Detection apparatus and method

Abstract

The invention is a method and apparatus for molecular testing to detect or measure the level of a target which may be a pathogenic disease or other biological material In some embodiments, the method is an isothermal or thermal cycling amplification method. In some embodiments, detection techniques aside from amplification are used. In some embodiments of the apparatus and method of the invention, the sample is gravity-fed into the apparatus. In some embodiments, the apparatus and method of the invention is based on amplifying under different conditions with different mass amounts of nucleic acid from the primary sample to determine the mass point where sample will or will not be detected. In some embodiments of the invention, the target pathogen contains RNA. The pathogen may be lysed to release the RNA, reverse transcribed to DNA and then amplified. In some embodiments of the invention, the target pathogen contains DNA. The target pathogen may be lysed and then the DNA may be amplified and detected. In some embodiments of the invention, the isothermal or thermocycling molecular test is used to measure the progression of a pathogenic disease.

Description

[0001] Detection Apparatus and Method

[0002] Field of the Invention

[0003] The apparatus and method of the invention allow detection and semi-quantification of a virus, cell or biological molecule present in a biological sample.

[0004] Background of the Invention

[0005] There are several companies that have introduced point of care testing instruments and kits for testing pathogens including viruses and including testing for COVID-19. Some devices depend on molecular detection which means they test for the pathogen RNA from a sample taken from an individual. A sample nose swab is taken, treated with reagents, and introduced into the measuring fixture of the kit. Lysis is performed releasing the RNA and then the RNA is reverse transcribed into DNA. Finally, DNA is amplified usually by an isothermal amplification. Usually, LAMP amplification is used currently. Detection after amplification may be optical, electrochemical, or chemical. The tests are called molecular tests because they measure directly the presence or absence of the nucleic acids, RNA or DNA in the biological sample.

[0006] The molecular tests on the market can detect low levels of the target pathogen, usually lower levels than by the antigen test.

[0007] The most common type of point of care testing technology is the antigen test, sometimes called a lateral flow test. In these tests, capillary action of a buffer is used to move a biological sample (usually from a nose swab) along the surface of a pad. Sample and buffer contact the end of the pad and capillary action is initiated. The pad contains reactive molecules in a strip perpendicular to flow that are sensitive to protein. Detection is of proteins specific to the pathogen and does not require amplification. The chemistry is based on an enzyme-linked immunosorbent assay. The tests have high specificity and are known to not give false positive results.

[0008] There is no amplification in the antigen test as found in most molecular tests. Detection is based on a concentrating effect of material as the target protein travels across the detection zone. In contrast, molecular tests amplify the pathogen nucleic acid and are usually very sensitive. Antigen tests are known to have lower sensitivity than molecular tests. Nevertheless, an individual who has an infectious disease is known to evaluate positive for both molecular and antigen tests if infected and will test negative if not infected. The advantage of the molecular test is that it has fewer false negatives than the antigen test.

[0009] During the progression of a disease, individuals may have different levels or concentrations of pathogens in their body. But these molecular and antigen tests only give a yes or no answer regardless of the concentration of the pathogens. qPCR molecular tests based on thermal cycling amplification can provide more analytical information using the threshold amplification time or digitized samples using Poisson mathematics to count the numbers of nucleic acid molecules in the original sample. But these types of analytical PCR molecular tests are more complicated and are generally performed in a laboratory. In addition, swab sampling provides different amounts of pathogen molecules to be analyzed from sample to sample.

[0010] There exists a need for a simplified molecular quantification method test to provide information to quantify a virus, cell or biological molecule present in samples and follow the progress of a disease in an individual. The method may be direct detection or detection after amplification.

[0011] Summary of the Invention

[0012] The invention incudes a method and apparatus for molecular testing to detect or measure the level of a target, including pathogenic disease or other biological material. In some embodiments of the invention, the pathogen contains RNA. The pathogen may be lysed to release the RNA, reverse transcribed to DNA and then amplified. In some embodiments of the invention, the pathogen contains DNA. The pathogen may be lysed and then the DNA may be amplified and detected. In some embodiments of the invention, the isothermal or thermocycling molecular test is used to measure the progression of a pathogenic disease.

[0013] In some embodiments, the apparatus and method of the invention is based on amplifying a target sequence (or sequences) from different mass amounts of nucleic acid from a primary sample. The amplification reactions are carried out with different mass amounts to determine the minimum mass at which the sample will be detected.

[0014] Isothermal molecular testing is characterized by circumstance and regimen. A sample from the individual is analyzed with an isothermal process that may include detection of a target from a virus, cell, or biomolecule nucleic acid. The testing is usually started at the onset of disease symptoms or after a known exposure to someone who has the disease or later found to have the disease. The answer provided by the test is either positive or negative. With a negative result, the testing may continue every day for a day or two or testing may be stopped. A positive result shows the individual is infected with the disease. The development of a disease is measured by taking another test, usually once a day or so, as the disease runs its course without knowing the severity of the disease. Once an individual tests negative for a disease, testing may continue, usually for a day or two to confirm the individual is now free of the disease. While this testing technology and regimen is effective, no information is produced about the amount of the pathogen present in the individual. And no information is available on whether any treatments are effective other than monitoring the overall symptoms and trends of the symptoms of the individual.

