Patent Application
20240238778
The instant application contains a Sequence Listing which has been submitted via USPTO's Patent Center and is hereby incorporated herein by reference in its entirety. Said ASCII copy, created on Jul. 24, 2023, is named 35022-49619_US_002US_ST25.txt, is 346,750 bytes in size, and is hereby incorporated by reference in its entirety.
The invention(s) described relate generally to multiplexed systems, kits, devices, and methods for reliably and efficiently assessing sexual health of a subject. Embodiments of the invention(s) described relate to consumer multiplexed, rapid test devices and kits that can be used to detect a target, such as the presence of two or more infections caused by a sexually transmitted pathogen, optionally in conjunction with the detection of pregnancy, fertility, and/or other sexual health conditions. Methods for producing the system(s) described are also described.
Sexually transmitted infections (STIs) remain an important focus area for global public health. There are over 1 million sexually transmitted infections (STIs) that are acquired each day and the numbers are growing as 75% of people infected are asymptomatic. However, a high morbidity is associated with STIs, such as the sequelae of reproductive tract infections, cervical cancer, congenital syphilis, ectopic pregnancy and infertility, as well as the morbidity of HIV-related illness and death from acquired immunodeficiency syndrome (AIDS). For example, pregnant women can be infected with sexually transmitted infections (STIs). STIs can complicate pregnancy and may have serious effects on both the pregnant individual and the developing baby. Some of these problems may be seen at birth; however, others may not be discovered until months or years later. In addition, it is well known that having an STI can make it easier for a person to get infected with HIV.
There is a need for consumer sensitive and multiplexed rapid tests to be able to detect two or more health conditions, for example a STI and a pregnancy test, in a single test. Such a device could prevent ectopic pregnancies, pelvic inflammatory disease, infertility and in some cases death.
The ability to rapidly and reliably assess health (e.g., sexual health) of a subject who is remote from a clinical setting can aid in early detection of abnormal health states. Additionally, users who need to test for multiple health conditions would benefit from an all-in-one test kit to easily and accessibly test for multiple health conditions. Design of system kit components to facilitate use by an end-user (e.g., in relation to sample provision and processing samples), as well as development of assay components that streamline sample processing to extract target material for multiplexed assays with reduced margin of error and contamination can enable such early detection, and thus prevent health complications. The system(s) and method(s) described herein include system components, assay materials, and additional elements for enabling rapid and reliable characterizations of two or more health conditions (e.g., sexual health conditions) of a subject in a multiplexed manner with reduced error and contamination.
In particular, embodiments of the invention(s) described can outperform existing tests for detection of sexual health statuses, in relation to comprehensively and efficiently testing for a panel of different pathogens in a streamlined, multiplexed format. In examples, embodiments of the invention can efficiently test for statuses of infections associated with the following agents in multiplexed formats: Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Treponema pallidum, Gardnerella vaginitis, Candida Albicans, Mycoplasma genitalium, estrogen, progesterone, testosterone, anti-mullerian hormone, follicle-stimulating hormone, luteinizing hormone, estradiol, thyroid-stimulating hormone, free thyroxine, prolactin, human immunodeficiency virus, human papillomavirus infection, Hepatitis B, and herpes simplex virus.
Embodiments of the invention(s) also produce increased sensitivity and/or specificity in test results by incorporating multiple test strips associated with different target material regions of the agents/biomarkers assessed, each independently assessed in their own respective testing container within a single device. Embodiments of the invention(s) also assess other sexual health conditions (e.g., statuses of pregnancy, statuses of fertility).
Embodiments of the invention(s) described also include custom extraction and processing buffer compositions for each health condition tested for that facilitate easy sample processing, especially in a consumer kit format. Embodiments of the invention(s) described also include effective reaction and testing substance compositions (e.g., aptamers, antibodies, etc.) that produce appropriate binding characteristics with sample target material and do not cross react, thereby enabling multiplexed format testing. In embodiments, the invention(s) the consumer kit includes a device designed in a simple, streamlined form factor for holding more than one test strip in a stable manner, separated from the adjacent strip(s) to avoid cross-contamination, while also positioning them for insertion at the appropriate depth into a specific corresponding well for that strip containing the relevant reagents and/or buffer along with the sample from the user for testing. The design of the device allows the user to easily place the ends of all strips into the corresponding wells simultaneously by simply placing the device on or over the wells, thus minimizing the chance that the user makes a mistake, such as inserting too much or too little of the strip into the well, inserting a strip into the incorrect well or cross-contaminating the strips, tipping over the wells and spilling the contents, etc.
In one or more aspects, a system for detecting status of one or more health conditions (e.g., health condition caused by an agent) includes: an extraction container for receiving a biological sample collected by a sample collection tool, the extraction container capable of containing a first buffer for extracting a target material associated with one or more health conditions from the sample to form a sample mixture comprising the target material and the first buffer; a testing container comprising: a holder comprising at least a first well configured to receive the sample mixture, wherein the first well comprises one or more additional buffers, that, when mixed with the sample mixture, form a first target sample solution in the first well, the one or more additional buffers associated with the one or more health conditions; and a base of the holder; a signal output device comprising a housing containing at least a first strip secured within the housing, wherein an end portion of the first strip is positioned within the signal output device for being placed into fluid communication with the first target sample solution of the first well, the signal output device configured to provide an indication of a status of the one or more health conditions for the first target sample solution. In some embodiments, the system is a sample health kit.
In one or more aspects, a system for detecting status of one or more health conditions (e.g., health condition caused by an agent) includes: an extraction container for receiving a biological sample collected by a sample collection tool, the extraction container capable of containing a first buffer for extracting a target material associated with one or more health conditions from the sample to form a sample mixture comprising the target material and the first buffer; a testing container comprising: a holder comprising at least a first and second well configured to receive the sample mixture, wherein the first and second well each comprise one or more additional buffers, that, when mixed with the sample mixture, form a first target sample solution in the first well, and a second target sample solution in the second well, the one or more additional buffers associated with the one or more health conditions; and a base of the holder; a signal output device comprising a housing containing at least a first and second strip secured within the housing, wherein an end portion of the first strip is positioned within the signal output device for being placed into fluid communication with the first target sample solution of the first well, and an end portion of the second strip is positioned within the signal output device for being placed into fluid communication with the second target sample solution of the second well, the signal output device configured to provide an indication of a status of the one or more health conditions for the first target sample solution and the second target sample solution.
In some embodiments, each of the one or more additional buffers within the first and second wells is associated with a health condition.
In some embodiments, the housing of the signal output device completely or partially surrounds the first and second strips.
In some embodiments, the housing is sized to fit over an upper portion of the target container, above the base, to allow the first and second strips to be lowered into the first and second well.
In some embodiments, the housing of the signal output device is configured to sit on top of the testing container.
In some embodiments, the housing of the signal output device comprises at least one wedge or peg configured to prevent movement or displacement of each of the at least first and second strips within the housing.
In some embodiments, further comprising one or more pressure points at one or more locations within the housing configured to prevent movement or displacement of the first and second strips.
In some embodiments, the one or more pressure points are configured to provide vertical fluid flow or horizontal fluid flow of the first and second target sample solution.
In some embodiments, the housing further comprises at least a first and second window for viewing the first and second strips and reading the indication of the status of the one or more health conditions.
In some embodiments, each strip comprises: an absorbent pad, a conjugate release pad, a wicking pad, a test line, and a control line.
In some embodiments, each strip further comprises a backing card and a sample pad. In some embodiments, each strip comprises one or more of: an absorbent pad, a conjugate release pad, a wicking pad, a test line, a control line, a backing card, and a sample pad. In some embodiments, each strip comprises one or more of: an absorbent pad, a conjugate release pad, a wicking pad, a membrane, a test line, a control line, a backing card, and a sample pad.
In some embodiments, each strip of the one or more strips comprises: a loading zone capable of receiving the target sample solution; a reaction zone coupled to the loading zone and comprising a first reaction substance conjugated to a first label, wherein the first reaction substance is capable of preferentially coupling to a first target of the target material within the target sample solution, a testing zone coupled to the reaction zone and comprising a first testing substance retained at a first region of the testing zone, wherein the first testing substance is capable of preferentially coupling to the first target of the target material within the target sample solution; and a control zone comprising a control substance retained at the control zone, wherein the control substance is not capable of preferentially coupling to the target material within the target sample solution.
In some embodiments, a health condition of the one or more health conditions is an infectious disease caused by at least one of a bacterial agent, viral agent, or a parasitic agent.
In some embodiments, a health condition of the one or more health conditions is a health condition associated with at least one of a Streptococcus pyogenes infection, Chlamydia trachomatis infection, a Neisseria gonorrhoeae infection, a Trichomonas vaginalis infection, a Treponema pallidum infection, a Gardnerella vaginitis infection, human immunodeficiency virus, human papillomavirus infection, Hepatitis B, herpes simplex virus, a fertility status, a pregnancy status, or a women's health status. In some embodiments, the system or kit of the present disclosure relates to detecting a women's health status. In some embodiments, the health condition is associated with at least one of a Streptococcus pyogenes infection, influenza, common cold, and the like. In some embodiments, the women's health condition is associated with at least one of: estrogen, progesterone, testosterone, anti-mullerian hormone, follicle-stimulating hormone, luteinizing hormone, estradiol, thyroid-stimulating hormone, free thyroxine, and prolactin. In some embodiments, the health condition is a women's health condition. In some embodiments, the health condition is associated with an infectious disease.
In some embodiments, comprising a dropper cap, the dropper cap fitted to the extraction container.
In some embodiments, further comprising a stirrer for stirring one or more of the sample mixture, the first target sample solution comprising the sample mixture and the one or more additional buffers, or the second target sample solution comprising the sample mixture and the one or more additional buffers.
In some embodiments, the stirrer comprises two or more arms.
In some embodiments, the stirrer comprises a first arm configured to stir the first target sample solution, and a second arm configured to stir the second target sample solution.
In some embodiments, the sample collection tool is configured to mix the first buffer and the sample to form the sample mixture.
In some embodiments, further comprising at least a third well configured to receive the sample mixture, wherein the third well comprises one or more additional buffers, that, when mixed with the sample mixture, form a third target sample solution in the third well, the one or more additional buffers associated with the one or more health conditions.
In some embodiments, the signal output device further comprises at least a third strip, wherein an end portion of the third strip is in fluid communication with the third target sample solution of the third well, the signal output device configured to provide an indication of a status of the one or more health conditions for the third target sample solution in the third well.
In some embodiments, further comprising the sample collection tool for collecting the sample.
In some embodiments, the sample is one of a vaginal fluid, a vaginal tissue, a vaginal washing, a vaginal swab, a vaginal discharge, a cervical swab, a cervical tissue urethral swab, a urethral discharge, a rectal swab, a rectal material, a rectal washing, urine, blood, serum, plasma, saliva, tears, a skin swab, semen, seminal fluid, sputum, bronchial fluid, bronchial washing, peritoneal fluid, peritoneal washing, pleural fluid, pleural washing, cerebrospinal fluid, eye fluid, eye tissue, lung fluid, lung tissue, liver fluid, liver tissue, heart fluid, heart tissue, brain fluid, brain tissue, kidney fluid, kidney tissue, spleen fluid, spleen tissue, muscle fluid, muscle tissue, nasal fluid, nasal swab, throat fluid, or throat swab.
