Analyte Detection Device

Abstract

The present invention relates to a sample contacting component of a sample analysis device or system, which has an electrochemical aptamer-based sensor associated therewith and contacts a sample located within or around the sensor.

Description

[Technical Field]

[0001] The present invention generally relates to the in vitro detection of analytes in clinical samples, which involves the collection of biological fluid from a subject or test subject for testing in a laboratory. The present invention also relates to the detection of analytes in clinical samples at the point-of-care. More particularly, the present invention relates to improvements in hardware used in such detection methods. [Background technology]

[0002] Laboratory-based analyte detection methods are certainly an important part of modern medicine: they provide clinicians with important information about their patients to help diagnose new medical conditions, manage existing conditions, aid in prognosis, or provide clinically relevant information.

[0003] In many cases, the detection method determines the level of an analyte in a blood sample taken from a patient. Exemplary analytes that can be detected in blood include glucose and hemoglobin A1c (for diabetes), calcium (for kidney disease), troponin and cholesterol / lipids (for heart disease), prostate-specific antigen (for prostate cancer), D-dimer (for coagulation disorders), and C-reactive protein (for infectious and inflammatory disorders).

[0004] Fluids other than blood can be used as samples for analytical methods, including urine, saliva, cell and tissue lysates, bronchoalveolar lavage fluid, cerebrospinal fluid, and semen.

[0005] Some analyte detection methods are not performed in the context of any disease state and include methods for detecting reproductive hormones during pregnancy, antibodies indicating seroconversion after vaccination, and dietary factors such as folate and vitamin D.

[0006] Analytes that are exogenous to the patient may be the subject of detection methods. Common analytes in this regard include viral and bacterial proteins, drugs (prescribed and illicit), environmental toxins, etc.

[0007] Typically, the first step is to obtain a clinical sample at the bedside or clinic, where the clinician prescribes one or more diagnostic methods to be performed on the sample. The sample is given a unique identifier and sent to a qualified laboratory for assay of the relevant analyte(s).

[0008] Today's state-of-the-art laboratories are essentially automated facilities, utilizing robotic technology for handling clinical specimens. Clinical samples typically pass through multiple robotic workstations to perform processes such as sample preparation, reagent addition, reagent removal, and test output acquisition. These processes include numerous liquid transfer steps (often via automated pipetting), incubation steps, wash steps, absorbance or photometric measurement steps, and sample transfer from one workstation to another.

[0009] Various infrastructure services required in the laboratory include a water treatment unit to produce DI water and dedicated plumbing to contain waste.

[0010] Each step typically requires specialized equipment and reagents that significantly increase the cost of laboratory investigations. Some reagents used in laboratory analyses are toxic, carcinogenic, or otherwise harmful to humans.

[0011] Each step in laboratory testing introduces the potential for error, potentially leading to erroneous information being provided to clinicians. Additionally, the multiple steps required result in significant delays in providing results.

[0012]

[0003] Further problems arise when detecting multiple analytes in a sample. In such situations, a relatively large volume of blood or other relevant fluid is typically obtained from a subject. In laboratories, this volume is divided into multiple aliquots, and each aliquot is tested for a single analyte. This approach is impractical when large volumes of sample are not available, and in any case adds cost and complexity.

[0013] An example of a medium-complexity laboratory-based test is found in the enzyme immunoassay (EIA) for antibodies to hepatitis B surface antigen (anti-HBs) in serum. This EIA is a solid-phase assay that relies on the "sandwich principle" to detect antibodies. A simplified version of the relevant protocol follows below.

[0014] Polystyrene beads coated with human hepatitis B surface antigen (HBsAg) are incubated with either a patient sample or an appropriate control. During the incubation, antibody, if present, immunologically binds to the solid-phase antigen. After removal of unbound material and washing the beads, biotin-tagged human HBsAg (B-HBsAg) and rabbit anti-biotin conjugated with horseradish peroxidase (anti-H-HRPO) are incubated with the antibody-antigen complex on the beads. The biotinylated surface antigen binds to this complex, forming an antigen-antibody-antigen "sandwich." Anti-biotin horseradish peroxidase binds to the biotin component of the "sandwich," forming a solid-phase network. Unbound conjugates are then removed, and the beads are washed. Next, an o-phenylenediamine (OPD) solution containing hydrogen peroxide is added to the beads. After incubation, a yellow color develops, proportional to the amount of anti-HBs bound to the beads. Within limits, the greater the amount of antibody in the sample, the higher the absorbance. The enzymatic reaction is stopped by adding acid. The absorbance of the control and samples is determined using a spectrophotometer with the wavelength set at 492 nm.

[0015] As will be readily appreciated, even the above simplified outline demonstrates the need for numerous reagents, multiple instruments, many dedicated steps, and a lengthy time to complete. The actual step-by-step protocol for the assay is significantly more involved and demonstrates even greater complexity.

[0016] High-throughput analytical laboratories comprise rooms filled with equipment, each with associated purchase / leasing, operating, maintenance, repair, and labor costs. Purchasing and storing reagents, cleaning solutions, and other consumables incurs additional costs. Thus, such facilities are very expensive to set up and operate, with associated costs being passed on to the relevant government health departments, health insurance companies, or consumers / patients.

[0017] A further problem in analytical laboratories is the lack of flexibility in established workflows. As an example, a sample for one subject may require the analysis of three analytes, in which case the sample is divided into three aliquots, and each aliquot is batched with other samples for the same test. In some cases, to ensure that results are provided in a timely manner, test batches must be run at less than the maximum number of available sample spaces. Running tests on samples with less than their full capacity wastes reagent and equipment resources.

[0018] A further problem is the need for point-of-care tests with simple procedures and rapidity for use in hospitals and other medical facilities. Patient-facing personnel typically lack the expertise, equipment, and time to perform complex analytical tests. For example, it may be desirable for a physician to immediately assess the level of a therapeutic drug in a patient's blood to ensure that the concentration is within the therapeutic window. The drug level is usually sent to an analytical laboratory, and the results are returned, perhaps after a delay of several hours. By the time the physician reviews the results, the drug has inevitably changed, so that corrective action may be too late.