[0015] Testing with an isothermal amplification or other direct detection methods produces only yes or no answers to the presence of disease. The elapsed time for pathogen detection normally will not provide any reliable quantitative information i.e. higher concentrations of the pathogen in the sample may or may not allow more rapid detection.

[0016] In the invention, a primary sample is taken and processed using a detection device and method. The sample is uniform meaning that a nasal swab, blood, saliva, or breath condensate sample is processed. The primary sample is split into two or more fractions and processed in two or more detection chambers. The target mass of the primary sample is reduced in each chamber. The reduction may be performed by reducing the detection chamber size or by adding a diluent to the primary sample. The detection limit in each detection chamber is different, e.g. with two detection chambers, one chamber will have a lower detection limit than the other chamber. Detection for viruses, cells, bacteria, fungi, and other pathogenic and biomolecular molecules can be is performed using nucleic acid amplification techniques (NAATs). PCR thermal cycling amplification can be used. A variety of isothermal amplification methods can be used including loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), helicase-dependent amplification (HDA), rolling circle amplification (RCA), and other methods such as CRISPR / Cas systems, fluorescence-based chemistries as examples. Targets may be detected by fluorescence, protein activity assay, or any spectroscopic or electrochemical method as further examples.

[0017] In some embodiments of the invention, one of the fractions’ analyte mass is reduced to produce a higher detection limit (i.e. less target is detected in the mass reduced sample or reduced concentration sample.) In some embodiments of the invention, one of the fractions is amplified in a smaller chamber than the other fraction to produce a higher detection limit (i.e. less target is detected in the smaller sample). In some embodiments, detection is performed without amplification. In some embodiments, multiple fractions of varied concentrations are present and can be processed or amplified in chambers of varying volumes. However, in all cases, the target including a virus, cell or biomolecule is only detected if it is in the sample at a high enough concentration or mass amount to be detected by the conditions of detection. In some embodiments detection is performed without amplification.

[0018] Each detection chamber reports results as negative or positive. In some embodiments of the invention, there may be an additional probe or additional detection chamber providing positive sampling and process probe information. A chamber having a low limit of detection (LLOD) would produce a positive indication even if only a small amount of virus, cell or biomolecule was present and detected. This is a highly sensitive detection limit. A chamber having a high value detection limit (HLOD) would detect pathogen only if a greater amount of virus, cell or biomolecule was present. This is an insensitive detection limit.

[0019] In the invention, a detection device will have two or more detection chambers having LLOD and a HLOD capability. Depiction of results from a two-detection chamber device would be the following:

[0020] 1 . Negative LLOD and Negative HLOD: virus not detected at any level.

[0021] 2. Positive LLOD and Negative HLOD: virus detected but it is present at low level.

[0022] 3. Positive LLOD and Positive HLOD: virus is detected and is at high level.

[0023] These can be depicted by colored lights or a script or message. Any number of detection chambers may be employed, each having different detection limits. Some devices of the invention can have 2 - 10 chambers. Some devices of the invention can have 10 - 50 chambers. In addition, there may be duplication of chambers to provide more reliable results. In some embodiments of the invention, the device is used for confirming the result and also may provide additional detection information.

[0024] One embodiment of the apparatus and method of invention is intended to provide diagnostic testing results that can estimate the level of virus, cell or biomolecule load or the severity of the disease. Another embodiment of the apparatus and method of the invention is to provide detection conditions to compare the molecular test result sensitivity to antigen test sensitivity. In some embodiments of the invention, the testing can be applied over a period of days or any time interval to follow the progression of the presence of the target including a virus, cell or biomolecule that may be pathogenic.

[0025] The apparatus and method of the invention is not a digital signal system for quantification.

[0026] The system does not digitize samples to detect and quantify by counting targets detected from a single molecule of target. The system of the invention does not report results based on Poisson digitization of samples where a sample is diluted to single reaction copies before detection or amplification and detection. The apparatus and method of the invention only reports if a signal above the detection limit is obtained. The mass amounts of primary samples are reduced by a series of reduction factors. For example, if the volume of one detection chamber is half the volume of another detection chamber, the reduction factor is 2. Similarly, for sample dilution, if two detection chambers hold the same volume but the sample is diluted 2-fold in one of the chambers, the reduction factor is 2. The number of sample chambers processed is much less than the number of reactions required / typical in digital PCR.

[0027] The goal of the invention is to provide yes / no information at different sensitivities. This is accomplished by adjusting the absolute mass amount of the analyte available in the sample by changing the reaction chamber size or by diluting the primary sample.

[0028] The detection limit of the method may be tuned by providing different mass amounts of the target including a virus, cell or biomolecule or products of the pathogen. One method of providing different mass amounts is by providing different reactions chamber sizes. However, the steps of lysing, reverse transcription and amplification may not have a linear relationship to the mass of the sample. In this way, for any one set of conditions, the results relate only to whether the signal indicates that the amount of target including nucleic acids in the primary sample is above the detection limit.

[0029] The detection limit of the method may be adjusted by providing different mass amounts or concentrations of the sample containing the target for detection, including a virus, cell or biomolecule or products of the pathogen. One method of providing different amounts is through dilution of the sample by various amounts. However, the steps of lysing, reverse transcription and amplification, or other means of detection, may not have a linear relationship to the concentration of the sample. In this way, for any one set of conditions, the results relate only to whether the signal indicates that the amount of analyte nucleic acid is above the detection limit or not above the detection limit.

