Factory and in vivo calibration of electrochemical aptamer sensors

Abstract

A method is provided for fabricating sensors having multiple aptamers and performing continuous measurements using the sensors. This method involves fabricating multiple sensors, each having at least one electrode on which multiple aptamers are attached to the electrode surface. A portion of the multiple sensors is calibrated, and a portion of the multiple aptamer sensors is identified as being of commercial quality ("product portion"). At least one set of calibration data collected from the calibration of the multiple sensors is mathematically associated with the product portion.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 531,047, filed on 7 August 2023, and U.S. Provisional Application No. 63 / 561,278, filed on 4 March 2024, which are incorporated herein by reference in their entirety.

[0002] The present invention relates in general to an aptamer sensor, and more specifically to an aptamer sensor that provides a calibrated measurement of at least one analyte. [Background technology]

[0003] This section is intended to introduce to the reader various aspects of the technology that may relate to various aspects of the invention described and / or claimed below. We believe that the descriptions in this section will help provide the reader with background information to better understand the various aspects of the invention. Therefore, these descriptions should be read in this context and not understood as an acknowledgment of prior art.

[0004] Aptamer sensors can identify the presence and / or concentration of an analyte of interest through the use of aptamer sequences that specifically bind to the analyte of interest. These sensors include aptamers attached to an electrode or optical surface, each aptamer having a redox-active molecule (redox tag) or a fluorescent molecule and a quencher (optical tag) attached to it. The redox pair can transfer charge to or from the electrode, and the fluorescent molecule can supply photons to a photodetector. When the analyte binds to the aptamer, the aptamer can change shape and alter electron transfer to the redox tag or fluorescence of the optical tag. This results in a measurable change, which can be translated into a measure of the presence or concentration of the analyte. When used in this manner, aptamers are an example of affinity-based biosensors. These aptamer sensors can be incorporated into wearable biosensors or even implantable biosensors, whose form factor and use case scenarios are similar to continuous glucose monitors.

[0005] A major unresolved challenge for aptamer sensors and other affinity-based biosensors is sensor calibration. Technologies proposed for "calibration-free" aptamer sensor operation recognize the need for methods to overcome the very large variability between sensors during fabrication and the significant sensor drift (accuracy degradation) caused by aptamer sensors during continuous operation. Historically, aptamer sensors have lacked reproducible manufacturability and have suffered from rapid degradation of aptamer sensors, leading R&D teams to focus on methods to avoid pre-calibration of sensors, as such approaches do not provide sufficient accuracy when using the sensors in real-world applications such as wearable or implantable continuous molecular monitoring.

[0006] Aptamer sensors and other affinity-based biosensors have additional challenges related to sensor calibration. Aptamer sensors differ from prior art sensors and calibration schemes, such as those used in continuous glucose monitors. Continuous glucose monitors have sensitivity (change in measurement versus change in glucose concentration) that is highly dependent on factors such as electrode area or enzyme area and diffusion rate through the protective membrane. Aptamer sensors also have sensitivity, but it is not necessarily highly dependent on factors such as electrode area or diffusion rate through the protective membrane. Therefore, aptamer sensors have different in vitro and in vivo calibration needs, for example. In glucose sensors, for example, a foreign body reaction can limit the glucose flux to the electrochemical sensor, reducing the sensor current even if the glucose concentration in the interstitial fluid remains unchanged. However, in aptamer sensors, the diffusion limiting factor affects the delay time to the analyte, and if the analyte changes concentration slowly in the body, such a change in delay time may not have a meaningful effect on sensor accuracy. Furthermore, in vivo calibration of aptamer sensors presents additional challenges not seen in glucose sensors. Aptamer sensors can measure analytes that are either large enough, have a sufficient density of cell receptors within the tissue, or are lipophilic or have other factors that further complicate in vivo correlation between blood and interstitial fluid, and therefore between blood and aptamer sensor measurements. Thus, aptamer sensors present a unique set of challenges that must be addressed for continuous monitoring applications, particularly when economical batch calibration should be achieved and when the device user wants to minimize, or ideally not require, the fingertip puncture calibration step during device use.Furthermore, degradation methods for aptamer sensors are unique compared to those for glucose sensors, as their degradation is often virtually multimodal, presenting additional challenges when attempting to predict degradation so that the sensor sensitivity can be adjusted in real time to maintain reasonably calibrated measurements.

[0007] Novel approaches to aptamer sensors that reduce or eliminate these drawbacks could offer significant advantages, enabling accurate monitoring devices for analytes beyond glucose. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] U.S. Patent Application Publication No. 2020 / 0182820A1 [Patent Document 2] International Publication No. WO2021067779A1 [Patent Document 3] International Publication No. WO2022067051A1 [Non-patent literature]

[0009] [Non-Patent Document 1] White et al., “Abeykoon SW, White RJ. Continuous Square Wave Voltammetry for High Information Content Interrogation of Conformation Switching Sensors. ACS Meas Sci Au. 2022.” [Non-Patent Document 2] Ferguson BS, Hoggarth DA, Maliniak D, Ploense K, White RJ, Woodward N, Hsieh K, Bonham AJ, Eisenstein M, Kippin TE, Plaxco KW, Soh HT, "Real-time, aptamer-based tracking of circulating therapeutic agents in living animals." [Overview of the project] [Means for solving the problem]

[0010] Several exemplary embodiments of the present invention are described below. These embodiments are presented merely to provide the reader with a brief summary of certain forms that the invention may take, and it should be understood that these embodiments are not intended to limit the scope of the invention. In fact, the invention may encompass various embodiments not expressly described below.

[0011] Many of the aforementioned shortcomings and limitations can be overcome by bringing novel and advanced interactions of chemicals, materials, sensors, electronics, microfluidics, algorithms, computing, software, systems, and other features or designs into an affordable, effective, simple, intelligent, or highly reliable way to bring sensing technologies closer to biofluids and analytes.

[0012] Some embodiments of the disclosed invention involve sensors as simple, individual elements. It is understood that many sensors require two or more electrodes, counter electrodes, reference electrodes, or additional support techniques or features not captured in this description. Sensors measure the properties of an analyte. Sensors are preferably substantially electrical, but may also include optical, chemical, mechanical, or other known biosensing mechanisms. Sensors can be dual, triple, or more, thereby providing improved data and readings. Sensors may provide continuous or discrete data and / or readings. Some embodiments of the disclosed invention show subcomponents of sensing devices that would have more subcomponents necessary for the use of the device in various applications (e.g., batteries, antennas, adhesives), and for the purpose of brevity and focusing on aspects of the invention, such components may not be explicitly shown in the figures or described in the embodiments of the disclosed invention. For example, while electrochemical aptamer sensors typically require separate working electrodes, counter electrodes, and reference electrodes, the present invention focuses on a working electrode that is simply coupled to a sensing-transducing bioreceptor element in the form of a mixed monolayer of aptamer and block molecules.

[0013] In one aspect of the present invention, a method is provided for fabricating a sensor using multiple aptamers and performing continuous measurements using the sensor. This method involves fabricating multiple sensors. Each sensor has at least one electrode, the electrode having an electrode surface. Next, multiple aptamers are attached to the electrode surface. Each aptamer has one or more attached redox tags, which enable electron transfer to and from the electrode. Next, a blocking layer is applied to the electrode surface. The blocking layer has multiple paths that facilitate electron transfer between the blocking layer surface and the electrode and the redox tags. Next, a portion of the multiple sensors ("calibration portion") is calibrated to generate at least one set of calibration data. Next, a portion of the multiple aptamer sensors is identified as being of commercial quality ("product portion"). The multiple sensors are fabricated in a batch process. At least one set of calibration data collected from the calibration of the multiple sensors is mathematically associated with the product portion. The product portion can provide accurate or precise continuous measurements.

[0014] In one embodiment, the calibration portion and the product portion are the same portion. In another embodiment, the calibration portion and the product portion are the same portion and are a single sensor. In one embodiment, the calibration portion includes at least three sensors. In another embodiment, the calibration data collected from the calibration portion is at least part in vitro data. In one embodiment, the calibration data collected from the calibration portion is at least part in vivo data.

[0015] In another aspect of the present invention, a device for continuous measurement of at least one analyte in a test fluid is provided. This device includes one or more sensors fabricated using the method described above. In one embodiment, the electrode areas are 0.02, 0.01, 0.05, and 0.02 μm².2 The device has a standard deviation smaller than a value selected from the group consisting of 20%, 10%, 5%, and 2% of the electrode area. In one embodiment, the device also includes at least one time point for calibration during the manufacturing of the device, the time point being after sensor manufacturing. In another embodiment, the device also includes at least one time point for calibration during the manufacturing of the device, the time point being after sensor shelf-stabilization. In one embodiment, the device also includes at least one time point for calibration during the manufacturing of the device, the time point being after sensor sterilization.

[0016] In another embodiment, the calibration data is data collected over a period selected from the group consisting of 1 day, 3 days, 7 days, 10 days, and 14 days. In one embodiment, the calibration data consists of redox tag currents for either a single or multiple time points in time. In another embodiment, the calibration data consists of redox peak potentials for either a single or multiple time points in time. In one embodiment, the calibration data consists of oxygen reduction currents for either a single or multiple time points in time. In another embodiment, the calibration data consists of redox tag density or the total number of redox tags for either a single or multiple time points in time. In one embodiment, the calibration data consists of capacitance or electrical impedance for either a single or multiple time points in time. In another embodiment, the calibration data consists of the time or frequency response of redox tag currents for either a single or multiple time points in time.

[0017] In one embodiment, the calibration data consists of chronoamperometric responses for either a single time point or a plurality of time points over time. In another embodiment, the calibration data consists of electrode surface areas for either a single time point or a plurality of time points over time. In one embodiment, the calibration data consists of titration responses for either a single time point or a plurality of time points over time. In another embodiment, the calibration data includes statistical data. In one embodiment, the continuous measurement includes a continuous measurement method selected from the group consisting of square wave voltammetry, continuous square wave voltammetry, kinetic differential measurement, calibration-free measurement, impedance spectroscopy, differential pulse voltammetry, chronoamperometry, and combinations thereof.

[0018] In one embodiment, the calibration data includes at least one variable in vivo parameter. In another embodiment, the calibration data includes at least one in vivo correlation between interstitial fluid and blood. In one embodiment, the calibration data further includes calibration data collected during use of a product portion of a sensor. In another embodiment, the method also includes automatic calibration data collected during use of a product portion of a sensor. In one embodiment, the method also includes automatic calibration data collected during use of a product portion of an aptamer sensor, wherein the automatic calibration data is the frequency or time response of the sensor collected during use of the sensor.

[0019] In another embodiment, the method also includes automatic calibration data collected during use of the product portion of the aptamer sensor, wherein the automatic calibration data is electrode surface area. In one embodiment, the calibration data further includes in vivo calibration data collected after or near the time point of the steady-state concentration of the analyte in the interstitial fluid. In another embodiment, the calibration data further includes a plurality of sets of calibration data collected at a plurality of analyte concentrations. In one embodiment, the method also includes at least one measurement waveform that changes electron transfer properties when passing through the blocking layer, and thus further includes at least a first stabilization period. The calibration data is at least partially composed of at least one measurement value from the first stabilization period. In another embodiment, the method also includes a plurality of waveforms, at least one of the plurality of waveforms changes electron transfer properties when passing through the monomolecular layer, and further includes a second stabilization period occurring after each application of at least one of the plurality of waveforms.

[0020] In one embodiment, at least one of the waveforms is an analyte measurement waveform, a sensor diagnostic waveform, or a sensor storage waveform. In another embodiment, the multiple waveforms are periodic. In one embodiment, the method also includes a calibration portion having a standard deviation of <±2% when continuous biosensing is performed over a period selected from the group consisting of 7, 14, 21, 30, and 40 days in interstitial fluid or serum or an equivalent surrogate test fluid at temperatures reached during operation for the product. In another embodiment, the method also includes a calibration portion having a standard deviation of <±3% when continuous biosensing is performed over a period selected from the group consisting of 7, 14, 21, 30, and 40 days in interstitial fluid or serum or an equivalent surrogate test fluid at temperatures reached during operation for the product. In one embodiment, the method also includes a calibration portion having a standard deviation of <±5% when continuous biosensing is performed on the product version over a period selected from the group consisting of 7, 14, 21, 30, and 40 days in interstitial fluid or serum or an equivalent substitute test fluid at temperatures reached during operation.

[0021] In another embodiment, the method also includes a calibration portion having a standard deviation of <±10% when continuous biosensing is performed on the product version in interstitial fluid or serum or an equivalent surrogate test fluid at operating temperatures over a period selected from the group consisting of 7, 14, 21, 30, and 40 days. In one embodiment, the method also includes a calibration portion having a standard deviation of <±20% when continuous biosensing is performed on the product version in interstitial fluid or serum or an equivalent surrogate test fluid at operating temperatures over a period selected from the group consisting of 7, 14, 21, 30, and 40 days. In another embodiment, the method also includes a product portion having an accuracy of <±5% when continuous biosensing is performed on the product version in interstitial fluid or serum or an equivalent surrogate test fluid at operating temperatures over a period selected from the group consisting of 7, 14, 21, 30, and 40 days.

[0022] In one embodiment, the method also includes a product portion having an accuracy of <±10% when continuous biosensing is performed on the product in interstitial fluid or serum or an equivalent surrogate test fluid at temperatures reached during operation over a period selected from the group consisting of 7, 14, 21, 30, and 40 days. In another embodiment, the method also includes a product portion having an accuracy of <±20% when continuous biosensing is performed on the product in interstitial fluid or serum or an equivalent surrogate test fluid at temperatures reached during operation over a period selected from the group consisting of 7, 14, 21, 30, and 40 days. In one embodiment, the method also includes a product portion of an aptamer sensor having a change in zero gain frequency (Zero Gain Frequency) less than at least one of <±5%, <±10%, <±20%, <±40%, <±80% when continuous biosensing is performed on the product version in interstitial fluid or serum or an equivalent surrogate test fluid at operating temperatures for a period selected from the group consisting of 7, 14, 21, 30, and 40 days. In another embodiment, the method also includes an oxygen reduction current measurable at a potential of -0.5V when continuous biosensing is performed on the product version in interstitial fluid or serum or an equivalent surrogate test fluid at operating temperatures for a period selected from the group consisting of 7, 14, 21, 30, and 40 days, increasing by at least one of <10%, <20%, <50%, <100% of the initial oxygen reduction current.