In some embodiments, the signal output device is capable of being signal processed using at least one of an optical sensor, optical reader, or a smartphone.
In some embodiments, the at least first and second wells are separately housed within the testing container, and wherein the at least first and second wells are not fluidically connected.
In some embodiments, the housing further comprises at least a first chamber configured to hold the first strip, and a second chamber configured to hold the second strip, and wherein the first and second chambers are not fluidically connected.
Aspects of the present disclosure provide a sample kit comprising: an extraction container for receiving a sample collection tool for mixing of the sample with a buffer within the extraction container to form a sample mixture and for performing a first step in an extraction process to extract from the sample, where present, the following: a first target material from one or more agents associated with a first health condition, and a second target material from one or more agents associated with a second health condition; a testing container for receiving, from the extraction container, the sample mixture comprising the buffer mixed with the sample, the testing container having at least the following: a first well containing one or more first reagents specific to the first health condition, the first well for receiving a portion of the sample mixture to combine with the one or more first reagents as a first target sample solution, and a second well containing one or more second reagents specific to the second health condition, the second well for receiving a portion of the sample mixture to combine with the one or more second reagents as a second target sample solution; and a signal output device having a housing containing at least a first test strip and a second test strip, wherein, upon coupling of the housing to the testing container, the first test strip fluidically contacts the first target sample solution and the second test strip contacts the second target sample solution, the first test strip including one or more zones with substances for interaction with the first target material to indicate whether the first health condition is present, and the second test strip including one or more zones with substances for interaction with the second target material to indicate whether the second health condition is present.
In some embodiments, the kit further comprises the sample collection tool for collecting the biological sample from a subject to test the subject for at least the first health condition and a second health condition.
In some embodiments, the testing container comprises at least a third well containing one or more third reagents specific to a third health condition, the third well for receiving a portion of the sample mixture to combine with the one or more second reagents as a third target sample solution.
In some embodiments, the signal output device further comprises a third test strip, wherein, upon coupling of the housing to the testing container, the third test strip fluidically contacts the third target sample solution, the third test strip including one or more zones with substances for interaction with the third target material to indicate whether the third health condition is present.
In some embodiments, the one or more reagents in each of the first and second wells comprise one or more buffers for being held within each of the first and second wells.
In some embodiments, the target material comprises a biomarker of a pathogen that causes the sexually transmitted infection, wherein the buffer within the extraction container, the testing container, or both the extraction container and testing container comprises one or more of: sodium hydroxide, potassium hydroxide, mM sodium chloride, mM potassium chloride, sulfuric acid or nitric acid or hydrochloric acid, PBS (phosphate buffered saline), TBS (Tris buffered saline), tricine, HBS (HEPES buffered saline), 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate, BugBuster™, octylthioglucoside, Triton X-100, octyl glucoside, chicken albumin, casein, bovine albumin, a crowding agent such as 1000-100000 dextran, 3500-20000 polyethylene glycol, Polyamines, amino acids, a non-toxic surfactant such as Tergitol, Tween-20, CHAPS, TAPSO, detergents like OTG, an antibacterial agent such as thimerosal, proclin-300, sodium azide, phosphate buffered saline, Tris-45 buffered saline, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline, and an extraction substance such as 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate, a protein extraction reagent, octyl-50 thioglucoside, and octyl glucoside.
In some embodiments, the target material comprises a biomarker of a pathogen that causes the sexually transmitted infection, wherein the buffer within the extraction container, the testing container, or both the extraction container and testing container comprises one or more of: 5-500 mM sodium hydroxide, 5-500 mM potassium hydroxide, 1-2000 mM sodium chloride, 1-2000 mM potassium chloride, 5-500 mM sulfuric acid or 5-500 mM nitric acid or 5-500 mM hydrochloric acid, 1-50% PBS (phosphate buffered saline), 1-50% TBS (Tris buffered saline), 1-50% tricine, 1-50% HBS (HEPES buffered saline), 00.1%-10% 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate, 0.1%-10% BugBuster™, 0.01%-10% octylthioglucoside, 0.01%-10% Triton X-100, and 0.01%-10% octyl glucoside.
In some embodiments, each strip of the one or more strips comprises:
a loading zone capable of receiving the target sample solution; a reaction zone coupled to the loading zone and comprising a first reaction substance conjugated to a first label, wherein the first reaction substance is capable of preferentially coupling to a first target of the target material within the target sample solution, a testing zone coupled to the reaction zone and comprising a first testing substance retained at a first region of the testing zone, wherein the first testing substance is capable of preferentially coupling to the first target of the target material within the target sample solution; and a control zone comprising a control substance retained at the control zone, wherein the control substance is not capable of preferentially coupling to the target material within the target sample solution.
Aspects of the present disclosure provides a method comprising: receiving, from a first device, a request for a sample kit of Claim 25, the sample kit to test a subject for at least a first health condition; receiving, from a second device, an image of a result of the sample kit; reading the result of the sample health kit from the image of the result; providing for display the result on a user interface of the first device; and receiving, from the first device, a request for a prescription for the subject based, in part, on the result.
In some embodiments, reading the result comprises reading a signal outputted by a signal output device of the sample kit using labels.
In some embodiments, at least one label of the labels is one of a visual label, gold nanoparticles, colored latex beads, magnetic particles, carbon nanoparticles, cellulose nano beads, selenium nanoparticles, silver nanoparticles, quantum dots, up converting phosphors, organic fluorophores, textile dyes, enzymes, liposomes or similar labels.
Aspects of the present disclosure provides a device for determining the presence or absence of one or more health conditions, comprising: a signal output device comprising a housing containing at least a first strip, wherein an end portion of the first strip is capable of being placed into fluid communication with a first target sample solution of a first well of an testing container, the signal output device configured to provide an indication of a status of the one or more health conditions for the first target sample solution.
Aspects of the present disclosure provides a device for determining the presence or absence of one or more health conditions, comprising: a signal output device comprising a housing containing at least a first strip, wherein an end portion of the first strip is capable of being placed into fluid communication with a first target sample solution of a first well of an testing container, the signal output device configured to provide an indication of a status of the one or more health conditions for the first target sample solution.
Aspects of the present disclosure provides a device for determining the presence or absence of one or more health conditions, comprising: a signal output device comprising a housing containing at least a first and second strip, wherein an end portion of the first strip is capable of being placed into fluid communication with a first target sample solution of a first well of an testing container, and an end portion of the second strip is capable of being placed into fluid communication with a second target sample solution of a second well of the testing container, the signal output device configured to provide an indication of a status of the one or more health conditions for the first target sample solution and the second target sample solution.
In some embodiments, the housing of the signal output device completely or partially surrounds the first and second strips.
In some embodiments, the housing is sized to couple over an upper portion of the target container, above the base, to allow the first and second strips to be lowered into the first and second well.
In some embodiments, the housing of the signal output device is configured to sit on top of the testing container.
In some embodiments, the housing of the signal output device comprises a wedge or peg configured to prevent movement or displacement of each of the at least first and second strips within the housing.
In some embodiments, further comprising one or more pressure points at one or more locations within the housing configured to prevent movement or displacement of the first and second strips.
In some embodiments, the one or more pressure points is configured to provide vertical flow or horizontal fluid flow of the first and second target sample solution.
In some embodiments, the housing further comprises at least a first and second window for viewing the first and second strips and reading the indication of the status of the one or more health conditions.
In some embodiments, each strip comprises: an absorbent pad, a conjugate release pad, a wicking pad, a test line, and a control line.
In some embodiments, each strip further comprises: a sample pad, and a backing card.
In some embodiments, each strip of the one or more strips comprises: a loading zone capable of receiving the target sample solution; a reaction zone coupled to the loading zone and comprising a first reaction substance conjugated to a first label, wherein the first reaction substance is capable of preferentially coupling to a first target of the target material within the target sample solution, a testing zone coupled to the reaction zone and comprising a first testing substance retained at a first region of the testing zone, wherein the first testing substance is capable of preferentially coupling to the first target of the target material within the target sample solution; and a control zone comprising a control substance retained at the control zone, wherein the control substance is not capable of preferentially coupling to the target material within the target sample solution.
The figures depict various embodiments of the present invention for purposes of illustration only. One skilled in the art will readily recognize from the following discussion that alternative embodiments of the structures and methods illustrated herein may be employed without departing from the principles of the invention described herein.
The system and methods can be adapted for the detection status of a health condition or panels including, but not limited to, infectious diseases (including sexually transmitted infections) caused by either bacterial, viral, parasitic agents including, Streptococcus pyogenes, Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Treponema pallidum, Gardnerella vaginitis, human immunodeficiency virus, human papillomavirus infection, Hepatitis B, herpes simplex virus, Streptococcus pyogenes and/or the relation to pregnancy or fertility. Additional non-limiting health conditions panels include but are not limited to Candida Albicans, Mycoplasma genitalium, estrogen, progesterone, testosterone, anti-mullerian hormone, follicle-stimulating hormone, luteinizing hormone, estradiol, thyroid-stimulating hormone, free thyroxine, and prolactin.
In an embodiment shown in
In some embodiments, the sample kit can be designed to include all the components needed for at least 2 tests, at least 3 tests, at least 4 tests, at least 5 tests, at least 6 tests, at least 7 tests, at least 8 tests, at least 9 tests, or at least 10 tests.
In some embodiments, each test is performed on a separate test strip within the signal output device. In some embodiments, the user tests for the same health condition for each test strip. In other embodiments, the user tests for different health conditions for each test strip within the sample kit.
In some embodiments, the sample collecting tool 105 is a swab, such as a vaginal swab, urethral swab, or an endocervical swab. Non-limiting examples of a sample collected by the sample collecting tool 105 include vaginal fluid, vaginal tissue, vaginal washing, vaginal swab, vaginal discharge, cervical swab, cervical tissue urethral swab, urethral discharge, rectal swab, rectal material, rectal washing, urine, blood, serum, plasma, saliva, tears, skin swab, semen, seminal fluid, sputum, bronchial fluid, bronchial washing, peritoneal fluid, peritoneal washing, pleural fluid, pleural washing, cerebrospinal fluid, nasal swabs and/or fluids, throat swabs and/or fluids eye fluid and/or tissue, fluid and/or tissue from lung, liver, heart, brain, kidney, spleen or muscle and any combination thereof. In some embodiments, the sample is a blood sample, and the sample collecting tool is, e.g., a lancet for piercing a finger and a container or slide for collecting the blood sample). In some embodiments, the sample is a urine sample, and the sample collecting tube is, e.g., a urine collection cup, urine swab, etc. In some embodiments, the sample is a vaginal discharge or a penile discharge, and the sample collecting tool is a swab, cotton pad, sample container, etc. In some embodiments, the sample is obtained from contacting an ulcer in genital area, and the sample collecting tool is a swab, cotton pad, sample container, etc.
In some embodiments, the sample can be preabsorbed, e.g., to reduce or minimize cross-reactivity and/or background. As nonlimiting examples, in some embodiments, the biological sample can be preabsorbed with a lysate of bacteria expressing glutathione-S-transferase (GST) and/or a lysate of normal (e.g., non-pathogen infected mammalian cells). In some embodiments, absorption of the sample can be with a lysate of pathogen-infected mammalian cells, to remove and/or block chlamydial antigen-specific antibodies from human samples, which can help confirm the specificity of human antibody binding to a test analyte.