[0030] In one embodiment of the invention, the sensitivity of the molecular detection conditions is adjusted to mimic antigen testing. In one embodiment of the invention, the sensitivity of the molecular detection conditions is adjusted to determine if the individual who tests positive for a pathogen is of low, medium or high infectiousness.

[0031] The apparatus and method of the invention is based on being able to detect or amplify a target and detect a specified amount of target, including a virus, cell or biomolecule, pathogen. In one embodiment of the invention, the sensitivity of the molecular detection conditions is adjusted to determine if breath exhaled from an individual contains pathogens at a low, medium or high infectiousness. In one embodiment of the invention, the molecular detection conditions are adjusted to determine the extent of viral shedding or viral load from a given sampling method. In one embodiment of the invention, this estimate may be monitored over time to track the progression of an infection.

[0032] Brief Description of the Figures

[0033] Embodiments of the invention as described in detail below with reference to the accompanying drawings, in which:

[0034] FIG. 1 is a schematic diagram of a gravity-fed analyte mass dilution apparatus according to an embodiment of the invention;

[0035] FIG. 2 is a schematic diagram of a gravity-fed apparatus to detect sample with varying chamber volumes and analyte mass; and

[0036] FIG. 3 is a flow diagram of a detection method that is an embodiment of the invention.

[0037] Definitions

[0038] Reaction chamber or detection chamber: Device chamber in which reactions, amplification and / or detection take place.

[0039] A chamber having a low limit of detection (LLOD): This is a highly sensitive detection chamber or detection reactor. A positive indication signal is given when even a small amount of pathogen is present and detected within a sample. Or, only a small amount of nucleic acid target is needed for detection within a given volume.

[0040] A chamber having a medium limit of detection (MLOD): Detection chamber or other reactor that produces a positive indication signal when medium amount of a target such as a pathogen is present and detected. Medium level detection limit detection capability is chosen to be between a low value detecting low concentrations (or low amounts of targets such as pathogens) and a high value detecting only high concentrations or high amounts of pathogen present.

[0041] A chamber having a high limit of detection (HLOD): This is an insensitive detection chamber or other reactor. This detection chamber produces a positive signal only when a higher amount of target such as a pathogen is present and detected. A higher amount of field sample target is needed for detection. A molecular test might be detuned (or made less sensitive) to detect high levels of antigen for example.

[0042] Sample containing target including a virus, cell or biomolecule: The sample, e.g. nasal swab, saliva, breath, breath condensate, etc. containing a target, such as a virus, cell or biomolecule. The sample may contain a pathogen or a part or component of a pathogen.

[0043] Number of reaction chambers: Each detection chamber can have a different detection limit for detecting a target or other entities. Each reaction chamber having a specific detection limit may have duplicate reaction chambers for confirmation of a positive or negative signal.

[0044] Detection: Generally, detection is of a target, including a pathogen or pathogens or component of a pathogen. But the invention can detect any molecule, combination of molecules or parts of molecules using any type of detector or sensor.

[0045] Detection limit, or limit of detection: The amount of a target that can be consistently detected. For example, the same amount of sample can be tested / analyzed multiple times under the same conditions. This is related to the original field sample. The amount of target can be defined by a number such as a copy number or by a concentration. This amount may be determined empirically as the amount of the target that is detected when present effectively 100% of the time, 99% of the time after 100 trials, 95% of the time after 19 of 20 trials, 80% of the time after 4 of 5 trials, or 75% of the time after or 3 of 4 trials.

[0046] Reaction mass amount: Amount of the target detection, including pathogen, virus, cell or biomolecule in the detection reaction chamber undergoing detection. The reaction chamber size can be adjusted to change the sample reaction mass amount. Or the detection reaction chamber analyte produced by dilution of the primary sample with a diluent can be adjusted to change the reaction analyte mass amount.

[0047] Reaction analyte mass concentration: Concentration of the target, including a virus, cell, biomolecule or pathogen in the detector reaction chamber undergoing detection. For this invention, target, virus, cell, biomolecule or pathogen terms are used interchangeably.

[0048] Pathogen lysing: Process of breaking up a pathogen including a virus, a cell, a cell wall, a bacterial or spore wall, envelope or any type of barrier to release nucleic acid.

[0049] Primary sample: Any sample taken from an individual or taken from biological material. This could be from a biological fluid or tissue, blood, breath, breath condensate, saliva, nose or cheek swab, etc. This is the primary sample that undergoes mass analyte dilution in the method of the invention. Mass analyte reduction factor. The mass amount that of the primary sample is reduced in a detection chamber. Reduction of the mass amount can be obtained by dilution to reduce concentration in the chamber or by reducing the volume of the chamber. The sample solution or liquid that undergoes mass analyte dilution in the method of the invention.

[0050] Reduction step: Any process from one chamber to another that reduces the mass of the target within the sample. This may be achieved by adding diluent to the sample in one chamber, or by adding a smaller volume of the primary sample. Reduction steps may be repeated to further compound the total mass reduction.