[0019] One aspect of the present invention is to provide an improvement over prior art laboratory-based or point-of-care analyte detection. A further aspect of the present invention is to provide a useful alternative to prior art laboratory-based or point-of-care analyte detection.

[0020] The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. No suggestion or representation is made that any or all of these matters formed part of the prior art or were general knowledge in the art relevant to this invention prior to each priority date claimed by this application. Summary of the Invention

[0021] In a first aspect, and not necessarily in its broadest aspect, the present invention provides a sample contacting component of a sample analysis device or system, the sample contacting component having an electrochemical aptamer-based (EAB) sensor associated with the sample contacting component, the sensor configured to contact a sample within or surrounding the sensor.

[0022] In one embodiment of the first aspect, the sample contacting component is a consumable, disposable, removable or replaceable component of a sample analysis device or system.

[0023] In one embodiment of the first aspect, the sample contacting component is a container, a fluid conduit or a probe of a sample analysis device or system.

[0024] In one embodiment of the first aspect, the vessel is a tube or well and the liquid conduit is a pipette.

[0025] In one embodiment of the first aspect, the EAB sensor comprises a working electrode.

[0026] In one embodiment of the first aspect, the working electrode is in the form of a wire, liner, membrane, plate, grid, cage, pin or needle.

[0027] In one embodiment of the first aspect, the sample contacting component comprises an electrical conduit in electrical communication with the working electrode.

[0028] In one embodiment of the first aspect, the electrical conduit extends from the working electrode to the edge of the sample contacting component.

[0029] In one embodiment of the first aspect, the electrical conduit has a terminus distal to the working electrode and a terminus forming or in electrical communication with a first interface portion, the first interface portion configured to form an electrical connection with a second interface portion of a mounting component of the device or system, the mounting component configured as a mount to the sample contacting component.

[0030] In an embodiment of the first aspect, the first interface portion and the second interface portion are configured to form an electrical connection.

[0031] In one embodiment of the first aspect, the electrical connection is a push-on / pull-off type or a pull-on / push-off type pressure fit.

[0032] In one embodiment of the first aspect, the electrical connection is a twist-on / twist-off type threaded connection.

[0033] In an embodiment of the first aspect, the first interface portion comprises a plate, a biasing means, a plug, a plug socket, a male portion, a female portion, or a threaded portion.

[0034] In an embodiment of the first aspect, the sample contacting component is configured to form a substantially liquid-tight and / or gas-tight connection with a mounting portion of the device or system.

[0035] In one embodiment of the first aspect, the sample contacting component comprises a sealing surface or structure that provides a seal with a mounting part of the device or system.

[0036] In one embodiment of the first aspect, the sample contacting component is made from a rigid or semi-rigid and / or elastically deformable material.

[0037] In one embodiment of the first aspect, the sample contacting component is manufactured from a synthetic polymer.

[0038] In one embodiment of the first aspect, the sample contacting component comprises a wall that substantially blocks the passage of liquid and / or gas.

[0039] In a second aspect, there is provided a sample analysis device or system comprising a mounting portion, the mounting portion comprising an electrical interface portion configured to form an electrical connection with an electrical interface portion of a sample contacting component configured to be mounted on the mounting portion.

[0040] In one embodiment of the second aspect, the sample contacting component is a consumable, disposable, removable or replaceable component of a sample analysis device or system.

[0041] In one embodiment of the second aspect, the sample contacting component is a container, a fluid conduit or a probe of a sample analysis device or system.

[0042] In one embodiment of the second aspect, the vessel is a tube or well and the liquid conduit is a pipette.

[0043] In an embodiment of the second aspect, the electrical interface portion is configured to form an electrical connection with the sample contacting component.

[0044] In one embodiment of the second aspect, the electrical connection is a push-on / pull-off type or a pull-on / push-off type pressure fit electrical connection.

[0045] In one embodiment of the second aspect, the electrical connection is a twist-on / twist-off type threaded electrical connection.

[0046] In an embodiment of the second aspect, the electrical interface portion comprises a plate, a biasing means, a plug, a plug socket, a male portion, a female portion, or a threaded portion.

[0047] In one embodiment of the second aspect, the mounting portion is configured to form a substantially liquid-tight and / or gas-tight connection with the sample contacting component.

[0048] In one embodiment of the second aspect, the mounting portion comprises a sealing surface or structure that forms a seal with the sample contacting component.

[0049] In one embodiment of the second aspect, the attachment portion is made from a rigid or semi-rigid and / or elastically deformable material.

[0050] In one embodiment of the second aspect, the attachment portion is made from a synthetic polymer.

[0051] In one embodiment of the second aspect, the fitting is configured to allow gas to pass through the fitting so as to cause the sample contacting component to aspirate or dispense a liquid sample.

[0052] In one embodiment of the second aspect, the device or system comprises a processor, the processor having access to program instructions configured to input a current value output by the EAB sensor and convert the current output value into a clinically relevant value.

[0053] In one embodiment of the second aspect, the processor is in electrical communication with the electrical interface portion of the mounting portion.

[0054] In a third aspect, the present invention provides a combination of a sample-contacting component according to any embodiment of the first aspect and a device or system according to any embodiment of the second aspect.

[0055] In one embodiment of the third aspect, the sample contacting component is mounted on the mounting portion such that the EAB sensor is in electrical communication with the processor.

[0056] In an embodiment of the third aspect, the sample contacting component is sealingly mounted on the mounting portion. [Brief explanation of the drawings]

[0057] [Figure 1] FIG. 1 is a cross-sectional view of a pipette of the present invention with an associated EAB sensor.

[0058] [Figure 2] FIG. 2 is a cross-sectional view of a pipette of the present invention having a complementary mount for a sample analyzer.