[0023] Another aspect of the present invention provides a method for producing a factory-calibrated batch of aptamer sensors. This method involves manufacturing the aptamer sensors in batches, then calibrating a portion of the aptamer sensors ("calibration portion") to produce at least one set of calibration data. Next, the portion of the aptamer sensors is identified as being of commercial quality ("product portion"). Then, the calibration data is assigned to the product portion of the aptamer sensors. In one embodiment, the calibration data includes sensor responses measured over several days. In another embodiment, the calibration data includes a single-point calibration captured after sensor operation over a period selected from the group consisting of 1 hour, 2 hours, and 3 hours.

[0024] In one embodiment, the calibration data includes a single-point calibration and a predictive calibration curve based on previous or historical measurement data. In another embodiment, the calibration data further includes measurements selected from the group consisting of aptamer density, frequency maps, square wave voltammetry, cyclic voltammetry, intermittent pulse amperometry, chronoamperometry, continuous square wave voltammetry, electrical impedance, oxygen reduction current, and combinations thereof. In one embodiment, the calibration data is corrected in use using automatic calibration, and for factory-calibrated aptamer sensors, it further includes known, expected, or predicted standard deviations to the sensor response, where automatic calibration is performed when the sensor response deviates by more than at least one of the standard deviation of the sensor response or twice the standard deviation. In another embodiment, automatic calibration shifts the frequency response used to identify at least one of the signal OFF frequency, zero or non-responding frequency, or signal ON frequency with the maximum sensor response. In one embodiment, the calibration data includes a frequency response, and during use, the device measures at least one of the following to recalibrate the frequency response: change in aptamer density, electrical impedance, or oxygen reduction current. In another embodiment, the calibration dataset is acquired in humans only after or near the point when the concentration of the analyte in the interstitial fluid reaches a steady state.

[0025] In one embodiment, the factory calibration of the sensor is performed immediately after the sensor is manufactured. In another embodiment, the factory calibration of the sensor is performed after the sensor has been stabilized for storage. In one embodiment, the factory calibration of the sensor is performed after it has been mounted on an electronic device. In another embodiment, the factory calibration of the sensor is performed after sterilization. In one embodiment, the factory calibration of the sensor is performed at one or more time points and one or more scales to provide a sensor lifetime calibration dataset. In another embodiment, the factory calibration of the sensor is performed at a single point, and the calibration is captured in operation at at least one of 1, 2, or 3 hours later.

[0026] In one embodiment, the calibration data includes at least one measurement parameter. In another embodiment, the measurement parameter is sensitivity. In one embodiment, the sensitivity includes multiple different sensitivities to multiple different measurements.

[0027] The disclosed objects and advantages of the present invention will be further understood in light of the following detailed description and drawings. [Brief explanation of the drawing]

[0028] [Figure 1] This is a schematic diagram of one embodiment of a device based on the principle of the present invention. [Figure 2] This is a schematic diagram of one embodiment of a device based on the principle of the present invention. [Figure 3] This is a schematic diagram of one embodiment of a device based on the principle of the present invention. [Figure 4] This is a schematic diagram of one embodiment of a plurality of sensors based on the principle of the present invention. [Figure 5A] This is an illustration of a square wave voltammogram of redox tag current for several different electrode regions. [Figure 5B]This is a diagram illustrating the square wave voltammogram of redox tag currents for several different electrode regions, which have been normalized. [Figure 6] This is a diagram illustrating the Langmuir isothermal adsorption curve of redox peak current versus concentration with a standard deviation. [Figure 7] This is a flowchart of the factory calibration process. [Figure 8A] This is a diagram illustrating how sensor readings change over time. [Figure 8B] This is a diagram illustrating how sensor readings change over time. [Figure 9] This diagram illustrates how sensor readings change over time, including multiple manual recalibration events. [Figure 10] This is a diagram illustrating a graph that shows how the scale of an analyte changes over time. [Figure 11A] This is a diagram illustrating a graph showing the sensor response to square wave frequencies over a 24-hour period. [Figure 11B] This is a diagram illustrating the sensor response to the square wave frequency over a 3-day period. [Figure 12A] This is a set of datasets showing the parameters used in Equation 1. [Figure 12B] This is a set of datasets showing the parameters used in Equation 1. [Figure 12C] This is a set of datasets showing the parameters used in Equation 1. [Figure 12D] This is a set of datasets showing the parameters used in Equation 1. [Figure 13A] This is a diagram illustrating a graph showing the analytes measured over time. [Figure 13B] This is a diagram illustrating a graph showing the analytes measured over time. [Figure 14] This is a diagram illustrating a graph showing the analytes measured over time. [Figure 15A] This is a diagram illustrating a graph showing the analytes measured over time. [Figure 15B] This is a diagram illustrating a graph showing the analytes measured over time. [Figure 16] This figure shows exemplary data for Figure 15. [Figure 17] This figure shows exemplary data for factory calibration. [Figure 18] This is a system-level diagram of an exemplary system. [Figure 19] This is a device-level diagram of an exemplary device. [Figure 20] This graph shows in vivo data collected for a cortisol sensor subcutaneously implanted in rats. [Figure 21] This is a diagram of Equation 1. [Modes for carrying out the invention]

[0029] definition As used herein, “continuous sensing” using a “continuous sensor” means a sensor that changes in response to a change in the concentration of at least one solute in a solution, such as an analyte. Similarly, as used herein, “continuous monitoring” means the ability of a device to provide multiple measurements of an analyte that change over time. “Continuous measurement” may also be used in this context.

[0030] As used herein, the phrase “about” means, when referring to a value or quantity of mass, weight, time, volume, pH, size, concentration, or percentage, to include variations from the specified quantity such as ±20% in some embodiments, ±10% in some embodiments, ±5% in some embodiments, ±1% in some embodiments, ±0.5% in some embodiments, and ±0.1% in some embodiments, that are appropriate for carrying out the disclosed method.

[0031] As used herein, the term “aptamer” means a molecule that undergoes a structural or binding change upon binding of an analyte to a molecule and follows the general operating principles of sensing methods as described herein. Such molecules are, for example, native or modified DNA, RNA, or XNA oligonucleotide sequences, Spiegelmers, peptide aptamers, affimers, and other forms of affinity-based biosensors. Modifications may include substituting native bases with non-native nucleic acid bases within the aptamer sequence, replacing a native sequence with a non-native sequence, or other preferred modifications that improve sensor function but exhibit behavior similar to that of conventional aptamers. Two or more aptamers bound together may also be referred to as aptamers (i.e., not separated in solution). Aptamers may have molecular weights of at least 1 kDa, 10 kDa, or 100 kDa.

[0032] As used herein, the term “sensing monolayer” means at least several aptamers on the working electrode, which may also include multiple molecules or mixtures of molecules forming a non-monolayer protective layer or monolayer protective layer on the surface, and the protection is protection from interfering substances, decomposition, or other factors that may reduce the accuracy and / or lifespan of the sensor.

[0033] As used herein, “protective film” refers to one or more layers or materials that protect the sensor blocking layer from fouling and are permeable to at least charge transfer. The protective film may optionally be selectively permeable to additional components in the test fluid, such as at least one analyte, the presence of at least one analyte, which allows the sensor to operate properly while protecting the protective film from performance degradation resulting from fouling or some combination thereof.

[0034] As used herein, the term “continuous measurement” means data collected by a device that records multiple readings over a period of time during which sensing occurs. Continuous measurements include measurements ranging from constant to intermittent and periodic (for example, from 5 a.m. to 10 a.m. every day).

[0035] As used herein, the term “analyte” means any solute in a biological fluid, either inside or from the body, that can be measured using a sensor. The analyte may be a small molecule in a solution or fluid, a protein, a peptide, an electrolyte, an acid, a base, an antibody, a molecule to which a small molecule has been bound, DNA, RNA, a drug, a chemical, a contaminant, or any other solute.

[0036] As used herein, “sensor,” “sensing device,” or “device” includes at least one sensor based on at least one aptamer and at least one sample solution when the sensor is in use. A device can sense multiple samples and may take on multiple configurations, such as a device for measuring blood, or a microneedle or indwelling sensor needle for measuring interstitial fluid, or a device for measuring saliva, tears, or sweat, or a urine sensor, or a device for measuring water contaminants or food processing solutes, or other devices for measuring at least one analyte found in the sample solution.

[0037] As used herein, “redox current” or “redox signal” includes the total redox current between a plurality of redox tags attached to an aptamer on a sensor and an electrode to a given sensor, an electrode typically referred to as the working electrode, as measured using techniques such as square wave voltammetry, chronoamperometry, or other preferred methods. The redox current is measured as the amplitude of the Faraday redox tag peak current minus the background current amplitude outside the redox peak. In other words, if the measurement is a voltammogram from square wave voltammetry, the redox current is the peak height of the redox current measured above the background current as if there were no redox tags.

[0038] As used herein, “signal gain” or “sensor response” includes changes in redox current or other types of signals resulting from changes in the concentration of an analyte, such as those measured using techniques such as square wave voltammetry, chronoamperometry, or other preferred methods.

[0039] As used herein, “background current” includes measured currents that are not redox currents. Background currents may be due to capacitive charging, Faraday currents, oxygen reduction currents, other redox active species, etc.

[0040] As used herein, “normalized current” is a measured current normalized to a certain current value. For example, it may be normalized to the redox current at the start of the sensor test (t=0s when the sensor is placed in a test solution such as serum, or t=0s when data collection begins even if the sensor is already in serum). Alternatively, for example, the background current for a batch of sensors may be normalized to a common value of the background current for all sensors.

[0041] As used herein, “electron transfer rate” includes the measured rate or time at which electrons are transferred between the redox tag and the working electrode.

[0042] As used herein, “frequency response” refers to the change in redox current or sensor response measured from the device as a function of the measurement frequency, such as the frequency used in square wave voltammetry measurements. Changes in frequency response may also relate to changes in electron transfer velocity. Frequency response can also be interpreted from chronoamperometric curves, such as those used in continuous square wave voltammetry measurement methods.

[0043] As used herein, the “non-response frequency” is the measurement frequency at which the redox current does not respond to increases or decreases in the concentration of the analyte. The non-response frequency can also be interpreted from a chronoamperometry curve, such as those used in continuous square wave voltammetry measurement methods.

[0044] As used herein, “sensor accuracy” is the maximum difference between the actual value (which must be measured by a primary or good secondary standard) and the indicated value at the sensor output. Accuracy may be expressed either as a percentage of the full scale or as an absolute term. Additional statistical methods may be included, as necessary, to interpret the accuracy.

[0045] As used herein, “test fluid” refers to interstitial fluid or a suitable substitute for the test fluid, such as serum.

[0046] As used herein, the “calibration portion” of an aptamer sensor is a batch (which may also be referred to as a batch) of sensors fabricated together or using the same or similar processes, which, after fabrication, is tested for at least one measurement in vitro or in vivo at the factory to provide calibration data to improve the use of the sensor when it is used as a product outside the factory. After fabrication, the testing may be a series of measurements, which may be preferable in many cases where the sensor degrades in an unpredictable manner that can reduce its accuracy during use. Calibration may mean supporting accuracy and / or precision over the duration of use of the sensor, or other means that help maintain the accuracy or precision of the sensor in use. For example, a sensor for hormones may only need to maintain precision in the rate of change up or down of hormone levels, rather than representing the exact absolute concentration.

[0047] As used herein, the “product portion” of an aptamer sensor refers to a batch of sensors manufactured together, or using the same or similar processes, that, after manufacturing, are used as a product by the end user for continuous measurement. The term product does not necessarily require commercial sale and is generally limited only to the use of the sensor outside the factory in the application. Accuracy is determined at least in part by the standard deviation, and therefore the standard deviation is important for both accuracy and precision.

[0048] As used herein, “calibration data” may be a single data point, a curve, multiple data points, a code, an algorithm, a formula, or any other mathematical correction factor or other type of correction that enables factory calibration of the sensor. Calibration data may be represented, stored, or used in a number of ways, including, for example, adjustable resistors or capacitors, as long as it serves the purpose of enabling factory calibration of the sensor.

[0049] As used herein, “electrode area” refers to the area of ​​a working electrode that is electrochemically active and therefore capable of assisting redox electron transfer. Rough or porous gold electrodes may have an electrode area several times larger than the linearly calculated area observable from the product of the electrode length and width, or 3.14 multiplied by the square of the radius, or other measurements.

[0050] As used herein, “batch process” means a process used to produce or manufacture multiple sensors using the same or similar process so that the sensors perform the same or similar behavior during use. Same or similar behavior may include accuracy, precision, degradation, or other measures during use.

[0051] As used herein, “sensor preservation stabilization” means any method, material, design, or other element or feature used to preserve the sensor in order to reduce degradation during the period between the manufacture of the sensor and the end use of the sensor. For example, this period may include the time the sensor is stored in a warehouse, or in a delivery truck, or in a commercial store such as a pharmacy.

[0052] As used herein, “sensor sterilization” means any method, material, design, or other element or feature used to sterilize the sensor before final use. For example, sensor sterilization may include electron beam, gamma ray irradiation, steam, ultraviolet irradiation, heat, ethylene oxide, or other suitable methods.

[0053] As used herein, “Automatic Calibration Data” means data collected during use of a sensor that is used to improve, maintain, correct, warn about, or otherwise influence the accuracy, precision, or other performance characteristics of the sensor. For example, automatic calibration data may include frequency or time responses to find zero or non-responding frequencies that may change during use (collected automatic calibration data is used to automatically calibrate measured and / or reported data). For example, automatic calibration data may include direct or indirect measurements of electrode surface area, such as capacitance or cyclic voltammograms, to collect all redox charges and calculate the number of aptamers with redox tags on the sensor. For example, automatic calibration data may also be used to provide warnings to the user that the sensor has lost accuracy or precision or other performance characteristics, or to include them in stored data. For example, automatic calibration data may also be used to continuously report direct measures or estimates of the accuracy, precision, or other performance characteristics of the sensor during use (collected automatic calibration data automatically calibrates the reporting of the sensor's performance).