[0051] Total reduction factor: The total reduction factor is the multiplication of all reduction steps. Each reduction factor is multiplied by the next reduction factor to get total reduction factor.

[0052] Detailed Description of the Invention

[0053] The goal of the apparatus and method of the invention is to provide a series of detection conditions for a molecular test using a gravity-fed liquid flow process of a primary sample. The test has a progression of detection limits within the apparatus and method of the invention. In some embodiments, there are only two reaction detection chambers with two detection limits. In some embodiments there are three or more detection chambers having conditions for three or more detection limits. In other embodiments of the invention, there are four, five, six, seven, eight, nine, ten, twelve, fourteen, fifteen, sixteen, eighteen, twenty, twenty-five, thirty, forty or more detection chambers. If a target, including a virus, cell, or biomolecule that is present in a sample taken from the field results in a positive signal in any one detection chamber, then it is above the minimum amount of target needed in any one or more of the detection chamber reactions. Other reaction chambers in the detection device may be present for positive controls for the apparatus and method of the invention.

[0054] In some embodiments of the invention, detection is of the nucleic acid. In some embodiments of the invention a nucleic acid target is amplified and detected. In some embodiments, more than one target can be amplified simultaneously. In some embodiments of the invention, detection is on a part or component of the virus, cell or biomolecule.

[0055] Yes / no diagnostic methods are designed to give a yes answer when any target material is reliably detected. They are designed to work at the highest sensitivity possible. This is the reason that antigen tests for Covid-19 for example are much less sensitive than molecular tests for Covid-19. The apparatus and method of the invention is purposely detuned to give progressively lower sensitivity results. However, a series of reaction chambers are used with progressive detuning. The primary sample mass is reduced by a gravity-fed process to reduce the analyte mass amount. This process can be called detuning because it makes the test less able to detect a target, including a virus, cell or biomolecule. Detuning may be linear or any form desired. In principle, any type of yes / no diagnostic test will work for this invention as long as the test can be detuned to reduce the sensitivity, thereby increasing the detection limit.

[0056] The invention can be applied to any yes / no detection device where the reaction chamber can be designed or tuned to be progressively less sensitive to have higher detection limits and a series of two, three, five, ten, twenty or more reaction detection chambers. In some embodiments of the invention, detection may be by sensor without amplification needed. These sensors may be provided in a chamber or on a surface.

[0057] In some embodiments of the invention the target, including virus, cell or biomolecule that is tested contains DNA. The target is lysed, amplified and detected. In some embodiments of the invention, the sample tested contains RNA. The sample is lysed to release RNA. RNA is reverse transcribed to DNA. In some embodiments of the invention, amplification is isothermal amplification. In some embodiments of the invention, amplification is thermal cycling amplification. The relative sensitivity of the method is tuned by providing different mass amounts of the sample containing cells, virus, pathogens, biomolecules or products to each reaction / detection chamber. In one embodiment of the invention, different mass amounts are achieved through serial dilution of the target with a diluent to produce a series of sample concentrations in a series of chamber reactors. Each concentration provides a different detection limit in terms of the concentration of the target in the primary sample.

[0058] In another embodiment of the invention the sample and detection reagent mixture concentrations are kept constant and are filled into a series of reaction chamber with varying volumes. Larger reaction chambers have lower detection limits because more sample nucleic acid product mass is available for amplification or detection. Smaller reaction chambers necessarily have higher detection limits in terms of concentration because less nucleic acid analyte mass is available to amplify or detect. Reaction chambers of different sizes may or may not have the same detection limit in terms of total number of copies of template.

[0059] The molecular reaction chamber’s detection limits can be compared to the amount of pathogen needed to be detected by antigen or lateral flow text. In one embodiment of the invention a method of controlling the detection limit of a reaction chamber is to control the pathogen mass amount. This can be done by providing different reaction chamber sizes. Another way of doing this is to provide different dilutions of the pathogen to reaction chambers of the same size. However, the efficiency of the steps of lysing, reverse transcription and amplification may cause the result not to be linear or proportional to the amount of target in the sample. For any one set of conditions, the results relate only to whether the signal resulting from the target’s presence is above the detection limit.

[0060] As noted before, each detection chamber results can be reported as negative or positive. In some embodiments of the invention, three-level detection limit detection chambers are used. A chamber having a low limit of detection (LLOD) would produce a positive indication even if only a small amount of target, including virus, cell or biomolecule were present and detected. This detection chamber has the highest ability to detect even if only a small amount of target is in the field sample. A chamber having a medium limit of detection (MLOD) would produce a positive indication even if only an intermediate amount of target was present and detected from the field sample. A chamber having a high limit of detection (HLOD) would detect pathogen only if a very high amount of target was present.

[0061] In the invention, a detection device may have three detection chambers having LLOD, MLOD and HLOD capability. Depiction of results from a three-detection chamber device would be the following:

[0062] 1. Negative LLOD, Negative MLOD, Negative HLOD: Target, including virus, cell, pathogen or other biomolecule not detected at any level.

[0063] 2. Positive LLOD, Negative MLOD, Negative HLOD: Target, including virus, etc., detected but it is present at low level.

[0064] 3. Positive LLOD, Positive MLOD, Negative HLOD: Target, including virus, etc. detected and is at a higher level.