In some embodiments, the sample collecting tool comprises a fluid collecting container. In some embodiments, the fluid collecting container comprises a tube. In some embodiments, the tube is serum tube or a plasma tube.
In some embodiments, the sample is or is recommended to be collected at a specific time or in a specific period of time. In some embodiments, the sample is or is recommended to be collected in the morning. In some embodiments, the sample is or is recommended to be collected within 1, 2, 3, 4, 5, 6, or more hours before urinating. In some embodiments, the sample is or is recommended to be collected at noon. In some embodiments, the sample is or is recommended to be collected in the evening. In some embodiments, the sample is or is recommended to be collected before the shower. In some embodiments, the sample is or is recommended to be collected before the individual having sex. In some embodiments, the sample is or is recommended to be collected after the individual having sex. In some embodiments, the sample is or is recommended to be collected within 1, 2, 3, 4, 5, 6 or more hours before or after the individual having sex. In some embodiments, the sample is or is recommended to be collected at least 4, 5, 6, 7, 8, 9, 10, 12 days after the individual ovulates.
In some embodiments, the biological sample is stable at room temperature for at least 1, 2, 4, 8, 12, 16, 20, 24 hours after obtained. In some embodiments, the sample is or is recommended to be tested within 1, 2, 3, 4, 5, 6 hours after it is obtained. In some embodiments, the sample is or is recommended to be tested shortly after it is obtained (for example, within an hour).
In some embodiments, the extraction of a target material from one or more agents associated with a health condition is accomplished in a single extraction step. In other embodiments, the extraction of a target material from one or more agents associated with a health condition is accomplished in at least one or more steps (e.g. at least one step, at least two steps, at least three steps, etc.). As a non-limiting example, the extraction can include an “A-B” buffer system or in a two- or more step reaction or reagent addition, or alternatively a single buffer system or in a one or two or more step reaction or reagent addition. The reagents can be buffers solutions or other types of reagents, including small molecule or chemical reagents, large molecule reagents (e.g., enzymes, antibodies, DNA, RNA, oligonucleotides, primers, probes, labels, tags, etc.).
In the A-B buffer system, upon collection of the sample via the sample collecting tool 105, the swab or biospecimen is extracted in buffer A, such as a universal buffer, in the extraction container. The extraction container may be, for example, a dropper tube 110. In some embodiments, the dropper tube 110 is prefilled with buffer A. In other embodiments, buffer A is provided in a separate container, and a user may pour buffer A into the dropper tube 110 before and/or during use of the test kit. In some embodiments, buffer A settles to the bottom of the dropper tube 110. The extraction container can be designed in formats other than a dropper tube/dropper cap. For example, the container could have a dropper that sits inside and is pulled out of the container and then used to deliver drops of the material. As another example, the container could be designed with a controlled pouring spot to pour out certain amounts of material, or with a plunger and stopper to eject a certain amount of material from the container, or as a simple test tube, beaker, or flask design. The description below is focused on the dropper design for illustration, but it also is applicable to alternative extraction container designs.
After the sample is placed in the dropper tube 110 with buffer A, a user may place the dropper cap 115 onto the dropper tube 110 (
In some embodiments, the extraction container or dropper tube 110 includes a stirring component, such as a ball or weight that can be shaken inside to mix. The dropper tube 110 can also include a magnetic stir bar to allow for stirring by use of a magnet.
The dropper tube 110 and dropper cap 115 can be made of various materials including plastic, glass, rubber, etc., and can be various sizes or designed to hold various volumes of liquid or amounts of solid material. The dropper tube can include measurement delineations or marks on the tube to allow the user to measure certain amounts of components to be added to the tube or to track an amount of sample added. In some cases, the kit can include more than one dropper tube 110 and dropper cap 115.
In some embodiments, the volume of the sample mixture dispensed into each of the wells of the testing container is a volume amount that is effective for testing the presence or absence of the one or more health conditions. In some embodiments, the volume of sample mixture dispensed into each well of the testing container ranges from 5 μl to 80 μl (e.g., such as 10 μl to 30 μl, 30 μl to 60 μl, 20 μl to 70 μl, 20 μl to 60 μl, 30 μl to 80 μl, 30 μl to 70 μl, and the like). In some embodiments, the volume of the sample mixture dispensed into each well is 5 μl or more, 10 μl or more, 15 μl or more, 20 μl or more, 25 μl or more, 30 μl or more, 35 μl or more, 40 μl or more, 45 μl or more, 50 μl or more, 55 μl or more, 60 μl or more, 65 μl or more, 70 μl or more, 75 μl or more, or 80 μl or more.
The wells in the testing container 120 can each contain buffers or other reagents, including small molecule or chemical reagents, large molecule reagents (e.g., aptamers, enzymes, antibodies, DNA, RNA, oligonucleotides, primers, probes, labels, tags, antibodies, molecular beacons, etc.). In some embodiments, there are different buffer solutions or reagents in each well. For example, each well may include one or more buffer solutions or other reagents specific to a particular health condition (e.g., a first health condition, a second health condition, a third health condition etc.) for which a subject or user is being tested. The subject might be tested for two or more different sexually transmitted infections (STIs), or might be tested for pregnancy in addition to one or more STIs. Similarly, the wells could be specific to different fertility tests including testing for levels of progesterone or estrogen, or there can be a combination of any of the above in a single testing container with each well being specific to a different health condition. In other embodiments, there are the same buffer solutions or reagents in each well or the same in some of the wells. In some embodiments, the extraction buffer (e.g., Buffer A) is the same as one or more buffers in one or more wells (e.g., Buffer B) of the testing container. In other embodiments, the extraction buffer (e.g., Buffer A) is different from one or more buffers (e.g. Buffer B) in the one or more wells.
In some embodiments, the buffer in the extraction container comprises one or more buffers selected from: sodium hydroxide, potassium hydroxide, sodium chloride, potassium chloride, sulfuric acid, nitric acid, hydrochloric acid, PBS (phosphate buffered saline), TBS (Tris buffered saline), tricine, and/or HBS (HEPES buffered saline), 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate BugBuster™, octylthioglucoside, Triton X-100, and octyl glucoside.
In some embodiments, the extraction container, testing container (e.g. one or more wells of the testing container), or both the extraction container and testing container comprises a buffer with a protein component such as, but not limited to: chicken albumin, 0.001%-1% casein or 0.001%-1% bovine albumin. In some embodiments, the buffer may contain a crowding agent such as 0.1%-10% 1000-100000 dextran or 3500-20000 polyethylene glycol or Polyamines or aminoacids. In some embodiments, the buffer may contain a non-ionic surfactant like Tergitol, Tween-20, CHAPS, TAPSO or detergents like OTG. In some embodiments, the buffer may contain an antibacterial agent as thimerosal, 0.01%-1% proclin-300 or sodium azide. phosphate buffered saline, Tris-45 buffered saline, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline, and an extraction substance comprising one or more of: 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate, a protein extraction reagent, octyl-50 thioglucoside, sodium hydroxide, Triton X-100, or octyl glucoside.
In some embodiments, the buffer in the extraction container, the testing container (e.g. one or more wells of the testing container), or both the extraction container and testing container can include one or more buffers selected from: 5-500 mM sodium hydroxide, 5-500 mM potassium hydroxide, 1-2000 mM sodium chloride, 1-2000 mM potassium chloride, 5-500 mM sulfuric acid or 5-500 mM nitric acid or 5-500 mM hydrochloric acid, 1-50% PBS (phosphate buffered saline), 1-50% TBS (Tris buffered saline), 1-50% tricine, 1-50% HBS (HEPES buffered saline), 00.1%-10% 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate, 0.1%-10% BugBuster™, 0.01%-10% octylthioglucoside, 0.01%-10% Triton X-100, and 0.01%-10% octyl glucoside.
In some embodiments, the extraction buffer is selected from one or more of: 5-500 mM sodium hydroxide, 5-500 mM potassium hydroxide, 1-2000 mM sodium chloride, 1-2000 mM potassium chloride, 5-500 mM sulfuric acid or 5-500 mM nitric acid or 5-500 mM hydrochloric acid, 1-50% PBS (phosphate buffered saline), 1-50% TBS (Tris buffered saline), 1-50% tricine, and/or 1-50% HBS (HEPES buffered saline). Extraction substances including, but not limited to: 00.1%-10% 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate, 0.1%-10% BugBuster™, 0.01%-10% octylthioglucoside, 0.01%-10% Triton X-100, and 0.01%-10% octyl glucoside
In some embodiments, one or more wells of the testing container includes one or more buffers selected from: 5-500 mM sodium hydroxide, 5-500 mM potassium hydroxide, 1-2000 mM sodium chloride, 1-2000 mM potassium chloride, 5-500 mM sulfuric acid or 5-500 mM nitric acid or 5-500 mM hydrochloric acid, 1-50% PBS (phosphate buffered saline), 1-50% TBS (Tris buffered saline), 1-50% tricine, 1-50% HBS (HEPES buffered saline), 00.1%-10% 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate, 0.1%-10% BugBuster™, 0.01%-10% octylthioglucoside, 0.01%-10% Triton X-100, and 0.01%-10% octyl glucoside.
In some embodiments, buffer B in a well of the testing container may comprise of a substance that is selected from one or more: 1-50% PBS (phosphate buffered saline), 1-50% TBS (Tris buffered saline), 1-50% tricine, and/or 1-50% HBS (HEPES buffered saline). Buffer B may also contain an extraction substance including, but not limited to: 00.1%-10% 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate, 0.1%-10% BugBuster™ 0.01%-10% octylthioglucoside, 0.01%-10% Triton X-100, or 0.01%-10% octyl glucoside.
In some embodiments, the buffer in the extraction container, the buffer in the testing container, or the buffer in the extraction container and the testing container comprises one or more buffers selected from: sodium hydroxide, potassium hydroxide, sodium chloride, potassium chloride, sulfuric acid, nitric acid, hydrochloric acid, PBS (phosphate buffered saline), TBS (Tris buffered saline), tricine, and/or HBS (HEPES buffered saline), 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate BugBuster™, octylthioglucoside, Triton X-100, octyl glucoside, chicken albumin, casein, bovine albumin, 1000-100000 dextran, 3500-20000 polyethylene glycol, Polyamines, amino acids, Tergitol, Tween-20, CHAPS, TAPSO, OTG, an antibacterial agent as thimerosal, 0.01%-1% proclin-300, sodium azide, phosphate buffered saline, Tris-45 buffered saline, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline, 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate, a protein extraction reagent, octyl-50 thioglucoside, and octyl glucoside.