[0059] [Figure 3] FIG. 3 shows a stand-alone sample analysis device incorporating a pipette of the present invention with an associated EAB sensor.

[0060] [Figure 4] FIG. 4 shows a handheld sample analyzer incorporating a pipette of the present invention with an associated EAB sensor.

[0061] [Figure 5] FIG. 5 shows an exemplary connector that forms an electrical connection between a pipette and a sample analyzer.

[0062] [Figure 6]Figure 6 illustrates a method and apparatus of the present invention for analyzing multiple blood samples from a group of patients, each requiring an assay for a different analyte.

[0063] [Figure 7] Figure 7 illustrates the generation of a customized pipette set from a pipette library. The customized set includes a combination of pipette types, each capable of detecting a specific analyte via an associated EAB sensor.

[0064] [Figure 8] FIG. 8 shows the integration of an EAB sensor into the floor of a multiwell.

[0065] [Figure 9] FIG. 9 shows the incorporation of an EAB sensor into a probe.

[0066] [Figure 10] FIG. 10 shows the probe of FIG. 9 immersed in a sample held in a multiwell plate.

[0067] [Figure 11] FIG. 11 shows the probe of FIG. 9 immersed in a sample held in a tube.

[0068] [Figure 12] FIG. 12 shows a tube equipped with electrodes and electrically connectable to a processor of a sample analyzer.

[0069] [Figure 13] FIG. 13 shows a system for analyte detection that uses a conventional disposable pipette into which the sample is aspirated and held during analysis.

[0070] [Figure 14] FIG. 14 shows a sensing head useful in the system shown in FIG.

[0071] [Figure 15] FIG. 15 shows a tray system useful for storing a sensing head such as that shown in FIG.

[0072] Unless otherwise indicated herein, features in the drawings that are given the same number are considered to be identical features, or at least functionally similar features, even when used between different drawings.

[0073] The drawings are not made to any particular scale or dimension and are not intended to be an entirely accurate representation of the various embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0074] After considering this description, it will be apparent to one skilled in the art how to implement the present invention in various alternative embodiments and alternative applications. However, while various embodiments of the present invention are described herein, it will be understood that these embodiments are presented by way of example, not limitation. As such, this description of various alternative embodiments should not be construed as limiting the scope or breadth of the present invention. Moreover, statements about advantages or other aspects may apply to particular example embodiments, and not necessarily to all embodiments, or to any embodiments included in the claims.

[0075] Throughout the description and claims of this specification, the terms "comprise" and variations of words such as "comprise" and "have" are not intended to exclude other additives, components, integers or steps.

[0076] References throughout this specification to "one embodiment" or "an embodiment" mean that the particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment, although they may.

[0077] As used herein, terms relating to position such as "lateral," "across," "above," "below," "higher," "lower," "upward," "downward," "plan view," and the like, should be considered with reference to the analyzer being used in its normal upright position, allowing the liquid sample to flow downward under the force of gravity.

[0078] The present invention is based, at least in part, on the inventors' discovery that the use of an electrochemical aptamer-based (EAB) sensor associated with a pipette or other sample-contacting component of a sample analysis device or system provides significant advantages to the operation of the sample analysis device or system. For example, an EAB sensor associated with a pipette allows for the use of only a single contact step with the sample to determine analyte concentration. This contrasts with the lengthy, multi-step testing methods used by prior art high-throughput devices and systems, making the present invention highly suitable for application in analytical laboratories that process a very large number of samples per day. Furthermore, equipment requirements are significantly reduced, and some versions of the present invention enable high-throughput processing using only a single item of equipment. Another potential advantage is that laboratory workflows can be modified to more efficiently process a varying number of different tests offered. A further advantage is that pipettes with associated EAB sensors can be used in point-of-care devices that enable rapid and procedurally simple bedside determination of analyte concentration.

[0079] Potentially useful EAB sensors in the context of the present invention may be potentiometric, amperometric, or conductometric. In potentiometric sensors, local equilibrium is established at the sensor interface, either the electrode or membrane potential is measured, and information about the sample is derived from the potential difference between the two electrodes. Amperometric sensors rely on a potential applied between a reference electrode and a working electrode, thereby causing the oxidation or reduction of a redox-active species, and the resulting current is measured. Conductometric sensors rely on measuring electrical conductivity over a range of frequencies.

[0080] EAB sensors have been shown to be capable of reliably and specifically detecting drugs in patient fluids. These types of sensors are typically amperometric, with an aptamer (such as DNA, RNA, or XNA) attached to a working electrode. Gold is often used as the probe surface for the working electrode. The aptamer has an associated redox-active species that acts as a reporter. The redox reporter is often methylene blue. Upon target (drug) binding, the aptamer undergoes a conformational change, bringing the redox reporter closer to the working electrode surface. This increased proximity increases electron transfer from the redox reporter to the electrode. The increased rate of electron transfer contributes to a change in faradaic current, which is detected by a potentiostat.

[0081] Aptamers are small (usually 20-60 nucleotides), single-stranded RNA, DNA, or XNA oligonucleotides that can bind to drug targets with high affinity and specificity. Aptamers may be thought of as the nucleotide analogue of antibodies, but their production is an in vitro cell-free process that is much easier and cheaper than producing antibodies by cell culture or in vivo methods.

[0082] Aptamers are typically selected from combinatorial libraries with a vast number (up to 10) of different oligonucleotides. RNA aptamers offer significantly greater structural diversity compared to DNA aptamers, but their application is complicated by stability issues in the presence of RNases, high temperatures, and unfavorable pH.

[0083] The selection of aptamers selective for a given drug can be facilitated by a process known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment). This process can be viewed as two alternating stages. In the first stage, library oligonucleotides are amplified to the desired concentration by polymerase chain reaction (PCR). For the selection of RNA aptamers, single-stranded oligoribonucleotides are generated by in vitro transcription of double-stranded DNA using T7 RNA polymerase. For DNA aptamers, a pool of single-stranded oligodeoxyribonucleotides is generated by strand separation of double-stranded PCR products. In the second stage, the amplified products are incubated with the target drug, and oligonucleotides that bind to the drug are used in the next round of SELEX.

