrodes can be used in several ways to selectively measure one or more properties of the interstitial fluid. One may be arranged and prepared. These properties include, but are not limited to, concentrations of biomolecules such as biochemical analytes, and / or physicochemical properties such as pH or concentration of dissolved gas. Such measurements may be continuous in nature and may reliably track any temporal changes in these concentrations until the stratum corneum is repaired. As described in more detail below, the preparation of the electrode in each cell may include an encapsulation that protects the reaction surface before initiating the desired measurement. Encapsulation is selectively permeable, depending on the target biomolecule, for example adhesive membranes, binding membranes, charged membranes, conductive membranes, impermeable membranes and reactive membranes, or selected for different applications. It may be realized by a membrane.

  In certain embodiments, the material comprising the biological fluid to be analyzed has already been collected from a variety of samples including, but not limited to, food, water, air, whole blood, urine, saliva, chemical reactants, and media. Also good. In such embodiments, a small amount of a substance containing biological fluid may be applied to the surface of device 12 either statically or by continuous flow across the surface. The controller 14 may be configured to open one or several selective detection cells 18 and begin monitoring a specific concentration of biological fluid. For example, blood collection can be analyzed immediately at the point of care, or air and water samples can be continuously monitored to obtain chemical and biological purity. Furthermore, when the cell 18 is exhausted or a new contamination threat is identified, the cell 18 can be replaced by opening the new cell 18 in its predetermined position. System 10 can be configured to provide applications specific to analysis and monitoring systems.

Sampling FIG. 3 is a schematic top view of a portion of an embodiment of a single cell 18 that may be one of many on device 12. In this embodiment, the cell 18 is configured for sampling, analysis, and delivery, and components for both sampling and analysis are exposed on the surface of the flexible substrate 40. In applications where the device 12 is attached to a non-flat surface, the substrate is flexible so that the device 12 can conform to the surface of the non-flat surface. A cavity 28 that may contain an embedded encapsulation, i.e., a delivery material, is shown in outline. The conductive path 30 that is finally connected to the conductive path shown in FIG. 2 is covered by a thin insulating layer and forms part of the selective working electrode 32 and the verification electrode 34. As shown in FIG. 3, the exposed electrical resistive element portion 36 is coupled to an exposed area 38 of another conductive path 39.

  FIG. 4A is a schematic top view of an embodiment of the sampling and analysis cell 18. In this arrangement, the selective working electrode 32 and the reference electrode 34 are joined by an exposed area of the conductive path 38 of the second conductive path, resulting in stratum corneum rupture and chronoamperometry analysis. Measurements or other electrical property analysis measurements can be made with the same electrodes 32, 34.

  FIG. 5 shows a cross section along AB of the embodiment of FIG. The cross section shows the resistive element 36 in the vicinity of the analysis electrode, which may be a selective working electrode 32 on a portion of the flexible substrate 40.

  FIG. 6 is a schematic cross section of a modification of the configuration shown in FIG. There, the analysis electrodes 32 and 34 are encapsulated within a cavity in the substrate 40 and are at sampling points on a thin film of substrate material. The cavity 28 may be filled with material that is delivered to the sampling site, or the cavity 28 may be empty.

  FIG. 7 shows the same cross section as FIG. There, a tear 42 is formed in the thin film of the substrate due to the operation of the resistance element. This tear 42 will expose the analytical electrodes 32, 34 to the substance to be analyzed and also allow any encapsulated delivery material to be delivered from the cavity 28 to the sampling site.

Rupture In one of several analytical applications, the device 12 is held in contact with the target skin or some other tissue or film. FIG. 11 shows the cell of FIG. 6 in contact with tissue such as skin, to schematically show the scaly stratum corneum 48 and the living epidermal tissue 50 with the interstitial fluid located beneath it. It has been simplified.

  In certain embodiments, the stratum corneum of interest may then be ruptured by applying a series of voltage pulses of about 2V lasting less than 1 second. The exact sequence and voltage required must be adjusted with respect to the particular object and the location and nature of the tissue to be ruptured. The reduction in heat and voltage between the sample electrodes does not remove dead cells in the stratum corneum but helps to bond between them, thereby creating a capillary opening. Capillary openings serve to exhale interstitial fluid from the living epidermis to the sample electrode, and sufficient fluid transfer maintains equilibrium with the interstitial fluid in the living tissue beneath the stratum corneum and maintains the equilibrium dynamically Make it possible.

  Barrier tissue rupture differs from the prior art where processes such as ablation and poration by mechanical means (eg, needles) are commonly used. Ablation refers to the removal of specific target cells. Barrier cells are often non-biological cells, and may be more scaly and flatter than biological shapes, packed close together and equiangularly, and bonded to each other by chemical bonds between biomolecules that make up the cell wall. Yes. The process of rupture refers to the process whereby the connections between the packed barrier cells are thereby gradually broken, allowing the formation of capillary openings between the cells. The cells themselves remain largely intact. At a point in time when the size of the capillary increases, there is a conductive path through which interstitial fluid is drawn to the surface. It was observed that the rate and nature of efflux is independent of, for example, basal metabolic blood pressure, and there is no internal pressure that directs interstitial fluid outward. Interstitial fluid flow can be enhanced using special hydrophilic surface treatments of sampling points on the sampling device and in the capillary structure or microenvelope. The capillary structure or micro-encasement is patterned on the surface in the case of sampling devices.

  This unique technique for reliable rupture of barrier tissue involves a combination of electrical energy pulses on a sampling electrode that is individually addressed and individually addressed. In particular, in human forearm skin, one or more conductive thin film resistors between the sampling electrodes were used. For multiple resistors, the resistance value is selected to cascade from high resistance (eg, 200 Ohm) to medium resistance to low resistance (50 Ohm). The size of each resistor corresponds to the size of the barrier cell that will be removed from its vicinity. This means that for a 50 micron stratum corneum cell, the gap between the electrodes should be less than 100 microns. An electrical voltage step of 0 to 2V may be applied to the electrode in short bursts of less than 1 second, typically rising 0.2V for each pulse. The device may be manufactured such that the conductive wire leading from the connector to the electrode is embedded other than at the resistor.

  In some embodiments, a moderate electric field up to 4 MV / m may be applied. Next, the temperature of the minimum resistance element is 2.2 V and may reach 140 ° C. in a very short time, and then the resistor may be opened. The opening is determined by the precision manufacturing material, dimensions, and order of the resistor and the underlying sampling device material (often a low melting temperature polymer film such as polyethylene or methacrylate). It breaks when the material is deformed. Thermal profile measurements show that the temperature drops to below 80 ° C. across the 50 micron thick stratum corneum. If there is only one resistor, the electrodes will be in open circuit, but the electric field will still be pulsed between the electrodes. For multiple resistors, the voltage process may continue until all resistors have braked and opened the circuit. Here, the electrode may be left alone to enable electrochemical measurement between the detection electrode and the verification electrode.

  In embodiments where it is desirable to ablate cells rather than rupture cells, a heater may be provided on the surface of the device. The heater may be configured, for example, such that 50 mJ pulses of thermal energy are applied to the stratum corneum cells in order to ablate the cells. Such a device is described, for example, in US Pat. No. 6,887,202, which is incorporated herein by reference.

  FIG. 12 is a schematic diagram illustrating the cell of FIG. 11 after rupturing of the barrier cells in region 52. FIG. An osmotic fluid path is provided to allow interstitial fluid 54 from viable epidermal cells 50 to be pushed and submerged by capillary forces to the sampling site. As described below, the interstitial fluid 54 soaking the electrodes may be analyzed by the selective electrodes 32 and 34.

  In some embodiments, the tearing electrode itself may be used for electrochemical measurements, thereby providing small and economical manufacturing, high packing density, and simplification of connection and control circuitry.

  In some applications, particularly those where the material to be sampled is already placed on the top surface of the target object, the device may not even include a mechanism for cell rupture.

Analysis In one embodiment of the invention, two sample electrodes are supported by a substrate 40 and are coupled by a resistive element. The electron conductive enzyme immobilization layer may cover at least one part of the sample electrode, and the protective layer may cover the entire device other than the vicinity of the sample electrode.

  In the embodiment relating to the analysis of interstitial fluid, any target biomolecule in the interstitial fluid extracted in the vicinity of the electron conductive enzyme immobilization layer interacts with the immobilized enzyme. This interaction may be detected, for example, by chronoamperometric measurements performed using a voltage applied across the sample electrode. Of course, the electrodes may be configured such that other types of electrical properties such as resistance, capacitance, etc. may be measured.

  FIG. 9 shows a cross section of FIG. There, the effective surface area of the analysis electrodes 32 and 36 is increased to achieve a corresponding increase in electrical signal and a decrease in the signal relative to the noise ratio. The increase in area can be achieved both by covering an additional area on the cavity wall and by using a protected sponge porous electrode material.

  FIG. 10 shows a cross section of FIG. There, the effective surface area of the analysis electrodes 32 and 34 is increased to achieve a corresponding increase in electrical signal and a decrease in signal relative to the noise ratio. The increase in area can be achieved by coating the surface of the working electrode 32 with a protected high density nanomaterial prepared with the desired selective chemistry. The preparation of such nanomaterials is described in more detail below.

  FIG. 15 shows a cell used for sampling body fluid that is not located on the opposite side of the protective membrane. The cell of FIG. 6 is in direct contact with the stagnant or fluid 56 to be analyzed. FIG. 16 shows the cell of FIG. 15 opened to allow body fluid to enter the cavity 28. Electrodes 32 and 34 are disposed in the cavity. The electrodes 32 and 34 may be exposed to body fluid, and a voltage may be applied to the electrodes and the resulting electrical properties may be measured.

  FIG. 17 shows a cell used for sampling solids or powders. The cell of FIG. 8 with the dried sample 58 to be analyzed applied or collected there. FIG. 18 shows the cell of FIG. 17 opened to allow the contained body fluid to soak and dissolve the dried sample 58, thereby allowing a concentration to be present at the analysis electrode.

  The illustrated embodiments of cells and electrodes described above are not intended to be limiting in any way, but instead are intended to provide examples of possible configurations.

  For example, in embodiments where it is desirable to measure gas concentrations including but not limited to oxygen, carbon dioxide, carbon monoxide (harmful), and nitrous oxide dissolved in biological fluids such as interstitial fluid, The device 12 may be configured as follows. In certain embodiments, the device 12 may be placed on the body to gain access to interstitial fluid. In another embodiment, the device 12 exposes the device 12 to inhaled air and exhaled air, thereby allowing measurement of a gas component to be performed in a manner such as at or near the nasal cavity. It may be placed in the pulmonary ventilation pathway.

  Of the device 12, such as those described in US Pat. Nos. 6,270,651 and 7001,405 and US Patent Application Publication No. 20010052459, the entire contents of which are hereby incorporated by reference. There are many ways in which the sensing electrode may be made sensitive to selective precision measurement of these gases. The sensing electrode of device 12 may be prepared using specific metal and electrolyte thin films. The specific metals and electrolytes selectively generate an electrochemical voltage proportional to the logarithm of the gas partial pressure when exposed to a gas. Some electrolyte materials that may be used are slightly water soluble, as shown by the layer 58 in FIG. 18, which is hydrophobic but allows gas molecules to pass through the layer to reach the detection electrode. It may be protected by a thin film of polymer coating that allows

  In another method of detecting a gas, such as oxygen blood gas, the sensing electrode of device 12 may be bound by a thin semiconductor polycrystalline layer of material such as tin oxide or indium tin oxide. Oxygen from the interstitial fluid or exhaled air may be absorbed into the semiconductor polycrystalline material and its electrical conductivity between the electrodes may be modified depending on the gas partial pressure. Further, a thin film of a semiconductor or an insulator (including a polymer) that selectively absorbs target gas molecules is used, and the amount of absorption is electrically changed as a change in capacitance between the electrodes 32 and 34 in the cell 18 of the device 12. May be measured.

  In certain embodiments of the invention, a piezoelectric detection device may be made in the cavity 28 of the device 12 to allow detection of antibody-antigen or Ab-Ag. Ab-Ag detection may be facilitated by monitoring resonance frequency shifts associated with binding events. In certain embodiments, a piezoelectric device includes a cantilever and a membrane structure that incorporates a piezoelectric material to detect an analyte bound to its receptor. For cantilever beams, two detection modes may be used. First, when a binding event occurs, the resulting surface stress may be measured by measuring the voltage generated by the stress piezoelectric film. Second, a shift in the resonant frequency of the cantilever beam as a result of the change in amount due to the coupling event may be detected. For membrane structures incorporating piezoelectric materials, Ab-Ag complex formation may be determined by measuring the resonant frequency shift.

  In certain embodiments, the device may mount a piezoelectric film within the device structure to generate a current that can be stored on the capacitive element. The device may wear out on the body (forearm, wrist, buttocks, etc.) where the natural body movement causes the patch to bend. By using piezoelectric elements, any bending pressure can be converted into electrical energy. The ability of a solid to convert and return mechanical pressure to an electrical signal is based on a non-uniform charge distribution within the solid. Although the entire material remains neutral, there are numerous dipole moments that can create a detectable internal potential. This electrical signal is maximized when the piezoelectric material first undergoes a polarization process known as poling. Before the polarization process, the dipoles are randomly distributed from one end of the solid to the other, but when polarized by an external electric field and temperature, the dipoles reset the internal dipoles to be in an ordered state and nearly aligned To the state. Mechanical compression reduces the magnitude of the dipole moment, thus increasing the magnitude of the dipole moment and increasing the voltage signal while simultaneously reducing the voltage.

