Patent Application
20170067889
The present invention will be further explained with reference to the attached drawings, wherein like structures are referred to by like numerals throughout the several views. The drawings shown are not necessarily to scale, with emphasis instead generally being placed upon illustrating the principles of the present invention. Further, some features may be exaggerated to show details of particular components.
The figures constitute a part of this specification and include illustrative embodiments of the present invention and illustrate various objects and features thereof. Further, the figures are not necessarily to scale, some features may be exaggerated to show details of particular components. In addition, any measurements, specifications and the like shown in the figures are intended to be illustrative, and not restrictive. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for teaching one skilled in the art to variously employ the present invention.
Among those benefits and improvements that have been disclosed, other objects and advantages of this invention will become apparent from the following description taken in conjunction with the accompanying figures. Detailed embodiments of the present invention are disclosed herein; however, it is to be understood that the disclosed embodiments are merely illustrative of the invention that may be embodied in various forms. In addition, each of the examples given in connection with the various embodiments of the invention which are intended to be illustrative, and not restrictive.
Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrases “in one embodiment” and “in some embodiments” as used herein do not necessarily refer to the same embodiment(s), though it may. Furthermore, the phrases “in another embodiment” and “in some other embodiments” as used herein do not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention.
In addition, as used herein, the term “or” is an inclusive “or” operator, and is equivalent to the term “and/or,” unless the context clearly dictates otherwise. The term “based on” is not exclusive and allows for being based on additional factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meaning of “a,” “an,” and “the” include plural references. The meaning of “in” includes “in” and “on.”
Lateral flow devices have been extensively used for rapid testing and diagnosis of a variety of clinical and pathological conditions in both point-of-care (POC) and home settings. Although these lateral flow tests (LFTs) have many advantages, such as being based on a mature technology that enjoys wide user acceptance and recognition, clear development & regulatory paths, established manufacturing processes, and is both cost-effective and scalable, they have several disadvantages that limit their wide-spread use, especially for self-testing. These disadvantages include, among other: challenging test performance protocols, especially in the hands of untrained individuals; compromised and user-dependent test accuracy and reproducibility; ambiguity in test results detection—LFTs are mostly qualitative in nature and depend on visual signal detection for their interpretation; problematic results interpretation and correlation—consumers are often confused about what to do with RDT results and how these would correlate with test results obtained at the POC or hospital lab. Additionally, results traceability and their communication to the healthcare professional are challenging with current RDTs.
Recently, advances in optical reader device technologies, including reduced optical components cost and their miniaturization have significantly improved LFT readout accuracy, also promoting the transition of LFT from qualitative to quantitative tests and transfer of such readers from POC to patients' homes for self-testing. Among such technological advancements, most notable are LFT-integrated optical readers, such as in the Clearblue pregnancy test, and the use of mobile phones (together with the required additional hardware and software) for LFT signal detection and quantification.
A different approach to devices for biological and clinical sample diagnosis combines microfluidics technologies for sample processing with electronic biosensors specific for individual analytes. Such devices, generally referred to as “Lab-On-Chip” (LOC), have gained wide popularity and acceptance in hospital laboratory settings and POCs, partially replacing traditional and high-throughput automatic diagnostic platforms. Although these LOC devices provide accurate diagnostic test results for multiple analytes in a relatively short period of time, their cost and the significant user expertise required for their proper operation has generally rendered them unsuitable for testing outside the POC.
The field of electronic biosensors is well-developed with many examples of their successful integration into diagnostic products (e.g., blood glucose monitoring). Amperometric biosensors generally rely on electrochemical reactions mediated by specific enzymes (e.g., glucose oxidase) for analyte detection and quantification, and are less optimal for detecting and quantifying binding events at the biosensor surface. Potentiometric biosensors, such as Field-Effect Transistors (FET), have the capability of detecting molecular binding events due to their effect on the electric field sensed by the biosensor's channel/gate, which influences source-drain current. However, non-specific interactions between the channel's surface material and the myriad of biochemical entities present in biological samples may overwhelm specific interactions with the analyte of choice, limiting FETs signal-to-noise ratio (S/N) and rendering them sub-optimal for clinical diagnostic applications. In order to minimize such non-specific interactions, potentiometric biosensor-based POC devices (e.g., iSTAT by Abbott) rely on elaborate and relatively expensive microfluidic technologies, limiting their application to professional, LOC-based systems.