[0065] 4. Positive LLOD, Positive MLOD, Positive HLOD: Target, including virus, etc. is detected and is at high level.

[0066] Any number of detection chambers that have different detection limits may be provided to provide information about the sample. The information is from the sample collected and information is limited to how effectively the sample was taken.

[0067] Due to the statistical nature of the distribution of the targets within the sample as well as the extent of the steps of detection, it is possible, albeit improbable, for a chamber with a higher limit of detection to detect a target while one with a lower limit of detection does not. In one embodiment of the invention, this may constitute an invalid result, and users may be prompted to repeat the test. However, since multiple measurements are performed on the sample, an outlier result may be ignored. In embodiments of the invention with a large difference in limits of detection such as 10-, 100-, or 1000-fold difference, and small numbers of detection chambers, such as 2, 3, or 4 chambers, a discrepancy between which wells amplify and their limits of detection could constitute an invalid result. A mathematical algorithm could be applied to account for this probability and the test will remain valid. In other embodiments of the invention, especially those with 5 or more, 10 or more, 20 or more or 50 or more detection chambers, and those with relatively similar limits of detection such as a 1.5-fold, 2-fold, or 3-fold difference between limits of detection, results out of sequence are more likely to occur due to statistical variation. In these cases, such an out-of-sequence result may not constitute an invalid test. Such results may be displayed directly or may be interpreted by automated software and displayed.

[0068] Depending on the sample and the test, increasing the number of detection chambers may provide more information and more accurate quantification. But in some cases, there may be a practical limit of chambers, depending on the device, such as 10 or 50 chambers. Chamber types may be duplicated or replicated to provide more reliable results or additional information. The detection limits can have different names such as level 1, 2, 3 etc. sensitivity or molecular level sensitivity, antigen level sensitivity, or high sensitivity, medium sensitivity and low sensitivity, etc.

[0069] In one embodiment of the invention, detection chambers may have a 2-fold difference in limits of detection produced by a 2-fold reduction in analyte mass. In this case, the pathogen may be detected in some chambers below their limit of detection, while pathogen might not be detected in other chambers with a lower limit of detection. In some embodiments, this overlap in the range of limits of detection may be undesirable, posing a practical limitation on how similar the limits of detection can be. Depending on the reliability of the detection method, limits of detection may require greater than a 1.5-fold difference, greater than a 2- fold difference, greater than a 3-fold difference, greater than a 4-fold difference, greater than a 5-fold difference, or greater than a 10-fold difference in each step of analyte mass reduction by gravity-fed liquid flow.

[0070] In one embodiment of the invention, 50 detection chambers may each have a 2-fold difference in the limit of detection. This creates a dynamic range of more than 1015-fold difference in limits of detection. This dynamic range is several orders of magnitude higher than the total number of virus particles typically found in an infected person’s body and is therefore impractical in detecting pathogens from human samples. In some embodiments of the invention, the number of detection chambers may be limited to less than 50 detection chambers, less than 40 detection chambers, less than 30 detection chambers, less than 25 detection chambers, less than 20 detection chambers, less than 15 detection chambers, less than 10 detection chambers or less than 5 detection chambers.

[0071] One embodiment of the apparatus and method of invention is intended to provide diagnostic testing results that indicate the concentration of the virus, cell or biomolecule within the sample by determining if a target, including a virus, cell or biomolecule is present in a concentration or amount above a specified or measured detection limit. Another embodiment of the apparatus and method of the invention is to provide detection conditions to compare the molecular test result sensitivity to antigen test sensitivity.

[0072] In some embodiments of the invention, the apparatus and method of the invention is based on amplifying a nucleic acid target under different conditions with different mass amounts of target to determine the concentration of the target at which the sample is consistently detected i.e. , a positive readable signal is produced. In other embodiments, amplification is not necessary for detection of the target.

[0073] The efficiency of any one step of the molecular testing is likely not to be 100%. Therefore, the prediction of the detection limit of a particular reaction chamber must be measured. Limits of detection are based on the probability of a target being present, as well as being detected if it is present. A statistical chance of detection remains well below the standard limit of detection. Detection limits for each chamber are determined empirically, as is the statistical chance that amounts of target above or below this threshold may be detected. Because there is a chance that target is detected below the threshold, or that it may not be detected despite being above the threshold, calculated target concentrations must be considered an estimate.

[0074] Even with this, the sampling method and the ability to capture a target, including a virus, cell or biomolecule sample or pathogen sample for processing will affect the testing results. For example, saliva may contain more pathogen entities than a nasal swab. The amount of pathogen entities in a nasal swab may depend on how the sample is collected by the individual. A breath condensate sample can be collected for different amounts of time. The efficiency to capture breath condensate liquid from exhaled breath may vary.

[0075] Sample nucleic acid containing reaction chambers can be used to amplify virus, cell, biomolecule or pathogen nucleic acid to achieve a yes / no answer for conditions with a series of different detection limits. In some detection conditions, yes / no detection data can mimic an antigen test. In some detection conditions, yes / no detection data can be measured with higher sensitivity than an antigen test. In some detection conditions, yes / no detection data can be measured with less sensitivity than an antigen test. In some embodiments of the invention, there are reaction chambers with different sensitivities. Two or more of these reaction chambers could be used for positive controls. The intent is to show the level of target in a sample, not by measuring the exact quantity of the target, but by estimating the copy number or the amount. This is accomplished by determining if the identical sample is above a detection limit with a progressive change of the reaction chamber volume. In the cases where the reaction volume is varied, the detection limit in terms of copy number stays relatively consistent, while the detection limit in terms of concentration is inversely proportional to volume.