  In a similar manner, a voltage applied at the same polarity as the piezoelectric material stretches the material while an applied voltage at the opposite polarity compresses the material. In one configuration of the device, a layer of polymeric piezoelectric material similar to polyvinylidene difluoride, or PVDF, is incorporated into or deposited on the substrate. One use of the device is to measure the potential and use it as a measure of the deformation of the sampling device. A second independent non-conflicting application includes control and communication devices 14 and 16. The potential generates a current and the controller 14 may rectify the potential and use it to charge either a capacitance or a built-in rechargeable flexible polymer lithium battery.

Delivery FIG. 4B is a schematic top view of an embodiment of a cell 18 configured to deliver material to a substance. In this arrangement, an embedded encapsulation or cavity 28 containing a delivery material is placed behind a pair of exposed areas 38 of the conductive path that are joined by an exposed resistive element portion of the conductive path. Yes. In this way, stratum corneum rupture may be used to achieve delivery to the interstitial fluid of the material within the embedded encapsulation 28. The material may be in gaseous, liquid, or solid form and may include drugs, chemicals, cells, biochemicals, biomolecules, proteins, peptides, genetic material, and the like.

  FIG. 4C shows a schematic top view of a portion of another embodiment of cell 18. The cell 18 may be one of many on the device 12. In this embodiment, both the sampling device and the analysis device are exposed on the surface of the flexible substrate 40, and the electrodes 32, 34 are located on the exposed portion of the conductive path, thereby resulting in a conductive There are cases where the number of control paths is reduced and the control function needs to be increased.

  Within separate encapsulations at the measurement point, to allow the desired concentration of chemical to dissolve in the interstitial fluid and re-access the body through the locally ruptured stratum corneum In addition, the controlled diffusion by sequential controlled cleavage through the encapsulating wall material or through the crevice of the encapsulating wall portion may result in biochemicals, dry powders or aqueous solutions in the gas phase. It may be saved and released. As an analytical chemistry tool, a chemical can be selected to react and modify the composition of the local interstitial fluid, and the reaction product may be measured by multiple electrodes. Alternatively, the encapsulated chemical sample may be a calibration standard. Alone, a selected measuring electrode or a newly selected measuring electrode can be used to continuously track a subject's response to an administered chemical, thus quantifying effectiveness and personal And can safely detect and prevent side effects with minimal dosage.

  FIG. 8 shows the cross section of FIG. 6 with the cavity 28 filled with delivery material 44 at the sample location. Embodiments relating to how the cavities may be filled are described in further detail below. The working electrodes 32 and 34 may be configured to set the concentration of the delivery material 44 and thereby measure how much material remains in the cavity and how much has been delivered to the sampling site. Good.

  FIG. 13 shows the cell of FIG. 8 in contact with tissue such as skin. The figure is simplified to schematically show the scaly stratum corneum 48 and the living epidermal tissue 50 under which the interstitial fluid is located.

  FIG. 14 shows a schematic diagram of the cell of FIG. 13 after rupture of the membrane that seals the barrier tissue and cavity 28. An osmotic fluid path is provided so that interstitial fluid 54 can flow out into the sampling site by capillary force and be immersed therein. The delivery material 44 mixes or dissolves in the interstitial fluid, returns to the living tissue 50 through the stratum corneum 48 that has been ruptured due to the resulting high concentration gradient, and diffuses from there to other parts of the body. .

  By designing individual sampling sites on the device 12 to a size of 10 micrometers or less, the intracellular fluid of individual cells can be analyzed by locally rupturing the cell membrane, and from the surface of the sampling device The therapeutic substance can be injected into the cell prior to releasing.

  As described above, in some embodiments, the tearing of the thin film that seals the cavity may be achieved by heat generated by the electrode and / or the resistive element that achieves the tearing of the stratum corneum. In such embodiments, to create a hot melt dehiscence, the thin film may be cleaved essentially by melting the thin film to the point where a fissure occurs in the film. In another embodiment, the hollow film may be cleaved by mechanical forces applied to the film to rupture, tear, and / or shear the film. Such mechanical force may be generated, for example, by generating bubbles inside the cavity by electrolysis using an electrode disposed in the cavity. The pressure inside the cavity due to foam formation can rupture the thin film and can be high enough for the material 44 encapsulated in the cavity 28 to flow out of the cavity 28 at the cleaved portion. Such examples relating to tearing the membrane and creating a flow path for material to exit the cavity or for material to enter the cavity should in no way be considered limiting.

Manufacturing During manufacture, the electron conducting layer on the electrode 32 may be electrochemically activated using an immobilized enzyme that modifies the target biomolecule. For example, when glucose is the target biomolecule, the enzyme is not limited thereto, but may be glucose oxidase. When lactate is the target biomolecule, the enzyme may be, but is not limited to, lactate oxidase, If bilirubin is the target biomolecule, the enzyme may be bilirubin oxidase. In some embodiments, the electron conducting layer may be electrochemically activated using an antibody. Different cells 18 placed on the same device 12 may be configured to detect different biomolecules by selectively electrochemically activating electrodes with specific enzymes and antibodies. .

  In one embodiment, device 12 may be a flexible patch-like chip with a multi-layered polymer metal laminate structure, with SU-8, Teflon-AF as the structural layer. It may be manufactured using a release layer, polymethyl methacrylate (PMMA), polypyrrole (PPy), and glucose oxidase (GOD). For simplicity, combining electrodes used for tearing of the stratum corneum with a detection electrochemical electrode and a reference electrochemical electrode without encapsulating and protective membrane tearing properties for clarity A specific embodiment of a device that has been selected will be described.

  In one embodiment, the sampling, analysis and chemical delivery device manufacturing process uses SU8 as the primary structural material and generally consists of five steps. This process is a subset of the early technology developed for the polymeric material polydimethylsiloxane (PDMS). The first step was the deposition of a Teflon release layer on the glass substrate. The glass substrate allows the multilayer multi-polymer device to be easily removed from the glass after manufacture. A thin layer of SU8 was formed by spin coating and served as a base layer (10μ) for the rest of the device, resulting in adhesion to Teflon. The third step in the manufacturing process consisted of spin coating a thick (150μ) SU8 layer. This thick layer provided structural struts for the device. A chrome / gold electrode / heater metallization (0.5μ) was sputtered, evaporated and patterned on top of a thick SU8 (150μ) layer. 10 μM PMMA was then spin coated as a protective layer for selective deposition of PPy and enzyme. To prevent the electrode pad from being covered by PMMA, tape was applied on the electrode pad prior to PMMA spin coating and removed prior to the PMMA baking process. The PMMA layer was further selectively plasma etched in such a way that only one of the electrodes was exposed and the other electrode was coated. The metal was patterned using positive photoresist and wet chemical etching. Prior to sputter deposition, a plasma surface treatment was used to improve the adhesion between SU8 and the metal layer. The device was then released from the glass substrate using a razor blade. The release layer was formed by spin coating an amorphous fluoropolymer solution diluted with a perfluorosolvent.

  The enzyme prototype glucose oxidase (GOD) was electrochemically absorbed onto the polypyrrole (PPy) layer using a potentiostat with 0.1 M electrolyte solution. The 0.1 M electrolyte solution was obtained by treating PPy and KCl with 8 V for 2 minutes. 0.1 M hexacyanoferrate and 8001 units / ml GOD (18 μl GOD and 48 μl potassium ferricyanide (K3FeCN6) in 10 ml phosphate buffer) were further added to the electrolyte solution for GOD deposition. . Redox electron mediators such as potassium ferricyanide increase the sensitivity of the resulting measurements for electrical properties by maximizing current conversion. A selective deposition of PPy + GOD is then performed on one of the exposed electrodes of the sampling, analysis and chemical delivery device cell (FIG. 3). A chronoamperometric dose response was recorded, which revealed that the sensor had good linearity with 0-10 mM glucose with a sensitivity of 2.9 mA / mM. For the lactate sensor chip, the same process was used except that GOD was replaced with lactate oxidase.

  The foregoing embodiments are in no way intended to be limiting, but are provided as examples. It is contemplated to make the device 12 using other materials and processes. Furthermore, the cavities 28 and microcapillaries may be made in the thick support layer when the thick support layer is grown.

  Microcapillaries may be used to manipulate the sampled material, such as interstitial fluid and / or material within the cavity, depending on where the capillary is located. The surface of the device 12 that can interface with bodily fluids is selectively treated by plasma surface treatment and / or chemical surface treatment to create a hydrophobic or hydrophilic surface at precisely controlled points in the device 12. The The bodily fluid is either a substance to be sampled and / or analyzed, or a material delivered from the cavity 28. For example, to create a hydrophilic surface on the surface of SU-8, PDMS, silicon, or aluminum, the surface may be treated with a silane derivative, with contact angles on gold, silver, or copper. In order to create a low hydrophilic surface, the surface may be treated with alkane thiols. Such examples of methods of changing the wetting behavior and transfer capability of a particular material are in no way intended to be limiting.

  In some embodiments, the cell of the device includes three layers of PDMS that are molded, metallized and bonded. Although only a single cell of the device is described herein, it is understood that other cells of the device may be manufactured in substantially the same manner. The intermediate support layer of PDMS includes a cavity and a pair of micro capillaries. The upper layer of PDMS seals the cavity, but includes a pair of microcapillaries that are substantially aligned with the microcapillaries of the intermediate layer. Embodiments in which the bottom layer of the PDMS is configured to ablate the stratum corneum for electrode and resistance element or interstitial fluid utilization used to seal the bottom of the cavity and rupture the cells of the stratum corneum The heating element may be included. The bottom layer may also include a pair of microcapillaries that are substantially in line with the other layers of microcapillaries.

  For each layer of PDMS, molds made from SU-8 may be used to create cavities and microcapillaries. The SU-8 mold is generally spin coated with SU-8 on a surface such as glass, and negative photoresist SU-8 is clearly shown in it (corresponding to cavities and microcapillaries). It may be made by exposing and developing in a state where it is applied. Once the mold is made, the mold may be used in a press, PDMS may be applied to the mold inside the press, and then cured in a furnace. Once cured, the PDMS may be removed from the press and mold. Other features of the cell, such as electrodes, may then be created by a metallization process in an appropriate layer of PDMS. After each layer of PDMS has the proper characteristics, the layers may be combined such that the microcapillaries are substantially in line with each other.

  In addition, during the fabrication of layers of various polymeric materials such as SU-8 and PDMS, sacrificial layers of various materials are used to undeformally release such layers from glass substrates or similar substrates. Also good. For example, in SU-8, polystyrene, PDMS, positive photoresist, and aluminum may be used as a release layer. Polystyrene may be dissolved in toluene, thereby releasing the SU-8 layer from the glass substrate or similar substrate. PDMS may be used to assist the resist in absorbing acetone in order to dissolve the positive photoresist in acetone and release the SU-8 layer. Aluminum may be etched to release the SU-8 layer. With respect to PDMS, sacrificial release layers that may be used to release PDMS from the aforementioned SU-8 mold are photoresist (etched with acetone for release), aluminum (with phosphoric acid). Etched), silver (K12 etchant for release), and copper (K12 etchant for release). In addition, fluoropolymers such as CYTOP® may also be used to trim the surface that receives the structural layer of the polymer, thereby enhancing the peel characteristics of the polymer and minimizing deformation. May be. By using a sacrificial layer of material and / or trimming the surface, deformation of the target structural layer may be minimized.

  Of course, other variations of device cell fabrication may be used, and the above-described embodiments are in no way intended to be limiting. For example, any layer of the device, including but not limited to polymeric materials having relatively low flexibility factors such as polyolefins, polyesters, methacrylates, and polyimides, and stiffer polymeric materials such as polycarbonate and polystyrene, etc. It is contemplated that other polymeric materials may be used.

  Further, the working electrode may be made using techniques in addition to those described above. For example, if a support layer is created for the device, the support layer may be wired with electrical connections (by metallization). The electrical connection extends from a zero insertion force (ZIF) connector located on the front side, side edge side, or back side of the support layer to a particular desired location on the electrode. Also, the metal component of the electrode may be electrodeposited before, during or after making the electrical connection. ZIF connectors located on the front side, side edge side, or back side of the support layer may then be connected to a controller that is programmed with a particular arrangement and configuration of the device's cells. When exposing the working electrode to a particular polymeric material that may include an enzyme or antibody, the controller provides a signal to the selected cell. The selected cell will receive that particular polymeric material at a particular location of the electrical connection termination. For example, the controller applies a specific electrochemical potential to the selected electrode with respect to ground, creating an anode or positively charged site. The selected electrode may then be electrochemically grown with a coating or film made of the polymeric matrix, thereby encapsulating the working electrode. The process may be repeated with different polymer matrices until all working electrodes are coated. This allows each cell to have an electrode coated with a specific detection coating based on the overall device configuration. The sensing coating may be adapted to sense electrical properties including but not limited to electrochemical current, capacitance, resistance, voltage-like potential, or electromotive force. In some embodiments, all working electrodes on a single device may have the same coating. In some embodiments, half of the working electrode may have one type of coating, while the other half may have a second type of coating or the like.