A relatively new development in the electronic biosensor area is the ability to directly detect analyte binding to a sensor electrode employing techniques such as electrochemical impedance spectroscopy (EIS). EIS potentially enables highly-sensitive, label-free detection of biomolecular binding events at the electrode's surface by measuring frequency-dependent changes in electrical impedance. Although EIS has been extensively investigated in the laboratory, it has so far gained limited utilization in POC diagnostic applications and products, putatively due to its intrinsic low signal-to-noise ratio (S/N) in detecting analytes in biological samples, combined with requirement for sophisticated and expensive instrumentation for signal detection and analysis.
Taken together, the above limitations have until now restricted the use of the different electronic sensing technologies in diagnostic applications where target recognition is achieved by detecting its specific interaction with an immobilized capture reagent. Furthermore, implementing these different electronic sensing modalities within the framework of disposable lateral flow tests raises additional cost and performance issues that have until now hampered the adoption of such diagnostic devices. One of the main issues to address when attempting the utilization of these and other biosensor technologies is their S/N, which typically limits their analyte cutoff value (also known as limit of detection (LOD)) to the nM range. Cutoff improvement has been addressed by different approaches, including: i) increasing the surface area of the sensing electrode/gate by different modifications (e.g., depositing gold nanoparticles (GNP), carbon nanotubes (CNT) and other conducting entities on the biosensor surface); ii) employing enzyme-mediated amplification schemes to generate species that diffuse to the electrode surface and become involved in a redox reaction (e.g., alkaline phosphatase, used in the iSTAT cardiac troponin I (cTnI) diagnostic device to generate a redox species); iii) reducing the molecular distance between analyte biding site and the biosensor electrode surface—especially for EIS and FET applications where the effect of such binding on the electric field and/or current sensed by the gate/electrode is exponentially dependent on such distance. Such distance reduction was attempted using fragments of the analyte-capturing entity (e.g., Fab fragments of antibodies) or shorter analogs thereof (e.g., aptamers); iv) using electroactive labels (e.g., gold nanoparticles (GNP), carbon nanotubes (CNT), etc.) to enhance the signal generated by the individual binding event, by binding such entities to the detection reagent used in ELISA detection schemes.
It is important to note that the above-mentioned approaches for LOD improvement had, as of yet, limited impact on the wide-spread adoption of such devices, with the exception of the mentioned iSTAT amperometric technology. EIS and FET technologies are still lagging behind in their utilization for commercial biosensor fabrication.
The present invention employs electroactive labels in combination with different strategies aimed at bringing such labels in close proximity to the sensing electrode surface. Such close proximity between the electroactive label and electrode surface maximizes the former's signal enhancement effect on specific analyte binding events. This approach is a variation of the “electrochemical ELISA” concept, where the analyte-specific conjugate used to label the analyte, allowing its electrochemical detection at the biosensor's capture zone, is being modified to allow its direct interaction with the biosensor surface. This unique approach combines employing high-affinity, specific interactions between the analyte and both the labeling and capture reagents, with lower-affinity interactions between the label and electrode surface that bring the latter two in close proximity. It is important to note that such lower-affinity interactions should be carefully engineered in order to minimize non-specific binding of the electroactive label to the electrode surface in the absence of the higher-affinity interactions between the analyte and both the labeling and capture reagents.
The device of the present invention also combines the advantages of LFT with those of electronic biosensors in an electronic lateral flow test (ELFT) format, thereby overcoming some of the major barriers for usage of such devices by untrained individuals and in settings where access to electricity and/or sophisticated instrumentation (such as dedicated readers) is limited or non-existent. Additionally, this unique ELFT platform allows detecting targets with increased sensitivity and specificity, without the need for visual or electro-optical sensors for results reading. This is achieved using the above-mentioned novel analyte labeling and detection schemes, combined with signal gating and acquisition protocols that take advantage of the passive capillary flow of reagents (including analyte) along the membrane that is part of the lateral flow component of this device. Transferring LFT signal detection mode from the traditional optical one into electronic has several advantages, such as user-independent and therefore unbiased test performance that should significantly reduce user-originated errors. Such reduced user dependence in test performance and results reading is expected to consequently also reduce false positive and false negative rates, thereby increasing test accuracy and reliability.