In some embodiments, the extraction container, testing container (e.g. one or more wells of the testing container), or both the extraction container and testing container comprises a buffer with a protein component such as, but not limited to: chicken albumin, 0.001%-1% casein or 0.001%-1% bovine albumin. In some embodiments, the buffer may contain a crowding agent such as 0.1%-10% 1000-100000 dextran or 3500-20000 polyethylene glycol or Polyamines or aminoacids. In some embodiments, the buffer may contain a non-ionic surfactant like Tergitol, Tween-20, CHAPS, TAPSO or detergents like OTG. In some embodiments, the buffer may contain an antibacterial agent as thimerosal, 0.01%-1% proclin-300 or sodium azide. phosphate buffered saline, Tris-45 buffered saline, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline, and an extraction substance comprising one or more of: 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate, a protein extraction reagent, octyl-50 thioglucoside, sodium hydroxide, Triton X-100, or octyl glucoside, 1-2000 mM sodium chloride or 1-2000 mM potassium chloride, chicken albumin, 0.001%-1% casein, 0.001%-1% bovine albumin, 0.1%-10% 1000-100000 dextran or 3500-20000 polyethylene glycol, Polyamines, amino acids, a non-ionic surfactant like Tergitol, 0.1%-5% TweePn-20, 0.1%-5% CHAPS, 0.1%-5% TAPSO or detergents like OTG, antibacterial agent as thimerosal, 0.01%-1% proclin-300 or sodium azide. In some embodiments, buffer B in a well of the testing container may also contain salt components, such as 1-2000 mM sodium chloride or 1-2000 mM potassium chloride. In some embodiments, buffer B may contain a protein component such as chicken albumin, 0.001%-1% casein or 0.001%-1% bovine albumin. In some embodiments, buffer B may contain a crowding agent such as 0.1%-10% 1000-100000 dextran or 3500-20000 polyethylene glycol or Polyamines or amino acids. In some embodiments, buffer B may contain a non-ionic surfactant like Tergitol, 0.1%-5% TweePn-20, 0.1%-5% CHAPS, 0.1%-5% TAPSO or detergents like OTG. In some embodiments, buffer B may contain an antibacterial agent as thimerosal, 0.01%-1% proclin-300 or sodium azide.
In some embodiments, Buffer A, Buffer B, or Buffer A and Buffer B in a well of the testing container may also contain salt components, such as 1-2000 mM sodium chloride or 1-2000 mM potassium chloride. In some embodiments, Buffer A, Buffer B, or Buffer A and Buffer B may contain a protein component such as chicken albumin, 0.001%-1% casein or 0.001%-1% bovine albumin. In some embodiments, Buffer A, Buffer B, or Buffer A and Buffer B may contain a crowding agent such as 0.1%-10% 1000-100000 dextran or 3500-20000 polyethylene glycol or Polyamines or amino acids. In some embodiments, Buffer A, Buffer B, or Buffer A and Buffer B may contain a non-ionic surfactant like Tergitol, 0.1%-5% TweePn-20, 0.1%-5% CHAPS, 0.1%-5% TAPSO or detergents like OTG. In some embodiments, Buffer A, Buffer B, or Buffer A and Buffer B may contain an antibacterial agent as thimerosal, 0.01%-1% proclin-300 or sodium azide.
The volume of buffer in each well can range from 50 μl to about 150 μl (e.g., 50 μl to 70 μl, 60 μl 100 μl, 70 μl to 100 μl, 70 μl to 120 μl, 80 to 120 μl, 90 μl to 120 μl, 100 μl to 110 μl, 110 μl to 120 μl). In some embodiments, the volume of buffer in each well is 70 μl or more, 75 μl or more, 80 μl or more, 85 μl or more, 90 μl or more, 95 μl or more, 100 μl or more, 105 μl or more, 110 μl or more, 115 μl or more, or 120 μl or more.
The testing container 120 is designed to scale based on the number of test strips that need to be evaluated and can be personalized for the assay to be as sensitive and specific without compromising or interfering with the other test strips. For example, if 3 health conditions are to be tested for, then the testing container can include 3 wells for each of the health conditions tested for, and the signal output device can include 3 test strips for placement into each respective well. The size configuration of the wells is designed to house the specific volume of buffer in the wells. Additionally, in some embodiments, the diameter and height of the well is also designed to allow each test strip to be lowered into each well such that the target sample mixture (sample mixture containing the target material mixed with buffer within the well) is in fluid communication with an end of the strip. There may also be more than one testing container 120 in a kit, each one containing different sets of reagents to run a particular set of tests.
The testing container may be constructed of various different materials, including plastic, rubber, glass, metal, etc. It may be disposable or it can be washed and reused in some designs. The testing container shown in the figure includes three wells, but it can alternatively include one or two wells, or it can include numbers above three (four, five, six, seven, eight, nine, ten, fifteen, twenty, or more). In some examples, it can be a sample plate or micro-well plate with numerous wells.
The testing container is designed to have a base portion 180 (
The mixing step if needed, may be further fulfilled by the stirrer 125 in the test kit (
The signal output device 130 includes a housing that contains the test strips. In one embodiment, the housing completely or partially surrounds the test strips. As shown in
In the embodiment shown in the figures, for example,
The housing can include one or more windows or apertures 210 for viewing the test strips and reading the results of the test. There can be a single larger window or multiple windows, one for each strip. In some cases, the test is a digital test that reads the test strips and presents the results for each test on one or more digital screens.
The side view shown in
To secure the strips in place and hold them in a stable position (avoid lateral motion and avoid slipping up or down inside the device), there are multiple pegs (or wedges or pressure points) on the front and back internal surfaces of the shell or housing. On the front 280 of the housing, there is an upper peg 224, a middle peg 235, and a lower peg 246 shown in the figure that all assist in holding the test strip in place. There is also a rounded peg 246 and a lower elongated rib 248 that has a groove in the middle, and these further help to hold the strips in place. In addition, the viewing windows have a lip 245 around the edge of the window that further rest on each strip to secure the strip in place. On the back 290 of the housing, there is a peg 255 and an elongated rib 265 that additional help to secure the strips in place.
Peg 225 holds the strip against the curvature of the back internal surface of the housing so that the upper portion of the strip is secured. The back surface of the housing curves inward at the top of the device and curves outward at the bottom of the device, such that the upper portion of the device has a smaller cross section from front to back than the lower portion of the device. The device tapers to a lower cross section from bottom to top of the device. Peg 225 sits on the back surface of the device opposite the uppermost lip 245 of the viewing window on the front surface, and these two parts 225 and 245 hold the strip in between securely in place. The elongated rib 265 on the back surface sits opposite the lower elongated rib 248 on the front surface to hold the strips in between at the lower end of the device. Thus, the lower portion of the strip is also secured. The pegs 235, 246, and rounded peg 247 rest on or near the strip to also help secure the strip in place from the front surface without a matching peg opposite them on the back surface.
In some embodiments, each test strip 135 includes one or more of a loading zone 8, a reaction zone 7, a testing zone 6, and/or a control zone 5. Test strips that can be used in the signal output device of the present system and methods can be found in U.S. Pat. No. 10,794,911, which is hereby incorporated by reference in its entirety.
In some embodiments, the loading zone 8, the reaction zone 7, the testing zone 6, and the control zone 5 (
In some embodiments, the sampling kit includes a process mode where, in the process mode, the sample output device 130 is placed over the testing container, and, once securely locked into or lowered into the testing container, the loading zone for each test strip can be in fluid communication with the buffers and/or reagents and sample mixture in each respective well of the testing container, and a sample generated upon extraction of an agent by the extraction buffer (e.g. sample mixture) flows against gravity through the loading zone, the reaction zone, the testing zone, and the control zone. In some embodiments, the retained orientation of the device (testing container coupled to the signal output device) thus positions the testing container vertically, such that the sample flows against gravity. In other embodiments, the retained orientation can be a non-vertical orientation (e.g. horizontal).
Test strips of Signal Output Device
The signal output device of the present disclosure includes at least a first strip and a second strip.
In some embodiments, the signal output device comprises at least one strip. In some embodiments, the signal output device comprises at least two strips, at least three strips, at least four strips, at least five strips, at least six strips, at least seven strips, at least eight strips, at least nine strips, or at least ten strips.
In some embodiments, each of the strips are placed in a fitted wedge of the housing of the signal output device that holds the strips in place. The configuration of the housing allows for each individual test strip to be in separate chambers, and not fluidically connected. In some embodiments, there are pressure points in the signal output device at multiple locations that keep the strip components in place. Additionally, the pressure points on the strips help hold and maintain flow of buffers throughout the strip until the strip is saturated.
The loading zone is located 8 at a distal end of the test strip 135 and is insertable into the testing container 120. When received by the loading zone, the target material flows to the reaction zone 7. In some embodiments, the loading zone can include or be composed of cellulose and/or glass fiber. In some embodiments, the loading zone 8 is capable of transporting the sample to other parts of the test strip 135 (e.g., by way of fluid coupling).
The strip of the signal output device and the wells of the testing container are designed such that a known volume of the target sample solution will be loaded or absorbed onto the loading zone of the strip that will facilitate detection of the presence or absence of a health condition. For example, as described in the present disclosure, each of the wells of the testing container can include a volume of the target sample solution ranging from 70 μl to 120 μl. The loading zone may be configured to receive a target sample solution of varying volumes, where in some instances the sample zone is configured to receive a sample having a volume ranging from 0.1 μl to 120 μl, such as 5 μl to 20 μl, such as 1 μl to 3 μl, such as 1 μl to 5 μl, such as 1 μl to 10 μl, such as 10 μl to 20 μl, such as 20 μl to 1200 μl, such as 30 μl to 1200 μl, and the like. In some embodiments, the sample zone is configured to receive a sample having a volume of at least 0.1 μl, at least 0.5 μl, at least 1 μl, at least 5 μl, at least 10 μl, at least 15 μl, at least 20 μl, at least 25 μl, at least 30 μl, at least 35 μl, at least 40 μl, at least 45 μl, at least 50 μl, at least 55 μl, at least 60 μl, at least 65 μl, at least 70 μl, at least 75 μl, at least 80 μl, at least 85 μl, at least 90 μl, at least 95 μl, at least 100 μl, at least 105 μl, at least 110 μl, at least 115 μl, or at least 120 μl.
In some embodiments, the transportation of the sample through different zones from the loading zone is in a continuous and/or homogenous manner. In some embodiments, the loading zone includes materials or reagents that pretreat the sample before its transportation. In some embodiments, the loading zone includes pretreating materials/reagents configured for one or more of: separation of sample components, removal of interfering materials, and/or adjustment of pH.
In relation to the extraction container 110 and the extraction buffer (e.g. “Buffer A”), the extraction buffer, upon contacting a sample from the subject, is configured to extract target materials associated with the health condition(s) of interest. Target materials can be associated with different regions of the same or different agents (e.g., infectious agents).
In some embodiments, the signal output device comprising at least a first strip and a second strip can include substrate materials and/or structures that provide for lateral flow of a sample from a loading zone to a testing zone. In some instances, the strip includes a bibulous material or member that readily absorbs liquid and provides for liquid flow through the member. Examples of bibulous materials include: organic or inorganic polymers and natural and synthetic polymers. More specific examples of suitable solid supports include, without limitation, glass fiber, cellulose, nylon, cross-linked dextran, various chromatographic papers and nitrocellulose. In some embodiments, the bibulous member includes a membrane, and in a specific example, the membrane is a nitrocellulose membrane. In some embodiments, the membrane is located in testing zone and/or control line zone, described in more detail below.