  When the entire device is manufactured, the same electrical connections used to grow the electrodes also span the detection and reference electrodes if the material is present and the electrical properties of the material may be measured. May be used to apply a potential. Electrical properties may include current, capacitance, resistance, voltage-like potential, or electromotive force, depending on the polymer matrix used to coat the electrode. Electrical properties may be correlated to specific properties of the material, such as the concentration of biomolecules in the material, or physicochemical properties such as pH.

  In certain embodiments, the detection electrode may be grown to include nanostructures on its surface, increasing the conductivity of the electrode. Polypyrrole (PPy), a conductive polymer, is a material currently used for nanowires and nanotubes. Nanowires and nanotubes form the basis for electrochemical biosensors and nanostructures because of their high environmental stability, electronic conductivity, ion exchange capacity, and biocompatibility. Taking full advantage of the already high surface area to volume ratio produced by a single nanotube or nanowire structure, nanostructured films resembling nanoglass have been made. Ppy is electrodeposited inside the small holes 250 of the template 252. The template is made from a sacrificial template media material such as aluminum oxide (alumina) shown in FIG. If the nanowire array 254 is grown from PPy, the sacrificial template 252 may be removed by etch or any other suitable process, thereby leaving an array of PPy nanotubes 256 in the electrode base layer. Using this nanotexturing method, it has been found that polypyrrole can yield better conductive performance than bulky PPy films, since the polymer chain alignment is consistently along the wire axis.

  Nanostructured membranes may be manufactured by methods similar to those described above, providing a simple and efficient biocompatible environment for incorporating proteins into the controlled matrix of grown PPy nanowires. Good. Conductive electroactive polymers including biologically active molecules (eg, enzymes, antibodies, cells, and DNA) may be widely used in biosensor applications and signal generation mechanisms. For conductive polymers, pulse amperometric detection and impedance spectroscopy have been found to be optimal for generating and analyzing antibody-antigen (Ab-Ag) signals. The PPy electrode was prepared by electropolymerizing monomer pyrrole with constant current on a Pt or Au electrode polished from an aqueous solution containing anti-human serum albumin (anti-HSA). Using cyclic voltammetry (CV), it was found that no reaction was obtained using a control polypyrrole that did not incorporate the antibody, whereas HSA interacted reliably with these anti-HSA detection layers. . The use of nanostructures makes it possible to immobilize larger amounts of enzymes and receptors, thereby increasing sensitivity.

  The stand-alone PPy nanotube array shown in FIG. 26 was configured as a means to circumvent three potential concerns with pure Ppy nanowire arrays. First, there may be a large series resistance observed in the nanowires due to its length (up to 50,000 nm) and due to the field effect induced by the immobilized antibody binding to the antigen. Gold nanowire cores are not affected by this. Second, because the elastic modulus of PPy (about 80 MPa) is at least an order of magnitude less than gold (1.6 GPa), the gold core structure is stiffer and less likely to collapse or stick to each other during processing or sampling. . Third, series resistance can also reduce the collected current and, consequently, the detection sensitivity, but during processing, during electrochemical deposition, the conductivity of the conductive base electrode It can be transferred to PPy nanostructures at the end that are not close but do not accept antibodies well.

  An alternative manufacturing scheme utilizes metal (Au) nuclei and can be described in four steps. 1) a step of forming an anode alumina template similar to the template shown in FIG. 26 on a gold film, and 2) a step of electrodepositing gold into the small holes of the alumina small holes from the underlying flat gold film electrode. And 3) selectively etching the alumina template to expose the gold nanowires, thereby leaving all metal nanostructures 258 as shown in FIG. 27, and 4) a Ppy film as shown in FIG. Electrodepositing 260 nanolayers onto all exposed gold surfaces. In effect, since the gold nanowires are incorporated to run electrically parallel to the conductive PPy nanotubes, this method can reduce the resistance path provided for electron flow inside the nanotubes. When compared to nanotubes, there appears to be an effect of reducing the resistance path for the nanowire, and for the same applied voltage there should be a greater amount of signal for the nanowire. In addition, a larger amount of signal improves the sensitivity and overall performance of the biosensor of device 12. Another variation of this process is that Au can be exchanged for carbon-based nanotubes. The carbon-based nanotubes are vertically deposited on a substrate using a standard growth process. This is followed by Ppy coating. One possible advantage of this process is that the carbon nanotubes are already conductive and do not require any template for their aligned vertical growth.

  Other manufacturing techniques that may be used to make nanopatterned electrodes are based on related but different technologies that are compatible with detection chip device manufacturing. Sensing chip device manufacturing includes, but is not limited to, dielectrophoresis, electrostatic deposition, and deposition (between two electrodes that are on the device or between one electrode on the device and the external reverse electrode). . The material so deposited is usually a polarized nanotube or a polarized nanowire. In one embodiment, the carbon nanotubes may be deposited on the detection electrodes and arranged to be parallel between the detection electrodes or perpendicular to the detection electrode plane. These nanopatterned materials deposited on the sensing electrode show large conductivity changes between the two probe electrodes when they absorb / adsorb specific analytes using specific receptors there is a possibility.

  In the related manufacturing techniques described above for forming PPY nanorods, patterning is applied to one tip of a very thin metal filament, including but not limited to aluminum, to form molded PPY nanorods or to deposit carbon nanotubes. can do. The resulting metal tip is covered with a dense deposit of nanostructured material that is aligned with the wire itself. The other end of the conductor is connected to the control and communication portions of the device. Mechanically, this material is flexible and easily deformable. Electrically, the material is extremely conductive. In the neurological applications described below, the nanopatterned electrode material mimics the ciliary structure that naturally occurs at the axon tip. Indeed, the nanopatterned material may be coated with a thin layer of PPY to which nerve growth factor is immobilized. Neural tissue may grow in the direction of the nanopatterned material and attach to the nanopatterned material in such a way that the cells live and function properly and indefinitely and maintain good electrical contact. .

Cavity Filling FIG. 21 illustrates an embodiment of an apparatus and method for filling a cavity 28 for a cell that includes the cavity 28. Cavity 28 may be formed by the molding process described above, or while a thick support layer (eg, SU8) 80 is spin-coated on a layer of another material, such as PDMS, that makes up thin film 82. Similarly, at least one capillary hole 84, preferably two capillary holes, may be made such that they extend through the support layer 80 parallel to the cavity 28. As shown in FIG. 21, the thin film 82 may also include capillary holes that are substantially aligned with the capillary holes 84 of the support substrate 80. Another layer of polymeric material, such as PDMS, may be used as the sealing layer 86 to seal the cavity 28 on top of it after the cavity 28 is filled with material from the material supply 88. When the thin film 82 is ruptured using the above-described foaming technique to provide access to the cavity 28 during use, the sealing layer 86 is preferably thicker than the thin film 82 so that the sealing layer 86 does not rupture.

  In certain embodiments, the sealing layer 86 also includes at least one capillary hole 90. The capillary holes are initially aligned with the cavity 28 so that the material to be supplied to the cavity 28 may pass therethrough and enter the cavity 28. In certain embodiments, the sealing layer 86 includes two capillary holes shown in FIG. 22 that are separated by the same distance as the two capillary holes 84 of the support layer 80. The availability of two capillary holes in the sealing layer 86 provides the option of using a push-pull mechanism during filling of the cavity. One of the two capillary holes may be used as an inlet for the material, and the other capillary hole may be used to remove excess air from the cavity 28. An interface may be created between the material supply and the capillary hole to fill the cavity 28 with material that will be delivered to the substance.

  As shown in FIG. 21, an interface 92 may be provided to facilitate filling the cavity 28 with material from the material supply 88. The interface 92 is disposed on one side of the glass carrier 94, which is configured to interface to a glass carrier 94 with a reservoir 96 and a capillary hole 98 made therein, a material supply 88. , A polymeric material layer 100, such as PDMS, and a release layer 102 on the opposite side of the glass carrier 94 that is configured to interface with the sealing layer 86 of the sampling, analysis and delivery device. As shown, the release layer 102 also includes at least one capillary hole 104 extending therethrough that is configured to be substantially aligned with the hole 90 of the sealing layer 86.

  The glass carrier 94 may be a photo-etchable glass, and the reservoir 96 and the holes 98 in the glass carrier are made by powder blasting. The layer of polymeric material 100 disposed on the top surface of the glass carrier 94 includes a hole 106 therethrough. As shown in part A of FIG. 21, the layer is shaped to receive a plug 108 at the end of the hose 110 that is connected to the material supply 88. This can help to ensure that the plug 110 is positioned and held in place when the cavity 28 is filled.

  The layer of polymeric material 100 may be bonded to the side of the glass carrier 94 that contains the reservoir 96, with the holes 106 in the layer of polymeric material 100 aligned with the reservoir 96 in the glass carrier 94. It may be arranged. This allows the material to be supplied by the material supply 88 to fill the reservoir 96, which is a cavity 28 through holes 98, 104, 90 in the various layers 94, 102, 86, respectively. It may be discharged inside.

  When material is supplied to the cavity 96, the sealing layer 86 of the device may move (eg, slide) relative to the support layer 80 to seal the cavity 28, as shown in portion B of FIG. . The capillary hole 90 of the sealing layer 86 is aligned with the capillary hole 84 of the support layer 82 as shown in part B of FIG. 21 to ensure that the sealing layer 86 moves to a position where the cavity 28 is securely sealed. May be. If it is determined that the sealing layer 86 is in place (eg, the cavity 28 is sealed), the sealing layer 86 may be coupled to the support layer 80 as shown in portion C of FIG. It may be peeled from 92.

  As described above, the cavity 28 of one cell 18 may be filled and sealed. In certain embodiments, the glass carrier may be configured to provide a channel to all cells 18. All cells include a cavity 28 located on the same device 12. In this way, the cavity 28 may be filled and sealed at the same time. 22-24 show top views of such a configuration. Specifically, FIG. 22 shows a single cavity 28 with a reservoir 96 shown as a square and a hole 106 in the polymeric material layer shown as a circle, thereby filling the cavity 28. And where the plug 108 is located relative to the cavity 28. Similarly, FIG. 23 shows five cavities 28 being filled simultaneously, and FIG. 24 shows 25 cavities 28 being filled simultaneously. In each of these figures, a channel 112 in a glass carrier (not shown in FIGS. 22-24) that routes material between the reservoir 96 and the through hole 98 terminates just above the cavity 28. 22 and 23, there are two capillary holes 84 in the support layer and parallel to each cavity 28. Although two reservoirs 96 are shown in each figure, as described above, one of the reservoirs may be used to receive air that is exhausted as material enters the cavity 28. The illustrated embodiment is in no way to be considered limiting, but is provided as an example of how the cavity is filled and sealed with material.

  For example, it is contemplated that the support layer 80 may interface directly with the release layer 102 without the sealing layer 86 being deposited on the support layer 80. In such embodiments, the cavity 28 may be filled and the sealing layer 86 may be applied to the support layer 80 after the support layer 80 is peeled from the interface 92 so that the cavity 28 may be sealed. Good.

Connector For simplicity, the implementation of a prototype of control functions in a simplified system is described here. The configuration of the sampling, analysis and chemical delivery device 12 was changed to fit a zero insertion force (ZIF) connector. The chip thickness was adjusted to 150 μm +/− 10 μm for insertion reproducibility, and the connector pad pinout was drawn to match the 300 pitch zigzag connector position. It is desirable to create a “bottom” tip contact that allows a flat connector and tip surface that can be pressed against the subject's body. That is, the connector body does not have a step that prevents good flat contact with the skin on the horizontal plane. In other embodiments, the chip contacts may be made on the front or side of the device. The main body of the connector is a dual-in-line package that is 1.8 mm high on the surface and can be attached, and is equipped with a ZIF slider mechanism that inserts the chip into place when correctly inserted. This allows the sensor chip of the sampling, analysis and chemical delivery device to be quickly and reproducibly modified, but the normal movement of the animal to accept multiple insertions and under experimental studies It was found to be robust enough to resist the force of trying to pull the chip from the socket out of the socket. The parts may be made in various widths corresponding to a range of 17 to 91 pin contacts. In our development studies, those corresponding to pins 31 and 61 were used. For example, a 61-pin connector may be coupled to a wire cable or the connector may be used with a flexible multi-conductor Kapton tape. In the integrated system, the connector may be firmly connected to the controller body. Connectors are available in both tape and open reel packages for economical automated assembly and manufacturing.