[0084] The separation of oligonucleotides with higher affinity for the target drug and the removal of unbound oligonucleotides are achieved by intense competition for binding sites. The selection pressure increases with each SELEX round. Maximum enrichment of the oligonucleotide pool with aptamers with the strongest affinity for the target molecule is usually achieved after 5-15 rounds.

[0085] EAB sensors are typically incorporated into a circuit with a reference electrode. The reference electrode is the site of a known chemical reaction with a known redox potential. For example, a reference electrode based on the silver-silver chloride (silver-silver chloride) redox couple has a fixed, known potential from which the redox potential of the working electrode is measured. The circuit also typically includes a counter electrode, which functions as the cathode or anode relative to the working electrode. Because the applied voltage bias does not pass through the reference electrode (due to the impedance of the potentiostat), any potential generated is attributed to the working electrode. Current is measured as the ratio of the potential of the interrogating electrode to the stable potential of the reference electrode. The potential difference generates a current in the circuit, thereby generating an output signal. The signal quantifies target binding by relying on electron transfer, ideally stoichiometrically proportional to target binding.

[0086] The present invention will be more fully described with reference to the following non-limiting embodiments.

[0087] Referring to Figure 1, one example of a sample contacting component of the present invention is an improved replaceable pipette tip 10. The pipette tip 10 includes a gold wire 15 running along the luminal surface of the pipette tip 10. The gold wire 15 is shown with a greatly exaggerated diameter for ease of illustration.

[0088] At its lower end (15a), the gold wire (15) has an analyte-specific aptamer attached to its surface, thereby forming the basis of a working electrode. As noted above, each aptamer has an associated redox reporter. Such methods for preparing aptamer-loaded working electrodes are known to those skilled in the art. Typically, the wire (15) is functionalized with the aptamer / reporter in a separate process before coupling to the pipette (10).

[0089] The portion of the wire 15b that is not coated with aptamer serves as an electrical conduit. Wire portion 15b does not need to be exposed to the sample within pipette 10 and, in this embodiment, is covered with adhesive to secure wire portion 15b to the inner luminal surface of pipette 10. The adhesive keeps wire portion 15b in place, and therefore aptamer-loaded portion 15a, at the bottom of pipette 10, ensuring contact with the sample within the lumen 10. Aptamer-loaded portion 15a may extend away from the luminal surface of pipette 10 to better contact the sample.

[0090] The gold wire 15 electrically connects at its upper end to a conductive plug 20. As shown in FIG. 2, the function of the plug 20 is to connect to a socket 105 in the pipette mount 110 of the sample analyzer 100. The socket 105 forms an electrical connection with the processor 115 of the sample analyzer 100 via a press-fit mechanism. The current resulting from interrogation of the working electrode (essentially the aptamer-coated portion 15a) is transmitted to the processor 115, where it is read as a current value and converted to a clinically relevant value, such as the concentration of the target analyte (present in the sample aspirated in accordance with the program instructions).

[0091] In addition to the electrical connections described above, the pipette 10 provides a pneumatic connection with the sample analyzer 10. The upper sealing portion 25 of the pipette 10 is shaped and sized to form a press fit with the complementary pipette mount 110 of the sample analyzer 100. A seal is formed, allowing the lumen of the pipette 10 to connect to a pneumatic system or device 120 via the pipette mount 110 and a channel 125 formed in the tubing 130.

[0092] In operation, the pipette 10 is attached to the mount 110 by lowering the pipette mount 110 onto the pipette 10. Typically, the pipette 10 is held in a rack or other support, allowing the mount 110 to resist the downward force of the mount 110, thereby forming a pressure fit therebetween. In many embodiments, a motor drive system under control of program instructions executed by the processor 115 urges a head comprising multiple mounts downward, each of which forms a pressure fit with its respective pipette.

[0093] When first attached to mount 110, pipette 10's lumen is initially empty. The tip of pipette 10 is lowered into a sample for testing, and pneumatic system 120, operated under control of a program instructed to be executed by processor 115, draws air from the lumen of pipette 10, thereby aspirating a volume of sample into the lumen of pipette 10. A motor drive system moves pipette 10 upward to empty a container holding the sample.

[0094] The aspirated sample contacts the working electrode (aptamer-loaded portion 15a) and the target analyte (if present) binds to the aptamer, creating a current in the gold wire (15).

[0095] Once the working electrode interrogation is complete, the mount is moved laterally by the motor drive system until it is positioned over a waste receptacle. The pipette 10 is expelled from the mount 110 by downwardly biasing the ejector 135. The ejector 135 is biased downward by a solenoid 140 under control of program instructions executed by the processor 115. The ejector 135 is returned to its original position (as depicted), allowing the mount 110 to accept a new pipette to perform a second run.

[0096] As will be appreciated, a working electrode requires a counter electrode for operation. The pipette (10) has a second wire (not shown) to which a second plug is electrically connected, and the pipette mount has a second socket (not shown) that receives the plug.

[0097] Additionally, a reference electrode (e.g., silver-silver chloride based) may be provided. In such embodiments, a third wire (not shown) and a third plug (not shown) are provided.

[0098] Operation of the EAB sensor requires a power source to provide the electrical potential required for the test. The power source is typically integral to the sample analyzer 100. Means for modulating the electrical potential, for example in the form of a square wave, can be provided for this purpose under the direction of the processor 115.

[0099] In some embodiments, the present invention is adapted for use in robotic high-throughput sample analysis systems. Such systems are capable of processing hundreds or thousands of samples per day and have a high level of automation. Multiple samples are processed simultaneously, typically in the form of racked tubes or multi-well plates.