[0054] Detailed description of the invention One or more specific embodiments of the present invention are described below. For the sake of brevity in describing these embodiments, not all features of actual implementations may be described herein. As in any engineering or design project, in developing such actual implementations, numerous implementation-specific decisions must be made to achieve the developer's specific goals, such as conforming to system-related and business-related constraints, which may differ for each implementation. Furthermore, while such development efforts may be complex and time-consuming, it should be understood that they are nonetheless routine design, fabrication, and manufacturing tasks for those skilled in the art utilizing this disclosure.

[0055] Some embodiments of the disclosed inventions show sensors as simple, individual elements. It is understood that many sensors require two or more electrodes, reference electrodes, or additional support techniques or features not covered herein. Sensors can be configured in dual, triple, or more arrangements, thereby providing improved data and readings. Sensors can provide continuous or discrete data and / or readings. Some embodiments of the disclosed inventions show subcomponents of what would be known sensing devices with many more subcomponents necessary for the use of the device in various applications (e.g., reference or counter electrodes, batteries, antennas, adhesives), and for the purpose of brevity and focusing on aspects of the invention, such components may not be explicitly shown in the figures or described in the embodiments of the disclosed invention. All ranges of parameters disclosed herein include the endpoints of those ranges.

[0056] Unless otherwise specified, similar numbers may refer to similar features throughout the figures presented herein.

[0057] Referring to Figure 1, in embodiments of the present invention, devices 111, 113, or 115 are configured to measure at least one analyte in the body, in interstitial fluid or blood, and are configured on the skin 12 (111, 113) or implanted in the body (115). Each device has a housing 110, 112, 114, which may house electronics and other necessary components for the sensing device. Each device has sensors 100, 102, 104. In the case of sensor 100, the sensor is inserted into the skin. In the case of sensor 102, the sensor 102 is coupled to a biofluid 14 in the skin via a porous or hollow microneedle 190. The sensors may be electrochemical or optical sensors, such as aptamer or other affinity-based sensors. The biofluid 14 may be another type of biofluid, such as interstitial fluid, blood, or cerebrospinal fluid or other biofluids.

[0058] Referring to Figure 2, similar numbers indicate similar features, and in embodiments of the present invention, a sensor 200 as shown in Figure 1 (e.g., 100, 102, 104) carries multiple aptamers 224 and uses electrochemical measurement of the sensor signal. An exemplary aptamer 224 is a vancomycin aptamer (hereinafter identified as "SEQ ID NO. 1") having a carbon thiol linker at the 5' end for attachment to the electrode 220 and a methylene blue redox tag 270 at the 3' end. Embodiments of the present invention may include, in place of or in addition to, the vancomycin aptamers disclosed above, other aptamers known to those skilled in the art, other than those disclosed herein. Embodiments of the present invention may include other aptamer tagging schemes, tagging methods, and methods for measuring tags in response to changing analyte concentrations, such as dual reporter tagging for compensating for sensor drift, tags having different redox potentials, two or more tags that can provide redox quenching, or other suitable tags. An exemplary working electrode 220 is gold and supports an aptamer 224 and a protective monolayer or blocking layer 222. In different embodiments, the protective monolayer or blocking layer 222 is mercaptohexanol or mercaptooctanol. The sensor 200 may further include a protective film or other protective material (not shown), such as polybetaine. The aptamer 224 and the blocking layer 222 together form a sensing monolayer. When the analyte 280 binds to the aptamer 224, the aptamer changes shape, changing the availability of the redox tag 270 for electron transfer with the electrode 220, resulting in a sensor response. As illustrated in Figure 2, increasing the analyte 280 results in an increase in the redox current relative to the sensor response. However, the present invention is not so limited to this particular example, and may include other sensor responses to a decrease in redox current or an increase in the analyte 280. Such a sensor can reliably measure the target analyte for a week or more after being manufactured.

[0059] Referring to Figure 3, similar numbers refer to similar features, and in embodiments of the present invention, a sensor 300 (e.g., 100, 102, 104) as shown in Figure 1 carries a plurality of aptamers 324 and uses optical measurement of the sensor signal. The aptamers 324 carry fluorescent tags 370 and fluorescent quenchers 372. Alternative fluorescent tags are possible, including those having other fluorescent tags that, together with the aptamers 324, can generate optical signals that change in response to changes in the concentration of the analyte 380, through resonance transmission, changes in the ratio of fluorescent tags, changes in fluorescence lifetime, and as a result. The fluorescent tags 370 are excited by photons of light 390 in a waveguide 350, which may be glass or polymer fiber, or any other suitable light guiding material and geometric shape. When the analyte 380 binds to the aptamer 324, the aptamer changes shape, and when excited by light from a light source 390, it changes the fluorescence of the fluorescent tag 370. The emitted fluorescence is coupled to the waveguide 350 and propagates to the photodetector (resulting in signal gain or sensor response). Such sensors are similar to molecular beacons used in assays. They can be created and used to continuously measure target analytes.

[0060] Referring to embodiments of the present invention, Figures 2 and 3 are non-limiting examples of aptamer sensors. These are provided to illustrate examples of types of aptamer sensors that are further presented in embodiments of the present invention.

[0061] Factory Calibration - Manufacturing and Characterization Methods Referring to Figure 4, where similar figures point to similar features, in embodiments of the present invention, multiple sensors 400 can be fabricated in batches on a substrate 416. In one embodiment, the substrate can be a 125 μm plastic sheet such as PET or Kapton. For example, an electrochemical sensor 200 can be fabricated in batches by depositing and patterning titanium as an adhesive layer and then gold as the working electrode surface 220. Preferably, an electrical insulator such as SU-8 or dielectric ink used in test strips of the electrochemical sensor can be deposited and patterned. Subsequently, the aptamer 224 and blocking molecule 222 are incubated and coated with a protective hydrogel such as UV-curable polybetaine, and optionally preserved with a trehalose coating for dry storage. These exemplary methods and designs for fabricating the sensors 400 are merely examples and are not limiting to the present invention. Sensor 400 can then be cut from substrate 416 using a laser, razor blade, or other tooling and then mounted on a device such as that illustrated in Figure 1. Alternatively, sensors can be fabricated individually if desired, but this may result in increased manufacturing costs. Sensors can also be virtually optical, as taught for Figure 3. For example, waveguide 350 could be a planar waveguide with a titanium adhesive layer formed on PET, then coated with an optical cladding coating of silicon dioxide (refractive index n=1.46) or Cytop fluoropolymer (n=1.34) using an adhesion promoter, then coated with polymethyl methacrylate (n=1.49) or SiON (n=1.8) as the waveguide core, and then coated with aptamer 324 and additional chemicals (anti-fouling chemicals, hydrogels, etc.) as needed for individual applications. In optical sensors, the term “current” as used herein can be replaced with the term “signal” or “intensity,” which measures photons of light instead of electrons.

[0062] Referring to Figure 5A in an embodiment of the present invention, similar numbers indicate similar features, where multiple sensors are fabricated in batches, and at least three sensors from the batch are stored for factory calibration measurements. For an electrochemical sensor as illustrated in Figure 2, Figure 5A further illustrates square wave voltammograms 560, 562, and 564 for redox tag 270 current versus background currents 560a, 562a, and 564a. Methylene blue typically has a redox peak current of around -0.3V relative to the Ag / AgCl reference electrode. As shown in Figure 5A, sensors from batch fabrication typically have different background currents 560a, 562a, and 564a due to different total surface areas of the electrode 220 or other factors. This variable background current can be addressed in one or more ways. For example, high-precision photolithography of an electrical insulator, which may be a photosensitive polymer such as SU-8 on vacuum-deposited gold, has a standard deviation of 0.02, 0.01, 0.05, and 0.02 μm. 2 317 μm × 316 μm or 0.1 μm, which are smaller than at least one of the above. 2 It can be used to achieve an electrode area that targets or is expressed as 20, 10, 5, or 2% of the electrode area. Alternative methods include laser ablation or other suitable electrode patterning methods. Alternatively, as shown in Figure 5B, the background current present in Figure 5A is optionally first mathematically normalized to a common value 561a, or i b It is referred to as such. The factory calibration process may proceed as illustrated in Figure 7. As a non-limiting example, the factory calibration process may be initiated immediately after fabrication, or after preservation stabilization with a material such as trehalose, or after installation on electronic equipment, or after sterilization, all of which may affect the sensor response after fabrication. For example, preservation stabilization with trehalose and electron beam sterilization may each cause a loss of 10-50% of the sensor response.

[0063] Next, in various embodiments of the present invention, the factory calibration process may include one or more of the following steps. (1) As illustrated in Figure 5B, the difference in background current is minimized by normalizing the high-precision electrode region during fabrication or by normalizing the sensor background current (561a). The background currents are then statistically merged and can be interpreted as a representative voltammogram 561 (e.g., statistical mean). Such normalization may not be necessary if the electrode area has high reproducibility and the monolayer sensing has high reproducibility across batches of sensors. (2) The calibration portion of the sensor is titrated over multiple concentrations in a test fluid such as human serum at 33°C (or other temperatures such as 35°C or 37°C) to capture a portion of the sensor response versus the analyte concentration, which increases or decreases the redox peak current as illustrated in 561b, 561c, and this can then be plotted as a statistically analyzed (e.g., mean) Langmuir isothermal adsorption curve 600 of redox peak current versus concentration 663 using a standard deviation 663a, as shown in Figure 6. Unlike exogenous analytes (e.g., drugs), for endogenous analytes (e.g., insulin), the serum may be depleted of the analyte using enzymes, antibody capture, denaturation, or other preferred methods. (3) Save the curve in Figure 6 as a calibration curve, dataset, algorithm, or other suitable calibration feature or element using a method such as digital archival storage. (4) A common code is assigned from the batch, and that code is associated with the calibration curve saved from the sensor in the calibration section. (5) Provide the user with the product portion of the sensor. When the sensor is activated for use, the user's worn electronic device, reader or sensor electronic device, or software or firmware or memory already has the code or calibration dataset. Alternatively, the code is entered or provided, and the calibration dataset is accessed as needed from the cloud, reader, or other suitable storage location for the calibration dataset. Alternatively, the calibration data is loaded into the device at the factory and used later when the device is used for measurement. Alternatively, if the manufacturing of the sensor has a very tight standard deviation, the software uploads a calibration curve without code. Alternatively, the calibration data exists and is used in the cloud. Alternatively, the calibration data exists and is used in another computing device other than the sensor or sensor reader. Other calibration methods that can access and utilize the calibration dataset, algorithm, or other calibration features or elements are suitable, even if not expressly stated herein. The calibration data is then ready for immediate use to maintain sensor accuracy, as illustrated later in examples such as Figure 13. (6) The product portion of the sensor (Figure 1) is used to measure the concentration of the analyte in the body. The raw sensor signal is calibrated to a precise or accurate measurement of the analyte via calibration data or a curve. For example, if the redox peak of the raw sensor signal is 3.3 μA and the calibration curve specifies that 3.3 μA corresponds to 53 μM vancomycin, the measurement is set to report 53 μM vancomycin. Based on a standard deviation of 663a or other suitable data, the sensor may also report accuracy, confidence, or other statistical measures related to the measurement. (7) Finally, report the calibrated measurements from the sensor to the cloud, the user, the physician, or any other end recipient of the calibrated measurements.

[0064] In one embodiment, the present invention relates to a method for creating a factory-calibrated batch of aptamer sensors. This method involves manufacturing multiple sensors in batches or in a similar manner. Next, calibration data is created from at least three sensors, including a calibration portion. Next, the calibration data is assigned to the product portion of the sensor. Finally, using the remaining sensors, quantitative measurements of at least one analyte are reported using the calibration data for a measurement period of at least 0.5 days, 1 day, 3 days, 7 days, 10 days, or 14 days.

[0065] The above example is a single-point factory calibration. For most forms of aptamer sensors, the sensor characteristics also change over time during operation. Two non-limiting examples are shown in Figures 8A and 8B. These examples may represent redox current, normalized redox current, kinetic differential measurements, calibration-free measurements (as described later in Figure 13), other measures, or other measurable responses such as chronoamperometry, which measures the change in time of redox electron transfer instead of the magnitude of the redox current. In Figure 8A, which represents an example using a 6-carbon linked monolayer and a polybetaine protective film, fouling by solutes such as albumin is suppressed, and during initial operation, the sensor redox peak current 865a increases as the gold surface and monolayer remodel during the initial (several hours) of the electrochemical measurement. Over longer periods (several days), the redox peak current typically gradually decreases as the sensor monolayer is lost from the sensor due to desorption. In Figure 8B, illustrating an example where an 8-carbon linked mercapto-octanol block layer is used and hydrogel protection is weak or minimal, fouling by a solute such as albumin occurs rapidly. During initial operation, the sensor redox peak current 865b decreases as the fouling material interferes with or alters redox electron transfer. Over longer periods (several days), the redox peak current also gradually decreases, typically as the aptamer is lost from the sensor due to desorption or as fouling continues to occur over time. Thus, in embodiments of the present invention, the calibration process described with respect to Figure 7 can be repeated at one or more time points and one or more scales to provide a sensor lifetime calibration dataset containing some or all of the curves 865a, 865b, which can be used to enable more accurate measurements over longer periods of sensor operation. Exemplary examples with respect to Figure 8 and Figure 13, which similarly apply to other embodiments of the present invention, are taught later.Accordingly, the present invention includes a method for calibration that includes a sensor response over at least one of the following periods: 1 day, 3 days, 7 days, 10 days, or 14 days. Alternatively, a single-point calibration may be captured after 1, 2, or at least 3 hours, which is suitable for sensors that are highly stable over the long term but have significantly large initial changes in response. In additional embodiments of the present invention, a sensor may have an initial response that can vary greatly but has a predictable long-term response (degradation or rate of change of sensor response). In this case, a single-point calibration may be used to initially calibrate the sensor, and since the long-term response of the sensor is predictable, it does not need to be calibrated for each batch of sensors, and consequently, the sensor calibration includes both a calibration curve for a factory-calibrated batch and a predicted calibration curve based on previous or historical measurement data.