  In some embodiments, the connector may be made in the following manner. The process may begin with a core substrate made from a flexible material such as polyimide or kapton. The core substrate is typically 0.001 inch thick (1 mil = 25 microns) and has a thin conductive copper film (9 microns) superimposed on both the top and bottom surfaces. As shown in FIGS. 29 and 30, device 12 may incorporate a microheater 270 having a 1 mil sweep width on the bottom or skin surface. The electrochemical sensing electrode represented by 272 in FIGS. 29 and 30 may be separated from the heater 270, but is in close proximity. Rupture of the stratum corneum by applying a current pulse to the microheater 270 may produce a temperature of about 130 ° C., which, if continued for about 30 milliseconds, will rupture individual stratum corneum cells. Sufficient to allow interstitial fluid to appear on the skin surface. The proximity of the two electrochemical electrodes 272 allows specific detection of the target biomolecule. One of the two electrochemical electrodes is gold while the other has electrodeposited polypyrrole embedded with biomolecular enzymes and redox mediators in some embodiments, or antibodies in other embodiments. The bottom dielectric layer 274 supports a support substrate 278 that may have a plurality of capillaries 276 extending therethrough. The copper layer 280 may be deposited on the top surface of the support substrate 278, but another copper layer 282 may be deposited on the bottom surface of the support substrate 278 prior to the deposition of the dielectric layer 274. The top layer of the flexible circuit 280 may have a bond pad 284 that may be electroplated with copper to provide a high ridge or mesa. The ridge 284 helps provide reliable electrical connectivity by the detection circuit. The detection circuit may be integrated into a second flexible circuit (not shown) that may be coupled to the top ridge 284 of the device 12. The sampling site comprises an array (eg 4 × 4 as shown in FIG. 29) with connecting wires for applying the proper voltage during sampling and during electrochemical detection and deposition (post-process). May be arranged. In the illustrated embodiment, the double-sided flexible substrate has functional sensors and interconnect wiring on the bottom side, while the top surface includes interconnects and contact pads for coupling to another flexible substrate. FIG. 31 shows how the device 12 may be placed over a large substrate area. The large area is about 10 inches × 12 inches. The thick lines between the devices 12 are connected together to form a common point for the charge that will be applied prior to device isolation. Also shown are interconnections for certain post-treatments, which may include conducting polymers immobilized with enzymes, and interconnections for imparting an appropriate charge during electrochemical detection.

The control device and communication device controller 14 is generally composed of two parts. The first is a computer interface wireless data collection system that can address up to 64 remote sensor nodes and can manage the authentication of each node while at the same time each node has its memory You can query the contents of the buffer. The second part is the sensor node. The sensor node consists of five functional parts such as RF communication (using unique authentication), microprocessor controller, multiplexer, analog circuit power supply and A / D converter, and multiple sensor inputs. The microprocessor may be programmed for a specific application. Analog circuitry, multiplexers, and input lines may be specially configured for the specific devices used in the system. The range of communication may be about 300 m or more. In real time, the controller 14 calculates the necessary series of electrical signals required to perform sampling, electrochemical analysis, and continuous logging of results and chemical emissions.

  The communication device 16 performs various operations including identifying the system, identifying the interrogator, transmitting stored information, and allowing reconfiguration of the control program. Allows an external device to query the control device 14. Reconfiguration can open several sampling cells 18 on the surface of the device 12 and test simultaneously on the same analyte or multiple different analytes, or choose to increase, decrease or stop chemical release, Or it may include changing the sampling frequency due to a detected stability or abrupt change in the measured concentration, or adjusting the details of the pulses used to rupture the stratum corneum. After an anticipated number of weeks of monitoring, the electrochemical detection cell 18 may all be used up, or all encapsulations may be opened and the disposable sampling, analysis and delivery device 12 must be discarded and the same Another device with a configuration or a different configuration may be inserted into the system 10.

  FIG. 20 is a block diagram illustrating some components of the controller 14 and the communication device 16. In the system 10, the material, body fluid, or tissue under test interacts with the sampling, analysis and delivery device 12. The device may be disposable, removable and manufacturing configurable. The components of the controller device 14 are a connector device, a multiplexer, an electrochemical potentio-galvano-stat, a break current detection circuit and a drive circuit, a controller, a measurement microprocessor, logic, adaptive sequencing, data storage, operation And providing power management for voltage regulation and battery charging / discharging. Components of the communication device 16 may include a receive antenna, a wireless communication module, RF power management, and battery power storage.

  The controller 14 and communication device 16 may be a single integrated system located within the same device, or the controller and communication device are separate devices that interact via wires or wirelessly. It is considered good. The embodiments described above are in no way intended to be limiting.

  FIG. 19 shows random addressing of cell elements using row addressing and column addressing. The addressing method includes a column address conductive path 60, a row address conductive path 62, and individually addressed sampling, analysis and delivery cells 64. This method is used instead of parallel addressing where each cell has its own conductive path connected to the controller shown in FIG. For example, 1024 cells organized in a matrix of 32 rows and 32 columns can be sequentially addressed to the controller by a 64-bit connector. There are 32 connections for the controller for row addresses and 32 for column addresses. In order to perform sampling and analysis simultaneously, the controller needs to continuously switch between the sample analysis cell and the delivery cell.

Example Applications Using System Embodiments In certain embodiments, the system 10 described above may be configured to monitor and treat diabetes. Type 1 diabetes (T1DM) is an autoimmune disease resulting from the destruction of insulin-producing pancreatic beta cells. To manage blood glucose, patients with T1DM must receive multiple daily insulin injections or check the glucose concentration several times a day using an insulin pump. Despite new and improved treatments, normoglycemia is difficult to achieve despite studies showing that strict glycemic control reduces the incidence of secondary complications of diabetes. One consistent normoglycemic limit is the technology currently available to patients to measure glucose concentrations. There are many competing techniques for collecting body fluids (eg, blood sweat, tears) and analyzing them with optical, electrochemical, and spectroscopic methods for glucose monitoring. However, all have varying degrees and types of invasiveness. In marked contrast to the available methods developed so far, the approach described in this application, coupled with dynamic sampling of interstitial fluid, is a superficial and highly controlled invasiveness (micro-temperature and cutaneous). Only with electric field-assisted tearing of the microscope section).

  The primary measurement target is glucose and the secondary targets are glycosylated hemoglobin and glycosylated protein, insulin, and cholesterol. Rupture of the skin allows about 10 nanoliters of interstitial fluid to diffuse to the stratum corneum surface with the aid of capillary forces. The glucose concentration in the blood and interstitial fluid averages about 90 mg / dl and changes from a dangerously low 40 mg / dl to a high one of 600 mg / dl. Become a healthy person. Minimally invasive sampling of interstitial fluid is performed at any one of several parts of the body, the most convenient inner arm or leg, and the trunk. The adhesive may hold the flexible device 12 in contact with the area intended for sampling. A small amount of interstitial fluid can be encapsulated in one of the cavities 18 and stored there for subsequent analysis. There is no need to modify the sample. Each electrochemical analysis is completed after about 10 seconds. More than two analyzes may be performed in parallel to improve accuracy and prove statistical measurement uncertainty. Rupture of the stratum corneum may be maintained for hours using periodic heating and voltage pulses, and optionally measurements can be performed accurately within this time frame. The time to equilibrate the concentration inside and outside the stratum corneum is on the order of seconds due to the very short diffusion path (<50 microns).

  In another embodiment, the monitoring device 12 may be configured to store a calibration sample of the analyte of interest in the device cavity 18. The calibration sample can be released and measured at the command of the control and communication system, and the accuracy of the measurement can be further improved.

  In another embodiment, the monitoring device 12 delivers a drug (such as about 1 unit dose), such as human insulin, that can be released one at a time under control and communication system commands, either in dry powder form or in water form. However, it may be configured to be stored in a plurality of device cavities 18. Drug administration and delivery decisions may be made by medical personnel, individual patients, or automatically. The latter configuration is called closed loop control. If the command and control system 14 is configured to maintain glucose within a certain range and the glucose monitoring device detects hyperglycemia, insulin may be administered in a certain order. First, a partial dose is administered by opening one particular cavity, releasing one unit of insulin. If hyperglycemia persists after several minutes, the second partial dose is administered until the complete dose is administered, and so on. This pulse mechanism mimics the mechanism of a healthy pancreas and releases insulin into the bloodstream in response to increasing glucose concentrations. It is possible to vary the complete dose based on the observed response to pulsed drug delivery, and if the individual response to insulin in diabetic patients being treated on a daily basis changes, they must do the same .

  In certain embodiments, the system may be configured to monitor a newborn's bilirubin concentration. Bilirubin is a naturally occurring toxic waste product of the body that is formed during the normal breakdown of hemoglobin. Hemoglobin is an iron-containing oxygen-carrying respiratory protein found in red blood cells. Neonates usually have a high rate of erythrocyte destruction, especially during the first few days after birth. This high rate can overwhelm the metabolic capacity of underdeveloped neonatal organs. A high erythrocyte destruction rate usually increases the bilirubin concentration, which can cause the newborn to have jaundice. Neonatal abnormally high bilirubin concentrations can lead to cerebral palsy, hearing loss, and other physical abnormalities. In extreme cases, not treating high levels of bilirubin can even cause death. Therefore, it is desirable to detect and treat an abnormal concentration of bilirubin as soon as possible. Currently, bilirubin is measured by taking a blood sample from a vein or via sputum lacing. Such invasive sampling methodologies in neonates are undesirable because they can cause significant discomfort. By configuring the system described above to measure neonatal bilirubin concentration, a non-invasive method may be provided. The non-invasive method may be performed by the newborn parent after the newborn is discharged.

  In such a system, the target biochemical to be measured in the substance is bilirubin, and the working conductive electrode of the device is immobilized within a polymer matrix of, for example, induced polypyrrole (Ppy), such as bilirubin oxidase. It is comprised so that a bio-recognition molecule may be included. Since bilirubin oxidase is an oxidizing agent that results in the production of positive ions when exposed to bilirubin, a local potential shift occurs. The shift is proportional to the concentration of bilirubin present in the substance coming into contact with the working electrode. The electrochemical cell described above may detect this potential shift and provide a signal to the controller. Once the sensor is calibrated, based on the signal from the cell, the controller can determine how much bilirubin is in the substance.

  In some embodiments, the system 10 is configured to monitor a human body's ability to efficiently utilize its fuel resources. The actual energy source used by muscles in any kind of activity is ATP (adenosine triphosphate), which is aerobic or anaerobic, i.e. with or without oxygen, respectively. Can be generated. An oxygen-free energy source is typically used at the start of exercise and when the exercise intensity is greater than that which can be tolerated by an available oxygen supply using an aerobic source. The point at which this occurs is called the “anoxic work threshold”, which is determined by the intensity of exercise / behavior, and because the body is unable to meet all its oxygen needs, Rises dramatically above the threshold. Phosphate and lactate systems are two energy sources in the anoxic system. Phosphate systems are for short-term energy explosions, while lactate systems are for strong long-term behavior. The lactate system uses sugars stored in muscle and blood, but the former is often utilized before the latter. Anaerobic degradation of glucose results in the formation of lactic acid in the muscle, but in the presence of oxygen, lactic acid is easily converted to ATP and fueled. Therefore, lactic acid may not accumulate if the exercise intensity is low enough to oxidize the lactic acid completely to make more ATP. If the production rate exceeds the removal rate, lactic acid begins to accumulate, the blood flow pH value (acidity) rises, the muscles do not function properly and begin to exhibit the familiar “depletion”. For highly trained athletes or for more leisure enthusiasts, it may be desirable to monitor stamina without reducing maximum performance.

  By using an embodiment of the device 12 that incorporates both glucose and lactate detection sites, the system 10 may be used to monitor the user's maximum output without compromising the anaerobic threshold. . The control and communication devices 14, 16 in communication with the device (see FIG. 1) provide information regarding the individual's glucose and lactate concentrations to their IPOD (registered trademark), portable information. It may be configured to relay to a terminal (PDA) or mobile phone and to show a graphical display that tracks the user's energy output with respect to anaerobic threshold. The device 12 is not intended for continuous use for applications in the athletic and food industries, professional and Olympic athletes, and occupations that require strong energy output (eg, firefighters, construction workers, etc.). Invasive non-intrusive monitoring may be provided and may be used for both animals, ie, motion animals (racing horses and race dogs) and work animals. By using the device 12 described above that includes a sensor cell configured to detect diffusion gases in the interstitial fluid, the pO2 concentration may also be monitored directly. Also, the output signal from such a sensor cell may be combined with an independent oximeter. The oximeter transmits its output to the controller 14 and the communication device 16 in real time. In addition, monitoring coronary signs in athletes (eg, monitoring C-reactive protein and / or homocysteine) allows team trainers and physicians to be aware of dangerous situations I think that. It may be possible to alert the team's health care workers to withdraw rivals before death or serious injury. In addition, testing drugs such as steroids has health benefits for athletes.

