Patent Application
20230408509
The present disclosure relates to a method of using electronic reporters of product and sample identity on a reagent, such as a micro sensor array. Such method is used to verify the integrity of produced values, which are capable for electronic calibration and the integrity of results, such as bioassay producing in-vitro diagnostic data, which can be linked to archival sample and to additional data, such as the identity of the individual or organism sampled, sampling time and other data related to the results.
Diagnostic laboratory systems are currently unable to decrease the time between the collection of an in-vitro sample and the time to return the diagnostic information for remedial action from better than 24 to 72 hours. This is due to the size of the equipment, the skill level needed to operate and the methods of collecting the biological sample. Point of care (POC) diagnostics systems can collect in-vitro diagnostic data directly from an individual's sample by immediately processing complex samples and determining the amounts of rare molecules. These systems decrease the time from collection to remedial action to less than 30 minutes (Pugia Anal Chem 2021).
Current POC systems require better verification of results and better data connections between the reagents and the analyzer to automatically identify product integrity. Several improvements have been made in the prior art. In Corey et al., U.S. Pat. No. 6,316,264, the use of an optical reporter on test strips for bioassay was demonstrated to detect validity of the diagnostic result by indicating when a strip was not properly placed in the system. In Alberella et al., U.S. Pat. No. 6,673,630, the use of optical reporters on test strips was demonstrated to provide a quality check of the system and reagent performance status as needed. In Zimmerle et al., U.S. Pat. No. 9,145,576, the use of an optical reporter on test strips was demonstrated to show moisture damage of the strip. In Howard III et al., U.S. Pat. No. 5,945,341, the use of optical reporters is often placed in a pre-defined location on a reagent disposable, such as a test strip or cartridge, to identify the reagent product automatically from a menu of potential reagent products.
Once the product identity has been verified, as it is also commonly known to those who practice in the art, a calibration code can be reported to an analyzer, such as through a bar code or RFID signal to provide additional calibration settings for that manufacturing lot. This code is often placed on the reagent as in a disposable form, such as a test strip, sensor, package, cartridge, or chip. These methods can automatically assign calibration settings to a system by providing data specific to the reagent lot from the time of manufacturing, which are traceable to standardization and allow calculation of analyte results. This is especially needed and used for quantitative bioassays, which require lot to lot adjustments.
Current POC systems need better methods to automatically identify and report integrity of samples capable of producing results. Several improvements have been made in the prior art. In Pugia, U.S. Pat. No. 5,374,561, the use of creatinine reagent on test strips for bioassay was demonstrated to detect validity of the urine sample as being sufficiently concentrated for producing a reagent result. In Kuo et al., U.S. Pat. No. 6,183,972, the use of optical reporters on immunochromatography test strips were demonstrated to provide a quality check of samples for correcting of a hook effect. In Pugia et al, Clin. Chem Lab Med. 2004, the use of optical reporters on immunochromatography test strips was demonstrated to provide a quality check of samples to a flow through device. However, these methods are not general to all sample types or issues, and do not correct for all interferents in all samples. This results in significant between-sample variability and all samples are not able to be automatically verified as being capable of producing accurate results.
A factor not addressed in the prior art is the ability to automatically connect the samples collected to: 1) reagent results; 2) an archived sample for repeat analysis; and 3) the additional data. This not only requires high density reading and writing of information but also requires a means to test the samples multiple times after collection, as well as automatic verification of the product and sample integrity and identity to produce results that are suitable for linking to additional data. Additional data can be any information, data, or record for the sample tested such as the identity of the sample, sampling time, origin of the sample such a patient, donor, organism, or environment and other data connected to the sample. Pugia Anal Chem 2021 demonstrates processes for collection of an initial sample that can be archived and allow repeat analysis using several types of immune and molecular assays. However, this process did not address product and sample integrity or suitability of reagent of the sample, or allowing automation of a system verification and calibration or for indicating suitable linking to additional data.