[0066] With respect to embodiments of the present invention, there are other factors that affect the calibration and accuracy of the sensor and may be measured during factory calibration. These factors include the electrochemically active electrode surface area, aptamer density (measured by the total redox charge in a cyclic voltammogram), frequency maps, measurement methods such as square wave voltammetry, cyclic voltammetry, intermittent pulse amperometry, chronoamperometry, and continuous square wave voltammetry, electrical impedance indicating that a low resistance or high capacitance value is a more porous blocking layer 222, the effect of fluctuating pH and salinity, the effect of temperature, oxygen reduction current exceeding -0.3V vs. Ag / AgCl reference electrode, and other measures.

[0067] Calibration-free operation vs. factory calibration In the context of this invention, there is a difference between "no-calibration" operation and "factory calibration." As detailed in later sections, no-calibration methods rely on the electron transfer characteristics between the redox tag and the working electrode. In practice, factory calibration is useful for no-calibration methods because the electron transfer characteristics and other factors for the sensor change over time in in vivo operation over durations ranging from days to weeks. When combined with factory calibration, the purpose of the no-calibration method almost certainly changes. It is no longer a method for maintaining the accuracy of the sensor, but rather simply provides superior data to supply to the factory calibration model, formula, algorithm, or method, and consequently maintains the accuracy of the sensor. Any calibration method depends on the appropriateness of the data supplied to it. For example, a calibration method that relies solely on changes in the measured absolute redox peak current cannot be said to have optimal accuracy because the absolute current has significantly large variability that reflects more than just the aptamer's reaction to the changing analyte concentration. Conversely, square wave voltammetry, when used with multiple frequencies or continuous square wave operation, can extract excellent data with less variability and greater dependence on the aptamer's response to changing analyte concentrations. After all, some of the leading factors that must be "calibrated" are temperature, concentration, in some cases pH or salinity, and the sensor's response to the waveform, as well as how these change over time. Calibration-free methods neither account for all modes in which the sensor changes over time, nor were they initially developed or intended to account for all modes in which the sensor changes over time. This is especially true in the field of aptamer sensors, at least, as it is only very recently that sensors with a sufficiently long lifespan for which long-term changes in sensor function and performance can first be observed have been manufactured.

[0068] Referring to embodiments of the present invention, the following measures are non-limiting examples of measurements that may be included in factory calibration, either as a one-time measurement for batch calibration or as measurements at multiple points in time, including an operating time period of up to two weeks or more.

[0069] A) Redox tag currents measured as redox peak versus baseline, as described in Figure 5B, at a single time point or time, and at multiple analyte concentrations or measurement parameters (such as square wave frequency).

[0070] B) Redox tag peak current potential at a single point in time or over time. Knowing the voltage position of the peak redox potential not only indicates the scanning window but can also provide an indicator of sensor degradation. For example, the redox peak may shift to a higher potential due to sensor fouling. In another example, when a protective monolayer such as mercaptooctanol is lost, the redox gauge peak potential may shift to a lower potential, or, for example, when the reference or counter electrode changes or degrades over time.

[0071] C) For example, the oxygen reduction current at a single point in time or over time, measured as an increase in background current at -0.5V. As the oxygen reduction current increases, it may be evidence that the protective monolayer is degrading or becoming more porous, which can shift the frequency response by allowing for a faster electron transfer rate. This may also change the interpretation of baseline current versus redox peak current, as the baseline is no longer flat.

[0072] D) For example, the redox tag density, or total number of redox tags, measured using cyclic voltammetry, which integrates the measured redox charge and then, by known electrode surface area, gives the redox tag density, and therefore the aptamer density. If aptamers in the redox current desorb over time, these may give a pseudo-signal decrease that is not due to a small amount of analyte, but simply due to a decrease in redox tags available for electron transfer.

[0073] E) Capacitance or impedance at a single point in time or over time that can give a measure of fouling and / or degradation of the protective monolayer, both of which may shift, for example, the frequency response to the sensor or the sensor response. Capacitance can be measured using a simple electrical impedance method, or capacitance measured within 1 ms or a few ms after a square wave pulse is applied can be determined from the dominant current.

[0074] F) Frequency response, electron transport velocity, sensor response at a sampling point in a continuous square wave, or other types of time or frequency response of redox tag current at a single point in time or against time that directly affect sensor accuracy as described herein.

[0075] G) Chronoamperometric response of redox tag current at a single time point or against time, which affects not only chronoamperometric measurements but also the accuracy of the selected sampling point for continuous square wave voltammetry.

[0076] H) Electrode surface area, also known as electrochemically active surface area, can be measured during manufacturing. For gold electrodes, a cyclic voltammogram is measured in sulfuric acid, thereby allowing the gold electrode surface area to be calculated from the charge passed through the redox peaks of gold. This measurement of electrode surface area is typically performed before the sensing monolayer is added. Other methods for measuring electrode surface area after the sensing monolayer is added often include, for example, capacitance, or cyclic voltammetry to obtain the total aptamers and redox tags on the sensor surface, which will represent the electrode surface area if deposited with a tight standard deviation across the entire batch of sensors (larger electrode surface area = greater total number of aptamers and redox tags = greater total charge transfer measured during the cyclic voltammetry cycle).

[0077] I) The sensor response versus analyte concentration, also known as the titration response at a single point in time or over time, can be measured using cyclic voltammetry, differential pulse voltammetry, square wave voltammetry, continuous square wave voltammetry, kinetic differential measurement between "signal on" and "signal off" sensor responses as illustrated in Figure 11, two or more frequencies as described in Figures 11A-11B and 12A-12D, chronoamperometry, or other preferred methods. Generally, it is important to calibrate the sensor response to changes in redox current using a single frequency (or timing sample point for continuous square wave voltammetry) or two or more frequencies or sample points (also known as kinetic differential measurement), and to compensate for changes in redox current that occur over time using batch calibration data. In addition, the frequency response shifts over time, as illustrated in Figures 11A and 11B, and can be similarly compensated for via calibration measurements and then during sensor use.

[0078] J) Standard deviation, precision, accuracy, or other statistical methods for any of the measures described above may be used to enable the sensing system to report ongoing precision, accuracy, or other statistical information in any other way that is important for determining the reliability of the data measured by the sensor, whether it reports to the end user, to a database, or otherwise.

[0079] Additional factory calibration measurements may include variable in vivo parameters such as salinity, temperature, or pH. However, if these measures are used for calibration, the sensor itself must have a suitable measurement method or a dedicated sensor for salinity, temperature, or pH. If temperature, pH, or salinity is unknown, their effects on the sensor response, which may change during operation, cannot be compensated for during sensor operation.

[0080] All of the above measurement data can be converted into formulas, algorithms, lookup tables, correction coefficients, datasets, or other preferred methods, which then allow the measurement, accuracy, precision, or other measurements related to the sensor measurement and operation to compensate for sensor drift, degradation, change, or other factors that reduce the reliability of the measured data.

[0081] Factory calibration may further include blood-to-interstitial fluid correlation for specific analytes, particularly large analytes that may be more diluted in interstitial fluid than in blood. For example, if the analyte is a protein, such as a cytokine, which has an interstitial fluid concentration that is only 50% of its blood concentration, factory calibration may involve limited testing on humans to determine this 50% dilution. The user could then receive a corrected or calibrated reading that is twice the factory-calibrated measurement tested in vitro in human serum (unlike in the body where interstitial fluid is present), where all the added cytokines used to determine the sensor response are available to the sensor. In practice, such blood-to-interstitial fluid correlation could be performed on every batch of manufactured sensors, but this is unlikely and would likely only be determined once at most with a limited set of subjects. For example, further blood samples could be taken from end-users of the device and analyzed, and such data could be fed back into the calibration algorithm over time.

[0082] Factory calibration may include further potentiostat (electronic device)-specific parameters that vary depending on the sensor type (e.g., protein vs. small molecule sensors, long aptamers vs. short aptamers), vary during sensor measurement in vivo, vary over time due to sensor changes, or for other reasons and uses. For example, a typical set of available parameter ranges for a potentiostat with square wave voltammetry or continuous square wave voltammetry may be as follows:

[0083] The initial potential (V) is between -10 and +10V, for example, -0.05V.

[0084] The final potential (V) between -10 and +10V, for example, -0.50V.

[0085] A stepped voltage increment, for example, 1mV or 5mV, with a potential step (V) of 0.001 to 0.05V.

[0086] Square wave amplitude, half-peak to half-peak, for example 35mV, with amplitude (V) between 0.001 and 0.5V.

[0087] Square wave frequencies are frequencies (Hz) between 1 and 100,000, such as 10 Hz, 40 Hz, or 300 Hz.

[0088] The resting time before potential scanning, which can range from 0 hours to any desired length of time, is a resting time (in seconds) between 0 and 100,000.

[0089] A sensitivity scale used when measuring (sampling) redox current, e.g., ~mm 2 For a working electrode with an area of ​​~10 -4 from 10 -7 A / V, 10 -12 Sensitivity (A / V) from 0.1.

[0090] Redox current measurements (samples) are collected and averaged (if applicable) over the duration, e.g., single data point, 5 data points, or any other number of data points, or the duration of the measurement, or the measurement width (seconds).

[0091] There are similar or other parameters commonly known for chronoamperometry, cyclic voltammetry, or other measurement techniques, and these do not need to be explicitly listed as being included in embodiments of the present invention.

[0092] Many commercial products such as glucose monitors do not require factory calibration of measurement parameters as listed above, because they do not need adjustment. However, aptamer sensors present several unique cases where factory calibration data includes at least one measurement parameter. For example, an aptamer sensor directly exposed to blood or interstitial fluid can change the redox current by about 10 times during the first few hours of use without fouling protection, which suggests that a 10-fold change in sensitivity is beneficial for maintaining the optimal precision, and ultimately the optimal accuracy, of redox current measurement. Therefore, the optimal sensitivity can be measured at the factory during calibration and used to adjust the measurement sensitivity during use as taught herein. For example, 10 -6 starting from the sensitivity of , over the first 6 hours of operation the 10 -7The sensitivity is adjusted non-linearly during scanning. As another example, aptamer sensors for lactate can use very small aptamers, and the acceptable aptamer density on the electrode is 10 times greater than that for protein aptamer sensors, which have considerably larger aptamers. Also, due to the size of the protein, overcrowding is more likely to occur on the sensor surface. Therefore, the sensitivity of lactate sensing can be 10 times lower than that of protein sensing, because lactate sensors can have redox currents that are more than 10 times greater. For example, during continuous square wave voltammetry (see the next section for details), multiple redox current measurements are sampled at time points such as 3 ms and 100 ms, where the magnitude of the redox currents differs by about 10 times. Therefore, the ideal measurement sensitivity would be 10 times lower for the 3 ms measurement than for the 100 ms measurement. For example, factory calibration data includes at least one measurement parameter which is the measurement sensitivity, and the measurement sensitivity includes multiple different sensitivities for multiple different measurements. In practice, implementing multiple sensitivities can be achieved by programming the electronics to adjust the sensitivity during measurement, for example, by using the first sensitivity for a redox current sampled at 3 ms and then increasing the sensitivity tenfold for a measurement sampled at 100 ms. Alternatively, it is possible to perform multiple measurements, such as measuring the redox current at 3 ms for a first square wave voltammetry measurement scan and then measuring the redox current at 100 ms for a second square wave voltammetry measurement scan, with the sensitivity being more than tenfold higher for the second measurement scan compared to the first.

[0093] Factory Calibration - Recalibration during use Referring to Figure 9 in embodiments of the present invention, the sensor may be manually calibrated (or “recalibrated”) at a time of care or use (calibration data is corrected using manual calibration at the time of use). For example, consider a non-limiting example of an NT-proBNP sensor for a hospital-based heart failure patient, as illustrated in Figure 9. NT-proBNP blood tests may be ordered regularly in the hospital for heart failure and used to improve the accuracy of the sensor. In addition, patients may also have appointments for home testing or follow-up after discharge, which may be used to calibrate the sensor. Furthermore, home care and long-term use of the sensor may require additional calibration, even with the use of home testing functions (such as finger-prick tests). Figure 9 illustrates sensor readings that change over time, including multiple manual recalibration events at three exemplary time points 967a, 967b, and 967c. At 967a, for example, the reported concentration for NT-proBNP increases because the sensor was underestimating the NT-proBNP concentration. Although Figure 9 does not show retrospective recalibration, it would also be possible to apply recalibration using averaging or other mathematical methods to ensure there are no discontinuities in the analyte measurement plot over time (this could appear as a smooth, continuous line that is appropriately shifted as needed due to recalibration events).

[0094] Factory Calibration - Automatic Calibration During Use Referring to embodiments of the present invention, manual calibration is highly feasible but less desirable than automatic calibration. Aptamer sensors are known to have several approaches that claim to provide “calibration-free” operation, such as those taught by Plaxco et al. in U.S. Patent Application Publication No. 2020 / 0182820A1. Among other possible methods, an additional such “calibration-free” method was recently taught by White et al., “Abeykoon SW, White RJ. Continuous Square Wave Voltammetry for High Information Content Interrogation of Conformation Switching Sensors. ACS Meas Sci Au. 2022.” Calibration provides a concentration value, which is calculated according to Equation 1 shown in Figure 21. In Equation 1, [T] is the concentration of the analyte (or target), and K D is the aptamer dissociation constant relative to the target, i is a constant including the redox peak current at the measurement frequency, and α is a constant including the ratio of the output signal at the minimum (or non-responding) frequency to the redox peak signal without the target at the measurement frequency, i NR γ is the redox peak at the minimum (or non-responding) frequency, and γ is a constant that includes the ratio of the analyte-saturated output signal to the output signal without the analyte. These measures and parameters are illustrated in Figures 12A–12D, as taught by Plaxco et al.

[0095] However, such calibration-free methods essentially assume a stable electron transport kinetic theory and stable peak and measurement non-response frequencies for square wave voltammetry (i or i NRFor the selected frequency, we assume it does not change, or for continuous square wave voltammetry, we assume a stable sampling time. Ultimately, in most aptamer sensors, these frequencies are not stable and shift during operation. Embodiments of the present invention teach a method that allows a measurement system to automatically recalibrate the accuracy of a “calibration-free” sensing method, as illustrated in Figures 10 and 11. Again, as previously stated, the purpose of using the “calibration-free” method in the present invention is not necessarily to avoid further calibration, but rather to obtain superior data acquisition that ultimately improves sensor accuracy.