  Based on measurements of a person's glucose concentration, it has been found that a direct correlation exists between an individual's arousal state and fatigue state. In fact, this debate has occurred with respect to chronic fatigue syndrome (CFS). Chronic fatigue syndrome is characterized by the onset of debilitating persistent fatigue and energy loss that lasts more than 6 months and is not due to any other medical or psychiatric disorder. It has been found that glucose inhibits certain types of glucose sensing neurons. The neurons produce a microprotein called orexin, which is a central regulator of consciousness. If the orexin neuron firing mechanism is affected primarily by very subtle changes in glucose, this can lead to changes in arousal, narcolepsy, and even obesity status. This raises the possibility that modulation of orexin cells by glucose has a broader behavioral role and contributes to continuous daily readjustment at wakefulness and consciousness levels. The previously described device 12 embodiments may provide clinical insights into CFS by non-intrusive monitoring of glucose concentration, and serve as an early warning indicator of emergency fatigue in the general public. It is contemplated that it may be done. This may be applied to occupations where it is most important to be careful. The occupations are, for example, military personnel, occupational pilots, school bus drivers, truck drivers, air traffic controllers, among others. In certain embodiments, the aforementioned system 10 may also include an alarm. If the device 12 being used to monitor the individual detects that the individual's glucose concentration has reached a concentration indicating that the individual is becoming fatigued, the alarm will start to keep the individual ready. May be.

  The potential for a successful resuscitation from severe traumatic hemorrhagic shock places importance on speed for both combat wounded and civilian trauma victims with hemoptysis. There are many research areas where non-invasive semi-continuous battlefield deployable monitors are thought to successfully increase the chances of resuscitation. First, with respect to both triage and effective treatment, there is an advantage in identifying the exact moment of shock time when an injured soldier is discovered by a physician. The typical hematocrit, plasma glucose, and lactate values observed during hemorrhagic shock are known and the following four progressive phases have been identified. 1) early compensatory phase (homeostasis maintenance mechanism), 2) maximum compensatory phase, 3) early decompensated phase (in blood replacement fluid, close to irreversibility), and 4) late decompensated phase (leads to death). If the resuscitation fluid can be administered before the late decompensated phase, the chances of survival are greatly improved if the kidney and liver are ischemic and severe acidosis occurs. Secondly, plasma ethanol and its metabolites can be time-monitored as a marker of the degree of liver function recovery by mixing extremely low concentration ethanol into the resuscitation fluid. From a research perspective, quasi-continuity (eg, read every 2 minutes) is believed to reveal the effectiveness of both the amount and composition of the resuscitation fluid used. It has been demonstrated that small doses of glucose (rather than blood) can alleviate systemic acidosis while delaying the onset of a fatal decompensated phase. Third, in order to know even more accurately, if an individual is in a compensatory / decompensated time course, the individual will be pre-injured with respect to their plasma glucose and lactate levels (and alcohol levels). They should have a “reference” and can vary significantly from normal resting values due to the extreme stress and effort encountered in combat.

  In this context, from combat, ie from rest to effort and possible injury, using the device 12 and system 10 described above, each individual's lactate, glucose, bilirubin, pyruvate, and (used as a marker) It is highly desirable to monitor ethanol non-invasively. Knowing both the bilirubin concentration as well as the rate at which ethanol is removed by the liver provides additional data on liver ischemia. In the case of a disaster in which the monitoring device functions as both triage and emergency medical equipment, only the frequency of sampling changes from low frequency to high frequency, quasi-continuous. When combined with other physiological devices, complete photos may be available to commanders, physicians, or military physicians. The body's response to trauma and large area burns is very similar to that of hemorrhagic shock, so the sensors described above assess the severity of injury, resuscitation, and response to treatment, especially the properness of the liver. As well as useful features.

  In some embodiments, the system 10 described above may be configured for use in monitoring multiple objects under the influence of the same conditions. For example, each platoon soldier may have the device 12 described above attached to his / her skin, and the device 12 may communicate with the central headquarters. In this way, each soldier may be individually monitored for multiple abnormal conditions. For example, glucose concentration and lactic acid concentration may be monitored. If the soldier is healthy, the individual's stationary reference can be measured and stored. Blood glucose and lactate levels can be monitored as soldiers work hard. Extreme effort can be recognized by hypoglycemia and elevated lactic acid levels. This physiological condition can affect a soldier's ability to act in combat or subsequent intense work situations. Thus, the command unit may decide to call the soldier to rest until it is detected that the glucose level and the lactic acid level have returned to normal values. Furthermore, if the soldier is injured, the sensor can be activated and measured quasi-continuously, and the system 10 may be used in the manner described above as a life-saving emergency medical device and triage device.

  In certain embodiments, the subject may be an animal in a herd or in a common environment. For example, the device 12 described above may be attached to some or all of the ears of a herd. The device may be configured to measure an animal's glucose level over time. The central command node may be placed on the gate at a distance from the flock, for example a quarter mile. The command node may be configured to send a signal to query all devices within its range to find those animals that may be suffering from the infection. If monitoring an animal's glucose level detects an increase in value, which indicates that the animal may be suffering, in order to more accurately determine the type of cause of the disease, Suffering animals may be separated from the herd, examined and tested. The device may be configured to use either antibody or enzyme coatings. Antibody or enzyme coatings target specific abnormalities in the subject, which are reflected in specific disease markers present in blood, milk, urine, and the like. In this way, the system may function as a wireless sampling point monitor. The monitor relays the analysis result to the command node in real time and can immediately signal the presence of the infection. In certain embodiments, the system may be used to analyze milk samples to ensure that the milk is safe for consumption before loading the milk into a refrigerated tanker at each collection point.

  In certain embodiments, the system may be configured to monitor the viability and functionality of organs and tissues prepared and stored for surgical implantation. For example, certain biomolecules that may be present in organs and tissues may be monitored, and it may be ensured that the organs and tissues are still suitable for transplantation. These biomolecules may be desired or unnecessary.

  The simplest test of the viability of most tissues is an assessment of glucose, lactate, blood oxygen gas, carbon dioxide, and pH values. As long as these concentrations are in range, the cells of the tissue can maintain normal metabolism. Other tests are appropriate for assessing the functionality of more complex tissues such as liver, heart, lung, kidney, and nerve tissue. For example, as described above, liver function can be monitored by analyzing the ability of the liver to remove bilirubin concentrations and molecules such as ethanol. The device 12 configured for these applications is similar to the structure described above with the possibility of exceptions. An exception is that if an organ or tissue is exposed during surgery, for example, the device 12 may be applied directly to the tissue to evaluate interstitial fluid without the complications of the stratum corneum-like barrier. Certain organs have outer membrane tissue that functions like a barrier. In this case, the barrier rupture may be achieved by adjusting the temperature level and the voltage level according to the characteristics of the outer membrane. In stopped organs that use artificial circulation to maintain function, device 12 may be used to monitor both input and output circulation fluids, and to monitor certain molecules such as ethanol in the liver. The ability to remove may be evaluated. In order to measure accurate electrical signals over time without damaging the neurons themselves, the neural tissue may require a special configuration of the device 12, as described below.

  Similarly, a small amount of tissue may be excised from the body by surgery or a minimally invasive probe. The small amount may be conveniently placed on the top surface of the device 12 for analysis of the contents of the interstitial fluid. The device 12 may measure the sample quickly (eg, in a few seconds) before the tissue begins to degrade, and may measure trace concentrations of biomolecules. The exact assessment panel performed on the biopsy sample will depend on what disease is under investigation. In most if not all cases, a very small volume (nl) of interstitial fluid to be analyzed should not disrupt the biological state of the specimen tissue.

  In certain embodiments, the system 10 described above may be configured to be used for early detection of cancer and diseases including but not limited to infections such as influenza, malaria, and dengue fever. In such embodiments, device 12 (eg, the detection electrode of the device) may be prepared with antibodies. The antibody binds to a particular antigen and its abnormal concentration is associated with an increased risk of disease or infection, or simply the presence of a growing mass or infection. In many diseases, the number of biochemical markers associated with the disease is increasing. In the case of prostate cancer and breast cancer, the specific antigens that are regularly tested are prostate specific antigen (PSA) and prostate membrane specific antigen (PMSA), estrogen, epidermal growth factor (EGF), and insulin growth Factor (IGF). Antibodies are available for all of these antigens and can be selectively immobilized on the electrodes of device 12. All antigens are smaller than 60 kDa and are found in the interstitial fluid. Therefore, tests available for these cancer markers are not only percutaneous aspirates, but also urine aspirates and chest aspirates. For maximum sensitivity to the lowest concentration, the method described above for nanopatterning the electrode may be used. For viral or spore infections, two different configurations of device 12 may be required. The first measures exposure to the virus or spore and uses an electrode with antibodies raised against the subject virus, such as the H5N1 virus. Spores are detected by looking for specific metabolites from the outside. The electrodes may be nanopatterned as described above to obtain the lowest concentration and to give the earliest warning regarding an individual's exposure to infectious agents. People do not get sick immediately upon exposure. Only when the external concentration of influenza virus is high, the virus begins to circulate in the blood. Viruses and spores are too large to be found in the interstitial fluid, but viral fragments (hemagglutinin and neuradminidase) are found in the interstitial fluid. Antibody detection is possible within a few minutes at interstitial fluid concentration values of 1 million fragments per ml using such a configuration of device 12. The interstitial fluid concentration value is low enough to respond to antiviral drug therapy.

  In some embodiments, the device 12 may be configured to monitor and treat malaria of a parasitic infection. In certain embodiments, device 12 may be configured to monitor both the presence of parasites and markers associated with organ functions such as liver, kidney, brain, and lung. In particular, the device is made to be highly sensitive using both antibodies and enzymes for characteristic metabolites. The metabolites are inter alia P.I. It is released by parasitic protozoa (Plasmodium spp.) such as falciparum histidine rich protein 2 (PfHRP2), parasite lactate dehydrogenase (pLDH). Malaria affects these organs, eventually damaging them, resulting in a further deterioration in health called the disease burden. Each organ marker is measured to determine how far the concentration is from normal and to estimate organ stress or damage. In another embodiment, the delivery cavity 18 of the device 12 can be combined with artemisinin, either sequentially or in combination with primaquine and older drugs, either individually or automatically by the control and communication portions of the device, as determined by a healthcare professional. It may be configured to deliver. Now, parasites are more durable against older drugs. In certain embodiments, the device may be similarly configured so that the device monitors the disease and its burden and administers the drug or combination of drugs, such as to monitor and treat other diseases such as HIV and tuberculosis. Good.

  In embodiments, the system may also be configured for the treatment of cancer and infectious diseases. Several types of conventional gene therapy have been developed, tested, and used to treat cancer, but are non-invasive that quantitatively monitor microbiomarkers during an individual's response to a treatment protocol And there are few sensitive measurement techniques or systems. Only a handful of these are based on biomolecular pathways common to almost all radiotherapeutic and / or chemotherapeutic drugs, all in critical periods and throughout the treatment period. Does not provide the possibility of continuous monitoring. Proteins present in a programmed cell death biological sequence for apoptosis may be specifically targeted using the methods described above to create a nanopatterned polymer on the detection electrode of the device. . This includes, but is not limited to, the 17 kDa subunit of cleaved caspase-3 at biologically relevant concentrations, ie between 1 ng / ml and 0.1 mg / ml. Some of the proteins in cell apoptosis, such as the p54 protein and the 17 kDa protein, have been found to be useful as surrogate markers of efficacy for the majority of cancer treatments that induce cell apoptosis.

  In an embodiment, the system may be configured for sampling of bodily fluids within the subject's mouth. For example, the system may be configured to detect bacteria, viruses, microbial membranes, inflammation, and caries, and normal hormones, proteins, and metabolites to assess the overall health of the subject. Human saliva components and other oral fluids contain a large number of analytes of interest for screening, diagnostic, and monitoring applications, and samples can be easily obtained using a variety of non-invasive low-risk methodologies . Saliva is produced from plasma and glands, as such, plasma proteins and drugs enter the tissue interstitial space based on Stirling force and vascular permeability. Similar forces regulate interstitial proteins, low molecular weight materials, drugs, ions, and water flow rates across the buccal and gingival mucosa. The buccal mucosa provides an easily accessible area to access the tissue gap and sample interstitial fluid. The relative rate of movement across the mouth membrane from plasma to saliva can be determined by simultaneously measuring the concentrations of drug, plasma protein, and other analytes in the two body fluids. In general, the systems described above may serve potential applications in monitoring drug pharmacokinetics, forbidden drug detection, and plasma / interstitial drug or mediator concentrations. Furthermore, it is known that various synergistic interactions of microorganisms in the gingival crevice activate inflammatory mechanisms that can lead to caries, gingivitis, and other oral lesions. Specific patterns of microbial specific proteins, metabolites, host proteins, and degradation products of interstitial connective tissue, dental tissue, and bone tissue may be identified. By performing standard proteomic analysis (liquid chromatography / mass spectrometry / mass spectrometry, ie LC-MS-MS) after sampling the clavicular fluid, the pattern of unique or denatured products can be transformed into disease specific biology. Traces may be defined. From this point of view, we use three manufacturing techniques for non-invasive sampling of such body fluids and biofilms to obtain information about pre-disease conditions.

  In one particular configuration, the sampling device can be augmented by electrodes and microfluidic channels adjacent to the sampling cavity. The device performs isoelectric focusing and capillary electrophoresis (2-D separation). The result of the separation may be detected electrically as described above, or the separated sample may be stored in a cavity for subsequent analysis outside the device using mass spectrometry.