Overcoming noise originating from non-specific binding events while amplifying specific ones is key to successful implementation of EIS- or FET-based biosensors in a diagnostic device. The device of the present invention makes use of several procedures and protocols, either individually or in different combinations, to achieve this goal in an ELFT format. Among these are:
Targeting the device of the present invention for use in point-of-care and home settings entails a significant cost sensitivity requirement. In order to achieve such cost saving goal, the device of the present invention optionally employs both disposable and reusable components. The reusable component contains a central processing unit (CPU) that is electrically-connected by detachable leads to the disposable LFT component that carries the biosensor integrated with the lateral flow test membrane and reagents. This novel design spares the CPU component for the performance of additional tests, while the rest of the device (disposable LFT component/cartridge) is being disposed-of following single use. The detached CPU can then be reconnected through its leads to a new disposable LFT cartridge for the purpose of new test performance. The detached CPU can also be connected through a USB port or by wireless communication to a personal computer or smart phone for results display, or combined with an integral liquid crystal display (LCD) that allows direct results visualization without the need for computer or smart phone connection. Further CPU features include: data storage capability, disposable LFT cartridge attachment detection and identification, self-calibration, etc. It should be noted however that the reusability of the CPU is just one preferred embodiment of the present invention and a fully-disposable (i.e., including CPU) device is also within the scope of the current invention.
In some embodiments, the device of the present invention is configured to measure the presence or absence of specific analytes in a biological sample. In some embodiments, the device comprises: (i) a sample pad, for use to directly collect a clinical sample (e.g., urine) or a pre-processed sample, (ii) a lateral flow membrane, for use in sample fractionation and analyte transfer to the device's biosensor, (iii) a labile analyte-specific, electroactively-labeled detection reagent that is incorporated within the conjugate pad of the lateral flow device, (iv) a biosensor embedded at the detection zone within such lateral flow device, serving to specifically detect analytes and produce an electric signal in response to analyte binding that is being amplified by direct interaction between the electroactive label and the biosensor surface, (v) an electronic processing unit serving to process the biosensor signal, transforming it into digital output, (vi) a display unit serving to visually display the diagnostic digital output of the processing unit.
In some embodiments, the method of the present invention is comprised of: (i) collecting a biological sample by the sample pad of the device; (ii) allowing the sample containing the analyte to passively fractionate along the lateral flow membrane, including filtration/separation of intact cells and cell debris, followed by solubilization and reaction of the carried analyte with the electroactively-labeled detection reagent in the conjugate pad and further flow of the formed complexes along the membrane, reaching the detection zone and interacting with the biosensor, thereby triggering signal acquisition; (iii) allowing the processing unit to process the signal generated by the electroactive label-amplified specific binding of analyte to the biosensor and provide a diagnostic readout; (iv) determining the presence, absence, amount or concentration of analyte in the clinical sample being analyzed according to the provided readout.
In some embodiments, the method of the present invention is comprised of: (i) manually collecting a biological sample, followed by sample extraction using an appropriate extraction buffer; (ii) transferring the extracted sample to the sample pad of the device of the present invention; (iii) allowing the extracted analyte solution to passively fractionate along the lateral flow membrane, solubilize and react with the electroactively-labeled detection reagent in the conjugate pad and to further flow along the membrane, reaching the detection zone, where the electroactively-labeled analyte complex specifically binds to the biosensor; (iv) allowing the processing unit to process the signal generated by the electroactive label-amplified binding of analyte to the biosensor and provide a diagnostic readout; (v) determining the presence, absence, amount or concentration of analyte in the clinical sample being analyzed according to the provided readout.
In some embodiments, the device of the present invention incorporates labile analyte-specific reagents, such as antibodies, DNA, aptamers, enzymes, receptors, etc., that are stably-labeled by electroactive markers. In some embodiments, such labile analyte-specific reagents are incorporated within a specific location (e.g., equivalent to what is known in the art as the “conjugate pad”) along the lateral flow membrane, preferably in between the sample pad and the analyte detection zone wherein the biosensor is embedded. Incorporation of such labile analyte-specific reagents can be either to a separate membrane (i.e., conjugate pad) that is in fluid contact with the lateral flow membrane, or within the lateral flow membrane itself. Reagent application to the membrane is achieved by liquid solution application techniques known in the art, such as spraying, spreading, stamping, dipping, etc., followed by solvent evaporation, leaving the labeled reagent physically adsorbed to the membrane. Multiple such labeled reagents can be applied, each targeting a specific analyte. During ELFT operation, the labeled analyte-specific reagent dissolves in the analyte solution or the buffer used as the LFT running/development media, and specifically reacts with its target analyte. Formed electroactively-labeled analyte complexes migrate by capillary flow forces along the LFT membrane until they reach the biosensor and bind to the specific capture reagent immobilized on the electrode's surface.