Improvements are needed for the method to upload bio-analysis data into the electronic medical record with a time and a patient record time stamp. This currently is performed routinely in clinical laboratories using diagnostics systems to upload data into the electronic medical record with a time and a patient record time stamp. Yet, this current method has noted the inability to trace or utilize in-vitro results over a time period, which is needed to measure the real time flux of biological systems. The inability of the current POC systems to verify product and sample integrity prohibits data connections between produced results and linking additional data.
Tracing the identity of organism producing samples over a given time period allows real time flux of the biological systems to be measured. Tracing one or more organisms or patients that are analyzed over time allows predictive models of biological systems for predicting complex outcomes. These outcomes can be used, for example, for the wellness of the host organism, managing the health of biomes of multiple organisms, improving the cost effectiveness of disease management, and protecting resources or containment of pathogenic organisms.
Diagnostic systems are currently unable to trace one or more organisms or access the time stamp for results from any given sample from an organism. This restricts the ability to improve the prediction of complex outcomes using algorithms developed by artificial intelligence and modeling. For example, the clinical outcome of a subject with a disease cannot be evaluated or improved to stall progression of disease over the life of the subject.
Currently, the clinical studies for evaluating disease progression of groups of subjects are lengthy and costly as many disease incidences or affect rates (e.g. death rate) are <5%/year, and it is difficult to predict the collection of samples and patients needed in advance. Assessments requiring lengthy sample collections of many subjects are often prohibitive and lengthy unless retrospective archival samples are used. Diagnostic systems able to identify the organisms providing the samples over time would significantly improve the predictions.
The protection and wellness of organisms and biomes often require diagnostic system data, which is timely and cost-effective, and provides answers for biological systems at the point where immediate actions can be taken. For example, an infectious disease spreading throughout a biome of multiple organisms often requires immediate isolation of one or more individual organisms to prevent spreading pathogens or destroying the wellness by causing imbalances of the biological systems (dysbiosis). The ability to read electronic records immediately and to trace or utilize in-vitro results over a time period is not currently possible with the current diagnostic systems.
IBRI's PCT/US2020/055931 (the “IBRI PCT”), which is incorporated by reference in its entirety, has recently demonstrated a device format that could perform multiplexed analysis of biomolecules directly on complex samples using multiple analyte detection microwells for electrochemical detection of target analytes. The analyte detection microwell includes a size exclusion filter with one or more pores, electrochemical detector, and affinity agents for a target analyte for capture and detection, which operates under a low hydrodynamic force. The affinity agent for detection is attached to a reagent capable of generating an electrochemical label. The affinity agent for capture is attached to a reagent capable of binding a surface in the microwell. The electrochemical label is detected by a working electrode and a counter/reference electrode placed in the microwell to measure labels formed by the affinity agent for detection. The format enables processing of biomolecule capture and immunoassay detection in a convenient format without needing user intervention to remove the filtration membrane to produce results.
The IBRI PCT design allows precise containment of small sample volumes in analyte detection microwells without loss of detection liquid, exposure to the environment, or the need for extraction and delivery into an analyzer. A microfluidic capillary stop placed underneath multiple microwells is used to hold the liquid in the microwells for capture and detection of the analyte to be measured. The device operates after manual dilution and mixing of the sample with liquids and affinity reagents, such as antibodies needed for immunoassays. The liquid reagents allow the diluted sample and reagents to be moved for capture and detection in the analyte detection microwell. Elimination of the manual dilution and mixing of the sample with liquids and affinity reagents is needed to allow the device to avoid any user intervention. Avoiding any user intervention is required for the design to be used in home testing and point-of-care testing settings. Avoiding the need for additional liquids, valves, and liquid dispensers is highly desired for miniaturization and ease of use.