[0096] A first example of automated calibration is as follows: In a first embodiment of automated calibration, the present invention requires that the sensor has a measured or known or expected or predicted standard deviation 1063a in addition to the actual sensor analyte measurement 1063. Secondly, in a first embodiment of automated calibration, the present invention requires that the sensor's sensor response or analyte measurement 1063 changes by at least a standard deviation 1063a, or at least twice the standard deviation 1063a, such as at time points 1068 and 1069. Thus, the calibration data can be corrected using automated calibration during use. In addition, a factory-calibrated aptamer sensor further includes a known or expected or predicted standard deviation for the sensor response, and automated calibration occurs when the sensor response changes by an amount greater than the standard deviation of the sensor response. As an example of this requirement, cortisol or melatonin or NT-proBNP have large diurnal variations that would satisfy this requirement for a cortisol or melatonin or NT-proBNP sensor. An example of this additional requirement is the dosage of a drug taken orally and measured by a sensor, which would also satisfy this requirement. As illustrated in Figures 11A and 11B, at t=1 day, the peak response frequency 1190a for signal OFF, the non-response frequency 1190b, and the peak response frequency 1190c for signal ON can all change to new frequencies at 3 days or other durations, as illustrated as 1191a, 1191b, and 1191c.When the sensor exhibits a change in response greater than the sensor's standard deviation (Figures 10, 1068, 1069) (resulting in a change in redox peak current), a full or partial scan of the sensor response versus frequency is collected and optionally plotted or analyzed as shown in Figures 11A–11B, and these frequencies are determined based on which frequencies have the largest change in redox peak current as the analyte concentration changes (1190a, 1191a, 1190c, 1191c) and which frequencies have the smallest change in redox peak current, or no such change at all, as the analyte concentration changes (1190b, 1191b). These new frequencies are then stored in the device, software, or elsewhere and can be used to reprogram and maintain the accuracy of the “calibration-free” method. In other words, non-responding frequencies such as 1190b and 1191b can be simply rediscovered or redetermined during device use by measuring at what frequencies the redox current remains unchanged when the response frequency confirms a change in the concentration of the analyte. In yet another embodiment, a measurement of only the shift of non-responding frequency 1191b can be obtained, again, since the sensor response should not be changing at non-responding frequencies 1090b and 1191b during periods of significantly large changes in sensor responses 1068 and 1069, and is therefore identifiable. The newly learned fact regarding the shift of non-responding frequency 1191b can then be used to shift the peak frequency relative to the signal OFF 1191a or signal ON 1191c frequency (assuming that the entire frequency response shifts similarly or predictably with respect to the shift of the non-responding frequency). For example, if the zero frequency is raised by 10%, it may also be assumed that the signal ON frequency shifts by 10% (or other predetermined value based on sensor aging tests).In this automated calibration method, which is identical except for being applied to continuous square wave voltammetry, the measurement is simply a time point (reciprocal of frequency), and the method and final results are sufficiently similar to those of the embodiments of the present invention. These same frequencies or time points can also be used to indicate kinetic differential measurements that increase the signal-to-noise ratio of a sensor by subtracting the signal-off response from the signal-on response, or by the ratio of the signal-on response to the signal-off response.

[0097] Referring to embodiments of the present invention, it will be seen that additional forms of automatic calibration may be available. A sensing device illustrated in Figure 1 may be attached or used to measure, in addition to the above, at least one of the following: changes in aptamer density that can affect the frequency response (via a cyclic voltammogram), electrical impedance that can affect the charge transfer velocity and frequency response (via electrical impedance measurements across multiple measurement frequencies), and capacitance background current, temperature or pH or salinity (using specialized sensors known to those skilled in the art) that can affect the sensor response, oxygen reduction current that can affect the background current and shows changes in blocking layer density that further affect the charge transfer velocity and frequency response and capacitance background current (via square wave voltammetry or amperometry). For example, in electrical impedance measurements, charge transfer resistance (R ct If the value decreases by 20%, the peak measurement frequency will shift by an increase of 55 Hz, and the sensor calibration of the redox peak current may decrease by 5%. All of this can be automatically used to compensate for the device calibration in real time as the sensor changes during operation over time.

[0098] Referring to embodiments of the present invention, embodiments of the present invention can also be applied to optical sensors such as those illustrated in Figure 3. For example, batch calibration is applied to optical sensors using a similar method in which the fluorescence signal or peak intensity replaces the redox peak current, such as normalizing the background fluorescence for all sensors in a batch to the minimum or maximum sensor response to a target analyte (zero or saturated target), or by taking three sensors from a batch of 12 sensors for factory calibration.

[0099] Referring to embodiments of the present invention, unlike diffusion flux-dependent glucose sensors, aptamer sensors are concentration equilibrium-dependent and may require calibration against analyte dilution of blood values ​​versus interstitial fluid values. For example, blood values ​​for an analyte can be measured from a single or multiple individuals, thereby obtaining an average value, and these blood concentrations can be used to calibrate the interstitial fluid sensing value to the blood value. For example, an albumin sensor for cardiovascular disease may have albumin levels in the interstitial fluid, which is about half the amount in the blood, depending on the patient. Therefore, first, a batch of sensors is calibrated as taught in a previous embodiment, and then one or more blood measurements of albumin can be used to calibrate the sensor response in the interstitial fluid (e.g., multiples of 2) so that the sensor can predict the blood concentration of albumin. Similar calibrations can also be used for Tmax and Cmax for, for example, the division of orally administered drugs. Because aptamers are affinity-based and concentration-dependent, sensor calibration may require a sufficient amount of time, such as several minutes or hours, for the concentration in the blood and interstitial fluid to reach a steady state or near-steady state (steady concentration or flux of the analyte throughout the body, or steady state after healing has occurred at the sensor insertion site) before calibration readings can be recorded, given that calibration may be attempted while the concentration is changing. Therefore, one embodiment of the present invention includes a factory calibration, which is obtained in a human only after or near the point in time when the concentration of the analyte in the interstitial fluid has reached a steady state.

[0100] Kinetic differential measurement for reduced sensor drift and higher sensor accuracy Referring to embodiments of the present invention, kinetic differential measurement may be used, as illustrated in Figure 13A. In the plot of Figure 13A, the normalized redox peak current is based on the signal-on redox current 1360a, but the present invention is not limited to that. Kinetic differential measurement provides a continuous measurement 1360a, which is the difference between the signal-on measurement 1362a and the signal-off measurement 1364a. Signal-on measurement 1362a and signal-off measurement 1364a typically decrease over time in redox current, but measurement 1360a is more stable and drifts less, as taught in Ferguson BS, Hoggarth DA, Maliniak D, Ploense K, White RJ, Woodward N, Hsieh K, Bonham AJ, Eisenstein M, Kippin TE, Plaxco KW, Soh HT, "Real-time, aptamer-based tracking of circulating therapeutic agents in living animals."

[0101] The present invention also provides time-dependent calibration for kinetic differential measurements, as illustrated in Figure 13B. For example, assuming the sensor in Figure 13A is the calibration portion of the sensor, the sensor in Figure 13B may be the product portion of the sensor shown for non-limiting examples of analytes that are not expected to change over time (e.g., drug monitors and sensing devices capturing medication compliance for drug users who are not using the drug). Simply put, calibration provides more accurate measurements over time 1360B. Calibration curves may also be captured and used for multiple sensor response levels corresponding to multiple analyte concentrations. An example of a curve is illustrated in Figure 14, where 1460-90 is a calibration curve for 90% of the maximum sensor response, 1460-50 is a calibration curve for 50% of the sensor response (sometimes called Kd or binding affinity to the sensor), and 1460-0 could be a calibration curve for no analyte, or an unmeasurable level of analyte, or 10% of the maximum sensor response.

[0102] Exemplary experimental data for two batches of sensors are shown in Figure 17, for an uncalibrated set of data consisting of a batch portion (n=3) and a product portion (n=1) of the sensor batch, plotted against a single stable concentration of the analyte for aptamer sensors, all fabricated as described herein and measured using a constant waveform, which cannot always be assumed, as will be discussed in the following section. In Figure 17, the outer dotted line for each plot represents the standard deviation.

[0103] Calibration and / or instructions for sensor stabilization One of the challenges of affinity biosensors, such as those described herein, is that it can take time for the measurement technique to stabilize to obtain accurate measurements. For example, electrochemical aptamer sensors, such as those described herein, often require several hours. Stabilization is a problem that has not been adequately addressed in the field of affinity biosensors for at least two reasons: (1) Academic or other research studies can allow for the stabilization of the device either in vitro or in vivo before accurate data can be collected, and are not subject to the commercial requirement of obtaining accurate data as quickly as possible after the sensor is put into use. (2) Affinity biosensors are notorious for having a short operating life, which makes it difficult to confirm the long-term need to re-stabilize the sensor after initial stabilization. Now that more stable sensor chemicals, such as 8-carbon linked monolayers (such as mercaptooctanol), can be implemented on optimized gold with appropriate fouling prevention measures, a new set of challenges arise regarding whether sensor stabilization is necessary. Two exemplary challenges are as follows:

[0104] The first challenge is initial sensor stabilization, as illustrated in Figure 15A. Sensor response curves for a given concentration of analyte are shown for initial placement of a sensor subject to fouling 1562 (e.g., without hydrogel protection) in serum or interstitial fluid. Fouling stabilization can be partially addressed by prefouling the sensor, as described in the application WO2021067779A1, titled "Shelf-Stable, Ready-to-use, Electrochemical Aptamer Sensors". However, long-lasting sensors (days to weeks or more) cannot tolerate fouling if they should be as accurate as possible in the measurement, and without fouling prevention, additional slow fouling will occur over a period of days or more. Even sensors that are foul-free and last for days to weeks may have initial stabilization, as illustrated in Figure 15A, compared to the fouling-free sensor response curve 1560. An example of such a sensor is an aptamer sensor on a sodium hydroxide roughened gold surface, which has a mercaptooctanol blocking layer and eight carbon linkages to the aptamer, and is protected by a polybetaine hydrogel.

[0105] The second challenge is the stabilization of the sensor in operation after initial stabilization, as illustrated in Figure 15B and shown with actual data in Figure 16. Figure 15B shows a sensor response curve representing an aptamer biosensor exposed to a first waveform 1591, which is an analyte measurement waveform, or a second waveform 1592, which is a sensor diagnostic waveform for determining the ongoing health of the sensor, for example, to compensate for sensor degradation and improve time-dependent calibration, such as the automated calibration taught herein. For example, waveform 1591 may be square wave voltammetry or continuous square wave voltammetry, while waveform 1592 may be a frequency response, as taught in Figure 11, or impedance spectroscopy for determining monolayer degradation through capacitance, or a DC voltage of -0.5V for determining monolayer degradation through the amount of oxygen reduction current in the sensor. Each time waveform 1592 is applied, it may disturb the sensor, particularly the sensing monolayer and any fouling material attached thereto, and it may otherwise be necessary to re-stabilize the sensor before accurate data can be collected. The downside to having to perform sensor stabilization is simple: the time available for accurate data collection is shorter. Without conventional sensor fouling by large solutes such as proteins, sensor stabilization can largely be attributed to the rearrangement of the sensing monolayer, including molecules bound to the surface, and solutes in the sample fluid, such as interstitial fluid, that incorporate the monolayer itself or nearby, but whose binding to the monolayer is irreversible or too small to hinder the free movement of aptamers. When the sensing monolayer or adjacent solutes are rearranged, this rearrangement alters the electron transfer properties through the monolayer.

[0106] In embodiments of the present invention, initial sensor stabilization, also referred to as the first stabilization period, can be resolved by calibrating the sensor stabilization response to at least one waveform. For example, in embodiments of the present invention, if the sensor does not suffer significant fouling and it is not beneficial to pre-foul the sensor (fouling-free sensor response curve 1560 in Figure 15A), the initial sensor response can be simply characterized from the calibration portion of the sensor and applied to the product portion of the sensor during use.

[0107] In one embodiment of the present invention, several additional examples and details are presented here of sensor stabilization after initial sensor stabilization, which may be referred to as a second sensor stabilization period. The waveform may be at least one of the following: analyte measurement waveform, sensor diagnostic waveform, sensor storage waveform, or another type of waveform. For example, an aptamer sensor may have a mixed monolayer of aptamers for two or more analytes, and two or more redox tag species at different redox potentials are used to distinguish each measurement of the aptamer population, requiring different waveforms 1591, 1592 for each type of aptamer, the waveforms being different and thus disturbing the sensor monolayer. Alternatively, for example, waveform 1592 could be a sensor diagnostic waveform such as impedance spectroscopy or a DC potential scale of oxygen reduction current. Alternatively, for example, waveform 1592 could be used to extend the sensor's lifespan by suppressing the oxidation of monolayer chemicals, and thus could be a DC potential of -0.2V, requiring less electrical computation or analog power compared to continuously operating more complex waveforms such as square wave voltammetry. The exact durations of waveforms 1591 and 1592 shown in Figure 15B, as well as their exact impact on the sensor response and sensor stabilization needs, are not limited to the example shown in Figure 15B, which is simply a single exemplary example. Furthermore, there may be more than just two waveforms (e.g., three different waveforms). Next, in embodiments of the present invention, sensor stabilization after initial sensor stabilization can be solved in at least two ways so that more data can be accurately collected. In the first method, sensor response 1591 is characterized from the calibration portion of the sensor and is used to compensate for the product portion of the sensor while the sensor is re-stabilized each time during use (e.g., re-stabilized after applying waveform 1592). In the second method, the waveform is periodic. For example, as shown in Figure 15B, if two or more waveforms are applied at periodic intervals, it can be assumed that the re-stabilization will be the same or similar each time over an operation of at least several hours, and more ideally several days or weeks.If the sensor re-stabilization is similar each time because the waveform is applied periodically, accurate data can be collected even during the period when the sensor re-stabilizes each time, as the trend toward sensor re-stabilization is known or is the same or similar each time.