  Salivary diagnostics uses microcapillary “joji” for oral cancer, cariogenic bacteria in the development of caries (cavities), efficacy of therapeutic agents, illegal drug addiction, and detection of HIV or herpes virus And may be achieved by sampling from the area between the gingiva and the cheek. Oral biofilm (plaque exfoliation) along with gingival crevicular fluid collection using microcapillaries attached to a dental probe device can help in the detection of periodontal disease, Helicobacter pylori, and bad breath. Using the embodiments of the non-invasive sample collection device 12 described above, interstitial fluid may be sampled from a relatively permeable mucosa lining the mouth, thereby determining systemic disease and drug Inspection becomes possible. A rapid clinical diagnostic test that may be configured to be used as a screening mechanism to determine certain pre-disease conditions such as gingivitis or other illnesses may be performed using system embodiments. Good. An embodiment of the system is in the form of a “stick bar with candy” based on saliva for home or in-house diagnosis.

  A dental system may be used for two specific diagnostic areas, including but not limited to them. First, interstitial fluid, saliva, and saliva, to determine molecular biological signatures or saliva “fingerprints” that correlate with many pathologies such as risk factors, disease susceptibility, and general health Sampling other oral fluids. The second diagnostic area focuses on the effects of saliva and salivary constituents in the development of oral diseases, cariogenic bacteria and subsequent caries, and the role of immunoglobulin A (IgA) in preventing acid-producing colonization It is intended for inclusion.

  Three types of micro-sampling devices may be used with each device being specific to the type of oral fluid being analyzed. First, a bundle of microcapillaries may sample saliva from between the cheek region and the gingival region, while a plurality of microcapillaries attached to the dental probe may sample gingival crevicular fluid. May be used. The third micro-sampling device is a variation of the system used for glucose sampling described above for sampling interstitial fluid through the mucosa. This sampling device, unlike the first two, contains a small battery and is mounted on a voltage reducer-like device. The sampling process may be initiated by pressing a button while the device is pressed against the cheek. When the button is released, the sample may be stored. This structure may be used to collect saliva and transmucosal fluid for detection of whole body markers. Numerous studies have shown a high correlation between serum constituents (systemic indicators) and oral fluid from the buccal mucosa.

  A microcapillary bundle sampling device device composed of a microcapillary assembly may be used to collect a saliva sample from between the cheek region and the gingival region. The microcapillary collection unit allows the sampling of a significant amount of saliva over a specific distribution area using a handheld device that resembles an eye drop container. Individual microcapillaries are commercially available and are made of micron scale glass tubes that are covered with a polyimide outer sheath layer to provide rigidity and durability. Both the inner and outer diameter dimensions vary for each of these microcapillaries, with an inner diameter as small as 30 microns and an outer diameter up to 350 microns.

  In an easy-to-use handheld sampling device 300, in the embodiment shown in FIG. 32, an individual microcapillary bundle 304 may be inserted into a valve pipette 302 having a flexible valve 303 at one end thereof. It may be held in place by "embedding" them in a silicone elastomer called polydimethylsiloxane or PDMS306. In the illustrated embodiment, the sampling bundle 304 is mounted in a molded pipette (not shown) similar to the pipette 302 shown in FIG. 32, and then the silicone PDMS embedding material 306 is poured and cured. The PDMS 306 does not cover the ends of the microcapillary 304 at both ends so that sampling can occur through the capillaries, while using a valve allows for aspiration and distribution onto the lab-on-a-chip device. Upon curing, the synthetic silicone and microcapillary structure 308 or PDMS plug, two of which are shown in FIG. 33, may be removed from the molded pipette, resulting in other similar bundles encased in PDMS. So that it can be made. For sampling purposes, the user may use the valve pipette 302, and after removing the valve 303, the PDMS plug 308 may be inserted as far as it can go into the pipette 302. The tapered nature of the valve pipette 302 ensures that the microcapillary 304 is in the correct position during sampling and seals the open end of the pipette 302. Valve 303 may be reattached and saliva sampling may occur between the cheek and teeth. The PDMS plug 308 may be removed and stored for later analysis, or the sampled body fluid may be distributed directly into the separation device. The purpose of the PDMS plug 308 is to provide a new holder or vial that is used for each sampling procedure. The illustrated embodiment is in no way intended to be limiting.

  In some embodiments, a microcapillary-based dental probe device 400 as shown in FIG. 34 is provided. Periodontal exploration is the most commonly used method for determining the degree of periodontal lesions and the degree of clinical association with gingival teeth. Overall, a depth of space of about 1 to 3 mm is considered acceptable. The purpose of this microcapillary based device 400 is to sample the gingival crevicular fluid during a standard periodontal examination and measure the degree of connective tissue pocket depth. In certain embodiments of the system, the platform may be configured to receive a commercially available disposable and easy-to-use periodontal probe 402 made of flexible polymeric plastic as a device substrate. The periodontal probe 402 may be modified by using a microcapillary 408 having a very small outer diameter (about 100 microns), may be mounted on the lateral surface of the tapered probe 402, and cured PDMS silicone. It may be held in place by a 25 micron thin layer of rubber 410. Since the distal end 406 of the probe 402 has a nominal diameter of 0.5 mm (or 500 microns), the attached microcapillary 408 and the PDMS layer 410 only increase the lateral dimension of the device by 0.25 mm. By using PDMS, the overall probe flexibility may be preserved and there should be no side effects when introduced in vivo for a short time. Gingival crevicular fluid may be passively transported into the two microcapillaries 408 by capillary action. In another configuration, the microcapillary 408 may be connected to a suction device (commonly found in dental clinics) to gradually draw gingival crevicular fluid into the microcapillary 408. In another embodiment, a single microcapillary may be placed inside the probe shaft by forming a probe around the microcapillary using a molding procedure. The illustrated embodiment is in no way intended to be limiting.

  In embodiments, the device 12 and the system 10 may be configured for use to help maintain a balance between caloric intake and caloric consumption over time. The balance is due to basal metabolism and physical exercise. In this case, the device 12 may be configured to measure physiological history and biological history throughout the day for weeks at a time. In one example, basal metabolism includes exercise, subcutaneous muscle flexion, heart rate, skin temperature, skin conductivity, interstitial fluid oxygen and carbon dioxide concentrations, and interstitial fluid glucose, lactate, insulin, ghrelin And by monitoring the concentration of leptin. Motion is best measured by incorporating a single-axis or multi-axis accelerometer in the command and control part of the device 12. Motion and muscle flexion, and small detection pulses of blood (and thus measuring heart rate) through the arteries and veins under the flexible sensing portion of the device, when the device 12 that adheres to the skin deforms in the skin, A voltage generated by a piezoelectric layer (ceramic polymer) embedded in the substrate is used. The temperature may be assessed by measuring the change in resistance of a material having a known resistance temperature coefficient deposited between the measuring electrodes, or by using, for example, the heater material shown in FIG.

  Also, skin resistance may be conveniently measured by either two electrode techniques or four electrode techniques. In the two electrode technique, the resistance may be calculated from either a DC or AC current of a specific frequency passing between two open electrodes in contact with the skin and from a known applied voltage. In this measurement, electrodes in one cell of device 12 are used in the same way as electrodes in different cells of the same device 12 to achieve a few millimeters of tear and local skin texture, characteristics, or composition variability You may make it remove the influence of. In the four electrode technology, a single electrode is ideally used at each of the four corners of the device 12, and a known DC or AC current flows through the skin between two adjacent corner electrodes, , Measured between two opposing electrodes. This technique may remove the normally small uncertainty in contact resistance between the electrode and the skin, resulting in a value specific to the skin. If the effort results in a large amount of sweating, conductive sweat can alter the resistance measurement. AC measurements taken at different vibration frequencies may be used to separate the contribution of sweat to the conductivity of the fatty tissue under the stratum corneum of the skin and cortex. Biochemical assessment may result in a continuous scale or metabolism experienced inside the body, which contributes to 60% of total calorie consumption. The measurements are not only individually collected and stored by the controller 14, but may also be interpreted to provide the best estimate of total caloric intake. This total value may be displayed in either an analog or digital format and notify the individual of cumulative calorie consumption.

  Also, caloric intake, appetite control, satiety, fat production and consumption may be quantifiable using measured values. The glucose and insulin concentrations measured during ingestion and digestion may provide a quantifiable assessment of how many calories have been absorbed in a single meal. The actual number of calories absorbed is quite different from the calories estimated from the calorie calculation of the food being eaten. Tracking the postprandial ghrelin level indicates satiety. By measuring an increase in ghrelin concentration, the onset of satiety may be detected before experiencing a feeling of physical satiety, thereby avoiding overeating. By tracking glucose and insulin concentrations before a meal, a physiological signal of appetite may be measured. By making a comparison between calorie consumption and caloric intake when feeling hungry, an intelligent decision may be made whether to eat and how much to eat.

  As is known, weight loss occurs when the body consumes more calories than the body absorbs over several weeks. By closely monitoring the calorie balance, a person can maintain a healthy deficit for fat loss on a daily basis. Leptin is one of many peptides in biological systems that produce, maintain, and remove fat by sending signals to the hypothalamus. Measuring leptin provides a direct measure of how the body controls its fat mass. In addition, the electrical resistance (impedance) at four points when conducted at various AC frequencies may be correlated with the amount of adipose tissue in the current path. This electrical analysis in conjunction with leptin concentration allows the system control and communication devices 14, 16 to post not only the state of the deposited adipose tissue at the analysis point, but also the rate at which its amount increases or decreases. Become. All of these are valuable tools currently unavailable to individuals to control their body weight and body composition in a non-intrusive manner through conscious intervention and to assist in autonomous mechanisms of energy storage and use.

  Although the diet contains a sufficient supply of protein, fat, and carbohydrates, food intake often lacks certain essential minerals, vitamins, and acids. In one non-limiting configuration, the cells 18 of the device 12 may be prepared to measure these available concentrations in the interstitial fluid. These molecules are small and easily found in the interstitial fluid. In certain embodiments, the cell 18 of the device 12 is made up of mineral ions such as calcium and potassium, vitamins such as vitamin A, and acids such as folic acid by electrochemical, enzymatic and antibody mechanisms. It may be prepared to measure the concentration. These nutrient deficiencies are not necessarily reflected in obesity or weight loss, but are thought to result in osteoporosis and neuropathy.

  In an embodiment, the system 10 generally described above may be used for heart, blood vessel, and stroke monitoring. A heart attack that is technically myocardial infarction or MI has been diagnosed in the past based on the presence or history of changes in waveform characteristics in chest pain and electrocardiogram (ECG). Recently, creatine kinase (CK and CK-MB) blood test results have been used for diagnosis, while cardiac troponin has begun to be used. The main advantage of troponin is that it is extremely sensitive even for monitoring damage to the heart. Troponin, a protein with three isotypes (I, T, and C), is released from dead and damaged cells in the myocardium, so that the increase in value is even during a heart attack, even if it is mild. It may indicate that there is damage that might occur. Another advantage of troponin over CK-MB testing, especially TnI and TnT, is that it remains in the bloodstream for days after a cardiac event, allowing increased diagnostic time, and CK and CK -MB is released exclusively from dead muscle cells (definition of infarction), while troponin is released from dead and severely damaged muscle cells. Studies have shown that people with elevated troponin but normal CK and CK-MB suffer from the same results as those who meet more traditional diagnostic criteria for diagnosing heart attacks.

  The presence of plaque in the heart artery that carries blood to the brain is associated with stroke, either by sending a thrombus to the brain or by stopping the blood flow and causing a stroke. Also, people with atherosclerotic carotid arteries are more likely to have atherosclerosis within the coronary arteries and throughout the circulatory system, thereby similarly prone to heart attacks. Tumor necrosis factor and its receptor are inflammatory markers that are elevated in the blood in various infections, inflammation, and autoimmune diseases. Experimental evidence suggests that immune processes and inflammation are involved in the development of arterial thickening. Increased levels of tumor necrosis factor receptor may be a reflection of inflammatory processes that affect plaque formation.

  For such monitoring applications, the system 10 may be configured to monitor a specimen panel. Specimen panels show healthy heart and circulatory function and may provide early presentation of abnormalities that can lead to heart attacks, circulatory disturbances, and stroke. In certain embodiments, the cell 18 of the device 12 may be prepared to simultaneously measure the concentration of the analyte in the interstitial fluid. Samples in the interstitial fluid include C-reactive protein, tumor necrosis factor receptors 1 and 2, creatine phosphokinase (CK and CK-MB), creatinine, troponin, interleukins 1, 2 and 6, interleukin- 2 receptors, as well as tumor necrosis factor-alpha.