The IBRI PCT device can be used with reagents for affinity assays such as electrochemical immunoassays (EC-IA), optical immunoassays (OP-IA), and mass spectrometric immunoassays (MS-IA) in the detection of cells and biomolecules trapped on the filtration membrane. In one example, polyclonal affinity reagents were used as a sandwich assay pair by placing an affinity label (biotin) on some of the polyclonal antibodies and placing a detection method (ALP) on the remaining polyclonal antibody. This format allows the biomolecules to be immediately captured on the filtration membrane using neutravidin attached to capture microparticles trapped on the membrane surface. For multiplexed analysis, the filtration membrane is divided into multiple microwells with a filtration membrane bottom. Descriptions of the affinity assay utilized may be found in Pugia, M. J. et al., “Multiplexed SIERRA Assay for the Culture-Free Detection of Gram-Negative and Gram-Positive Bacteria and Antimicrobial Resistance Genes” Anal Chem, 2021.
A solution is needed to allow identity and integrity of the product and sample to be verified that is easily integrated onto small reagent disposals of the IBRI PCT, and allow linking additional data to the analyte data collected from immediate testing or archival sample testing.
An object of an embodiment of the present disclosure is to provide one or more electrochemical reporters into a micro-filtration sensor to determine if the identity and integrity of the product and sample are suitable for use and linked to additional data. The sensor array is, additionally, used for electrochemical detection of analytes and includes one or more analyte detection microwells, size exclusion filters, electrochemical detectors, and affinity agents for capture and detection of a target analyte. The affinity agent for analyte detection is attached to a reagent capable of generating an electrochemical signal. Additional affinity agents are placed in the sensor array to capture the affinity agents and electrochemical reporters.
In non-limiting embodiments or examples, electrochemical reporters in one or more analyte detection microwell with or without captured and/or detected analytes, and the microwell can be removed and stored for additional analysis. The electrochemical reporter is used to report identity and integrity of the results, can also be used to identify the archival analyte detection microwell with the identity of the sample and the sampling time.
In non-limiting embodiments or examples, the electrochemical reporters are attached to a reagent capable of being affinity agents for capture or attached to a reagent capable of binding a surface in the analyte detection microwell. The capture or attached electrochemical label is detected by a working electrode and a reference electrode placed in the microwell to measure the electrochemical reporter. The electrochemical reporter of product identity disclosed herein includes producing electrochemical signals for calibration of a sensor. In some non-limiting embodiments or examples, the electrochemical reporter of product identity is placed in a specific position on an array of one or more analyte detection microwells. The microwell position and electrochemical labels detected are used as an indicator of product identity. In other non-limiting embodiments or examples, the amount of electrochemical reporter is varied as an indicator of product identity by detection of the amount of electrochemical reporter.
In non-limiting embodiments or examples, the affinity agents for capture of target analyte are not included in the sensor with the electrochemical reporter of the product identity and integrity. The electrochemical reporter is captured after the addition of the liquid to the analyte detection microwell and is not released as waste through the size exclusion filter. In non-limiting embodiments or examples, the affinity agents for capture of target analyte is included in the sensor with the electrochemical reporter of the product identity and integrity.
In non-limiting embodiments or examples, detection of an electrochemical reporter of the product and sample identity and integrity occurs after exposure to a sample or liquid which results in a signal of the electrochemical reporter. The observed signal is compared to the expected signal as an indicator of the integrity and identity of the product and sample as being capable of producing results. If the product is not identified as the expected product for the analyzer, a result is not allowed to be generated. If the product is does not have integrity such as product which is damaged or degraded, a result is not allowed to be generated. If the sample is not identified as the expected sample type to be use for the analyzer, for example a urine when a blood sample is expected, a result is not allowed to be generated. If the sample is does not have integrity such as sample which is adulterated or degraded, a result is not allowed to be generated. In other non-limiting embodiments or examples, detection of an electrochemical reporter of the product identity occurs before or after exposure to a sample and is indicative of the integrity of the product and sample as being capable of producing results suitable for linking the results to additional data.