[0100] A basic benchtop robotic system (300) is shown in FIG. 3, which shows a pipette (10) with an EAB sensor, a pipette mount (110) in pneumatic and electrical communication with the pipette (10), a waste container (130), a multiwell plate (135) holding multiple samples for analysis, a rack (140) holding multiple pipettes each with an EAB sensor, a first arm (145) for moving the pipette mount (110) in the x and y directions, and a second arm (150) for moving the pipette mount (110) in the z direction.

[0101] The present invention is also applicable to portable sample analyzers, which are devices that can be used at the point of care, such as a clinic, outpatient facility, or bedside. See FIG. 4 , which shows a handheld device 400 having a body 405 containing a pneumatic system. In use, the body 405 is grasped by the fingers of a user's hand, with the user's thumb resting on a button 410. The button 410 is actuated by the thumb to trigger the pneumatic system, which aspirates a sample through the pipette 10 when attached. The pipette 10 includes an EAB sensor, and thus the pipette mount 110 forms an electrical connection to the pipette 10. The pipette mount 110 also provides a pneumatic connection to the pipette 10, allowing the pneumatic system to aspirate a sample into the pipette. The ejector 135 is provided in the form of a slidable sleeve and is urged downward by manual depression of the ejector actuator 415. The electronics (including the processor) are located in the head portion 420. The test output values ​​are displayed on a screen 425.

[0102] The device (400) also includes a selector button (430) that allows the user to select a specific analyte for detection. In the illustrated embodiment, the device is selected to detect the metabolite creatinine. Of course, the user must select the correct pipette (10) that has a working electrode loaded with a creatinine-detecting aptamer. The selected analyte is used by the processor to determine the correct equation or standard curve to use in generating clinically relevant values ​​from the raw current values ​​output by the EAB sensor.

[0103] The type and placement of the working electrode may, of course, vary from that shown in the drawings. In one embodiment, the electrode may be in the form of a wire, liner, foil, plate, grid, cage, pin, or needle. The working electrode may be associated in some way with the pipette wall, or in other embodiments, may be centrally located within the pipette lumen. In other embodiments, the working electrode extends through the pipette wall into the lumen, with the electrical conduit running outside of or embedded within the pipette wall.

[0104] The plug and socket electrical connection between the pipette and the mounting portion is clearly exemplary only. With the benefit of this specification, those skilled in the art will find other configurations that are at least operable in the context of the present invention. For example, a system comprising two opposing conductive plates (optionally spring-loaded), or a rigid member biasing member (similar to a mains power outlet) could be used.

[0105] Connector configurations that eliminate the need to axially rotate the pipette to any particular position to enable connection to the fitting are generally preferred. In many embodiments, three separate electrical connections are required, one each for the working electrode, reference electrode, and counter electrode. Therefore, it is desirable for the pipette to be connectable to the fitting at any axial rotation and still function.

[0106] In some embodiments, a guide system is implemented to axially rotate the pipette into position as the pipette is moved onto the pipette mount so that the connector is accurately aligned.

[0107] In some embodiments, the processor is configured to detect which connector on the mounting portion is connected to which electrode (i.e., working electrode, counter electrode, or reference electrode). As an example, each connector on the mounting portion may have a light-sensitive optical detector adjacent to it configured to measure the reflectance of a surface (white, gray, or black) adjacent to the connector on the pipette.

[0108] In another example, the processor is configured to determine a unique electrical characteristic that is diagnostic of which connector (on the pipette side) is connected.

[0109] In yet another example, referring to FIG. 5, the connections are in the form of annular conductive tracks (500, 505, 510) located on the outer surface of the pipette mount and complementary annular conductive tracks (600, 605, 610) located on the inner surface of the pipette. Connectors (500), (505), and (510) connect to processor inputs for the working, counter, and reference electrodes, respectively. Connectors (600), (605), and (610) connect to the working, counter, and reference electrodes, respectively. When the pipette (10) is fully seated in the mount (110), the connectors align (500 and 600, 505 and 605, and 510 and 610) to ensure proper connections between the electrodes and the processor inputs.

[0110] The devices of the present invention (regardless of embodiment) typically include a potentiostat for controlling the potential applied to the working electrode.

[0111] The use of a pipette with an associated EAB sensor enables a device that is self-contained and capable of performing a wide range of analyte detection methods without the need for additional equipment. Furthermore, the device can detect target analytes with a single step of contacting the sample with the EAB sensor.

[0112] In this exemplary workflow, multiple subjects (715, 725, 735, ... n) each provide a blood sample in a vacutainer (720, 730, 740) for analysis of an analyte, such as a drug, metabolite, or antibody of particular specificity. Aliquots of blood from each vacutainer (720, 730, 740) are placed into wells of a 96-well multiwell plate (745), one aliquot per well.

[0113] The multiwell plate (745) is mounted on the tray (750) of the high-throughput analyte detection device (710). The device (710) includes an array of pipettes with EAB sensors (not shown in FIG. 1, but shown elsewhere at 10) arranged in a 12x8 grid, aligned with the wells of the multiwell plate (745). As described more fully below, the array of pipettes is first positioned above the multiwell plate (745) and lowered into the wells of the multiwell plate (745) for analysis, at which point sample from each well is aspirated into the respective pipette lumen where it comes into contact with the EAB sensor.

[0114] After the aptamer-based biosensors are tested, the analyte concentrations for each subject (715, 720, 725) are displayed on a screen (755) for review by the operator, if desired.

[0115] The analyte concentrations for each subject (715, 720, 725) are transmitted in an electronic file, data packet, or other manner to a laboratory server (800) and stored in the associated analytical sample laboratory relational database (805). The analyte concentrations are stored in linked association with an identifier such as the associated subject's name (optionally with date of birth) or other unique identifier such as a health insurance number, social security number, patient number, etc.

[0116] At the end of the analysis, the multiwell plate (745) is removed from the tray (750) and discarded.

[0117] The pipette tips can be removed from the high-throughput analyte detection device (700) and also discarded.