  In certain embodiments, the system 10 may be configured for monitoring substances such as smoking, drinking, anesthetics, and drug abuse, for example. Nicotine and its metabolic cotinine may be monitored transcutaneously using the system 10 described above. The oxidation of nicotine to cotinine is catalyzed by the cytochrome P-450 2A6 enzyme located in the cell mitochondria. Cotinine is often used to distinguish between smokers and non-smokers. Non-smokers are persons who are susceptible to passive smoking, also known as secondhand smoke (ETS). In contrast to mainstream smoke, sidestream smoke contains higher amounts of carcinogens such as ammonia, benzene, carbon monoxide, nicotine, and n-nitrosamine. Smoking supervision may be used by parents to ensure that children are never involved in tobacco abuse. More importantly, these two behaviors are the main single risk factor for sudden infant death syndrome (SIDS), so doctors may monitor pregnant women during pregnancy and after delivery. Also, more recent studies have shown that passive smoking components identified to date may affect respiratory neural control that may lead to sleep apnea and SIDS episodes. It is shown that there is. Studies have shown that the lungs of infants who die from SIDS have significantly higher concentrations of nicotine than control infants. Smoking may be continuously monitored using our non-invasive patch technology. System 10 may be incorporated with tamper-evident control. The tamper-evident control is likely to indicate that the device 12 has been removed from the user for an extended period of time while smoking. Device 12 may also be used on urine samples as a stand-alone in vitro tool. The urine sample is usually where cotinine is excreted from the body and is introduced into the sampling site on the device 12.

  Embodiments of the present invention use either enzyme detection using electrochemical reactions or specific fixed receptor sites that utilize antibody-antigen detection using resonant frequency shifts of oscillating pendant membranes. The device 12 may be used to monitor forbidden items, including athletic drugs, drugs of abuse (opiates, cocaine, narcotics), and alcohol abuse. Device 12 may incorporate several panels of detection cells to monitor individuals for various prohibited items, or may be specific for a particular substance.

  In certain embodiments, the system 10 is configured to utilize the vertically oriented microcapillaries previously described. The microcapillary is perpendicular to the skin surface. The sampled interstitial fluid may be withdrawn upward through the capillary due to capillary action. The same capillary may be filled with a gel such as polyacrylamide or agarose. The gel provides a semi-porous medium that allows sampled interstitial fluid to flow through itself. However, since capillary action may not be effective, the voltage may be applied using electrodes at the top and bottom of the capillary. Since the size of the capillary filled with gel is only a few hundred microns, the interstitial fluid is driven electrophoretically by applying a low level potential. Thus, since E = V / d, a low voltage is believed to generate a higher electric field, thereby allowing separation of biological components within the gel based on the charge / mass ratio. Once the device is removed from the user, the effective 1-D separation may then be further analyzed.

  In certain embodiments, the system may be configured for neural interfaces, short-term or long-term interfaces (eg, prosthetic control, treatment response. In addition, the system may be configured for neurological disorders such as depression, anxiety and A flexible, deformable nanopattern coated with nerve growth factor anchored in electrochemically deposited PPY may be configured to monitor and optionally treat multiple sclerosis The device may obtain a good biocompatible connection to a biological neuron by using the reinforced material on the thin wire electrode described above, the connection between the neuron and the filamentous nanoscale conductor. The electrical current flows through the conductor with the aid of a tunneling mechanism.

  In certain embodiments, the system may be configured for environmental biosecurity by monitoring animals, crops, water, and food supplies. The introduction of foot-and-mouth disease in the United States represents the single largest biological threat to our trillion dollar agricultural industry over the next 20 years. The described system may be configured to provide a scalable platform for determining disease distribution throughout the country or at specific locations. If individual monitoring is required, real-time, high-throughput non-invasive assessment may be combined with on-board testing for tentative determination of infection. If a particular individual is positive, subsequent efforts at the individual level may be required.

  In the relevant biosecurity area, devices may be configured for specific chemicals, such as specific insecticides. Although environmentally induced diseases affect more or less everyone, an individual's susceptibility may pre-tend to the extent of a group of addictive reactions in preference to another group. In particular, individuals in the developmental stage ranging from fetal stage to adolescence are particularly susceptible to such environmental stresses. This is because major physical functions have not yet matured to the level that can withstand, process and deal with such exposure. The use of biomarkers embedded within the device may provide continuous monitoring of the child's environmental health, early detection of toxins, and continuous monitoring to prevent functional impairment in the child's physical condition, eg insecticide A series of treatments for children who have been exposed to a toxic environment containing the agent may be determined. There are a number of different insecticides, but most affect the nervous system by disrupting enzymes that regulate the neurotransmitter acetylcholine. Insecticide monitoring includes, but is not limited to, optimizing drug treatment for production, ensuring that crops are never overtreated, and monitoring produce from vendors claiming not to use pesticides. It can also be used in crops for various reasons. If the soil sample can be placed at the detection site, the device may be used as a stand-alone device. Moisture contained in the sample may reach a sensor that includes a set of appropriate receptor panels for various pesticides, and an indicator may provide information about the concentration of pesticides in the environment .

  It is also contemplated that the system described above may be configured for use in detection and therapy in a wide variety of applications not described herein.

  As will be appreciated by those skilled in the art, the applications considered for the systems described above are broad and unlimited. The above examples of applications should in no way be considered limiting. Instead, they are provided as examples of the wide utility that embodiments of the present invention may provide.

  Although the present invention has been described in language specific to structural features and / or methodological actions, the invention as defined in the appended claims is not necessarily limited to the specific characteristics or acts described. I want you to understand. Rather, the specific features and acts are disclosed as exemplary forms of implementing the claimed invention.

FIG. 1 is a schematic diagram illustrating an embodiment of a biomolecule sampling and delivery system. FIG. 2 is a schematic diagram illustrating an embodiment of functional components of the sampling, analysis and delivery device of FIG. FIG. 3 is a schematic top view of a portion of a sampling, analysis and delivery cell structure according to an embodiment of the present invention. FIG. 4A is a schematic top view of a sampling and analysis cell structure according to an embodiment of the present invention. FIG. 4B is a schematic top view of the structure of a delivery cell according to an embodiment of the present invention. not listed. FIG. 5 shows a cross section along AB of the embodiment of FIG. FIG. 6 is a schematic cross section of a modification of the configuration shown in FIG. FIG. 7 shows the same cross section as FIG. 6 with a tear in the thin film of the substrate. FIG. 8 shows the cross section of FIG. 6 with a cavity filled with material for delivery at the sample site. FIG. 9 shows the cross section of FIG. 6 with the effective surface area of the analysis electrode increased. FIG. 10 shows the cross section of FIG. 6 with the effective surface area of the analysis electrode increased. FIG. 11 shows the cell of FIG. 6 in contact with tissue, such as skin, which has been simplified to schematically show scaly stratum corneum and living epidermal tissue. FIG. 12 is a schematic diagram showing the cell of FIG. 11 after barrier tissue rupture. FIG. 13 shows the cell of FIG. 8 in contact with tissue. FIG. 14 shows a schematic diagram of the cell of FIG. 11 after barrier tissue rupture. FIG. 15 shows a cell used for sampling body fluid. FIG. 16 shows the cell of FIG. 15 open to allow bodily fluids to enter the cavity in which the analysis electrode is located. FIG. 17 shows a cell used for solid or powder sampling. FIG. 18 shows the cell of FIG. 17 open to allow the contained body fluid to soak and dissolve the dried sample and to allow a concentration to be present at the analysis electrode. not listed FIG. 20 is a block diagram illustrating some components of the controller and wireless communication. FIG. 21 is a schematic side view of the cell portion of the device showing an embodiment that fills and seals the cavity of FIG. FIG. 22 is a schematic top view of an embodiment of an interface for filling a single cavity of a device. FIG. 23 is a schematic top view of an embodiment of an interface for simultaneously filling the five cavities of the device. FIG. 24 is a schematic top view of an embodiment of an interface for simultaneously filling the 25 cavities of the device. FIG. 25 is a flow diagram illustrating an embodiment of a method for monitoring and treating an abnormal biochemical state of a subject. FIG. 26 is a schematic diagram of an embodiment of a method for growing an electrode on a device. FIG. 27 is a schematic illustration of an embodiment of a nanotube array grown on an electrode of a device. FIG. 28 is a schematic view of the nanotube array after the nanotubes have been coated. FIG. 29 is a schematic top view of an embodiment of a device. 30 is a cross-sectional view taken along line XXX-XXX in FIG. 31 is a schematic top view of the devices of FIG. 29 being manufactured. FIG. 32 is a schematic diagram of an embodiment of a hand-held sampling device that may be used for dental applications. FIG. 33 is a schematic diagram of a pair of plug embodiments that may be used with the handheld sampling device of FIG. FIG. 34 is a schematic illustration of an embodiment of a microprobe-based dental probe device.

Claims (132)