In non-limiting embodiments or examples, additional data is the identity of the sample and donor, or previous results or data collected on the sample and donor or additional results collected on the sample and donor in the future. In non-limiting embodiments or examples, additional data is factory calibration setting. In non-limiting embodiments or examples, additional data is connected using near field communication (NFC) or Radio-frequency identification (RFID), Bluetooth, or wireless communication which requires a confirmation of detection of an electrochemical reporter of the product and sample identity before additional data can be utilized. In non-limiting embodiments or example this factory setting resides on a memory storage device on the sensor array.
In non-limiting embodiments or examples, a lack of confirmation of product and sample identity by detection of an electrochemical reporter prevents communications of additional data to be utilized. In non-limiting embodiments or examples, the break in communications of additional data is caused by a change of voltage or current to electrochemical reporters which prevents their operation.
Further non-limiting embodiments or examples are set forth in the following numbered clauses:
These and other features and characteristics of the present disclosure, as well as the methods of operation and functions of the related elements of structures and the combination of parts will become more apparent upon consideration of the following description and the appended claims with reference to the accompanying drawings, all of which form a part of this specification, wherein like reference numerals designate corresponding parts in the various figures. It is to be expressly understood, however, that the drawings are for the purpose of illustration and description only and are not intended as a definition of the limits of the disclosure.
To promote an understanding of the principles of the invention, reference will now be made to the non-limiting embodiments illustrated in the drawings, wherein like reference numbers correspond to like or functionally equivalent elements, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended. Any alterations and further modifications in the described embodiments, and any further applications of the principles of the invention as described herein are contemplated as would normally occur to one skilled in the art to which the invention relates. Certain embodiments of the invention are shown in detail, but some features that are well known, or that are not relevant to the present invention, may not be shown for the sake of conciseness and clarity. For purposes of the description hereinafter, the terms “end,” “upper,” “lower,” “right,” “left,” “vertical,” “horizontal,” “top,” “bottom,” “lateral,” “longitudinal,” “forward,” “reverse” and derivatives thereof shall relate to the example(s) as oriented in the drawing figures. However, it is to be understood that the example(s) may assume various alternative variations and step sequences, except where expressly specified to the contrary. It is also to be understood that the specific example(s) illustrated in the attached drawings, and described in the following specification, are simply exemplary examples or aspects of the invention. Hence, the specific examples or aspects disclosed herein are not to be construed as limiting. Moreover, as used in the specification and the claims, the singular form of terms include plural referents unless the context clearly dictates otherwise.
For purposes of the description hereinafter, the term integrity of product and sample means, for example, a product and sample capable of producing results and not having been compromised by conditions such as degradation, alteration, breakage, interference, or other factors that would alter results from expected values.
For purposes of the description hereinafter, the term identity of product and sample means, for example, source of these materials is determined as expected and is valid to be traceable to other data such as the collection location, manufacturing location, calibration setting, expected values, identity of sample donor, times of collection or manufacturing, and other data traceable to the materials.
An object of the disclosure, includes one or more electrochemical reporters of identity and integrity that are placed into a sensor array to produce an electrochemical response to the product and sample. The microfiltration sensor may be an array of analyte detection microwells which are also used for electrochemical detection of analytes with each of the one or more analyte detection microwells including a size exclusion filter, electrochemical detectors, and affinity agents for capture and detection of a target analyte. The electrochemical reporters do not interfere with the analysis of a target analyte, and are capable of generating an electrochemical reporter independent of a target analyte.
For purposes of the description hereinafter, an electrochemical reporter is a chemical that undergoes or catalyzes an oxidation and/or reduction during electrochemical reaction when placed between an anode electrode and a cathode electrode in an analyte detection microwell. Changes in current or resistance at defined voltages are used to detect the reduction or oxidation of the electrochemical response as a signal. Electrochemical reporters may include organic hydrocarbons and metals, which can be oxidized or reduced by accepting or donating electrons to change bond between atoms, such as C, O, N, S, P, H, and others, or valency of metals, such as +1, −1, etc. Electrochemical reporters may be chemicals such as enzymes, chelators, reductants, oxidants, mediators, and others can be used to enhance the signals produced.