[0076] In some embodiments of the invention, pumps can be used to fill the liquids into the detection chambers. In some embodiments, syringe plungers may be used to fill detection chambers. In some embodiments of the invention, the detection chambers can be filled by gravity flow of liquids.

[0077] Gravity feed can be used to fill chambers of different volumes or with different concentrations as shown in FIG. 1. In this example, a diluent reservoir 2 contains diluent 4 in sufficient quantity to fill all reaction chambers with a series of analyte solution with progressively lower concentrations. The reservoir is disposed at a higher elevation than the subsequent chambers. On the top of the reservoir is hydrophobic air vent 6, which allows air to pass through, while prohibiting liquid from entering or exiting the reservoir. The bottom of the reservoir is attached to fluidic channel 8, which terminates at pierceable seal 10.

[0078] A modified syringe serves as a sample collection device 12, depositing liquid biological sample into mixing chamber 14. Near the top of mixing chamber 14 is air vent 16 allowing a plunger to expel gas and liquid into the chamber, while only retaining the liquid. At the bottom of mixing chamber 14 are two openings. The first is sealed with check valve 18, which is attached to piercing needle 20. When needle 20 pierces seal 10, diluent flows from reservoir 2 to mixing chamber 14. The second opening consists of pierceable seal 22 below protruding fluidic channel 24. Pierceable seal 22 is pierced by piercing needle 26, allowing fluid to drain into second fluidic channel 28. When mixing chamber is pressed into the rest of the apparatus, needles 20 and 26 simultaneously pierce seals 10 and 22 respectively. This allows diluent 4 to flow into mixing chamber 14 and combine with the sample. When this combined volume rises above the elevation of protruding channel 24, it begins to flow into second channel 28.

[0079] The elevation of the liquid in mixing chamber 14 rises until it reaches the elevation of first loop 30 in fluidic channel 28. Once it reaches this elevation, the liquid drains into first analysis chamber 32. Each sequential analysis chamber is identical and includes air vent 34 allowing trapped gas to escape while retaining the liquid. As liquid rapidly drains from mixing chamber 14 into first analysis chamber 32, the liquid elevation falls below that of the first loop 30. This prevents the liquid from transferring to the next analysis chamber until more diluent flows from diluent reservoir 2 into mixing chamber 14. Once first analysis chamber 32 is filled, additional liquid will be unable to flow into the chamber, and it will instead flow along the channel to subsequent chambers 36. As only a fraction of the original sample is retained in mixing chamber 14 after each analysis chamber 36 is filled, each subsequent mixing chamber contains a more dilute solution and reduced analyte mass of the sample than previous chambers.

[0080] In FIG. 2, gravity feed is used to fill in parallel flow a series of detection reaction chambers of different volumes. The reaction chambers will have different analyte mass amounts and different analyte detection limits. A sample is prepared with a buffer and drains into the device through opening 40. The sample travels through fluidic channels 42 to multiple analysis chambers of varying volumes. In one instance, the largest chamber 44 has a volume of 1.6 mL, the next largest chamber 46 has a volume of 40 pL, and the smallest chamber 50 has a volume of 1 pL. A second 40 pL chamber 48 is used to detect the presence of a positive control sample. Air is able to escape from each chamber via a hydrophobic vent 52, which permits air but not liquid to pass through.

[0081] When a sample has less analyte mass, it is more difficult to detect. Therefore, a larger volume of the sample must be present to detect it. If the amount of analyte mass in the chamber is extremely low, it is only detected in the largest chamber 44. If the analyte mass is extremely high, it is detected in all three chambers, including the smallest 50. The volume range of 1.6 mL to 1 pL allows a 1,600-fold dynamic range of detection limits.

[0082] FIG. 3 is a flow diagram show steps in a method 100 for evaluating a breath condensate sample for the presence of a target that is an embodiment of the invention. The method 100 may use the apparatus shown in FIGs. 1 and 2. The method begins with a step 102 of obtaining a breath condensate sample, e.g. by collecting exhaled breath from a user in a sample collection device. The method continues with a step 104 of delivering the collected sample to a detection device, e.g. by using a plunger to push the collected sample out of the modified syringe discussed above. The method continues with a step 106 of conveying the sample into a plurality of detection chambers, e.g. using any of the gravity feed structures discussed above. The method continues with a step 108 of testing the sample in each of the detection chambers and then a step 110 of determining presence of the target based on the results of the testing, as explained in more detail below. In other instances, this dynamic range can be expanded by including larger and smaller chambers. Analyte mass amounts can be determined with more precision by increasing the number of chamber volumes between the maximum and minimum volumes.

[0083] Examples

[0084] Example 1

[0085] A series of isothermal LAMP reactions were carried out in varying reaction sizes ranging from 5 to 200 pL and with concentrations of single-stranded DNA template varying from 0.01 to 2.5 copies / pL. The reaction sizes and template concentrations are shown in Table 1 below, with the total copy count of each sample.