[0118] Figure 6 illustrates the generation of a customized pipette set from a pipette library. The customized set contains a combination of pipette types, each capable of detecting a specific analyte via an associated EAB sensor.

[0119] In preparation for the next analysis, a new set of pipette tips is placed into the high-throughput analyte detection device (710). The particular set of tips selected depends on the analyte to be detected in the next analysis. For example, if the target analyte is troponin, each pipette in the set will have an aptamer specific for troponin.

[0120] An analytical laboratory or point of care may have a "library" of pipettes from which an operator (or even a machine) may select according to the desired analyte. In some situations, pipettes specific to different analytes can be mixed to form a set of pipettes. See FIG. 7. For example, each sample may be tested for troponin and creatinine phosphokinase; in this case, each subject's blood is placed into two wells, with the contents of the first well aspirated into the troponin-sensing pipette and the contents of the second well aspirated into the creatinine phosphokinase-sensing pipette. Alternatively, a single pipette may contain aptamers capable of sensing more than one analyte.

[0121] A mixed set of pipettes can be used when each subject requires analysis of a single target analyte, but two different analytes need to be analyzed in one multiwell plate. For example, one group of subjects may require analysis of glucose, and a second group of subjects may require analysis of testosterone. In this case, the multiwell plate can be split in half, with one half for glucose and the other half for testosterone.

[0122] As can be seen from the above, the use of aptamer-loaded pipettes offers significant flexibility in analytical laboratory operations: highly customized pipette sets can be created to allow multiple selected analytes to be assayed on a single multiwell plate.

[0123] The use of aptamer-based biosensors in combination with pipettes enables this customization. Many prior art assays have significantly different analytical methods, preventing different assays from being run on the same plate or tube rack. For example, one method may be an enzyme-linked immunosorbent assay (ELISA) involving multiple steps, while another method may rely on magnetic bead technology. The vastly different process steps prevent prior art assays from being run simultaneously. In contrast, aptamer-based detection methods may rely on only a single contacting step, regardless of the target analyte. For example, the contacting step may be performed by simply aspirating a sample into a pipette so that the sample contacts the aptamer within the pipette lumen. At that point, the biosensor is interrogated by applying a potential (e.g., by square-wave voltammetry), providing a reliable current output proportional to the amount of analyte present within seconds.

[0124] The use of a single contacting step allows for significant time savings that dramatically improve analytical laboratory throughput and also simplifies analyte detection at the point of care. Thousands of samples can be analyzed because washing steps, the use of multiple reagents at different points in the detection method, and sample transfer between items or devices are eliminated. In this exemplary workflow, approximately 60, 70, or 80 samples can be analyzed using a 96-well microwell plate (typically, multiple wells are used for controls, standard curve generation, etc.).

[0125] Apart from the pipette, the EAB sensor may be associated with a well of a multi-well plate (45). Figure 8 shows three wires (15b, 15c, 15d) extending into the well, respectively: a test electrode (loaded with aptamer), a counter electrode, and a reference electrode. Each wire has a connector (one shown as 20a) built into the floor of the well. The plate holder (110a) has a complementary set of connectors (one marked 105a) that form electrical connections with the well connectors (20a). Conduits from each well connector (20a) connect to a processor.

[0126] In another alternative, an aptamer-loaded probe (900) is provided that can contact a sample held in a multiwell plate (FIG. 10) or a tube (FIG. 11). In this embodiment, the probe (900) is made of an electrically insulating material to insulate the conductive rings (905, 910, 915). Ring (905) is loaded with the aptamer and is the working electrode, ring (910) is the counter electrode, and ring (915) is the reference electrode.

[0127] Figure 12 shows tubing with associated working electrode (15b), reference electrode (15c), and counter electrode (15d). The tubing connector (labeled 20a) electrically connects with the tube rack connector (labeled 105a).

[0128] 13 illustrates an embodiment similar to the pipette-based embodiment described above, but which uses a conventional disposable pipette 1000. The analyte detection device 710 may provide some or all of the necessary electrical components, with the pipette serving only the purpose of holding the test sample in contact with the electrodes during analysis.

[0129] In the embodiment of Figure 13, the analyte detection device provides aptamer-loaded electrodes and counter electrodes in the form of pins (1005, 1010). The pins (1005, 1010) are molded into a barrel (1015) to provide an integrated sensing head. The diameter of the barrel (1015) is sized to form a pressure fit with the pipette (1000).

[0130] The pins (1005, 1010) extend beyond the upper surface of the barrel (1015) and provide connections (1005a, 1010a) that electrically connect with the electrical sockets (1020, 1025) of the intermediate portion (1030). The electrical connection thus formed can maintain the barrel (1015) together with the intermediate portion (1030). Alternatively, a press fit between the other portions of the barrel (1015) and the intermediate portion (1030) can be implemented. A magnetic connection is also a useful alternative.

[0131] A keying arrangement (1035) with a protrusion on one portion and a complementary recess on the other portion is incorporated to ensure a precise connection between the barrel (1015) and the mid-section (1030). The keying arrangement may also be used to provide a press fit that holds the barrel (1015) and the mid-section (1030) together.

[0132] The device interface portion 1040 is a permanent part of the device 710 and forms an electrical connection with the midsection 1030 and, therefore, the electrode pins 1005, 1010. The device interface portion 1040 forms the electrical connection interface of the device 710. The device interface portion 1040 provides an aspiration tube 1045 configured to pass through the midsection 1030 and the barrel 1015 to provide gas communication between the lumen of the pipette 1000 and the pneumatic system (not shown) of the analyte detection device 710.

[0133] In this embodiment, the pipette (100) is typically for single use only.

[0134] The electrode pins (1005, 1010) and barrel (1015) can be used 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more times. The aptamers on the working electrode pin (1005) can be purged of bound species from previous samples by a simple wash step or by applying an electric current.