In some embodiments, the lateral flow membrane of the present invention incorporates specific elements, positioned between the sample and conjugate pads, whose purpose is to filter cells and cell debris, bind potentially-contaminating materials, or otherwise separate sample components that interfere with optimal test performance. An example for such separation elements are membranes having asymmetric pore size that efficiently sieve blood cells, selectively allowing passage of soluble plasma components.
In some embodiments, the electroactive label described in the present invention is a molecule (e.g., polymer) or nanoparticle (e.g., noble metal nanoparticle), that are either: (i) conductive (e.g., gold nanoparticles (GNP) and carbon nanotubes (CNT)), (ii) exhibit a dielectric constant(s) different than the media surrounding the biosensor element, (iii) electrochemically reactive (e.g., may become involved in a redox reaction), (iv) catalyze the generation of electrochemically reactive species, or (v) highly-charged, e.g. polyelectrolytes.
In some embodiments, the device of the present invention incorporates an electronic biosensor embedded within a lateral flow membrane, thereby allowing direct fluid contact between the analyte solution and the biosensor surfaces. In some embodiments, the biosensor may be amperometric, potentiometric or impedimetric, in which the sensing element (i.e., electrode) has been modified to carry analyte-specific capture reagent on its surface. In some embodiments, analyte-specific capture reagent includes: antibodies, antigens, DNA, aptamers, enzymes, receptors, and generally any molecule that has micro-molar or higher affinity towards the target analyte.
In some embodiments, the biosensor incorporated in the device of the present invention is disposable and its sensing element is composed of an electrode array in which the electrodes are arranged parallel to each other and vertical to the direction of fluid flow through the lateral flow membrane. In some embodiments, the electrode array consists of co-planar interdigitated electrodes. In some embodiments, each electrode in the array is 100 nm to 100 μm wide, 100 μm to 10 mm long and spaced 100 nm to 1 mm apart. In some embodiments, the electrode's cross-section is essentially rectangular with a height of 10 nm to 10 μm above its underlying substrate. In some embodiments, the electrode is porous, with pore sizes ranging 1-100 nm.
In some embodiments, the electrodes that are part of the biosensor of the device of the present invention are constructed from conductive materials, such as (but not limited to): gold, copper, platinum, graphite, glassy carbon and conductive polymers (e.g., polypyrrole). In some embodiments the substrate that underlies the electrode is electrically semiconducting, dielectric or insulating. Prior to biosensor construction, the surface of these electrodes is cleaned, employing techniques known in the art and involving a combination of: mechanical and electrochemical polishing, chemical stripping, plasma treatment and various washing steps, etc., in order to remove contaminants and optimally achieve nascent, molecularly flat and chemically clean surfaces allowing further electrode manipulation steps required for biosensor construction.
In some embodiments, the external, clean surface of the electrodes of the device of the present invention is modified by various chemical reagents and techniques known in the art to fabricate chemically-reactive end groups—such as: maleimide, epoxy, and N-hydroxysuccinimide (NETS) ester—on such electrode surfaces. In some preferred embodiments, where the electrode's surface material is gold, the nascent electrode surface produced following surface cleaning is protected from undesired subsequent adsorption of contaminating chemical species by forming a densely-packed self-assembled monolayer (SAM) on its surface immediately following surface cleaning. Examples of appropriate SAM-forming substances are, but not limited to, organic sulfur-containing compounds that avidly bind to the gold surface via their sulfur atoms, such as: organic thiols, sulfides, thioesters and disulfides.