[0086] Table 1 : Reaction chamber size and template concentration

[0087] Each reaction sample contained Codex HiRev Isothermal DNA Polymerase, Codex HiRev Reaction Buffer and SYBR Green I dye at a concentration of 1X, per manufacturers guidelines, 1.1 mM dNTP mix, and 1.6 pM of FIP and BIP primers, 0.4 pM of FL and BL primers, and 0.2 pM of F3 and B3 primers. Single stranded DNA template was added to separate aliquots reach concentrations of 2.5, 0.25, 0.025, 0.01 copies / pL. These aliquots were then each subdivided into reaction volumes of 5, 10, 20, 50, 100, and 200 pL. They were incubated for 60 minutes at 65°C, with fluorescent measurements collected every 30 seconds. Following amplification, a melt-curve was performed to verify the specificity of the amplification.

[0088] The limit of detection or detection limit was determined by the lowest amount of DNA to amplify. In this case, every amount of DNA above the limit of detection amplified as well. The limits of detection are shown in the Table 2 below both in terms of concentration and total copy number. In this test, every sample with a copy number greater than 1.25 was successfully amplified, and no test with less than 1.25 copies amplified.

[0089] Table 2: Limits of detection for different reaction chamber volume sizes

[0090] Under these conditions, sensitivity in terms of total detection limit is constant across a wide range of reaction sizes. By increasing the total reaction volume size, the detection limit is proportionally decreased, allowing for detection of the target even in more dilute samples than a smaller volume would allow. This is shown in Table 3 below.

[0091] Table 3: Samples that amplified* by concentration and reaction chamber size

[0092] *Samples that amplified and were detected because they were above the detection limit.

[0093] Example 2

[0094] A sample of exhaled breath condensate was collected from an individual who was infected with Sars-CoV-2. The sample was tested with RT-qPCR and determined to contain approximately 30 copies of virus per pL. Another portion of this sample was diluted to volumes of 50, 25, 10, 5, 1, and 0.5 pL. Each of these samples was then tested using RT- LAMP, in an assay with a detection limit of 50 copies of virus. The protocol for this test was identical to the LAMP amplification in Example 1, with a total reaction volume of 100 pL. The results are shown in Table 4.

[0095] Table 4: LAMP results from Sars-CoV-2 sample dilutions

[0096] In each sample wherein the copy count exceeded the test’s detection limit, the viral RNA amplified. In the samples in which the copy count was below the detection limit, amplification did not take place within 30 minutes.

[0097] With a known detection limit of 50 copies of virus, the minimum possible concentration of virus can be estimated based on which dilutions amplified. The minimum concentration is defined as the detection limit divided by sample volume as shown in Table 5.

[0098] Table 5: The minimum analytic mass concentration of virus to amplify in varying sample volumes

[0099] Using this approach, the analyte mass concentration is predicted to be between 10 - 50 copies / pL, which agrees with the qPCR quantification of 30 copies / pL.

[0100] Example 3

[0101] In one embodiment of the invention, a known volume of biological sample is prepared. In various embodiments, this could be less than 10 pL, 10 pL, 20 pL, 25 pL, 30 pL, 40 pL, 50 pL, 60 pL, or more than 60 pL of sample. A known volume of diluent is added and mixed with the sample, and the same volume is transferred to the first reaction vessel for analysis. This volume may be more or less or equal to the volume of the sample. In this example, the reaction chambers are all the same volume. An additional volume of fresh diluent is then added to the remaining mixture of the sample and diluent, diluting it further, before the volume is transferred to the second reaction vessel for analysis. This process may be repeated 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 96, 100 times or more than 100 times. The dilution factor is defined as the ratio of the sample volume to the total volume of the sample with the diluent added.

[0102] In one instance, a 30 pL sample is prepared and diluted with 120 pL of diluent, creating a total mixed volume of 150 pL. 120 pL is transferred to analysis chamber 1 , leaving 30 pL of diluted sample behind. This sample in analysis chamber 1 is five times more dilute than the original sample. A fresh 120 pL of diluent is added to the diluted sample, becoming five times more diluted. 120 pL of this is transferred to analysis chamber 2. The process is repeated until 8 analysis chambers are full, each with five times lower mass amount of sample and target as the previous chamber. The overwhelming majority of the original sample is distributed between the 8 analysis chambers. With this arrangement and a detection limit of 50 copies of the target, it is possible to estimate the copy number from a range of 40 - 3.1 million copies in the original sample. In this example, the chamber with the highest detection limit that does detect the sample defines the minimum amount of target in the sample. The chamber with the lowest detection limit that does not detect the sample defines the maximum possible amount of target in the sample. This is shown in Table 6.