[0135] Intermediate portion 1030 may be used multiple times, but may require replacement when it wears out and is unable to form the necessary press fit, electrical connection, or other required function. Advantageously, intermediate portion 1030 is a serviceable part of device 710 and can be replaced by a technician or even a non-professional user.

[0136] 14, a separate sensing head is shown in which the third electrode pin (1012) is a reference electrode. As described below, space exists to incorporate additional working electrodes, each loaded with an aptamer for detecting additional analytes.

[0137] Because the aptamers in electrochemical sensors are unstable, consideration can be given to storing newly manufactured electrode sets in a ready-to-use condition. A general goal is to provide dry, substantially oxygen-free storage conditions. Preferably, the electrodes are also stored in a manner that allows for easy connection to a sample-analyte device.

[0138] Considering the above requirements, a storage system as shown in Fig. 15 is proposed, which comprises a tray (2000) having a plurality of recesses (one of which is designated 2005), each recess configured to receive and hold a sensing head (1005, 1010, 1015). The sensing heads (1005, 1010, 1015) are arranged such that the electrode pin portions (1005a, 1005b) extending beyond the barrel (1030) face upwards, thereby providing access to a complementary connector located directly above and which can be moved downwards towards the pin portions.

[0139] The barrel (1030) is provided with an annular seal (2010), which may be an O-ring or an overmolded gasket, that acts to seal against the upper inner wall of the recess (2005), thereby isolating the aptamer coated on the lower end of the working electrode pin (1005) from the atmosphere.

[0140] The sensing heads 1005, 1010, 1015 can be placed in the tray 2000 and sealed against the walls of the recess 2010 under oxygen- and moisture-free conditions. For example, the tray 2000 can be loaded with sensing heads in a dry nitrogen gas environment. Additionally or alternatively, each recess 2010 can include a perforated receptacle containing a desiccant (e.g., silica gel) and an oxygen absorber (e.g., iron powder).

[0141] The present invention is suitable for computer implementation given that the output of the EAB sensor is an electrical signal that is stored electronically as a numerical value (e.g., a current value) in volatile memory and manipulated and analyzed by an associated processor under software direction.

[0142] As will be appreciated by those skilled in the art, the present invention may be deployed, in part or in whole, via one or more processors that execute computer software, program code, and / or instructions thereon. The processor may be part of a server, client, network infrastructure, mobile computing platform, fixed computing platform, or other computing platform. The processor may be any type of computational or processing device capable of executing program instructions, code, binary instructions, etc. The processor may be or include a signal processor, digital processor, embedded processor, microprocessor, or any variant of coprocessor (such as a math coprocessor, graphics coprocessor, communication coprocessor, etc.), which may directly or indirectly facilitate the execution of program code or program instructions stored thereon.

[0143] Additionally, the processor may allow for the execution of multiple programs, threads, and codes.

[0144] Threads may be executed simultaneously to improve processor performance and facilitate simultaneous operation of applications. As an example, the methods, program code, program instructions, etc. described herein may be implemented with one or more threads. Threads may spawn other threads with priorities associated with them, and the processor may execute these threads based on priority or any other order based on instructions provided in the program code. The processor may include memory for storing the methods, code, instructions, and programs as described herein.

[0145] Any processor or mobile device or server may access a storage medium through an interface that may store methods, codes, and instructions as described herein and elsewhere. Storage media associated with a processor for storing methods, programs, codes, program instructions, or other types of instructions that may be executed by a computing or processing device may include solid-state memory and hard disk memory.

[0146] A processor may include one or more cores, which can increase the speed and performance of a multiprocessor. In some embodiments, a processor may be a dual-core processor, a quad-core processor, other chip-level multiprocessor, etc., that combines two or more independent cores (called dies).

[0147] The methods and systems described herein may be deployed, in part or in whole, through one or more hardware components executing software on a server, client, firewall, gateway, hub, router, or other such computer and / or network hardware. Software programs may be associated with a server, which may include a file server, print server, domain server, Internet server, intranet server, and other variations such as a secondary server, host server, distributed server, etc. A server may include one or more of the following: memory, processor, computer-readable medium, storage media, ports (physical and virtual), communication devices, and interfaces capable of accessing other servers, clients, computers, and devices via wired or wireless media. The methods, programs, or code described herein and elsewhere may be executed by a server. Additionally, other devices necessary for the execution of the methods described herein may be considered part of the infrastructure associated with the server.

[0148] A server may provide an interface to other devices, including, but not limited to, clients, other servers, printers, database servers, print servers, file servers, communication servers, distribution servers, etc. Furthermore, this coupling and / or connection can facilitate remote execution of programs over a network. Networking some or all of these devices can facilitate parallel processing of a program or method in one or more locations without departing from the scope of the present invention. Furthermore, any of the devices connected to a server via an interface can include at least one storage medium capable of storing methods, programs, code, and / or instructions. A central repository can provide program instructions to be executed on different devices. In this embodiment, the remote repository can function as a storage medium for program code, instructions, and programs.

[0149] A software program may be associated with a client, which may include a file client, a print client, a domain client, an Internet client, an intranet client, and other variations such as a secondary client, a host client, a distributed client, etc. A client may include one or more of: memory, a processor, a computer-readable medium, a storage medium, ports (physical and virtual), communication devices, and interfaces capable of accessing other clients, servers, computers, and devices via wired or wireless media. The methods, programs, or code described herein and elsewhere may be executed by a client. Additionally, other devices necessary for execution of the methods described herein may be considered part of the infrastructure associated with the client.

[0150] A client may provide an interface to other devices, including, but not limited to, a server, other clients, printers, database servers, print servers, file servers, communication servers, distributed servers, etc. Furthermore, this coupling and / or connection may facilitate remote execution of programs over a network. Networking some or all of these devices may facilitate parallel processing of a program or method in one or more locations without departing from the scope of the present invention. Furthermore, any of the devices connected to a client via an interface may include at least one storage medium capable of storing methods, programs, applications, code, and / or instructions. A central repository may provide program instructions to be executed on different devices. In this embodiment, the remote repository may serve as a storage medium for program code, instructions, and programs.