While the bibulous member and overall configuration of a lateral flow assay device implemented in embodiments of the system may vary, in certain embodiments the bibulous member can have a strip configuration, some embodiments of which are described in U.S. Pat. No. 10,794,911, which is hereby incorporated by reference in its entirety. Where the bibulous material is configured as a strip, the bibulous member can have a length that is longer than its width. In some examples, the length is longer than the width by 1.5 fold or more, such as 2-fold or more, e.g., 10 fold or more, including 20-fold or more. In some examples, the length of the bibulous member ranges from 0.5 to 20 cm, such as 1.0 to 15 cm, e.g., 2.0 to 10 cm, while the width ranges 0.1 to 10.0 cm, such as 0.5 to 2.5 cm, e.g., 1 to 2 cm. The thickness of the bibulous member may also vary, ranging in some instances from 0.01 to 0.05 cm, such as 0.1 to 0.4 cm, e.g., 0.1 to 0.25 cm.
Optionally, the signal output device can include an absorbent pad downstream from the reaction zone and any control region, e.g., at the end distal from the sample loading zone, where the absorbent pad is configured to absorb fluid and reagents present therein that have flowed through the bibulous member. While the configuration of the absorbent pad may vary, in some instances it is configured to absorb a volume of liquid that is substantially the same as the volume of sample that is applied to the sample loading zone during use.
In some embodiments, the signal output device can include a sample pad. In some embodiments, the signal output device can include a backing card. In some embodiments, the signal output device can include a membrane. In some embodiments, at least one strip of the signal output device can include one or more of: an absorbent pad, a conjugate release pad, a wicking pad, a test line, a control line, a sample pad, and a backing card. In some embodiments, the signal output device can include a sample pad. In some embodiments, the signal output device can include a backing card. In some embodiments, at least one strip of the signal output device can include one or more of: an absorbent pad, a conjugate release pad, a wicking pad, a test line, a control line, a membrane, a sample pad, and a backing card.
As such, embodiments of the loading zone can include a terminal zone (e.g., a most upstream zone) of the bibulous member, e.g., positioned closer to one end of the bibulous member. Alternatively, embodiments of the loading zone may be distinct from the bibulous member, but configured to provide for fluid communication of sample into the bibulous member upon application of sample to the sample loading zone. The loading zone may be configured to receive samples of varying volumes, where in some instances the sample zone is configured to receive a sample having a volume ranging from 0.1 ul to 20 ml such as 5 ul to 20 ml.
In some instances, the loading zone may include a metering device configured to meter a specific amount of sample into the bibulous member.
In some embodiments, the reaction zone is fluidly coupled to the loading zone and includes one or more reaction substances, such as one or more reaction substances listed in TABLE 1, TABLE 2, and/or TABLE 3. The one or more reaction substances may be conjugated to labels that are configured to enable detection of a target material associated with the health condition.
When received into the loading zone 8 of the signal output device 130, the target material flows to the reaction zone 7, where the reaction zone 7 includes a first reaction substance conjugated to a first label, wherein the first reaction substance preferentially couples to a first target of target material. The reaction zone can optionally include a second reaction substance conjugated to a second label, where the second reaction substance preferentially couples to a second target of a target material. In an embodiment, the first reaction substance and the second reaction substance are not immobilized within the reaction zone. In some embodiments, the reaction zone can include more than two reaction substances conjugated to respective labels, in relation to assays performed on different types of target material (e.g., related to different health conditions). In some embodiments, the reaction zone only includes one reaction substance conjugated to a label, wherein the reaction substance couples to a target of a target material.
Embodiments of the reaction zone can be positioned at some distance downstream from the loading zone. The distance between the loading zone and the reaction zone may vary. In some embodiments, the distance ranges from 0.1 to 10 cm, such as 0.1 to 3 cm and including 0.5 to 2 cm. In some embodiments, the reaction zone overlaps with the loading zone in a portion or full. In some embodiments, the reaction zone overlaps with the loading zone in about 25%, 50%, 75%, and 100%.
As described above, in some embodiments, the reaction substances implemented for detection are not immobilized at the reaction zone(s). In relation to immobilization, a substance and the bibulous member maintain their position relative to each other in space under the conditions of use, e.g., under the assay conditions. As such, a not immobilized reaction substance is not stably associated with the bibulous member and can migrate under the capillary pressure or other drivers of sample flow. In some embodiments, examples of which are described above, a reaction substance binds to a specific region of a target material of a pathogen or other agent.
In some embodiments (some of which are described above), the reaction zone includes two or more reaction substances that are conjugated to the same or different labels. In some embodiments, the two or more reaction substances are not immobilized. In some embodiments, the two or more substances each bind to the same specific region of the same target material of an agent or biomarker. In some embodiments, the two or more substances each bind to two or more different specific regions of the same target material of an agent or biomarker. In some embodiments, the two or more substances each bind to a specific region of two or more different target materials of an agent or biomarker. In some embodiments, the two or more substances each bind to a specific region of target materials of two or more different agents or biomarkers.
The reaction substances in the reaction zone that bind to specific agent/biomarker regions of target material of interest can include one or more of: a protein, a peptide or its analogs (e.g., an antibody, antigen, peptoid, D-peptide, beta-peptide), or a nucleic acid (e.g., an aptamer) or its analogs.
In some embodiments, the reaction substances each bind to one or more specific regions of target material of different pathogens or biomarkers (e.g., of sexual health). In examples, the reaction zone can include non-immobilized aptamers and/or antibodies listed in TABLE 1, TABLE 2, and/or TABLE 3, such that a sample that has undergone extraction can flow through the reaction zone and individual units of target material associated with each health condition can bind with their respective reaction substances before flowing to the testing zone.
In some embodiments, the reaction substance that binds to a specific region of an agent is an aptamer or aptamers. In some embodiments, the aptamer is generated by an in vitro process known as SELEX (systematic evolution of ligands by exponential enrichment). In some embodiments, the aptamer is an organic molecule.
In some embodiments, the aptamer has a molecular weight of about 50 to 100 Da, 50 to 200 Da, 50 to 500 Da, 50 to 1000 Da, 50 to 2,000 Da, 50 to 3,000 Da, 50 to 4,000 Da, 50 to 5,000 Da, 50 to 6,000 Da, 50 to 7,000 Da, 50 to 8,000 Da, 50 to 9,000 Da, 50 to 10,000 Da, 50 to 11,000 Da, 50 to 12,500 Da, 50 to 15,000 Da, 100 to 200 Da, 100 to 500 Da, 100 to 1000 Da, 100 to 2,000 Da, 100 to 3,000 Da, 100 to 4,000 Da, 100 to 5,000 Da, 100 to 6,000 Da, 100 to 7,000 Da, 100 to 8,000 Da, 100 to 9,000 Da, 100 to 10,000 Da, 100 to 11,000 Da, 100 to 12,500 Da, 100 to 15,000 Da, 200 Da to 500 Da, 200 to 1000 Da, 200 to 2,000 Da, 200 to 3,000 Da, 200 to 4,000 Da, 200 to 5,000 Da, 200 to 6,000 Da, 200 to 7,000 Da, 200 to 8,000 Da, 200 to 9,000 Da, 200 to 10,000 Da, 200 to 11,000 Da, 200 to 12,500 Da, 200 to 15,000 Da, 500 to 1000 Da, 500 to 2,000 Da, 500 to 3,000 Da, 500 to 4,000 Da, 500 to 5,000 Da, 500 to 6,000 Da, 500 to 7,000 Da, 500 to 8,000 Da, 500 to 9,000 Da, 500 to 10,000 Da, 500 to 11,000 Da, 500 to 12,500 Da, 500 to 15,000 Da, 1,000 to 2,000 Da, 1,000 to 3,000 Da, 1,000 to 4,000 Da, 1,000 to 5,000 Da, 1,000 to 6,000 Da, 1,000 to 7,000 Da, 1,000 to 8,000 Da, 1,000 to 9,000 Da, 1,000 to 10,000 Da, 1,000 to 11,000 Da, 1,000 to 12,500 Da, 1,000 to 15,000 Da, 2,000 to 3,000 Da, 2,000 to 4,000 Da, 2,000 to 5,000 Da, 2,000 to 6,000 Da, 2,000 to 7,000 Da, 2,000 to 8,000 Da, 2,000 to 9,000 Da, 2,000 to 10,000 Da, 2,000 to 11,000 Da, 2,000 to 12,500 Da, 2,000 to 15,000 Da, 3,000 Da to 4,000 Da, 3,000 to 5,000 Da, 3,000 to 6,000 Da, 3,000 to 7,000 Da, 3,000 to 8,000 Da, 3,000 to 9,000 Da, 3,000 to 10,000 Da, 3,000 to 11,000 Da, 3,000 to 12,500 Da, 3,000 to 15,000 Da, 4,000 to 5,000 Da, 4,000 to 6,000 Da, 4,000 to 7,000 Da, 4,000 to 8,000 Da, 4,000 to 9,000 Da, 4,000 to 10,000 Da, 4,000 to 11,000 Da, 4,000 to 12,500 Da, 4,000 to 15,000 Da, 5,000 to 6,000 Da, 5,000 to 7,000 Da, 5,000 to 8,000 Da, 5,000 to 9,000 Da, 5,000 to 10,000 Da, 5,000 to 11,000 Da, 5,000 to 12,500 Da, 5,000 to 15,000 Da, 6,000 to 7,000 Da, 6,000 to 8,000 Da, 6,000 to 9,000 Da, 6,000 to 10,000 Da, 6,000 to 11,000 Da, 6,000 to 12,500 Da, 6,000 to 15,000 Da, 7,000 to 8,000 Da, 7,000 to 9,000 Da, 7,000 to 10,000 Da, 7,000 to 11,000 Da, 7,000 to 12,500 Da, 7,000 to 15,000 Da, 8,000 to 9,000 Da, 8,000 to 10,000 Da, 8,000 to 11,000 Da, 8,000 to 12,500 Da, 8,000 to 15,000 Da, 9,000 to 10,000 Da, 9,000 to 11,000 Da, 9,000 to 12,500 Da, 9,000 to 15,000 Da, 10,000 to 11,000 Da, 10,000 to 12,500 Da, 10,000 to 15,000 Da, or 12,000 to 15,000 Da, each inclusive. In some embodiments, the aptamer has a molecular weight of about 100 to 10,000 Da.
In some embodiments (some of which are described above), the reaction zone includes reaction substances including aptamers listed in TABLES 1, 2, and 3.
In some embodiments, the reaction substance that binds to a specific region of an agent includes a molecular beacon or molecular beacons. Molecular beacons are a specific DNA hairpin structure with fluorophore at one end and quencher at the other end. Fluorophore cannot produce fluorescence in the absence of an analyte (e.g., a target substance of a pathogen) because of closely located quencher. When complementary DNA sequence (e.g., a target substance of a pathogen) is present as a target analyte, stem and loop portions of the beacons are opened as a result of a force and fluorescence signal is observed. In some embodiments, the molecular beacon binds to a target substance of a pathogen, wherein the target substance comprises a nucleic acid, a toxin, and/or a protein or peptide. In some embodiments, the molecular beacon comprises a loop region and/or a double stranded stem region. In some embodiments, the loop region is complementary to a target substance (e.g., a DNA, an mRNA, a toxin or a protein of a pathogen).