In some embodiments, the chemically-reactive end groups that are generated on the external surface of the biosensor electrode of the present invention are further reacted with an analyte-specific capture reagent, thereby covalently immobilizing such capture reagent to the electrode's external surface. Alternatively, the analyte-specific capture reagent may be directly immobilized to the biosensor surface through non-specific interactions, and/or specific linkers that are attached to the capture reagent and are reactive with the biosensor electrode's surface. An example for such specific linkers are bi-functional amine-reactive (e.g., NHS ester) organic molecules that incorporate a thiol functionality reactive with the gold electrode surface. Analyte binding to such electrode surface-immobilized capture reagent can be monitored by measuring the time-dependent electrochemical impedance signal (Z) between adjacent electrode pairs (working vs. reference electrode—Zwr). In some embodiments of the present invention, Zwr is compared with an equivalent signal—Zcr—measured between a control electrode, carrying a reagent that is a close analog of the capture reagent but non-specific to the analyte (e.g., host species- and isotype-matched antibody), and the reference electrode. Both Zwr and Zcr signals emanate from electrode pairs that are positioned to equivalently interact with the analyte solution—i.e., they occupy equivalent positions with respect to fluid flow along the LFT membrane. Real-time difference between these two signals in terms of their phase and intensity (ΔZ(t)=Zwr(t)−Zcr(t) is then taken to represent the specific, time-dependent binding of the analyte to its capture reagent on the working electrode. Measurement of the time-dependent differential electrochemical impedance signal (ΔZ(t)) helps in minimizing non-specific contributions to the electrode's impedance from temporal and spatial variations in buffer composition, temperature, pH and biochemical composition of the tested sample.
In some preferred embodiments, the binding of the capture reagent to the working electrode's surface is mediated through electrically-conducting molecular structures, such as a conductive polymers (e.g., polypyrrole), carbon nanotubes (CNT), electrically-conducting self-assembling monolayers (ecSAM), or combination of ecSAM and gold nanoparticles (GNP), naming a few examples.
In some embodiments, the electronic processing unit (CPU) of the device of the present invention measures biosensor impedance signal, either under open circuit (zero current) or electrochemical conditions, by applying a sinus-modulated AC potential of 5-10 mV at several distinct frequencies in the range of 0.1 Hz to 10 MHz across the working (test) and reference electrodes. The same modulated AC potential is simultaneously applied between the control (see above for definition) and reference electrodes (see also
In order for the advantages of the invention to be readily understood, a more particular description of the disclosure briefly described above will be rendered by reference to specific embodiments that are illustrated in the appended drawing(s). It is noted that the drawings of the disclosure may not be to scale and are merely schematic representations, not intended to portray specific parameters of the disclosure. It is to be understood that these drawing(s) depict only typical embodiments of the disclosure and are not therefore to be considered to be limiting of its scope, the disclosure will be described and explained with additional specificity and detail through the use of the accompanying drawing(s), in which:
In some embodiments, the device of the present invention is configured to permit conducting an immunoassay for a specific analyte or set of analytes that are extracted from a biological sample or present in a biological fluid, such as blood, urine, saliva etc. In some embodiments, the device of the present invention integrates traditional lateral flow immunochromatographic assay technologies with those of electronic biosensors in a single apparatus, employing electroactive labels that act to amplify the change in impedance signal at a biosensor surface following detection reagent immobilization. Additionally, proper selection and construction of the electroactive label relative to the biosensor surface prevents non-specific contact between the two, promoting such interaction only upon the specific contact between the detection reagent, the analyte and the capture reagent. Such integration is expected to reduce the complexity of diagnostic test performance, while increasing test sensitivity and specificity, due in part to the non-biased nature of the employed signal detection protocols, since these won't rely on subjectively determining if a visually-discernible signal is formed on the lateral flow test strip. In some embodiments, the device of the present invention employs electroactive labeling techniques to enhance the difference in impedance signal following binding of a molecule to the surface of a biosensor electrode.
It should be re-emphasized that proper selection and construction of the electroactive label vis-à-vis the biosensor surface are required in order to prevent non-specific interaction between the two, promoting such interaction only upon the specific interaction between the electroactively-labeled (19) detection reagent (18), the analyte (17) and the capture reagent (16). This is achieved by balancing such low-affinity interaction forces against the lateral flow forces that act to drive the electroactive label away from the surface of the biosensor electrode. Such delicate balance assures that lateral flow forces overcome these low-affinity interaction forces in the absence of specific binding of the analyte electroactive-label complex to the capture reagent.
While a number of embodiments of the present invention have been described, it is understood that these embodiments are illustrative only, and not restrictive, and that many modifications may become apparent to those of ordinary skill in the art. Further still, the various steps may be carried out in any desired order (and any desired steps may be added and/or any desired steps may be eliminated).
This application claims priority under 35 U.S.C. §119(e) to U.S. provisional application Ser. No. 62/214,048, filed Sep. 3, 2015, the entire disclosure of which is incorporated by this reference into the present U.S. patent application.
| Number | Date | Country | |
|---|---|---|---|
| 62214048 | Sep 2015 | US |