[0151] The methods and systems described herein may be deployed in part or in whole over a network infrastructure. The network infrastructure may include elements such as computing devices, servers, routers, hubs, firewalls, clients, personal computers, communication devices, routing devices, and other active and passive devices, modules, and / or components known in the art. Computing and / or non-computing devices associated with the network infrastructure may include storage media, apart from other components. The processes, methods, program codes, instructions described herein and elsewhere may be executed by one or more of the network infrastructure elements.

[0152] The methods, program codes, calculations, algorithms, and instructions described herein may be implemented on a cellular network having multiple cells. The cellular network may include mobile devices, cell sites, base stations, repeaters, antennas, towers, etc. The cell network may be a GSM, GPRS, 3G, 4G, 5G, EVDO, mesh, or other network type.

[0153] The methods, program codes, calculations, algorithms, and instructions described herein may be implemented on or via a mobile device. Mobile devices may include mobile phones, personal digital assistants, laptops, palmtops, netbooks, pagers, e-book readers, etc. These devices may include, among other components, storage media such as flash memory, buffers, RAM, ROM, and one or more computing devices. The computing devices associated with the mobile devices may be capable of executing program codes, methods, and instructions stored thereon.

[0154] Alternatively, the mobile device may be configured to execute instructions in cooperation with other devices. The mobile device may communicate with a base station interfaced with a server and configured to execute program code. The mobile device may communicate over a peer-to-peer network, a mesh network, or other communication network. The program code may be stored in a storage medium associated with the server and executed by a computing device embedded within the server. The base station may include a computing device and a storage medium. The storage device may store program code and instructions executed by the computing device associated with the base station.

[0155] The computer software, program code, and / or instructions may be stored and / or accessed on computer-readable media, which may include computer components, devices, and recording media that hold digital data used for computations for some interval of time, storage devices known as random access memory (RAM), and mass storage devices for more permanent storage, typically in the form of optical disks, magnetic storage devices like hard disks, and the like.

[0156] The methods and systems described herein can transform physical and / or intangible items from one state to another. The methods and systems described herein can also transform data representing physical and / or intangible items from one state to another.

[0157] The elements described and shown herein may imply logical boundaries between the elements. However, in accordance with software or hardware engineering practices, the shown elements and their functionality may be implemented on a computer through a computer-executable medium having a processor capable of executing program instructions stored thereon, as a monolithic software structure, as a stand-alone software module, or as a module employing external routines, code, services, etc., or any combination thereof; all such implementations may be within the scope of the present disclosure.

[0158] Additionally, the illustrated elements may be implemented on machines capable of executing program instructions. Thus, while this description describes functional aspects of the disclosed system, the specific configuration of software for implementing those functional aspects should not be inferred from these descriptions unless explicitly stated or apparent from the context. Similarly, it will be understood that the various steps identified and described above may be varied, and the order of steps may be adapted to suit particular applications of the technology disclosed herein. All such variations and modifications are intended to fall within the scope of the present disclosure. Thus, the depiction and / or description of the order of various steps should not be understood as requiring a particular order of performance of those steps unless required by a particular application or unless explicitly stated or apparent from the context.

[0159] The above-described methods and / or processes, and steps thereof, may be implemented in hardware, software, or any combination of hardware and software suitable for a particular application. Hardware may include general-purpose computers and / or special-purpose computing devices or specific computing devices or specific aspects or components of specific computing devices. The processes may be implemented in one or more microprocessors, microcontrollers, embedded microcontrollers, programmable digital signal processors, or other programmable devices, along with internal and / or external memory. The processes may also or instead be embodied in application-specific integrated circuits, programmable gate arrays, programmable array logic, or any other device or combination of devices that may be configured to process electronic signals. Furthermore, it will be understood that one or more of the processes may be implemented as computer-executable code capable of being executed on a computer-readable medium.

[0160] Application software may be written using a structured programming language such as C, an object-oriented programming language such as C++, or any other high-level or low-level programming language (including assembly language, hardware description languages, and database programming languages ​​and techniques) that can be stored, compiled, or interpreted for execution on one of the above devices, as well as heterogeneous combinations of processors, processor architectures, or combinations of different hardware and software, or any other machine capable of executing program instructions.

[0161] Thus, in one aspect, each of the methods described above and combinations thereof may be embodied in computer-executable code that, when executed on one or more computing devices, performs the steps. In another aspect, the methods may be embodied in a system that performs the steps, may be distributed in some manner across devices, or all of the functionality may be integrated into a dedicated, stand-alone device or other hardware. In another aspect, the means for performing the steps associated with the processes described above may include any of the hardware and / or software described above. All such permutations and combinations are intended to fall within the scope of the present disclosure.

[0162] Any of the methods disclosed herein may be implemented using Windows TM , Linux (registered trademark), Android TM , iOS TM The software may be executed by application software capable of running on any past, present, or future operating system of a processor-enabled device such as a . It will be understood that any software may be distributed across multiple devices or in a "software as a service" or "platform as a service" format, whereby participants need only some computer-based means of engaging with the software.

[0163] The present invention is described primarily with reference to the analysis of clinical samples taken from human subjects. It will be understood that the present invention is applicable to other applications requiring high-throughput testing of samples for one or more analytes, including, but not limited to, veterinary medicine, agriculture, scientific research, quality control and quality assurance in manufacturing environments, food safety, and the analysis of environmental samples such as water and soil.

[0164] Those skilled in the art will appreciate that the invention described herein is susceptible to further variations and modifications other than those specifically described, and it is to be understood that the invention includes all such variations and modifications that are within the spirit and scope of the invention.

[0165] Accordingly, the spirit and scope of the present invention is not intended to be limited by the foregoing examples, but is to be understood in the broadest sense permitted by law.

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