In some embodiments, the molecular beacon has about 10 to 15, 10 to 20, 10 to 25, 10 to 30, 10 to 35, 10 to 40, 10 to 45, 10 to 50, 15 to 20, 15 to 25, 15 to 30, 15 to 35, 15 to 40, 15 to 45, 15 to 50, 20 to 25, 20 to 30, 20 to 35, 20 to 40, 20 to 45, 20 to 50, 25 to 30, 25 to 35, 25 to 40, 25 to 45, 25 to 50, 30 to 35, 30 to 40, 30 to 45, 30 to 50, 35 to 40, 35 to 45, 35 to 50, 40 to 45, 40 to 50 or 45 to 50 base pairs in the loop region, each inclusive, wherein the loop is complimentary to a target substance. In some embodiments, the molecular beacon has about 15 to 30 base pairs in the loop, wherein the loop is complimentary to a target substance.
In some embodiments, the molecular beacon has about 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 4 to 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 6 to 7, 6 to 8, 6 to 9, 7 to 8, 7 to 9 or 8 to 9 base pairs at the double stranded stem region.
In some embodiments, the reaction substance that binds to a specific region of a pathogen includes a DNA probe.
In some embodiments, the reaction substances include antibodies. In some embodiments, an antibody used is a monoclonal antibody. In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is a bispecific antibody that binds to two separate regions of an agent, or two separate regions of two different agents. In some embodiments, the substance is a fragment or a variant of an antibody (e.g., Fab fragment or single chain variable fragment).
In some embodiments, the reaction substances include monoclonal antibodies that bind to a specific region of target substance on an agent (e.g., Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Treponema pallidum, Gardnerella vaginitis, Mycoplasma genitalium, estrogen, progesterone, testosterone, anti-mullerian hormone, follicle-stimulating hormone, luteinizing hormone, estradiol, thyroid-stimulating hormone, free thyroxine, prolactin, Candida Albicans (yeast infection), Human immunodeficiency virus (HIV), Human papillomavirus (HPV), Hepatitis C virus (HCV), Hepatitis B virus (HBV), Herpes simplex virus (HSV)). Such monoclonal antibodies can be generated using a hybridoma technique. For example, monoclonal antibodies can be produced from a single B-lymphocyte clone involving immunizing a certain species (e.g., a mouse, rat, rabbit, or goat) against the specific region on a target substance and obtaining the B-lymphocytes from the spleen of the species. The B-lymphocytes are then fused (by chemical- or virus-induced methods) with an immortal myeloma cell line lacking the hypoxanthine-guanine-phosphoribosyltransferase (HGPRT) gene and not containing any other immunoglobulin-producing cell. These hybridoma cells are then cultured in vitro in selective medium (i.e. medium containing hypoxanthine-aminopterinthymidine) where only the hybridomas (i.e. the fusion between the primary B-lymphocytes and myeloma cells) survive as they have inherited immortality from the myeloma cells and selective resistance from the primary B-lymphocytes (as the myeloma cells lack HGPRT, they cannot synthesize nucleotides de novo as this is inhibited by aminopterin in the selective medium). The initial culture of hybridomas contains a mixture of antibodies derived from many different primary B-lymphocyte clones, each secreting its own individual specific antibody into the culture medium (i.e. the antibodies are still polyclonal). Each individual clone can be separated by dilution into different culture wells. The cell culture medium can then be screened from many hundreds of different wells for the specific antibody activity required and the desired B-lymphocytes grown from the positive wells and then recloned and retested for activity. The positive hybridomas and monoclonal antibodies generated can then be stored away in liquid nitrogen.
Monoclonal antibodies can also be generated using phage display. This involves isolating B-lymphocytes from the blood of humans and then isolating the mRNA and converting it into cDNA using PCR to amplify all the VH and VL segments. These segments can then be cloned into a vector (usually as scFv) next to the PIII protein of a bacteriophage. This vector is then introduced into E. coli cells in order to generate a library containing approximately 1010 clones of antibody fragments. E. coli can then secrete the bacteriophage containing the VH and VL segments as part of the bacteriophage coat. Specific VH and VL segments against the target substance can then be selected and used to re-infect E. coli with the bacteriophage. Cells containing the plasmid can then be isolated and sequenced. Its advantages include: once the library is made, the same library can be used to generate new antibodies and does not have to be remade, no immunizations are required as the entire process is done in vitro, antibodies can be obtained much more quickly than the traditional hybridoma technique and the library can be used to generate antibodies to toxic target substances that could not be used to immunize an animal.
In some embodiments, monoclonal antibodies can also be improved in multiple aspects. For example, binding affinity to the target substance can be improved by using phage display libraries to isolate antibodies with strong affinities for the target substance.
In some embodiments, monoclonal antibodies are recovered and/or purified with a process comprising one or more of the following steps: 1) harvest antibodies with centrifugation/filtration thereby removing cells and cell debris; 2) protein A and/or protein G chromatograph which yields highly purified product in a single step; 3) low pH hold to inactivate endogenous/adventitious viruses; 4) additional chromatography steps to further remove impurities and viruses; 5) filtration to further remove endogenous/adventitious viruses; and 6) ultrafiltration/diafiltration.
In some embodiments (some of which are described above), the reaction zone includes reaction substances including antibodies listed in TABLES 1, 2, and 3.
In some embodiments (some of which are described above), the reaction zone further includes a substance that binds to a biomarker of pregnancy. In some embodiments, the biomarker(s) include one or more of: human chorionic gonadotropin (hCG), activin A, pregnancy-associated plasma protein-A (PAPP-A), human placental lactogen (hPL), A disintegrin and Metalloprotease-12 (ADAM-12), pregnancy-specific beta glycoprotein 1 (SP-1), placental mRNAs, progestrerone, Inhibin A, Vascular Endothelial Growth Factor (VEGF), Placental-like growth factor (PlGF), Leukemic Inhibitory Factor, Glycodelin, Mucin-1, Adrenomedullin, and other biomarkers.
In some embodiments (some of which are described above), the reaction zone further includes a substance that binds to a biomarker of fertility. In some embodiments, the biomarker(s) include one or more of: oestrone-3-glucuronide (E3G0, luteinizing hormone, follicle stimulating hormone (FSH), estrogen, progesterone, testosterone, anti-mullerian hormone, follicle-stimulating hormone, luteinizing hormone, estradiol, thyroid-stimulating hormone, free thyroxine, prolactin, dehydroepiandrosterone (DHEA), cortisol, sex hormone binding globulin (SHBG), triiodothyronine (T3), Thyroxine (T4), thyroid stimulating hormone (TSH), thyroid peroxidase antibodies (TPO antibodies), and other biomarkers.
In some embodiments, the reaction substances and/or testing substances configured for detection of Chlamydia trachomatis include individual or a cocktail of deoxyribonucleic acid (DNA)-based aptamers or any permutation of the sequences listed in TABLE 1 and/or individual or a cocktail of antibody clones listed in TABLE 1.
Neisseria gonorrhoeae
In some embodiments, the reaction substances and/or testing substances configured for detection of Neisseria gonorrhoeae include individual or a cocktail of deoxyribonucleic acid (DNA)-based aptamers or any permutation of the sequences listed in TABLE 2 and/or individual or a cocktail of antibody clones listed in TABLE 2.
In related embodiments, aptamers (e.g., aptamers listed in TABLE 2) for detection of Neisseria Gonorrhoeae are modified by a biotin or —SH group or any other modification at the 5′ and/or 3′ terminal of the aptamer.
In one embodiment, the system is configured to detect presence of Neisseria gonorrhoeae from a sample acquired from the subject, such that the target material includes one or more specific regions of biological material (e.g., tissue content, cellular content, protein content, amino acid content, nucleic acid content, etc.) of Neisseria gonorrhoeae, the reaction substances are configured to bind to specific regions of Neisseria gonorrhoeae, and/or the testing substances are configured to bind to specific regions of Neisseria gonorrhoeae.
In specific examples of this embodiment, the target material of Neisseria gonorrhoeae includes individual proteins (and homologs) or a cocktails of proteins (and homologs) including one or more of: Uniprot ID numbers: P95359 (SEQ ID NO 23), A0A1D3HF49 (SEQ ID NO 24), P05430 (SEQ ID NO 25), Q02219 (SEQ ID NO 26), Q51006 (SEQ ID NO 27), Q5F942 (SEQ ID NO 28), B4RQH9 (SEQ ID NO 29), Q5F6W5 (SEQ ID NO 30), P29842 (SEQ ID NO 31), Q5F542 (SEQ ID NO 32), B4RLT9 (SEQ ID NO 33), D6H5Z3 (SEQ ID NO 34), and Q5F651 (SEQ ID NO 35); and GenBank/NCBI Accession Numbers: YP_208979.1 (SEQ ID NO 75), KX124787.1 (SEQ ID NO 76), SCW17313.1 (SEQ ID NO 77), YP_209073.1 (SEQ ID NO 78), and YP_209148.1 (SEQ ID NO 79). In some embodiments, the target material of Neisseria gonorrhoeae includes lipoglycans or lipopolysaccharides of the Neisseria gonorrhoeae species.
In some embodiments, the reaction substances and/or testing substances configured for detection of Trichomonas vaginalis include individual or a cocktail of deoxyribonucleic acid (DNA)-based aptamers or any permutation of the sequences listed in TABLE 3 and/or individual or a cocktail of antibody clones listed in TABLE 3.
Trichomonas vaginalis
In related embodiments, aptamers (e.g., aptamers listed in TABLE 3) for detection of Trichomonas vaginalis are modified by a biotin or —SH group or any other modification at the 5′ and/or 3′ terminal of the aptamer.
In one embodiment, the system is configured to detect presence of Trichomonas vaginalis from a sample acquired from the subject, such that the target material includes one or more specific regions of biological material (e.g., tissue content, cellular content, protein content, amino acid content, nucleic acid content, etc.) of Trichomonas vaginalis, the reaction substances are configured to bind to specific regions of Trichomonas vaginalis, and/or the testing substances are configured to bind to specific regions of Trichomonas vaginalis.
In specific examples of this embodiment, the target material of Trichomonas vaginalis includes individual proteins (and homologs) or a cocktails of proteins (and homologs) including one or more of: NCBIGenBank Accession numbers: EAX87747.1 (SEQ ID NO 41), EAY21310.1 (SEQ ID NO 42), EAX96596.1 (SEQ ID NO 43), EAY19137.1 (SEQ ID NO 44), EAY01676.1 (SEQ ID NO 45), EAX86868.1 (SEQ ID NO 46), EAX98121.1 (SEQ ID NO 47), EAY18961.1 (SEQ ID NO 48), AAA91133.1 (SEQ ID NO 49), AAC48339.1 (SEQ ID NO 50), and AAC72899.1 (SEQ ID NO 51). In some embodiments, the target material of Trichomonas vaginalis includes lipoglycans or lipopolysaccharides of the Trichomonas vaginalis species.
In some embodiments, the labels used (e.g., labels to which reaction substances are conjugated) include one or more of: gold nanoparticles, colored latex beads, magnetic particles, carbon nanoparticles, cellulose nano beads, selenium nanoparticles, silver nanoparticles, quantum dots, up converting phosphors, organic fluorophores, textile dyes, enzymes, liposomes and labels.