[0108] Embodiments of the present invention may be applied to sensors that do not foul or show little change in sensor response to fouling, or to devices that foul and exhibit significant changes in sensor response due to fouling (such devices typically have a fairly short operating life or low measurement accuracy). Fouling material bonded to the monolayer of the sensor may also rearrange over time in response to waveforms, thus potentially necessitating stabilization.

[0109] Other aptamer sensors The sensing or working electrode is composed of an electrode material such as gold. The gold is then incubated with an aptamer via thiol deposition onto the gold electrode, and the aptamer contains a redox tag such as methylene blue. Between the aptamers, the gold is incubated with a protective monolayer such as mercaptohexanol, mercaptooctanol, or other suitable chemicals. To prevent fouling of the monolayer surface, a protective film such as polybetaine or other suitable material may be added. The working electrode may be preserved in a preservative such as trehalose to allow for dry storage. In a non-limiting but specific example, the binding of the aptamer to the target causes a shape-structure change that brings the redox tag closer to the electrode, resulting in increased electron transfer (increased current). As the concentration of the target increases, more binding of the target to the aptamer occurs, resulting in more electron transfer (higher measurable current). Conversely, as the concentration of the target decreases, the current decreases. Devices such as those taught herein can be inserted into the skin using methods commonly used for inserting glucose sensor needles into continuous glucose monitors (e.g., slotted insertion guides or other methods). Examples of device fabrication and testing are shown below.

[0110] Electrochemical measurements Electrochemical measurements may be performed using a miniaturized potentiostat or by a benchtop CHI 620E potentiostat (located in Austin, Texas) connected to a 64-channel multiplexer in a standard three-electrode system where an aptamer / alkylthiolate functionalized electrode functions as the working electrode. The counter and reference electrodes are inserted into the skin using a platinum counter electrode and an Ag / AgCl reference electrode, or alternatively, the counter and reference electrodes may be large gel electrode pad electrodes on the skin surface, as taught in the application International Publication No. WO2022067051A1, titled "Aptamer sensors with reference and counter voltage control". Cyclic voltammograms may be recorded at a scanning speed of 100 mV / s within a window of -0.1 V to -0.5 V. Square wave voltammetry can be performed with an amplitude of 25 mV at the optimal measurement frequency for each aptamer within a potential window of -0.1 V to -0.5 V.

[0111] To improve the calibration-free operation of the sensor, kinetic differential measurement, two-frequency measurement, or continuous square wave voltammetry may be used. Exemplary in vivo data collected for a cortisol sensor subcutaneously implanted in rats are shown in Figure 20, where cortisol bolus injections were performed at 5 mg / kg or 10 mg / kg.

[0112] Additional targets The present invention generally applies to aptamer sensors and other types of affinity biosensors, and is therefore not limited to specific examples as taught herein. Available electrochemical sensors for analytes such as cortisol, vancomycin, phenylalanine, insulin, BNP, NT-proBNP, IL-6, C-peptide, C-reactive protein, albumin, and sensors for other analyte targets can be incorporated into the present invention without limitation. Such aptamers can be obtained from the literature, by SELEX, or purchased from companies such as SOMAlogic or Base Pair Technologies, and can be adapted to aptamer sensors similar to those shown in Figure 3, or to other aptamer sensing configurations based on alternative switching mechanisms such as redox quenching, molecular pendulum, or other preferred methods.

[0113] Features of ultra-stable sensor performance Sensor performance can change significantly over time, but factory calibration can compensate for many of these changes. Aptamer sensors have unique calibration requirements compared to other types of continuous sensors. The most important performance characteristics of aptamer sensors that can maintain performance by leveraging factory calibration are as follows: 1) Maintain appropriate redox tag current versus background signal. For redox tag current, the aptamer and redox tag should remain attached to the monolayer, and for background signal, the blocking layer portion of the monolayer should remain unchanged so as not to substantially increase capacitive current and oxygen reduction current. 2) Maintain a predictable electron transfer rate between the redox tag and the electrode. This electron transfer rate can shift significantly if the sensing monolayer becomes more porous due to monolayer desorption or if electron transfer is hindered via fouling. 3) Potentially most importantly, while the above factors may vary to some extent, if they do vary in a predictable manner, batch calibration must maintain a strict standard deviation for the calibration portion of the sensor. This is particularly important because, when there is a large variation in performance between sensors, it requires a significant amount of testing resources and can prematurely or over time degrade the sensor product, making it quite difficult to use all sensors calibrated economically or practically during their intended service life (the calibration portion and the product portion are inseparable).

[0114] The present invention first provides, for the first time, ultra-stable standard deviations, capacitance, electron transfer velocity, and oxygen reduction current to support factory calibration and ultimately the real-world accuracy of the product portion of the aptamer sensor. The following tabular data shows sensor standard deviations that are dramatically more stable than those previously demonstrated for aptamer sensors and more stable than those often observed across continuous biosensors in general. The sensors are fabricated using methods such as those detailed herein. Example 2 shows one specific example of a sensor that enables ultra-stable performance.

[0115] In one embodiment, the present invention includes a calibration portion for an aptamer sensor having a standard deviation of <±2% in at least a 10X range of measurements when continuous biosensing is performed on a production version in interstitial fluid or serum or an equivalent substitute test fluid at temperatures reached during operation for at least one of 7 or 14 days.

[0116] In another embodiment, the present invention includes a calibration portion for an aptamer sensor having a standard deviation of <±3% in at least a 10X range of measurements when continuous biosensing is performed on a production version in interstitial fluid or serum or an equivalent surrogate test fluid at temperatures reached during operation for at least one of the following periods: 7, 14, 21, 30, or 40 days.

[0117] In one embodiment, the present invention includes a calibration portion for an aptamer sensor having a standard deviation of <±5% in at least a 10X range of measurements when continuous biosensing is performed on a production version in interstitial fluid or serum or an equivalent substitute test fluid at temperatures reached during operation for at least one of the following periods: 7, 14, 21, 30, or 40 days.

[0118] In another embodiment, the present invention includes a calibration portion for an aptamer sensor having a standard deviation of <±10% in at least a 10X range of measurements when continuous biosensing is performed over at least one period of 7, 14, 21, 30, or 40 days in interstitial fluid or serum or equivalent surrogate test fluid at temperatures reached during operation relative to the production version.

[0119] In one embodiment, the present invention includes a calibration portion for an aptamer sensor having a standard deviation of <±20% in at least a 10X range of measurements when continuous biosensing is performed on a production version in interstitial fluid or serum or an equivalent substitute test fluid at temperatures reached during operation for at least one of the following periods: 7, 14, 21, 30, or 40 days.

[0120] In another embodiment, the present invention includes a product portion of an aptamer sensor having an accuracy of <±5% in at least a 10X range of measurements when continuous biosensing is performed on the product version in interstitial fluid or serum or an equivalent surrogate test fluid at temperatures reached during operation for at least one of the following periods: 7, 14, 21, 30, or 40 days.

[0121] In one embodiment, the present invention includes a product portion of an aptamer sensor having an accuracy of <±10% in at least a 10X range of measurements when continuous biosensing is performed on the product version in interstitial fluid or serum or an equivalent substitute test fluid at temperatures reached during operation for at least one of the following periods: 7, 14, 21, 30, or 40 days.

[0122] In another embodiment, the present invention includes a product portion of an aptamer sensor having an accuracy of <±20% in at least a 10X range of measurements when continuous biosensing is performed on the product version in interstitial fluid or serum or an equivalent surrogate test fluid at temperatures reached during operation for at least one of the following periods: 7, 14, 21, 30, or 40 days.

[0123] In one embodiment, the present invention includes a product portion of an aptamer sensor having a change in zero gain frequency of at least one of <±5%, <±10%, <±20%, <±40%, <±80% of the zero gain frequency on day 1 when continuous biosensing is performed on the product version over 7, 14, 21, 30, and 40 days in interstitial fluid, serum, or equivalent surrogate test fluid at temperatures reached during operation.

[0124] In another embodiment, the present invention includes an oxygen reduction current measurable at a potential of -0.5V, which increases by at least one of <10%, <20%, <50%, and <100% of the initial oxygen reduction current when continuous biosensing is performed over 7, 14, 21, 30, and 40 days in interstitial fluid, serum, or equivalent surrogate test fluid at temperatures reached during operation relative to the production version.

[0125] The above example assumes that the batch has a very precise distribution of redox current magnitudes at a given concentration of the analyte. For example, assume that the sensor has both a signal-on response and a signal-off response, and has a non-response frequency at which the current does not change. Changes in electrode area, aptamer density, or other factors can affect this magnitude of current for each sensor. In factory calibration, if necessary, several additional measures may be taken to ensure that the performance as specified above is maintained even if the magnitude of the current varies between sensors in the batch. For example, non-limiting options for informing the magnitude of the current for each individual sensor may include: (1) During manufacturing, the magnitude of the current to each individual sensor in the product portion of the sensor can be directly measured at a non-response frequency or a response frequency where the analyte concentration in the test solution is known, or the magnitude of the current can be indirectly determined through measures such as electrode surface area or capacitance. (2) During in vivo use, the magnitude of the current can be measured by measuring the product portion of the sensor at non-responding frequencies. If necessary, the non-responding frequencies can be automatically determined during in vivo use for most analytes by determining when the concentration of the analyte is changing and measuring at what frequencies no resulting change in current occurs. (3) During in vivo use, the electrode active surface area may be measured for each sensor using capacitance or other methods during in vivo use and used indirectly to determine the magnitude of the current. Other methods may include, for example, cyclic voltammetry to obtain the total aptamers and redox tags on the sensor surface, which will represent the electrode surface area when deposited with a tight standard deviation across the entire batch of sensors (larger electrode surface area = greater total number of aptamers and redox tags = greater total charge transfer measured during the cyclic voltammetry cycle). For example, within the first few hours of in vivo operation, the electrode active surface area may be measured electrochemically or via capacitance, and this measurement is used to normalize the measured current as previously described with respect to Figure 5. (4) During in vivo use, the total redox tag density or the number of redox tags may be measured during the manufacture or operation of the sensor using cyclic voltammetry or other measures and may be used indirectly to determine the magnitude of the current. (5) Other methods, as required or available, for devices such as those described herein.

[0126] After the magnitude of the current is known for each sensor, the percentage change in current for each sensor is highly predictable with a small standard deviation (see the table above). Therefore, the current for any given sensor can then be normalized to the known magnitude of the current itself, and then the standard deviation can be maintained as described and specified in the table below.

[0127] Application to redox quenching The present invention may also be applied to redox quenching systems such as carminic acid or hemin-tagged aptamers tagged at the ends of hybridized aptamer chains, where binding of the analyte cleaves the chain and activates the redox activity of the redox tag. In this switching structure for aptamer sensors, non-response frequencies may also be applied to maintain calibration, as with other techniques described herein. One major difference from redox quenching systems from the viewpoint of the present invention is that the background redox tag current is very low or near zero, and the overall change in redox current due to analyte binding can be quite large. Thus, in one embodiment, the redox quenching system of the present invention is a plurality of aptamers, each containing one or more attached redox tags, the attached redox tags providing electron transfer by electrodes.

[0128] System Overview With a basic understanding of the sensors described above, we now refer to Figure 18, where similar numbers do not necessarily refer to similar features (the numbering for Figure 18 is in the 800s and is unrelated to Figure 8). Figure 18 is a schematic diagram of computer system 800 in various aspects of the present disclosure. Similar numbers in Figure 8 do not necessarily refer to similar features as in other figures. Computer system 800 includes a number of hardware processing devices (commonly designated by reference number 802) linked together by one or more networks (commonly designated by reference number 804).

[0129] Network 804 provides communication links between various processing devices 802 and may be supported by network components 806 that interconnect the processing devices 802, including, for example, routers, hubs, firewalls, network interfaces, wired or wireless communication links and corresponding interconnections, cellular stations and corresponding cellular conversion technologies (e.g., those that perform conversion between cellular and TCP / IP). Furthermore, network 804 may include connections using one or more intranets, extranets, local area networks (LANs), wide area networks (WANs), wireless networks (Wi-Fi), the Internet including the World Wide Web, and other deployment configurations to enable communication between cellular and / or processing devices 802 in real time or by other means (e.g., via time-shifted, batch processing, etc.).

[0130] Processing device 802 may be any device that can communicate with another processing device 802 over network 804, or using a combination thereof, for example, via Bluetooth, ultrawideband, near-field communication (NFC), via one or more radio frequencies (RF), or via any other form of wired or wireless communication.

[0131] Some examples of processing devices 802 include cellular devices (including cellular mobile phones (i.e., smartphones)), tablet computers, netbooks, notebook computers, personal computers, servers, cloud devices, and edge devices.

[0132] Furthermore, in several contexts and roles, the processing device 802 is intended to be a wearable monitoring device. Examples of wearable monitoring devices include purpose-driven appliances, Internet of Things (IoT) devices, and special-purpose devices. Implemented as a wearable monitoring device, the processing device 802 is schematically illustrated in Figure 8 simply as a wearable device attached to the patient's arm for illustrative purposes. In practical applications, the wearable monitoring device may be attached to other parts of the patient's body.

[0133] In some embodiments, the wearable monitoring device may communicate locally (for example, to a smartphone) via Bluetooth, ultrawideband, one or more radio frequencies (RF), or any other form of wired or wireless communication. In other embodiments, the wearable monitoring device may communicate over a network, for example via Wi-Fi, and / or locally to another processing device 802.

[0134] The exemplary computer system 800 also includes processing devices (e.g., a web server, a file server, and / or other processing devices) implemented as a server 812 that supports the analysis engine 814 and the corresponding data sources (collectively identified as data sources 816). The analysis engine 114 and data sources 116 provide resources for implementing and storing data related to collecting and aggregating data from wearable monitoring devices, captured events, combinations thereof, etc., as described in more detail herein.