[0103] Table 6: Required number of copies for detection in serial sample mass dilutions

[0104] Example 4

[0105] In one embodiment of the invention, the mixing and dilution of the primary sample into a progression of mass concentration dilutions is achieved through a gravity-fed mechanism as shown in FIG. 1. The diluent is stored in a reservoir at a higher elevation than the other chambers. The sample is placed in a mixing chamber at a lower elevation than the diluent reservoir, and several analysis chambers are disposed at a lower elevation. A fluidic channel connects the diluent reservoir to the mixing chamber. A second fluidic channel connects the mixing chamber to each analysis chamber in series, such that each chamber fills completely before the subsequent chamber begins to fill. The channel connecting the mixing chamber to the analysis chambers allows liquid to flow more quickly than the channel from the reservoir to the mixing chamber. This allows the mixing chamber to substantially drain into each successive analyte detection chamber. The fluidic channel exiting the mixing chamber protrudes vertically into the mixing chamber to a defined height, ensuring that the chamber never fully empties. The remaining liquid ensures that a fraction of the sample is retained to be diluted for subsequent analysis chambers. In this embodiment, the dilution factor is defined as the ratio of the retained volume to the total volume of the analysis chamber.

[0106] Example 5

[0107] In one embodiment of the invention, the target for quantification is a protein or other biomolecule. Proteins may be detected when the protein is consumed, combined with detection reagents, or detectable products are generated. As the primary protein sample mass is reduced between two or more chambers, the mass amount of the protein is reduced by the reduction factor. When the mass falls below the detection limit for a given reduction factor, the protein is not detected. In this case, the detection limit and reduction factor can be used to calculate the maximum concentration of the protein present. In the same manner, the mass reduction factor and detection limit can be used to calculate the minimum concentration of the protein that is present. The minimum and maximum concentrations of the target constitute the possible range of concentrations for the target.

Claims

Claims1. A method for evaluating a breath condensate sample for the presence of a target, the method comprising:(a) providing a primary breath condensate sample;(b) providing a device comprised of at least two detection chambers;(c) using gravity flow to fill the detection chambers with the breath condensate sample, wherein each detection chamber has a mass amount of breath condensate sample, wherein at least one detection chamber contains a smaller mass amount of breath condensate sample than the other detection chamber or chambers, wherein the breath condensate sample mass amounts in the chambers are related by a known reduction factor;(d) testing the breath condensate sample in the detection chambers for the presence of the target; and(e) scoring each detection chamber for the presence of the target.

2. The method of claiml , wherein the breath condensate contains nucleic acids and wherein the testing in step (d) is nucleic acid amplification.

3. The method of claim 2, wherein the nucleic acids in the breath condensate sample are amplified isothermally.

4. The method of claim 2, wherein the breath condensate sample is amplified by thermal cycling.

5. The method of claim 3 or claim 4, wherein the primary sample is diluted with a gravity-fed diluent in step (c) to obtain at least one detection chamber with a smaller mass amount of the breath condensate sample.

6. The method of any preceding claim, wherein at least one detection chamber has a smaller volume than the other detection chamber or chambers.

7. The method of any preceding claim further comprising, following step (e), estimating the target copy number for each detection chamber, wherein the lowest target copy number that produces a consistent positive signal in step (e) is the detection limit of the detection chamber.

8. The method of any preceding claim, wherein the volume of the primary sample is 20 pL or greater.

9. The method of any preceding claim, wherein step (d) is completed in 40 minutes or less.

10. The method of any claim 9, wherein step (d) is completed in 30 minutes or less.

11. The method of any claim 10, wherein step (d) is completed in 20 minutes or less.

12. The method of any claim 11, wherein step (d) is completed in 10 minutes or less.

13. The method of any preceding claim, wherein the known reduction factor is in the range of 2 to 10.

14. The method of any preceding claim, wherein the known reduction factor is in the range of 2 to 200 between any two detection chambers.

15. The method of any preceding claim, wherein total known reduction factor is 10 to 1016across all the detection chambers in the device.

16. The method of any preceding claim further comprising, following step (e), identifying the detection chamber with the smallest mass amount of breath condensate sample and a positive amplification result for the presence of target.

17. The method of claim 16 further comprising calculating the concentration of the target in the primary sample by multiplying the number of target copies in the detection chamber identified in claim 16 by the known reduction factor for that detection chamber.

18. The method of claim 7, wherein the target is detectable if the detection limit multiplied by the known reduction factor for the detection chamber is greater than the amount of target in the primary sample.

19. The method of claim 7, wherein the target is undetectable if the detection limit multiplied by the known reduction factor for the detection chamber is less than the amount of target in the primary sample.

20. An apparatus for evaluating a breath condensate sample for the presence of a target, the apparatus comprising: a receptacle for receiving a primary breath condensate sample; a detection device comprising at least two detection chambers; and a gravity feed structure providing a fluid communication path from the receptacle to the at least two detection chambers, wherein the gravity feed structure is configured to fill the detection chambers with a respective mass amount of the breath condensate sample, wherein at least one detection chamber is configured to contain a smaller mass amount of breath condensate sample than the other detection chamber or chambers, and wherein the breath condensate sample mass amounts in the chambers are related by a known reduction factor.

21. The apparatus of claim 20, wherein the detection chambers are configured to receive different volumes or different concentrations of the breath condensate sample.

22. The apparatus of claim 20 or 21 further comprising: a diluent reservoir containing a volume of diluent liquid; and a mixing chamber in fluid communication with the receptacle and the diluent reservoir and configured to provide a mixture of the primary breath condensate sample with the diluent liquid, wherein the gravity feed structure is configured to fill the detection chambers with a mixture from the mixing chamber.

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