In some embodiments, the conjugation of the substance that binds to a specific region of a pathogen and the label is stable for at least about 1, 3, 5, 7, 10, 12, or 14 or more days. In some embodiments, the conjugation is stable for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks. In some embodiments, the conjugation is stable for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months. In some embodiments, the conjugation is stable for at least about 1, 2, 3, 4, 5, or more months. The conjugation is stable when at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5% of the conjugates are functional (e.g., labeling the true positive and/or not labeling the false negative) and/or at most 30%, 25%, 20%, 15%, 10%, 5%, 2.5%, 1% or 0.5% of the conjugates are not functional (e.g., labeling the false positive and/or not labeling the true positive).
In some embodiments, the concentration of the label is at least about 10−12, 10−11, 10−10, 10−9, 10−8, 10−7, or 10−6 M. In some embodiments, the concentration of the label is at most about 10−11, 10−10, 10−9, 10−8, 10−7, 10−6, 10−5 or 10−4 M. In some embodiments, the concentration of the label is about 10−12, 10−11, 1010, 10−9, 10−1, 10−7, or 10−6 M.
In some embodiments, the label is capable of generate a direct signal after encountering the analyte (e.g., the specific region that the conjugated substance binds to). In some embodiments, the label generates a signal after an additional step.
In some embodiments, the device comprises more than one label. In some embodiments, the more than one label can be composed of same or different material(s). In some embodiments, the more than one label can generate same or different signals.
In some embodiments, the labels used (e.g., labels to which reaction substances are conjugated) include Cellulose NanoBeads. Cellulose NanoBeads (e.g., NanoAct™) are inert and spherical, which have high affinity to biomolecules and can be functionalized. Cellulose nanobeads are highly stable, deeply colored particles that have demonstrated appropriate performance.
In some embodiments, the labels used (e.g., labels to which reaction substances are conjugated) include latex beads. Latex beads are inert and spherical, which have high affinity to biomolecules and can be functionalized.
In some embodiments, the labels used (e.g., labels to which reaction substances are conjugated) include gold nanoparticles. In some embodiments, the gold nanoparticles include colloidal gold. Colloidal gold is inert and spherical, which have high affinity to biomolecules and can be functionalized. In some embodiments, the average diameter of the gold nanoparticles is about 5 to 150 nm. In some embodiments, the average diameter of the gold nanoparticles is no greater than 150 nm or 200 nm. In some embodiments, the average diameter of the gold nanoparticles is about 40 nm. In some embodiments, the average diameter of the gold nanoparticles is about 30 nm. In some embodiments, the average diameter of the gold nanoparticles is about 60 nm. In some embodiments, the average diameter of the gold nanoparticles is about 5 to 25, 5 to 50, 5 to 75, 5 to 100, 5 to 125, 5 to 150, 5 to 175, 5 to 200, 25 to 50, 25 to 75, 25 to 100, 25 to 125, 25 to 150, 25 to 175, 25 to 200, 50 to 75, 50 to 100, 50 to 125, 50 to 150, 50 to 175, 50 to 200, 75 to 100, 75 to 125, 75 to 150, 75 to 175, 75 to 200, 100 to 125, 100 to 150, 100 to 175, 100 to 200, 125 to 150, 125 to 175, 125 to 200, 150 to 175, 150 to 200, or 175 to 200 nm, each inclusive.
In some embodiments, the labels used (e.g., labels to which reaction substances are conjugated) include Europium ions. In some embodiments, Europium ion is chelated by isothiocyanate. Isothiocyanate can be functionalized and has high affinity to biomolecules. Europium ions are highly fluorescent and have demonstrated appropriate performance over standard labels in lateral flow applications.
In some embodiments, the labels used (e.g., labels to which reaction substances are conjugated) include a magnetic particle or aggregate. In some embodiments, the magnetic particle or aggregate can produce a signal, wherein the signal can be read by an optical strip reader or magnetic assay reader. In some embodiments, the magnetic particle or aggregate comprises one or more iron oxide particle. In some embodiments, the one or more iron particles comprise Fe3O4 particles. In some embodiments, the one or more iron oxide particles are modified with polyethylene glycol. In some embodiments, the one or more iron oxide particles are crosslinked with poly-L-lysine.
Fluorescent and/or Luminescent Materials
In some embodiments, the labels used (e.g., labels to which reaction substances are conjugated) include a fluorescent or luminescent material. In some embodiments, the label includes an organic fluorophore (e.g., rhodamine). In some embodiments, the label includes a fluorescent microsphere. In some embodiments, the label includes a nanomaterial. In some embodiments, the nanomaterial includes quantum dots. In some embodiments, the quantum dots are encapsulated into a nanobead, thereby improving the detection sensitivity.
In some embodiments, the labels used include at least two or more different quantum dots, wherein the different quantum dots generate different colors.
In some embodiments, the labels used include upconverting phosphors (UCP). In some embodiments, the UCP are characterized with their excitation in infra-red region and emission in high energy visible region. In some embodiments, the UCP are characterized with the absence of auto fluorescence or the absence a significant level of auto fluorescence. In some embodiments, the average diameter of UCP is about 10 nm to 1 um. In some embodiments, the average diameter of UCP is about 10 to 50, 10 to 100, 10 to 200, 10 to 300, 10 to 400, 10 to 500, 10 to 750, 50 to 100, 50 to 200, 50 to 300, 50 to 400, 50 to 500, 50 to 750, 50 to 1,000, 100 to 200, 100 to 300, 100 to 400, 100 to 500, 100 to 750, 100 to 1,000, 200 to 300, 200 to 400, 200 to 500, 200 to 750, 200 to 1,000, 300 to 400, 300 to 500, 300 to 750, 300 to 1,000, 400 to 500, 400 to 750, 400 to 1, 000, 500 to 750, 500 to 1,000, or 750 to 1,000 nm, each inclusive. In some embodiments, the average diameter of UCP is about 40 to 400 nm. In some embodiments, the average diameter of UCP is about 40 nm.
In some embodiments, the labels used include fluorescent europium nanoparticles. In some embodiments, the fluorescent europium nanoparticles comprise europium III nanoparticles. In some embodiments, the average diameter of europium nanoparticles is about 100 to 1,000 nm. In some embodiments, the average diameter of europium nanoparticles is about 400 to 600 nm. In some embodiments, the average diameter of europium nanoparticles is about 500 nm (e.g., 520 nm).
In some embodiments, the labels used include silica nanoparticles. In some embodiments, the label comprises lanthanide chelate-loaded silica nanoparticles. In some embodiments, the labels used include a fluorescent microsphere.
In some embodiments, the labels used (e.g., labels to which reaction substances are conjugated) include an enzyme. In some embodiments, the enzyme is horse-radish peroxidase (HRP). In some embodiments, the enzyme is alkaline phosphatase (AP). In some embodiments, the enzyme is Glucose oxidase. In some embodiments, the enzyme is Urease. In some embodiments, the amplification of the detectable signal is obtained by reacting the enzyme with one or more substrates or additional enzymes and substrates to produce a detectable reaction product.
In some embodiments, the labels used (e.g., labels to which reaction substances are conjugated) include colloidal carbon. Unstabilized carbon can be used to produce carbon sols suitable for protein adsorption. Their carbon sols are formed by suspending carbon particles of well-defined particle sizes in distilled water or low ionic strength buffers, sonicated or vigorously agitated, followed by centrifugation. These unstabilized carbon sols were flocculated easily by salt. However, when coated with macromolecules such as antibodies, they were “protected” from flocculation. In practice, increasing amounts of a macromolecule are incubated with a fixed amount of non-stabilized carbon aqueous sol under defined conditions to determine the “minimal protective amount”. The optimal pH for adsorption can be determined by one of ordinary skill in the art. Unlike colloidal gold, in which the conjugation of protein to colloidal gold is near instantaneous, adsorption onto colloidal carbon takes a longer time from one to several hours. Colloidal carbon has appropriate properties in terms of stability and high color contrast on a membrane.
In some embodiments, the testing zone is fluidly coupled to the reaction zone and includes one or more testing substances corresponding to the target material in the target sample solution. In some embodiments, the testing zone includes one or more immobilized substances, such as one or more substances listed in TABLES 1-3. In some embodiments, the immobilized substances bind to specific regions of target material of agents or biomarkers associated with the health condition(s) of interest. In some embodiments, the specific region(s) are the same as the region(s) that the non-immobilized (i.e. capable of migrating downstream) substance in the reaction zone binds to. In some embodiments, the specific region(s) are different from the region(s) that the non-immobilized (i.e. capable of migrating downstream) substance(s) in the reaction zone bind to.
In some embodiments, the target material-reaction substance complexes flow from the reaction zone to the testing zone, where the testing zone includes a first testing substance retained at a first region of the testing zone, where the first testing substance preferentially couples to the first target of target material. The testing zone can optionally include a second testing substance retained at a second region of the testing zone, where the second testing substance preferentially couples to the second target of target material. In some embodiments, the first testing substance and the second testing substance are thus immobilized within the reaction zone, and the first and the second regions are test lines within the testing zone. However, the first and the second regions can alternatively be defined in another manner (e.g., as dots, as areas, as patterns, etc.) within the testing zone. Furthermore, in embodiments, the testing zone can include more than two testing substances, in relation to assays performed on different types of target material (e.g., related to different health conditions). In other embodiments, the testing zone only includes a single testing substance coupled to a target of a target material when the target material is present.
In some embodiments, the affinity between the immobilized or non-immobilized substance(s) and the target material to which the substance(s) specifically bind when they are specifically bound to each other in a binding complex is characterized by a KD (dissociation constant) of 10−5 M or less, 10−6 M or less, such as 10−7 M or less, including 10−8 M or less, e.g., 10−9 M or less, 10−10 M or less, 10−11 M or less, 10−12 M or less, 10−13 M or less, 10−14 M or less, 10−15 M or less, including 10−16 M or less. “Affinity” refers to the strength of binding, increased binding affinity being correlated with a lower KD.
In some embodiments, the affinity between the immobilized testing substance in the testing zone and the target material of interest from the sample is about equal to or stronger than the affinity between the non-immobilized reaction substance in the reaction zone and the same target material. In some embodiments, the affinity between the immobilized testing substance in the testing zone and the target material is at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold than the affinity between the non-immobilized reaction substance in the reaction zone and the same target material.
In some embodiments, the affinity between the immobilized testing substance in the testing zone and the target material of interest from the sample is about equal to or weaker than the affinity between the non-immobilized reaction substance in the reaction zone and the same target material. In some embodiments, the affinity between the immobilized testing substance in the testing zone and the target material is at most about 90%, 80%, 70%, 60%, or 50% of the affinity between the non-immobilized reaction substance in the reaction zone and the same target material.
In some embodiments, the testing zone further includes a substance that binds to a biomarker of pregnancy. In some embodiments, the biomarker(s) include one or more of: human chorionic gonadotropin (hCG), activin A, pregnancy-associated plasma protein-A (PAPP-A), human placental lactogen (hPL), A disintegrin and Metalloprotease-12 (ADAM-12), pregnancy-specific beta glycoprotein 1 (SP-1), placental mRNAs, progestrerone, Inhibin A, Vascular Endothelial Growth Factor (VEGF), Placental-like growth factor (PlGF), Leukemic Inhibitory Factor, Glycodelin, Mucin-1, Adrenomedullin, and other biomarkers.