[0135] In one exemplary implementation, the data source 116 is implemented by a collection of databases that store various types of information. As just one example, the data source 816 may include data related to a wearable monitoring device, such as device data 818, e.g., configuration data, version data, software version control and control, and data generated from wearing the wearable monitoring device. The data source 816 may also include medical data 820, e.g., medical research data, used to calibrate, tune, design, modify, etc., the wearable monitoring device. The data source 816 may optionally also include user data 822, e.g., data about a patient wearing the wearable monitoring device, and such data may be collected. As a further example, the data source 816 may include platform data 824, e.g., data used by the analysis engine 814, e.g., computer drivers, GUI information, algorithms for processing physiological states, etc. As yet another example, the data source 816 may optionally include a variety of miscellaneous data 826, for example, any data required by the analysis engine 814 not otherwise described above.

[0136] Considering Figure 18 as an environment used by a wearable monitoring device, in some embodiments, processing of physiological data of a corresponding patient wearing the wearable monitoring device (e.g., biochemical sensing with additional sensing modalities to enhance patient care or health and wellness) may be performed on a processing device 802 (such as the wearable monitoring device itself), on a processing device 802 such as a smartphone, by an analysis engine 814, or via a combination thereof (e.g., by distributing the processing task across two or more processing devices).

[0137] With respect to processing device 802 implemented as a wearable monitoring device (see processing device 802 shown in the diagram as being attached to the patient's arm), it is sometimes desirable for the wearable monitoring device itself to perform all processing. In this regard, compatible devices such as smartphones can optionally provide a graphical user interface for displaying measurement results on a dashboard, but all processing is performed by the wearable monitoring device itself.

[0138] In other embodiments, the smartphone may perform several operations, for example, to execute algorithms, rules, or other operations, as more fully described herein, to compare calculated data with thresholds on a dashboard.

[0139] In yet another embodiment, the analysis engine 814 can collect data from each wearable monitoring device, such as data for trend analysis of patient data, data for device status for health monitoring (for example, data for detecting malfunctions of the wearable device itself), data for monitoring battery charge levels, and data for version control (for example, data for performing software updates).

[0140] In some embodiments, the analysis engine 814 is controlled by a third party, for example, a manufacturer of a wearable monitoring device implemented in the environment.

[0141] In some embodiments, the analysis engine 814 schematically represents integration into an electronic health record system, which connects a patient to their physician so that, for example, the physician can access the electronic data generated by the corresponding wearable monitoring device.

[0142] Calibration codes or data and methods as taught herein may be stored in any device within System 800 that can store memories and transmit the calibration by communication in such a manner that the final data shared with the user is corrected by the calibration before the data is shared with the user.

[0143] Monitoring devices Referring now to Figure 19, an exemplary wearable monitoring device 900 is graphically illustrated according to an aspect of the disclosure of the present invention, where similar numbers do not refer to similar features (the numbering in Figure 19 uses numbers in the 900s and has no relation to Figure 9). The wearable monitoring device 900 may represent an exemplary embodiment of the processing device 802 (Figure 18), such as the wearable monitoring device described earlier.

[0144] The wearable monitoring device 900 includes a housing 910 that is attached to the patient. The housing can be attached to the patient via adhesive 904, a strap, or other fasteners.

[0145] The wearable monitoring device 900 also includes at least a first working electrode 920, a second working electrode 922, and may further include a third working electrode 924 or more working electrodes. Alternatively, electrodes 922 and 924 may be a counter electrode and a reference electrode. In some embodiments, one or more electrodes include an analyte detection material, such as an aptamer, so that continuous sensing can be performed. Electrode 950 may be a gel electrode pad, as previously described, and serves as a reference electrode and a counter electrode.

[0146] In practical applications, the housing 910 is coupleable to electrodes 920, 922, and 924. As used herein, “coupleable” should be interpreted broadly, unless otherwise specified, to mean permanently coupled, detachably coupled, temporarily coupled, user-installable, user-removable, user-installable and removable, factory-installable, factory-removable, user-installable, factory-installable and removable, or any combination thereof.

[0147] As illustrated, the housing 910 includes a potentiostat 991 which is communicatively coupled to electrodes 920, 922, 924 (or a combination thereof) using an optional multiplexer 990, or alternatively, each of electrodes 920, 922, and 924 may receive a direct, dedicated connection to the potentiostat 991. In practical applications, the term “potentiostat” should be interpreted broadly and is not limited to a specific number of sensors. For example, a potentiostat may be implemented as a bipotentiostat, polypotentiostat, etc., depending on the sensor configuration provided by the wearable monitoring device 900.

[0148] In addition, the wearable monitoring device 900 includes a controller 993 which is communicatively coupled to a memory 992. The controller 993 is also communicatively coupled to a communication interface 994.

[0149] The controller 993 includes the necessary electronic components that enable the controller 993 to perform the intended functions of the wearable monitoring device. For example, the controller 993 may include a processor, bus interface, ports, registers, memory, etc., which enables the wearable monitoring device 900 to perform the functions that are described more fully herein.

[0150] Furthermore, as illustrated, the controller 992 is communicably coupled to one or more of the following: an optional multiplexer 990, a potentiostat 991, a memory 992, a transceiver 994, an optional variety of sensors 995, an optional display / output 996, or a combination thereof.

[0151] The communication interface 994 may include at least one transceiver that communicates via, for example, Bluetooth, Wi-Fi, ultrawideband, short-range wireless communication, or a combination thereof.

[0152] The optional display / output 996 may include a display screen, a dimensionally limited display screen, a touchscreen, a haptic output, a light output, a speaker / alarm, or a combination thereof.

[0153] The controller 993 uses a potentiostat 991 to collect measurements from electrodes 920a, 920b, and 920c and stores the collected measurements in memory 992. The controller 993 may further provide filtering, analysis, control, authorization, authentication, and other controller-specific functions. The communication interface 994 facilitates the coupling of the wearable monitoring device 900 with an external computing device, such as a smartphone or cloud computer. In this regard, the communication interface 994 may include one or more modalities, each having different data and / or authorization. For example, a patient may access data from the wearable monitoring device 900 on a smartphone 1006, as illustrated in Figure 10, while a physician may access more detailed information from a cloud server and / or through electronic health records (see Figure 9). In this regard, multiple modalities of communication may be utilized with the wearable monitoring device 900.

[0154] In some embodiments, the adhesive 904 of the wearable monitoring device 900 is a gel electrode 950 connected to at least one of the potentiostat 991, controller 993, or sensor 995, or includes a gel electrode 950. For example, the gel electrode 950 could also be a counter electrode or reference electrode for electrodes 920, 922, and 924.

[0155] (Examples) material Sulfuric acid (96%, pa), sodium hydroxide (98%, pelletized), pulverized phosphate-buffered saline (PBS, pH 7.4), Tris-EDTA solution (TE buffer, pH 8), bovine serum, Tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 98%), sodium azide (99.5%), 1,6-d6-mercapto-1-hexanol (MCH, 98%), and 8-mercapto-1-octanol (MCO, 97%) were obtained from Sigma Aldrich, Inc. (located in the USA). 2-Hydroxy-2-methylpropiophenone (photoinitiator, purity: >96%) was purchased from TCI Chemicals. [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (monomer, purity: >95%) was obtained from Chem-Impex INC. (located in the USA). Anhydrous ethanol (100%, anhydrous) was purchased from Fisher Scientific, Inc. (USA). Vancomycin hydrochloride (94.6%) and ethylene glycol dimethacrylate (crosslinking agent, purity: >98%) were obtained from Alfa Aesar, Inc. 3'-methylene blue and 5'-thiol modified oligonucleotide sequences were synthesized by Integrated DNA Technologies, Inc. (IDT, USA). Exemplary aptamer sequences are as follows:

[0156] [Table 1]

[0157] Sensor preparation Gold electrodes with a titanium adhesive layer are deposited onto a PET or Kapton strip providing a support for the electrode, such as support 460 in Figure 4. The gold is patterned via photolithography and chemical etching to provide a photosensitive or screen-printable electrical insulator. The gold may be further electroplated with additional gold, electrochemically roughened or cleaned, or used as is before aptamer and protective layer incubation. Electrochemical cleaning can be performed in a standard three-electrode electrochemical cell consisting of a gold working electrode, a platinum counter electrode, and an Ag / AgCl reference electrode by performing 700 cyclic voltammetry scans from -1V to -1.6V at a scanning speed of 1V / s in 0.5M NaOH, followed by 150 scans from 0V to 1.6V at a scanning speed of 1V / s in a 0.5M H2SO4 solution. After electrochemical washing is complete, the electrodes are thoroughly rinsed with DI water and dried under a nitrogen atmosphere (99.999% purity) and can be used for subsequent incubation. The lyophilized pellets of the modified aptamers can be diluted with TE buffer to a 100 μM stock solution and kept at -20°C until use. Preparation of the aptamer dilution standard solution can be carried out by first mixing an aliquot of the 100 μM aptamer stock solution with an equal volume of 0.5 M TCEP dissolved in Milli-Q water. This mixture is allowed to stand for 1 hour to ensure complete reduction of the disulfide aptamer molecules. The resulting solution is then diluted with 1x PBS / 2 mM MgCl2 buffer to an intermediate concentration of ~4 μM, and the concentration is confirmed using absorbance measured at 260 nm using a Nanodrop UV / Vis Spectrophotometer. This solution can then be diluted to 500 nM with 1x PBS / 2 mM MgCl2 buffer for a 1-hour incubation of the aptamer. The aptamer-functionalized electrode can then be rinsed with DI water and incubated overnight at room temperature in 5 mM MCH or MCO prepared with 1x PBS. The functionalized sensor can then be rinsed with DI water before coating with trehalose for storage and finally used for measurement.If electrochemical roughening and hydrogel protection are desired, prior to incubation with the aptamer and MCH or MCO, the electrodes may be immersed in a 5M NaOH solution and exposed to alternating potential steps of -5V and +0.8V (vs. Hg / Hg2SO4, saturated Na2SO4) for a total duration of 6000 seconds in an electrochemical cell consisting of a Kapton®-carbon counter electrode and a saturated Hg / Hg2SO4 reference electrode, with a total potential of 20 ms. After roughening is complete, the electrodes may be rinsed with large amounts of DI water and the aptamer and MCH or MCO, and then incubated as described above. Modification of the sensor with an anti-biofouling amphoteric polybetaine hydrogel can be performed by drop-casting 1 μL of an aqueous mixture consisting of monomer / crosslinking agent / photopolymerization initiator (2.8 g / 1.8 μL / 36 μL each dissolved in 1 mL of DI water) onto the sensor and irradiating it with UV light (λ: 280-450 nm, Bluewave LEDPrime UVA, Dynamax, USA) for 45 minutes.

[0158] (Example 1) The following are non-limiting embodiments of the present invention that incorporate some of the techniques described above for maintaining sensor calibration during in vivo use.

[0159] 1) 500 sensors are manufactured in one batch, then stored, stabilized, and sterilized. 12 sensors are reserved for sensor calibration, and 488 sensors are reserved for the final product.

[0160] 2a) The calibration portion of the sensor is then tested in the factory in serum at 34°C for 15 days, and the intended product portion of the sensor is used for up to 14 days. The sensor is simultaneously tested at a 50% analyte concentration, and data is captured every 5 minutes using kinetic differential measurement and continuous square wave voltammetry at 10 Hz, merged as mean and standard deviation, and the maximum signal ON, signal OFF, and non-response currents are measured and their corresponding points in time (frequency) of measurement are obtained (e.g., corresponding to 10, 40, 300 Hz).

[0161] 2b) On days 0, 1, 3, 5, 7, 10, and 14, the titration curve is captured for a sensor with 10 titration points, increasing in 10% increments from 0% to 90% of the sensor response range, which is typically about 80 times the concentration change.

[0162] 3) The magnitude of the current associated with a 50% analyte concentration is stored as a calibration curve, and the sensor response calibration curve is created based on fitting it to the titration curves generated on days 0, 1, 3, 5, 7, 10, and 14 (titration does not need to be measured continuously to create a calibration curve that includes the sensor response over the titrated range).

[0163] 4) The product portion of the sensor is then assigned a code that relates in reverse to the data captured for the calibration portion of the sensor. This code retrieves the complete calibration data from the cloud (server). Alternatively, the sensor may be fully programmed at the factory using a calibration curve. The product portion of the sensor may also be assigned calibration data for in vivo blood correlation, which is tested for each batch of sensors using a limited number of subjects, or tested less frequently, or in some cases not tested at all.

[0164] 5) The product portion of the sensor is then applied to the user, and after 30 minutes of stabilization, the sensor captures the total aptamer tag current three times sequentially in short intervals using cyclic voltammetry, obtaining an average value that shows a measure of the current magnitude for each individual sensor. Optionally, 12 hours after sensor stabilization, the sensor initiates an automatic calibration process, beginning to look for changes in analyte concentration sufficient to determine the non-responding frequency and the current magnitude at the non-responding frequency, and using that value to further calibrate the accuracy of the sensor over time. Continuous square wave voltammetry can capture all frequency data in a single 10Hz scan, so the 10Hz frequency does not need to be changed during product use to capture non-responding frequencies.

[0165] (Example 2) The sensor according to the present invention was fabricated as follows: A cortisol aptamer was linked to electrochemically roughened gold with NaOH via an 8-carbon (8-methyl group) linker-equipped thiol, and then coated with an MCO blocking layer. A UV-curable polybetaine hydrogel was applied. The sensor was continuously tested in serum at 33°C and measured using square wave kinetic differential assays at 10 Hz for signal OFF and 300 Hz for signal, and titrated on days 7, 14, 21, 30, 40, and 50. The cortisol concentration in micromolars is "conc.", the standard deviation is "sd", and the kinetic differential assay is "KDM". The cortisol concentration was high for this particular sensor, and for the product portion of the sensor, there would be a known zero concentration of cortisol between the administration of cortisol boluses. The measurement range was from 0.5 micromoles to 500 micromoles. The resulting data are shown below.

[0166] [Table 2]

[0167] [Table 3]

[0168] [Table 4]

[0169] [Table 5]

[0170] [Table 6]

[0171] [Table 7]

[0172] Several aspects of this ultrastable dataset are highly suggestive. The 5 micromolar data is located near the center of the sensor range (half of the maximum redox tag current measured) and near the sensor's binding affinity value for cortisol.

[0173] [Table 8]

[0174] Other steps not described in detail herein but readily apparent from the disclosed embodiments or incorporated together with the disclosed embodiments are also included as part of the invention. The embodiments described herein provide specific examples for illustrating the elements of the invention, but do not necessarily cover all possible embodiments commonly known to those skilled in the art.