For purposes of the description hereinafter, an analyte detection microwell for electrochemical detection of target analytes is as described in accordance with the IBRI PCT. The target analyte, size exclusion filter, detection microwell, electrochemical detector, and affinity agents for target analyte capture and detection are defined as terms and examples in accordance the IBRI PCT. The materials and methods described herein are useful with any of a broad variety of target analytes. The target analytes include a wide range of target molecules and target cells. In addition, the target analytes may comprise one or more target variants, as described hereafter.
In non-limiting embodiments or examples, the measurements of the electrochemical reporter (13) is taken from one or more microwells (6) positioned in an array of multiple microwells (6) in a micro-filtration sensor (14) used for a set of assays for different markers, as shown for example in
In non-limiting embodiments or examples, once an acceptable identity and integrity of the product and sample have been confirmed by measuring the electrochemical reporter (13), analyte results are indicated as being suitable for linking additional information. Additional data is added to the measured set of analytes (2) by a microprocessor capable of linking data together such as in a programmable controller board (20). Examples of additional data can be any information, data, or record for the sample (7) tested such as the identity of the sample (7), sampling time, origin of the sample (7), such a patient, donor, organism, or environment, and other data connected to the sample (7). Additional data can be any information, data or record for the reagent and system used such as the identity of the system, location of the system, reagent lot, time and date of analysis, identity of the operator, calibration settings for the system and reagent and other data connected to the reagent and system.
In non-limiting embodiments or examples, the additional data linked is provided by a phone, smart device, clouds, hard disk, solid state disk, RFID encoders, NFC encoders, microprocessor chip (EEPROM), computers, voice, fingerprint, image, bar code, analyzer firmware, or other electronic methods. In some embodiments or examples, multiple data may be linked across a time period of the same origin. For example, the data can be linked to archived samples or to the same patient. The combined data can be connected over time to improve predictions of outcomes. For example, the data sets and analysis of biological material or biological system as signal, behavior, action, or characteristic of biological systems.
In practice, the invention can make use of the same signal generating reagent (10) used for the immunoassay electrochemical response (11) as the signal generating reagent (10) for the electrochemical reporter (13). For example, use of the electrochemical immunoassay (EC-IA), as previously described as an example (Pugia, Anal. Chem. 2021 or Pugia Anal Chem 2006), where enzyme, like alkaline phosphatase, is used to generate redox probe, like para-amino phenol as the electrochemical response (11) from an enzyme substrate, like para-amino-phenyl phosphate, as the signal generating reagent (10) or a nanoparticle is used a electrochemical response (11) from ferrous cyanide or other redox probes as the signal generating reagent (10). When enzymes, or nanoparticles are attached to a separate microwell (6), it can also be used as the electrochemical reporter (13) to generate electrochemical response (11) independent of the analyte (2). Both system, enzymes, and nanoparticles can be used in the same microwell (6), for electrochemical reporter (13) and analyte detection method (2) as long as the redox probe can be detected independently. These reagent methods can collect a sample and analyze the sample initially by reporting EC-IA results that are discussed in IBRI PCT.
coli (MyBioSource, San Diego, CA, USA), K. pneumoniae (Thermo Fisher
Unless otherwise noted all other materials were purchased from Sigma Aldrich or Thermo Fisher Scientific.
Micro-filtration sensors (14) with arrays of microwells (6) of either 110 or 200 μm diameters and 300 μm depths were made using standard microfabrication photolithography techniques with <0.1 μm dimensional tolerance. Microwells (6) were patterned with the arrays inside a 6.5-mm diameter of 35 mm 2 or the size conventional ELISA plate well. In brief, film layers (4 to 20 μm) of dense, high-quality thermal SiO2, Cu or Au were patterned with a slotted membrane (14) grid (9.0×21.0 μm pores) by photolithography and dry etch processes. A 200-nm layer of gold was added to the slotted membrane (15) grid by vapor deposition or gold coating to serve as a gold electrode. A second layer of 300 μm thickness was made with silicon (110 or 200 μm wells) by photolithography and dry etch processes order to create the array of microwells (6). The fabricated microwells layer was then mounted with the filtration membrane face up on the “top side” and was further processed for electrode with via gold electroplating of the micro-filtration sensor (5). The layers were mounted with the microwell (6) opening on the “top side” and was further processed by etching electrode current lines (16) and filling with copper via electroplating and covering the lines with a protective layer to keep each microwell (6) readable.