A method for monitoring a biological state of a subject comprising:
Exposing the sensor of the sampling, analysis and delivery device to a substance comprising the target biomolecule;
Supplying an electrical signal to the sensor;
Measuring electrical characteristics at the sensor in response to the electrical signal;
Associating the measured electrical property with the biological state;
Determining whether the measured biological condition is normal or abnormal;
Generating a signal if the measured biological condition is abnormal. 3. The method of claim 2, further comprising delivering material from a cavity within the sampling, analysis and delivery device to the substance in response to the generated signal. 4. The method of claim 3, further comprising analyzing the substance for the effectiveness of the material. The step of determining whether the measured biological state is normal or abnormal comprises comparing the measured biological state to a normal health baseline condition. The method of claim 1 comprising: The method of claim 1, wherein the target biomolecule is selected from the group consisting of glucose, lactate, bilirubin, ethanol, pyruvate, and cytochrome P-450 2A6 enzyme. The target biomolecules include glycosylated hemoglobin and glycosylated protein, insulin, cholesterol, C-reactive protein, homocysteine, orexin, P.I. falciparum histidine rich protein 2, parasite lactate dehydrogenase, prostate specific antigen, prostate membrane specific antigen, estrogen, epidermal growth factor, insulin growth factor, hemagglutinin, neuradminidase, cleaved caspase-3 17 kDa Subunit, p54-like protein, immunoglobulin, anesthetic, leptin, ghrelin, vitamin, folic acid, creatine kinase, troponin, C-reactive protein, tumor necrosis factor receptors 1 and 2, creatine phosphokinase, creatinine, troponin , Interleukins 1, 2 and 6, interleukin-2 receptor, tumor necrosis factor-alpha, n-nitrosamine, nicotine, cotinine, opiates, cocaine, and spore metabolites. Recorded in Method of. 2. The method of claim 1, wherein the target biomolecule is selected from the group consisting of influenza virus, multiple sclerosis viruses, dengue fever, malaria, HIV, and tuberculosis. The method of claim 1, wherein the electrical property is selected from the group consisting of current, capacitance, resistance, voltage-like potential, and electromotive force. The method of claim 8, wherein the electrical property is current. Sampling substances including biochemical substances;
Analyzing the substance using a pair of electrodes, the pair of electrodes being disposed on a support substrate; and
Using a controller to control the sampling and analyzing steps;
Communicating information between the controller and a remote device. The method according to claim 10, wherein the sampling step includes a step of tearing a protective member using the pair of electrodes. The method according to claim 10, wherein the sampling includes tearing the protective film using another pair of electrodes. The method of claim 12, wherein the protective film comprises a barrier tissue. The method according to claim 12, wherein the protective film includes a stratum corneum. The method of claim 10, wherein the support substrate includes a cavity, and the sampling step includes expelling the substance into the cavity. The method of claim 15, wherein the electrode is at least partially disposed within the cavity. The method of claim 10, further comprising delivering a material to the substance. The method of claim 17, wherein the material comprises at least one from the group consisting of chemicals, drugs, biomolecules, proteins, peptides, and genetic material. The method of claim 17, wherein the material is reactive with the substance. The method of claim 17, wherein the support substrate comprises a cavity, and the method further comprises storing the material in the cavity until the delivering step. The method of claim 10, wherein the communicating comprises wirelessly communicating with the remote device. The method of claim 10, wherein the information includes a result of the analyzing step from the controller to the remote device. The method of claim 10, wherein the information includes instructions from the remote device to the controller. A system for sampling, analyzing and / or delivering at least one biochemical substance comprising:
A support substrate;
A plurality of cells supported by the support substrate and arranged in a plurality of columns and a plurality of rows, each of the plurality of cells sampling, analyzing, and / or delivering the chemical. A plurality of cells configured in
A plurality of column address conductive paths, a plurality of row address conductive paths, and a plurality of zero insertions are configured to control the interaction between each of the plurality of cells and a substance associated with the biochemical substance. A controller connected to the plurality of cells via a force connector;
A communication device configured to communicate with the controller and an external device. 25. The system of claim 24, wherein each of the cells includes a pair of electrodes supported by the support substrate. 26. The system of claim 25, wherein at least some of the pair of electrodes are configured to rupture a protective film disposed between the support substrate and the material. 27. The system of claim 26, wherein a resistive element is disposed between the at least some of the pair of electrodes and configured to rupture the protective film. At least some of the cells each include a sensor that includes a working electrode and a reference electrode, the working electrode configured to be electrochemically activated and responsive to the substance, and a voltage across the electrode. Applied, and at least when the working electrode is exposed to the material, an electrical property corresponding to the property of the material is generated, and the controller is configured to control a series of electrical signals to the electrode. 26. The system of claim 25, wherein the generated electrical characteristic is received. 30. The system of claim 28, wherein the electrical property is a current generated between the electrodes. 30. The system of claim 28, wherein the property of the substance is the level of a biochemical analyte in the substance. 30. The system of claim 28, wherein the property of the substance is a physicochemical property. 25. The system of claim 24, wherein the support substrate is flexible and is configured to adhere to a protective film. The system of claim 32, wherein the support substrate comprises a polymer. The system according to claim 32, wherein the protective film is a stratum corneum, and the substance is an interstitial fluid located under the stratum corneum. 33. The system of claim 32, wherein the protective film is a barrier tissue. 25. The system of claim 24, wherein at least some of the plurality of cells include cavities within the support substrate. 40. The system of claim 36, wherein the cavity is configured to receive a sample of the material. 40. The system of claim 36, wherein the cavity is configured to store a material comprising at least one from the group consisting of chemicals, drugs, biomolecules, proteins, peptides, and genetic material. 40. The system of claim 38, wherein the material is reactive with the substance. 40. The system of claim 38, wherein the material is a calibration standard. 40. The system of claim 38, wherein the material is a gas, liquid, or solid. 25. The system of claim 24, wherein the controller is configured to control a voltage applied to each of the plurality of cells. 43. The system of claim 42, wherein the controller is configured to apply the voltage to only one of the plurality of cells at a time. 43. The system of claim 42, wherein the controller is configured to apply the voltage to two or more of the plurality of cells at a time. The system of claim 24, further comprising a power supply, wherein the controller is configured to manage the power supply. 46. The system of claim 45, wherein the power source is a battery. 25. The system of claim 24, wherein the communication device includes at least one wireless communication module configured to wirelessly communicate with the external device. The system of claim 24, wherein the external device is a computer. A system for analyzing the properties of a substance,
A support substrate;
A plurality of sensors supported by the support substrate,
Each sensor is supported by the substrate and includes a pair of electrodes including a working electrode and a reference electrode, the working electrode configured to be electrochemically activated and responsive to the substance;
A plurality of sensors, wherein when a voltage is applied across the electrode and at least the working electrode is exposed to the material, an electrical property corresponding to the property of the material is generated;
A controller for controlling a series of electrical signals to the electrodes and receiving the generated electrical properties;
Including the system. 50. The system of claim 49, further comprising a communication device for communicating with the controller and an external device. 51. The system of claim 50, wherein the communication device includes at least one wireless communication module configured to wirelessly communicate with the external device. 51. The system of claim 50, wherein the external device is a computer. 50. The system of claim 49, wherein the electrical property is a current generated between the electrodes. 50. The system of claim 49, wherein the property of the substance is the level of a biochemical analyte in the substance. 55. The system of claim 54, wherein the biochemical analyte is glucose. 55. The system of claim 54, wherein the biochemical analyte is lactate. 55. The system of claim 54, wherein the biochemical analyte is bilirubin. 50. The system of claim 49, wherein the property of the substance is a physicochemical property. 59. The system of claim 58, wherein the physicochemical property is the pH of the substance. 59. The system of claim 58, wherein the physicochemical property is a dissolved gas level in the material. 50. The support substrate according to claim 49, wherein the support substrate is flexible and configured to adhere to a protective film, and the substance is disposed on a surface of the protective film opposite the support substrate. The system described. Each sensor further includes a resistive element disposed between the electrodes, the electrode and the resistive element tearing the protective film in contact with the respective sensors, on the opposite surface of the protective film 62. The system of claim 61, configured to allow the material being disposed to contact at least the working electrode. The system according to claim 61, wherein the protective film is a stratum corneum and the substance is an interstitial fluid located under the stratum corneum. 62. The system of claim 61, wherein the protective film is a barrier tissue. Each sensor further includes a cavity within the support layer, wherein at least a portion of the electrode is disposed within the cavity, and the cavity communicates with the material disposed on an opposite surface of the protective film. 64. The system of claim 61, wherein the cavity is configured to be opened by the resistive element to allow. 66. The system of claim 65, wherein the cavity is configured to receive a sample of the material. Each sensor further includes a cavity within the support substrate, the cavity configured to store a material including at least one from the group consisting of chemicals, drugs, biomolecules, proteins, peptides, and genetic material. 50. The system of claim 49, wherein: 68. The system of claim 67, wherein the material is reactive with the substance. 68. The system of claim 67, wherein the material is a calibration standard. 68. The system of claim 67, wherein the material is a gas, a liquid, or a solid. 50. The system of claim 49, wherein the controller is configured to control the voltage applied to each of the plurality of sensors. 72. The system of claim 71, wherein the controller is configured to apply the voltage to only one of the plurality of sensors at a time. 72. The system of claim 71, wherein the controller is configured to apply the voltage to two or more of the plurality of sensors at a time. 50. The system of claim 49, further comprising a power supply, wherein the controller is configured to manage the power supply. 75. The system of claim 74, wherein the power source is a battery. The plurality of sensors are arranged on the support substrate in a grid including a plurality of columns and a plurality of rows, and the system includes a plurality of column address conductive paths that connect the plurality of sensors to the controller. 50. The system of claim 49, further comprising: and a plurality of row address conductive paths. 50. The system of claim 49, further comprising a plurality of zero insertion force connectors configured to connect the plurality of sensors to the controller. A sensor that senses the characteristics of a substance containing a biomolecule,
A pair of electrodes supported by a substrate and including a working electrode and a reference electrode, the working electrode being electrochemically activated and configured to react to the substance; Including
When a voltage is applied across the electrode and at least the working electrode is exposed to the material, an electrical property corresponding to the property of the material is generated.
Sensor. 79. The sensor of claim 78, wherein the working electrode is electrochemically activated using an enzyme. 80. The sensor of claim 79, wherein the biomolecule is selected from the group consisting of glucose, lactate, bilirubin, ethanol, pyruvate, and cytochrome P-450 2A6 enzyme. 79. The sensor of claim 78, wherein the working electrode is electrochemically activated using an antibody. The biomolecules include glycosylated hemoglobin and glycosylated protein, insulin, cholesterol, C-reactive protein, homocysteine, orexin, P.I. falciparum histidine rich protein 2, parasite lactate dehydrogenase, prostate specific antigen, prostate membrane specific antigen, estrogen, epidermal growth factor, insulin growth factor, hemagglutinin, neuradminidase, cleaved caspase-3 17 kDa Subunit, p54-like protein, immunoglobulin, anesthetic, leptin, ghrelin, vitamin, folic acid, creatine kinase, troponin, C-reactive protein, tumor necrosis factor receptors 1 and 2, creatine phosphokinase, creatinine, troponin 84. selected from the group consisting of: interleukins 1, 2 and 6, interleukin-2 receptor, tumor necrosis factor-alpha, n-nitrosamine, nicotine, cotinine, opiates, cocaine, and spore metabolites. In Sensor mounting. 82. The sensor of claim 81, wherein the biomolecule is selected from the group consisting of influenza virus, multiple sclerosis viruses, dengue fever, malaria, HIV, and tuberculosis. 79. The sensor of claim 78, wherein the electrical characteristic is selected from the group consisting of current, capacitance, resistance, voltage-like potential, and electromotive force. A resistance element disposed between the electrodes, wherein the electrode and the resistance element are at least a protection located between the working electrode and the material such that the working electrode is exposed to the material; 79. The sensor according to claim 78, configured to rupture the membrane. 86. The sensor of claim 85, wherein the protective film includes dead skin cells of the stratum corneum that are in contact with the sensor, and the substance includes interstitial fluid located under the stratum corneum. 86. The sensor according to claim 85, wherein the substrate is a flexible substrate having an adhesive on one side thereof, and the adhesive is configured to adhere to the protective film. 90. The sensor of claim 87, wherein the substrate comprises a polymer. 90. The sensor of claim 88, wherein the polymer is SU-8. 90. The sensor of claim 88, wherein the polymer is polymethyl methacrylate. 90. The sensor of claim 88, wherein the polymer is polydimethylsiloxane. The substrate further includes a cavity, and at least a portion of the electrode is disposed within the cavity, the cavity being opened by the resistive element to allow the cavity to communicate with the material. 86. The sensor of claim 85, configured as follows. And further comprising a material contained within the cavity, the cavity configured to deliver the material as a working electrode to a position on an opposite surface of the protective film after the cavity is opened. 94. The sensor of claim 92. 94. The sensor of claim 93, wherein the material comprises at least one from the group consisting of chemicals, drugs, biomolecules, proteins, peptides, and genetic material. 94. The system of claim 93, wherein the material is reactive with the substance. 94. The system of claim 93, wherein the material is a calibration standard. 94. The system of claim 93, wherein the material is a gas, liquid, or solid. A sampling device for sampling substances containing biochemical substances,
A support substrate;
A sampling device comprising a pair of electrodes supported by the substrate and configured to rupture a protective film located between the support substrate and the material. 99. The sampling device of claim 98, further comprising a cavity within the support substrate, the cavity configured to receive a sample of the material. 100. The sampling device of claim 99, wherein at least a portion of the pair of electrodes is exposed to the cavity. 99. The sampling device of claim 98, further comprising a resistive element disposed between the electrodes, wherein the resistive element is configured to rupture the protective film. 99. The sampling device of claim 98, wherein the protective film is a barrier tissue. The sampling device according to claim 98, wherein the protective film is a stratum corneum. 99. The sampling device of claim 98, wherein the support substrate is flexible. 105. The sampling device of claim 104, wherein the support substrate comprises a polymer. 99. The sampling device of claim 98, further comprising a zero insertion force connector configured to connect the pair of electrodes to a controller. 107. The sampling device of claim 106, wherein the controller is configured to control a voltage applied to the electrode by a power source. 108. The sampling device of claim 107, wherein the power source comprises a battery. A method for analyzing biochemical substances in a substance,
Exposing at least the working electrode of the sensor to the substance;
Applying a voltage across the working and reference electrodes;
Measuring electrical properties generated as a result of applying the voltage across the electrode;
Correlating the measured electrical property with the property of the material. 110. The method of claim 109, wherein the property of the substance is the level of a biochemical analyte in the substance. 110. The method of claim 109, wherein the property of the substance is a physicochemical property. 110. The method of claim 109, wherein the electrical property is selected from the group consisting of current, capacitance, resistance, voltage-like potential, and electromotive force. 113. The method of claim 112, wherein the electrical property is the current. 110. The method according to claim 109, further comprising: prior to the exposing step, creating a capillary through the protective film by applying a series of voltage pulses to the electrode and the resistive element disposed between the electrodes. The method described. 115. The method of claim 114, further comprising the step of exhaling the substance from under the protective film through the capillary. 115. The method of claim 114, wherein the protective film comprises barrier tissue. 115. The method of claim 114, wherein the protective film comprises a stratum corneum. 110. The method of claim 109, further comprising releasing material to the substance. 119. The method of claim 118, wherein the material comprises at least one from the group consisting of chemicals, drugs, biomolecules, proteins, peptides, and genetic material. 120. The method of claim 119, wherein the material is reactive with the substance. 110. The method of claim 109, further comprising wirelessly communicating the property of the substance to a remote device. A method for filling a cavity in a support layer of a delivery device comprising:
Disposing a sealing layer having the holes therein on the support layer such that the holes are located above the cavity;
Filling the cavity with a material;
Placing the sealing layer relative to the support layer such that the holes are spaced from the cavity and the cavity is covered by the sealing layer;
Bonding the sealing layer to the support layer. The filling of the cavity has a reservoir and the holes therein for the sealing layer such that the holes in the glass carrier are substantially aligned with the holes in the sealing layer. Placing the glass carrier, and filling the reservoir with the material, and the material flows out of the reservoir through the holes in the glass carrier and the holes in the sealing layer to fill the cavity. 123. The method of claim 122, comprising a step. The support layer includes at least one capillary hole parallel to the cavity, and the step of disposing the sealing layer relative to the support layer moves the sealing layer to position the hole in the sealing layer. 123. The method of claim 122, comprising the step of aligning with the capillary holes in the support layer. A method of manufacturing a device for analyzing a property of a substance, comprising:
Connecting between a plurality of connectors and a plurality of electrodes arranged on the support substrate;
Exposing the plurality of electrodes to a polymer matrix;
Applying an electrochemical potential to an electrode selected from the plurality of electrodes with respect to ground;
Coating the selected electrode with the polymeric matrix. Exposing the plurality of electrodes to a second polymer matrix different from the first polymer matrix;
Applying an electrochemical potential from the plurality of electrodes to another selected electrode with respect to ground;
126. The method of claim 125, further comprising coating the selected electrode with the second polymeric matrix. 126. The method of claim 125, wherein the connector is a zero insertion force connector. 128. The method of claim 127, wherein the zero insertion force connector is disposed on a front surface, a rear surface, and / or a side surface of the support substrate. 126. The method of claim 125, wherein the macromolecular matrix comprises an antibody for reacting with the substance. 126. The method of claim 125, wherein the polymer matrix comprises an enzyme for reacting with the substance. 126. The method of claim 125, wherein the polymeric matrix comprises a redox electron mediator. 129. The method of claim 129, wherein the connector is also used to supply a voltage to the electrode when the device is being used to analyze the property of the substance.

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