[0175] array

[0176]

number

[0177] 12 Skin 14. Biofluids 100, 102, 104 sensors 110, 112, 114 Housing Devices 111, 113, and 115 190 microneedles 200 sensors 220 electrode 222 Protective monolayer or blocking layer 224 Aptamer 280 Analytes 270 Redox Tags 300 sensors 324 Aptamer 350 waveguides 370 fluorescent tags 372 Fluorescent Quencher 390 Light from a light source 400 sensors 416 circuit boards 560, 562, 564 square wave voltammograms 560a, 562a, 564a background current 561a Common Value 663 Redox Peak Current vs. Concentration 663a standard deviation 800 Computer Systems 802 Processing Device 804 Network 806 Network Component 812 Servers 814 Analysis Engine 816 Data Sources 818 Device Data 822 User Data 826 Various miscellaneous data 865b Sensor Redox Peak Current 900 Wearable Monitoring Devices 904 Adhesive 910 Housing 920 First working electrode 920a, 920b, 920c electrode 922 Second working electrode 924 Third working electrode 950 electrode 950 Gel Electrodes 967a, 967b, 967c 991 Potentiostat 992 memory 993 Controller 994 Communication Interface 994 Transceiver 995 Various miscellaneous sensors 996 Optional display / output 1063 Analyte measurement values 1063a standard deviation As of 1068, 1069 1090b, 1191b Non-response frequencies 1190a Peak response frequency for signal OFF 1190b Non-response frequency 1190c Peak response frequency to signal ON 1191b frequency 1360a Signal ON redox current, continuous measurement value 1360B hours 1362a Signal ON measurement value 1364a Signal OFF Measurement Value 1560 Fouling-free sensor response curve 1591 First waveform 1592 Second waveform

Claims

1. A method for fabricating a sensor containing multiple aptamers and performing continuous measurements using the sensor, A step of manufacturing multiple sensors, wherein each sensor includes at least one electrode, and the electrode has an electrode surface, A step of attaching a plurality of aptamers to the electrode surface, wherein the aptamers include one or more attached redox tags, and the attached redox tags enable electron transfer to and from the electrode. A step of applying a blocking layer to the electrode surface, wherein the blocking layer includes a plurality of paths that facilitate electron transfer between the blocking layer surface and the electrode and the redox tag, The steps include: calibrating a portion of the aforementioned multiple sensors ("calibration portion") and generating at least one set of calibration data; The step includes identifying a portion of the plurality of aptamer sensors as being of commercial quality ("product portion"), The plurality of sensors are manufactured in a batch process, and furthermore, at least one set of calibration data collected from the calibration of the plurality of sensors is mathematically associated with the product part, and the product part can provide accurate or precise continuous measurements.

2. The method according to claim 1, wherein the calibration portion and the product portion are the same portion.

3. The method according to claim 1, wherein the calibration portion and the product portion are the same portion and are a single sensor.

4. The method according to claim 1, wherein the calibration portion includes at least three sensors.

5. The method according to claim 1, wherein the calibration data collected from the calibration section is at least part in vitro data.

6. The method according to claim 1, wherein the calibration data collected from the calibration section is at least part in vivo data.

7. A device for continuous measurement of at least one analyte in a test fluid, comprising one or more sensors, A step of manufacturing multiple sensors, wherein each sensor includes at least one electrode, and the electrode has an electrode surface, A step of attaching a plurality of aptamers to the electrode surface, wherein the aptamers include one or more attached redox tags, and the attached redox tags enable electron transfer to and from the electrode. A step of applying a blocking layer to the electrode surface, wherein the blocking layer includes a plurality of paths that facilitate electron transfer between the blocking layer surface and the electrode and the redox tag, The steps include: calibrating a portion of the aforementioned multiple sensors ("calibration portion") and generating at least one set of calibration data; The method is manufactured using a method that includes the step of identifying a portion of the plurality of aptamer sensors as being of commercial quality ("product portion"), The plurality of sensors are manufactured in a batch process, and furthermore, at least one set of calibration data collected from the calibration of the plurality of sensors is mathematically associated with the product part, and the product part is a device capable of providing accurate or precise continuous measurement.

8. The electrode areas are 0.02, 0.01, 0.05, and 0.02 μm. 2 The device according to claim 7, having a standard deviation smaller than a value selected from the group consisting of the following.

9. The device according to claim 7, wherein the electrode area has a standard deviation smaller than a value selected from the group consisting of 20%, 10%, 5%, and 2% of the electrode area.

10. The device according to claim 7, further comprising at least one point in time for calibration during the manufacturing of the device, wherein the point in time is after the manufacturing of the sensor.

11. The device according to claim 7, further comprising at least one point in time for calibration during the manufacturing of the device, wherein the point in time is after sensor storage stabilization.

12. The device according to claim 7, further comprising at least one point in time for calibration during the manufacturing of the device, wherein the point in time is after sensor sterilization.

13. The method according to claim 1, wherein the calibration data is data collected over a period selected from the group consisting of 1 day, 3 days, 7 days, 10 days, and 14 days.

14. The method according to claim 1, wherein the calibration data comprises redox tag currents for either a single time point or multiple time points in time.

15. The method according to claim 1, wherein the calibration data consists of redox peak potentials for a single time point or for any of multiple time points in time.

16. The method according to claim 1, wherein the calibration data comprises oxygen reduction currents for either a single time point or multiple time points in time.

17. The method according to claim 1, wherein the calibration data comprises the redox tag density or the total number of redox tags for a single time point or for any of multiple time points in time.

18. The method according to claim 1, wherein the calibration data comprises capacitance or electrical impedance at either a single time point or multiple time points in time.

19. The method according to claim 1, wherein the calibration data comprises the time or frequency response of the redox tag current to either a single time point or a plurality of time points in time.

20. The method according to claim 1, wherein the calibration data comprises a chronoamperometric response to either a single time point or a plurality of time points with respect to time.

21. The method according to claim 1, wherein the calibration data consists of electrode surface area for a single time point or for any of multiple time points in time.

22. The method according to claim 1, wherein the calibration data comprises a titration response to either a single time point or a plurality of time points with respect to time.

23. The method according to claim 1, wherein the calibration data includes statistical data.

24. The method according to claim 1, wherein the continuous measurement includes a continuous measurement method selected from the group consisting of square wave voltammetry, continuous square wave voltammetry, kinetic differential measurement, calibration-free measurement, impedance spectroscopy, differential pulse voltammetry, chronoamperometry, and combinations thereof.

25. The method according to claim 1, wherein the calibration data includes at least one variable in vivo parameter.

26. The method according to claim 1, wherein the calibration data includes at least one in vivo correlation between interstitial fluid and blood.

27. The method according to claim 1, further comprising calibration data collected during use of the product portion of the sensor.

28. The method according to claim 1, further comprising automatic calibration data collected during use of the product portion of the sensor.

29. The method according to claim 1, further comprising automatic calibration data collected during use of the product portion of the aptamer sensor, wherein the automatic calibration data is the frequency or time response of the sensor collected during use.

30. The method according to claim 1, further comprising automatic calibration data collected during use of the product portion of the aptamer sensor, wherein the automatic calibration data is the electrode surface area.

31. The method according to claim 1, further comprising in vivo calibration data collected after or near the time point of steady-state concentration of the analyte in interstitial fluid.

32. The method according to claim 1, further comprising multiple sets of calibration data collected at multiple analyte concentrations.

33. The method further includes at least one measurement waveform that changes the electron transfer characteristics as it passes through the blocking layer, and therefore further includes at least a first stabilization period. The method according to claim 1, wherein the calibration data comprises at least a portion of at least one measurement taken during the first stabilization period.

34. The method according to claim 1, further comprising a plurality of waveforms, at least one of which alters the electron transfer characteristics as it passes through the monolayer, and at least further comprising a second stabilization period occurring after each application of at least one of the plurality of waveforms.

35. The method according to claim 34, wherein at least one of the waveforms is an analyte measurement waveform, a sensor diagnostic waveform, or a sensor storage waveform.

36. The method according to claim 34, wherein the plurality of waveforms are periodic.

37. The method according to claim 1, further comprising a calibration portion of a calibration portion having a standard deviation of <±2% when continuous biosensing is performed on the product version in interstitial fluid or serum or an equivalent substitute test fluid at temperatures reached during operation for a period selected from the group consisting of 7, 14, 21, 30, and 40 days.

38. The method according to claim 1, further comprising a calibration portion of a calibration portion having a standard deviation of <±3% when continuous biosensing is performed on the product version in interstitial fluid or serum or an equivalent surrogate test fluid at temperatures reached during operation for a period selected from the group consisting of 7, 14, 21, 30, and 40 days.

39. The method according to claim 1, further comprising a calibration portion of a calibration portion having a standard deviation of <±5% when continuous biosensing is performed on the product version in interstitial fluid or serum or an equivalent substitute test fluid at temperatures reached during operation for a period selected from the group consisting of 7, 14, 21, 30, and 40 days.

40. The method according to claim 1, further comprising a calibration portion of a calibration portion having a standard deviation of <±10% when continuous biosensing is performed on the product version in interstitial fluid or serum or an equivalent substitute test fluid at temperatures reached during operation for a period selected from the group consisting of 7, 14, 21, 30, and 40 days.

41. The method according to claim 1, further comprising a calibration portion of a calibration portion having a standard deviation of <±20% when continuous biosensing is performed on the product version in interstitial fluid or serum or an equivalent surrogate test fluid at temperatures reached during operation for a period selected from the group consisting of 7, 14, 21, 30, and 40 days.

42. The method according to claim 1, further comprising a product portion having an accuracy of <±5% when continuous biosensing is performed on the product version over a period selected from the group consisting of 7, 14, 21, 30, and 40 days in interstitial fluid, serum, or an equivalent substitute test fluid at temperatures reached during operation.

43. The method according to claim 1, further comprising a product portion having an accuracy of <±10% when continuous biosensing is performed on the product version over a period selected from the group consisting of 7, 14, 21, 30, and 40 days in interstitial fluid, serum, or an equivalent substitute test fluid at temperatures reached during operation.

44. The method according to claim 1, further comprising a product portion having an accuracy of <±20% when continuous biosensing is performed on the product version over a period selected from the group consisting of 7, 14, 21, 30, and 40 days in interstitial fluid, serum, or an equivalent substitute test fluid at temperatures reached during operation.

45. The method according to claim 1, further comprising a product portion of an aptamer sensor having a change in zero gain frequency of less than at least one of <±5%, <±10%, <±20%, <±40%, <±80% of the zero gain frequency when continuous biosensing is performed on the product version over a period selected from the group consisting of 7, 14, 21, 30, and 40 days in interstitial fluid, serum, or equivalent surrogate test fluid at temperatures reached during operation, the product version having a change in zero gain frequency of less than at least one of <±5%, <±10%, <±20%, <±40%, <±80% of the zero gain frequency.

46. The method according to claim 1, further comprising: an oxygen reduction current measurable at a potential of -0.5V, which increases by at least one of <10%, <20%, <50%, <100% of the initial oxygen reduction current when continuous biosensing is performed on the product version over a period selected from the group consisting of 7, 14, 21, 30, and 40 days in interstitial fluid, serum, or an equivalent substitute test fluid at a temperature reached during operation;

47. A method for creating a factory-calibrated batch of aptamer sensors, The steps include manufacturing the aforementioned aptamer sensors in batches, The steps include: calibrating a portion of the aptamer sensor ("calibration portion") and generating at least one set of calibration data; The steps include identifying a portion of the aptamer sensor as being of commercial quality ("product portion"), A method comprising the step of assigning the calibration data to the product portion of the aptamer sensor.

48. The method according to claim 47, wherein the calibration data includes sensor responses measured over several days.

49. The method according to claim 47, wherein the calibration data includes a single-point calibration captured after sensor operation over a period selected from the group consisting of 1 hour, 2 hours, and 3 hours.

50. The method according to claim 47, wherein the calibration data includes a single-point calibration and a predictive calibration curve based on previous or past measurement data.

51. The method according to claim 47, wherein the calibration data further comprises measurements selected from the group consisting of aptamer density, frequency map, square wave voltammetry, cyclic voltammetry, intermittent pulse amperometry, chronoamperometry, continuous square wave voltammetry, electrical impedance, oxygen reduction current, and combinations thereof.

52. The method according to claim 47, wherein the calibration data is corrected using automatic calibration during use, and the factory-calibrated aptamer sensor further includes known, expected, or predicted standard deviations for the sensor response, and the automatic calibration is performed when the sensor response has changed by more than at least one of the standard deviation of the sensor response or twice the standard deviation.

53. The method according to claim 52, wherein the automatic calibration shifts the frequency response used to identify at least one of the signal OFF frequency having the maximum sensor response, the zero or non-responding frequency, or the signal ON frequency having the maximum response.

54. The method according to claim 47, wherein the calibration data includes a frequency response, and during use, the device measures at least one of a change in aptamer density, electrical impedance, and oxygen reduction current to recalibrate the frequency response.

55. The method according to claim 47, wherein the calibration dataset is obtained in humans only after or near the time when the concentration of the analyte in the interstitial fluid reaches a steady state.

56. The method according to claim 47, wherein the factory calibration of the sensor is performed immediately after the manufacture of the sensor.

57. The method according to claim 47, wherein the factory calibration of the sensor is performed after the storage stabilization of the sensor.

58. The method according to claim 47, wherein the factory calibration of the sensor is performed after installation on an electronic device.

59. The method according to claim 47, wherein the factory calibration of the sensor is performed after sterilization.

60. The method according to claim 47, wherein the factory calibration of the sensor is performed at one or more time points and one or more scales in order to provide a sensor lifetime calibration dataset.

61. The method according to claim 47, wherein the factory calibration of the sensor is performed at a single point, and the calibration is captured in operation at at least one of 1, 2, or 3 hours later.

62. The method according to claim 1, wherein the calibration data includes at least one measurement parameter.

63. The method according to claim 62, wherein the measurement parameter is sensitivity.

64. The method according to claim 63, wherein the sensitivity includes a plurality of different sensitivities for a plurality of different measurements.

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