The neutravidin was linked to the gold surface of the slotted membrane (15) using the following functionalization procedure. The modification of the working electrode to functionalize the surface with neutravidin was performed by the 11-MUA, EDC, and HHSS method. This fabrication starts with dissolving 1.0 mM of 11-Mercapotundecanoic acid (11-MUA) into 50 mM phosphate buffer solution at pH 10. Next, 150 μL of the solution is added to each well and allowed to sit overnight. The wells were washed with water five times and heated at 37° C. until dry. The terminal carboxylic groups (of 11-MUA) were then activated for one hour by applying 150 μL of mM N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and 15 mM (N-hydroxy-succinimide ester (NHSS) in 50 mM phosphate buffer solution at pH 6.1. The sensor was washed with water five times and heated at 37° C. until dry. Next, the surface of the working electrode is treated with 0.5 μL of neutravidin (Thermo fisher Prod. 31000) dissolved at 10.0 mg/mL into 50 mM phosphate buffer and reacted for 30 minutes to immobilize at 37° C. until dry. The sensor was washed with water five times and heated at 37° C. until dry. In non-limiting examples, the neutravidin was replaced with alkaline phosphatase (1.7 mg/ml) and directly linked to the microwell.
After functionalization the micro-filtration sensors were blocked with 200 μL solution of blocking buffer. The blocking buffer was made with 112.5 mL of water containing 10% Candor (Candor Bioscience, Cat. #110125), 3.18 g MOPSO, 1.50 g BSA (Bovine Serum Albumin (Fraction V), and 60 uL Proclin 200 and adjusted to pH 7.5 with 10 N sodium hydroxide and the buffer. After blocking overnight, the micro-filtration sensors were washed five times with 200 μL of TBS-T (Tris buffered saline with 0.05% Tween-20) and allowed to air dry.
The analyzer included fluids for reactions with liquid reagents (23) and electrodes (12) and electrochemical reader (26) for detection of electrochemical responses (11) to the captured analyte (2) or electrochemical reporter (13). An Arduino controller with a menu-driven program (Adafruit Industries, New York, NY, USA) was used as the programmable controller board (20). A motor driver circuit board (18) was used to monitor and regulate the vacuum pressure for filtration (10-100 mbar negative pressure±10%). An MPXV5050DP analog differential pressure sensor (19) (Mouser Electronics, Mansfield, TX, USA) was used to measure the pressure in a conical 50-mL Falcon tube or 5-ml Eppendorf tube as the waste collection chamber (21). This Arduino-based vacuum-driven fluidic control system including proportional-integral-derivative (PID), maintained a user-defined pressure in the waste collection chamber (21). The control loop drives a DC diaphragm pump (22000.011, Boxer Pumps, Ottobeuren, Germany) through a DRV8838 brushed DC motor driver (Texas Instruments, Dallas, TX, USA) as the vacuum pump (17) to evacuate the air from the waste collection chamber (21). The vacuum pump (17) and the pressure sensor (19) were connected to waste collection chamber (21) using appropriate fluidic connectors (IDEX Health & Science, Oak Harbor, WA, USA). The dispensing of liquid reagents (23) was controlled using the same Arduino controller board (20) and three peristaltic dispensing pumps (24) with linear actuator motors to pump liquid reagents (23) into the micro-filtration sensor (14) for delivery (100 uL±1%) through steel needles as liquid dispensers (24).