In some embodiments, the testing zone further includes a substance that binds to a biomarker of fertility. In some embodiments, the biomarker(s) include one or more of: oestrone-3-glucuronide (E3G0, luteinizing hormone, follicle stimulating hormone (FSH), estrogen, progesterone, testosterone, anti-mullerian hormone, follicle-stimulating hormone, luteinizing hormone, estradiol, thyroid-stimulating hormone, free thyroxine, prolactin, dehydroepiandrosterone (DHEA), cortisol, sex hormone binding globulin (SHBG), triiodothyronine (T3), Thyroxine (T4), thyroid stimulating hormone (TSH), thyroid peroxidase antibodies (TPO antibodies), and other biomarkers.
In some embodiments (some of which are described above), the strip also includes a control zone. When present, the control zone is located downstream from the loading zone. In some embodiments, the control zone is located upstream or downstream from, or overlaps with the reaction zone. In some embodiments, the control zone is located upstream or downstream, or overlaps from the testing zone. In some embodiments, the control zone is located downstream from both the reaction zone and the testing zone.
The control zone may include a control substance retained at the control zone. In some embodiments, the control substance is immobilized. In some embodiments, the control substance binds to any particle. In some embodiments, the substance binds to a mobile control binding agent (a control binding agent that is not immobilized). In some embodiments, the mobile control binding agent is or includes the non-immobilized reaction substance in the reaction zone. In some embodiments, the mobile control binding agent is or includes an agent in the sample or a solution the sample is prepared in. In some embodiments, the control zone includes two or more substances that bind to two or more mobile control binding agents. In some embodiments, the two or more mobile control binding agents are from different sources (e.g., one is from the sample or a solution the sample is prepared in, and another from the non-immobilized substance originally in the reaction zone).
In an embodiment, when received into the loading zone of the test strip 135, the target material flows to the reaction zone. The target material-reaction substance complexes flow from the reaction zone to the testing zone. The sample material including the target material and/or other material flows from the testing zone to the control zone, which includes a control substance retained at the control zone.
In some embodiments, the sample material including the target material and/or other material flows from the testing zone to the control zone, which includes a control substance retained at the control zone, where the control substance does not preferentially couple to the target material and/or binds to any substance (e.g., binds non-specifically to different materials). As such, the control substance is immobilized at the control zone and can be immobilized along a line of the control zone or in another manner (e.g., as a dot, as an area, as a pattern).
In some embodiments, the system achieves a sensitivity of detecting the presence of a specific agent or biomarker in each of the strips of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82.5%, 85%, 90%, 92.5%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the system detects two or more different agents or biomarkers with a sensitivity for at least two agents/biomarkers being both about or more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82.5%, 85%, 90%, 92.5%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the system detects three or more different agents or biomarkers with a sensitivity for at least three agents/biomarkers being both about or more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82.5%, 85%, 90%, 92.5%, 95%, 96%, 97%, 98%, or 99%.
In some embodiments, the system achieves a specificity of detecting the presence of a specific agent or biomarker of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82.5%, 85%, 90%, 92.5%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the system detects two or more different agents or biomarkers with a specificity for at least two agents/biomarkers being both about or more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82.5%, 85%, 90%, 92.5%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the system detects three or more different agents or biomarkers with specificity for at least three agents/biomarkers being both about or more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82.5%, 85%, 90%, 92.5%, 95%, 96%, 97%, 98%, or 99%.
In some embodiments, the signa includes, is coupled to, or otherwise communicates with a detection system. In some embodiments, the signal output device is coupled to the detection system. In some embodiments, the detection system includes an optical reader (e.g., an optical strip reader). In some embodiments, the optical reader measures the intensity of colors produced at test and control lines. In some embodiments, the intensity of colors is recorded by an imaging software (e.g., an application on a computer, such as a mobile app). In some embodiments, the intensity of colors are recorded by a camera and then processed by an imaging software. In some embodiments, the optical system comprises a source of light. In some embodiments, the source of light comprises a monochromatic light. In some embodiments, the optical system is an automated system. In some embodiments, the optical system is a manual system.
In some embodiments, the detection system includes a fluorescence reader (e.g., a fluorescence strip reader). In some embodiments, the fluorescence reader measures the fluorescence intensity of test and control lines.
In some embodiments, the detection system includes a photoelectric sensor. In some embodiments, the photoelectric sensor measures photoelectrons produced as a result of the colloidal gold being exposed to a light source.
In some embodiments, the detection system includes a magnetic reader (e.g., a magnetic strip reader). In some embodiments, the detection system comprises an electrochemical detector. In some embodiments, the detection system comprises a digital device, such as, but not limited to, a mobile device, tablet, computer, and the like.
In some embodiments, the system does not include an external detection system. In some embodiments, the systems described herein produce a signal that can be assessed by the eye (e.g., with visual observation).
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The signal may be interpreted by the eye. In some embodiments, the signal output device may be further signal processed using a camera on the smartphone or optical reader or fluorimeter. In the case of the smartphone, the data can be viewed by the user or be sent to a provider for next steps in patient care via telehealth. The provider can in turn provide necessary guidance to the user and be able to send the prescription directly to the user.
In various embodiments, each test kit has an ID associated with it. This ID would encapsulate multiple variables which could identify the device some of which would include Lot ID, Batch ID, Test ID, Expiration Date, Seller, Re-seller information, etc. Capture of the ID could be either based on QR code or user input. The ID can help provide additional services to the end user. Some of the services may include, but are not limited to, connecting to telehealth, various helplines, clinics, prescriptions. Once the user completes the test, if the signal is processed using a camera on the smartphone and after the ID is captured or entered, the test results could be sent out to the right provider and patient services could be customized.
In some embodiments, the signal output device is read using a smartphone or computer device. For example, the smartphone can include a software used to take an image of the signal output device windows showing the test strips, and the smartphone can provide the result to the user indicating whether the user tested positive or negative, or indicating a particular level of an agent present in the sample. The smartphone or computer device can be used to read the signal provided by a label or tag on the signal output device. Non-limiting examples of a label and/or tag include a visual label, a fluorophore label, a gold label, a CNB label, or the like. For example, an image can be taken of the windows showing the test strips, and the label/tag may be visible in the image as a readable test result for the user or the phone may translate the test result for the user. In some embodiments, the phone includes a mobile software application stored on a computer readable medium and executable by a processor. The mobile application can be an application corresponding to the sample kit including a user interface that allows the user to take images or otherwise input information into a user interface, and to be presented with test results.
The user's family members, physician, nurse, or members of the medical team can also have access to a medical side mobile or computer application that receives information from the user's mobile application about the user's test. In some embodiments, the medical team can read the results, send comments to the user, send an interpretation of the test results, among other interactions. In one embodiment, the sample kit includes a telehealth system that allows the physician to interact with the user including scheduling a video conference or phone call to discuss the test results. In one embodiment, the medical team can prescribe medication or therapy to treat the condition if the user has a positive test. The physician can send the prescription to the pharmacy based on the test results received through the mobile application. The user can coordinate the delivery or pickup of the prescription through the mobile application, and coordinate refills of the prescription with the medical team and pharmacy. In one embodiment, the system is an end-to-end computer telehealth system where a medical team can prescribe to the user a test in which the user receives the sampling kit in the mail or from the store. The user takes the test, and uses the mobile application to read the test results. The mobile application sends the test results over a network or the internet to the medical team. The medical team takes various actions based on the results including providing a prescription to the pharmacy, and the user then receives the prescription and can continue to be monitored by the medical team through the mobile application.
The foregoing description of the embodiments has been presented for the purpose of illustration; it is not intended to be exhaustive or to limit the patent rights to the precise forms disclosed. Persons skilled in the relevant art can appreciate that many modifications and variations are possible in light of the above disclosure.
Any of the steps, operations, or processes described herein may be performed or implemented with one or more hardware or software modules, alone or in combination with other devices.
Embodiments may also relate to an apparatus for performing the operations herein. This apparatus may be specially constructed for the required purposes, and/or it may comprise a general-purpose computing device selectively activated or reconfigured by a computer program stored in the computer. Such a computer program may be stored in a non-transitory, tangible computer readable storage medium, or any type of media suitable for storing electronic instructions, which may be coupled to a computer system bus. Furthermore, any computing systems referred to in the specification may include a single processor or may be architectures employing multiple processor designs for increased computing capability.
Finally, the language used in the specification has been principally selected for readability and instructional purposes, and it may not have been selected to delineate or circumscribe the patent rights. It is therefore intended that the scope of the patent rights be limited not by this detailed description, but rather by any claims that issue on an application based hereon. Accordingly, the disclosure of the embodiments is intended to be illustrative, but not limiting, of the scope of the patent rights, one implementation of which is set forth in the following claims.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); μl, picoliter(s); microliters (μl), s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.
In order to provide proof of concept, precision, and reproducibility of the sample test kit, the present inventors performed precision and reproducibility testing for various test panels.
For all test panels shown below, the “control solution” represents the result output of the sample health kit as described in the present disclosure, and the “control limit” represents the actual, true result based on the known sample tested.
Additionally, for all test panels, the indication status of the test panel is assessed visually by the naked eye for the presence or absence of a health condition. In this example, a test zone that shows a visual, physical line on the test zone “window” will indicate that the result is positive (e.g., the sample does include the analyte/health condition being tested for). A test zone that does not show any visual, physical line on the test zone “window” will indicate that the result is negative (e.g., the sample does not include the analyte being tested for).
Test Panel—Chlamydia trachomatis
Precision testing was performed on a total of 180 tests per each test panel (Chlamydia trachomatis). Data analysis included the computation of percent agreement and lower 95% confidence interval. The results are provided in Table 4, reproduced below:
Reproducibility for the Chlamydia trachomatis test panel was performed using over a period of 5 days for a total of 450 tests (5 repeats (reps)sol×3 lots×5 days×3 solutions×2 users=N=number of 450 tests).
Precision: Precision testing was performed total of 180 tests per each test panel (Trichomonas Vaginalis). Data analysis included the computation of percent agreement and lower 95% confidence interval
Reproducibility was performed using over a period of 5 days for a total of 450 test (5 reps/sol×3 lots×5 days×3 solutions×2 users=N of 450 tests).
Precision: Precision testing was performed total of 180 tests per each test panel (Neisseria Gonorrhea). Data analysis included the computation of percent agreement and lower 95% confidence interval
Reproducibility was performed using over a period of 5 days for a total of 450 test (5 reps/sol×3 lots×5 days×3 solutions×2 users=N of 450 tests).
The results of the precision and reproducibility studies for the 3 test panels tested in the sample kit show that the sample health kit of the present application is efficacious in providing, with high precision and reproducibility, the true and accurate indication (e.g., positive, or negative) of the status of the health condition tested, as shown when the result output of the sample kit (control solution) is the same as the control (control limits with known indication status of the sample)
This application is a § 371 of International Application No. PCT/US2021/044091 filed Jul. 31, 2021, which claims the benefit of U.S. Provisional Application No. 63/060,041 filed Aug. 1, 2020, the disclosures of which are hereby incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/044091 | 7/31/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63060041 | Aug 2020 | US |