For analysis of the analyte (2), biotinylated antibody reagents for capture and alkaline phosphate (ALP) labeled antibody reagents for detection in buffer are manually added to a complex sample containing the analyte (2) in a microwell (6). The antibodies used are specific for the analyte (2) to be detected. For example, polyclonal antibodies recognizing S. aureus (Thermo Fisher Scientific), E. coli (MyBioSource, San Diego, CA, USA), K. pneumoniae (Thermo Fisher Scientific) and P. aeruginosa (Abcam, Cambridge, UK) may be added. The analyte (2) for each example were bacterial lysate at 5×10{circumflex over ( )}3 to 5×10{circumflex over ( )}4 cell equivalents/mL prepared as described in Pugia Anal Chem 2021 and detected by square wave voltammetry as described on the micro-filtration sensor (5). In other examples the alkaline phosphate (ALP) labeled antibody reagents were replaced with nanoparticle (NP) labeled antibody reagents as described in Pugia Anal Chem 2006 and detected by electrochemical impedance spectroscopy (EIS) as described in Pugia (See Papers 1-3 Anal Chem 2006 etc) on the micro-filtration sensor (5).
As an example of analysis of electrochemical reporter (13), biotinylated alkaline phosphate (ALP) in buffer at 32, 64, and 96 pM are manually added to a complex sample (7) containing or lacking analyte (2) in a microwell (6). In this example, the electrochemical reporters (13) of different concentrations are kept separated in different microwells (6) and also are kept separated from the microwells (6) used for analysis of analytes (2).
The analyzer shown in
As another example of electrochemical reporter (13), biotinylated nanoparticles (NP) in buffer at 1, 3, and 10 pM are manually added to a complex sample (7) containing or lacking analyte (2) in a microwell (6). The electrochemical reporters (13) of different concentrations in this example are kept separated in different microwells (6) and also are kept separated from the microwells (6) used for analysis of analytes (2). The same analyzer process describe above is used, except that para-amino phenol (pAP) solution was changed to 0.5 mL Tween-20 10%, 0.625 mL of potassium ferricyanide 200 mM, 0.625 mL of potassium ferrocyanide 200 mM, and 2.5 mL of Tris-HCl 1 M pH 8 with ca. 15 mL of water in a 25 mL volumetric flask electrochemical response (11) and allows electrochemical impedance measurements with the electrochemical reader (26) by the electrodes (12) in each microwell (6).
Microparticle suspensions of 250 μL of 0.1% v/w streptavidin polystyrene beads (101 μm diameter; range of 90.0 to 105 μm; 0.03 mmol biotin FITC per mg) were added to 250 μL of blocking buffer and incubated at 37° C. overnight with constant shaking (˜800 rpm). The suspension was centrifuged at 1,000 rcf for five minutes to remove the supernatant, washed twice with 500 μL of PBS, and finally re-suspended in 500 μL TBS. The number of microparticles per μL was determined by phase contrast imaging (BioTek, Lionheart System). Additionally, the streptavidin polystyrene 2 microparticle could be replaced with streptavidin hydrogel particles, and 21 microparticle could additionally be optically labeled with Atto 550-Biotin or FluoSpheres™.
Blocked microparticles were captured into the blocked microwells (6) using the analyzer shown in
According to a non-limiting example, affinity reagents (3 and 8) used for analyte (2) detection were added to assigned microwells (6) locations of the micro-filtration sensor (14). The electrochemical reporter (13) were loaded into the remaining microwells (6), where the locations of electrochemical reporters (13) were known at time of manufacture. All microwells (6) of the micro-filtration sensor (14) are able to process the sample (7) and generate an electrochemical signal and the electrochemical response (11) of these locations allowed comparison to expected values to identify micro-filtration sensor (14) product.
Each electrochemical reporter (13) can have unique electrochemical label concentration. For example, the four electrochemical reporter (13) concentrations as shown in
In non-limiting embodiments or examples, to verify samples integrity with analyte specific capture reagents, the response electrochemical reporters (13) exposed to the sample as well, and then compared against the value known at time of manufacture for a typical sample. If the response of the electrochemical reporters (13) were within an allowable range of the expected electrochemical response (11), the analyte results are calculated. When the electrochemical reporters (13) are impacted by the sample or product stability, then analyte results can be compensated for using the results of electrochemical reporters (13) as a live calibration. When the electrochemical reporters (13) are so impacted by the sample or product stability, that analyte results cannot be compensated then the sample is not viable and analyte results are prevented.
The calibration curve equation and correlation parameters at the time of manufacture are electronically uploaded for calculating results based on a typical sample and based on lot information to calculate the expected results using the electrochemical signals generated by analyte specific affinity reagents (3, 8) and the linked factory data.
The electrochemical reporters (13) exposed to the sample were used to correct the current for sample integrity when a difference in the sample background and response is observed from the typical sample. Since the electrochemical reporters and analyte specific capture reagents use the same electrochemical signal generating reagent (10), the electrochemical response (11) can be used to re-calibrate all analyte specific capture reagents and serve multiple analyte correction factors. Additionally, any impacted factor in the electrochemical reporters during the sample measurement will be corrected, such as temperature, degradation of reagent, humidity, and others.
Sample variation is shown in Table 1 across the five different urines. In the middle column, only one calibration is used based on the typical sample and without use of electrochemical reporters (13). The observed variation across these samples is shown in Table 1 and ranged from 9% to 72% and indicate a lack of sample integrity for most samples. The same five urines were tested in the presence of the electrochemical reporter (13) and the observed electrochemical reporter (13) values were used to correct the calibration based on the typical sample. A significant improvement is shown by the reduction in variation (CV %) using the correction by the electrochemical reporters (13). After correction, all samples were comparable to a typical sample and had passing integrity.
When electrochemical reporters (13) demonstrate the integrity and identity of the product and sample is as expected then analytes results were produced and indicated as suitability for linking the results to other sample and product data. Additional data connections can be linked between the results produced such as medical records, factory calibration setting, previous test results, patient, sample origin, time/date stamp, identity of, and archival sample among other examples of linkable data. Additionally, when the electrochemical reporters (13) indicate poor integrity of sample or product or cannot identify the sample or product, suitability for linking the results to other sample and product data can be prevented. Voltage or current above the electrodes resistance will damage the electrode (12), electrochemical reporter (13) and signal generation of the second affinity agent (8). Current and voltage can be applied to the microwell to prevent to the microwell (6) from generating an electrochemical response (11) to the electrochemical reporter (13) and to the second affinity agent (8) for a target analyte detection. This failsafe operation that prevents downloaded of additional data.
In non-limiting embodiments or examples, to verify samples with analyte specific capture reagents, the response electrochemical reporters (13) exposed to the sample first, and then compared against the value known at time of manufacture for a typical sample. If the response of the electrochemical reporters (13) were within an allowable range of the expected electrochemical response (11), the analyte results are calculated.
The calibration curve equation and correlation parameters at the time of manufacture are electronically uploaded for calculating results based on a typical sample and based on lot information to calculate the expected results using the electrochemical signals generated by analyte specific affinity reagents (3, 8) and the linked factory data (
The electrochemical reporters (13) are demonstrated as indications of integrity and identity of the product and sample as the location and expected results were produced for the sample and reagent product when electrochemical reporters (13) were used. Expected results indicated the suitability for linking the results to other sample and product data. Additionally, and unexpectedly, the electrochemical reporters (13) allowed for improving samples of poor integrity to allow measurements of the analytes (2) by correcting calibration.
While the invention has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only the representative embodiments have been shown and described and that all changes, equivalents, and modifications that come within the spirit of the inventions defined by the claims are desired to be protected. All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference and set forth in its entirety herein.
This application is the United States national phase of International Application No. PCT/US21/54998 filed Oct. 14, 2021, and claims priority to U.S. Provisional Patent Application No. 63/092,890, filed Oct. 16, 2020, the disclosures of which are hereby incorporated by reference in their entireties.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US21/54998 | 10/14/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63092890 | Oct 2020 | US |
