WO2012149203A1 - Methods and systems of non-invasive detection - Google Patents

Abstract

Methods and systems for creating a reagent to sense analyte(s) within a gas sample can include providing a reagent that changes color when exposed to the analyte of interest and exposing the reagent to the analyte of interest. The reagent can be mixed with a material chosen to allow easy passage of gas flow through and over the reagent to increase the exposure of reagent to the analyte of interest.

Description

METHODS AND SYSTEMS OF NON-INVASIVE ANALYTE DETECTION

PRIORITY CLAIM

[001] This application claims the benefit of U.S. Provisional Application No. 61/479,313, filed April 26, 2011, which is incorporated herein by reference in its entirety.

FIELD

[002] The disclosure pertains to apparatuses and methods for collecting and analyzing breath samples to detect the presence of various substances, including those that are related to or indicative of physical conditions or diseases.

BACKGROUND

[003] Various diagnostic screening and testing methods are available to identify or quantify a medical or physical condition of an individual. Generally, these methods require the collection of a fluid sample (e.g. , blood, plasma, and urine) from a patient and the submission of that fluid sample to a laboratory for analysis. For example, there are diagnostic tests available for the quantification of the end products associated with lipid peroxidation. Lipid peroxidation is the process whereby free radicals cause cell damage in the body by removing electrons from lipids in cell membranes. Free radicals are often associated with the consumption of processed foods, alcohol, and the use of tobacco products, and have been implicated as a potential cause or aggravating factor in numerous disease processes. It is also commonly believed that organisms age, at least in part, because cells in the body accumulate free radical damage over time.

[004] Conventional diagnostic tests for lipid peroxidation typically require the collection of a blood, plasma, or urine sample from a patient. Such conventional diagnostic tests are somewhat undesirable, however, since they require the collection of a sample in a relatively invasive manner from the patient. Moreover, such conventional diagnostic tests can be expensive and time-consuming, since they typically involve labor-intensive laboratory analysis of the collected samples.

[005] Testing methods that are based on breath samples are particularly desirable since, unlike blood, urine, or other physical samples, breath samples can be easily obtained from an individual in a simple and non-invasive manner. For example, U.S. Patent No. 7,285,246, which is incorporated herein by reference in its entirety, discloses a hand-held fluid analyzer for detecting alcohol or other preselected substances in the fluids present in the exhaled breath of a test subject. The '246 patent relies on visual inspection of an indicator reagent to determine whether the preselected substance is present and, as a result, is limited in its ability to detect specific amounts or ranges of a preselected substance in the sample.

SUMMARY

[006] In some embodiments, a substantially dry Schiff reagent is provided. This component can be produced using lyophilization (e.g., instead of and/or in addition to heated drying) to combine an aqueous phase Schiff reagent with a material such as silica beads which allows for the gas being blown through the reagent to have lots of surface area for contact, and prevents the dry reagent from completely impeding gas flow.

[007] In some embodiments, laboratory samples can be delivered by email, digital stream, text stream, image stream for analysis at a location apart from the location where the physical test is being performed. If desired, the results of the analysis can be returned to the location of the physical test by email, text, or other such remote delivery.

[008] The methods and systems described herein can be used to detect and/or determine the amount of aldehydes in breath, which can be relevant in measuring an amount of oxidative stress.

[009] The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures. BRIEF DESCRIPTION OF THE FIGURES

[010] FIG. 1 illustrates the structure of an aldehyde, with the R group representing any of many carbon chains or rings.

[Oi l] FIGS. 2 and 3 are graphs depicting breath malondialdehyde increases as persons climb to altitude and decline, illustrating a direct correlation of breath malondialdehyde with the development of acute mountain sickness symptoms.

[012] FIGS. 4 and 5 are graphs depicting exhaled malondialdehyde (as an assessment of oxidative injury) correlates to exhaled levels of cobalt and chromium and Tungsten (not shown).

[013] FIG. 6 is a graph depicting a breath malondialdehyde increases after exercise in conditions of exposure to particulate air pollution.

[014] FIG. 7 is a graph depicting blood malondialdehyde, as assessed by TBARS post-exercise.

[015] FIG. 8 is a graph depicting two standard curves for propanaldehyde

(propanal) using the reagent technology and colorimetric measurement systems described herein.

[016] FIG. 9A illustrates the appearance of FRED 2S (Freddie 2 Short) Reagent (on paper).

[017] FIG. 9B illustrates the appearance of FRED 2S reagent in current production FRED tubes.

[018] FIG. 10 depicts a Fred 2S heating unit.

[019] FIG. 11 is a graph depicting a FRED 2 reagent responsiveness at PPM range.

[020] FIG. 12A is a graph depicting a FRED 2S and propanal.

[021] FIG. 12B is a graph depicting a FRED 2S and Acetaldehyde.

[022] FIG. 12C is a graph depicting a FRED 2S and Butanal.

[023] FIG. 12D is a graph depicting a FRED 2S and nonanal [024] FIG. 13A is a graph depicting a FRED 2S standard curve illustrating a relevant human breath PPB range.

[025] FIG. 13B is a graph depicting atmospherically exposed reagent sensitivity to relevant aldehyde concentrations.

[026] FIG. 14 is a graph depicting a graphical presentation of potential utility of 5 minute heating time, as opposed to 20 minute heating time, for FRED 2S assay.

[027] FIG. 15 is an appearance of FRED 2S reagent after heating. Left tube— humidified 0 ppb artificial breath gas. Right tube, Humidified 1000 ppb propanal

[028] FIG. 16 is a graph depicting a FRED 2S raw absorbance score for 11 subjects, back to back assays.

[029] FIG. 17 is a graph depicting a FRED 2S human subject scores, converted to PPB using propanal as standard.

[030] FIG. 18A is a graph depicting a FRED 2S mean raw score (absorbance) vs. Body Mass Index in 7 subjects.

[031] FIG. 18B is a graph depicting a FRED 2S raw score (absorbance) vs. Body Mass Index in 7 subjects with all replicates presented as opposed to simply the means.

[032] FIG. 19 is a graph depicting a FRED 2L low PPB standard curve.

[033] FIG. 20 is a graph depicting a FRED 2L extended standard curve.

[034] FIG. 21 is a graph depicting a FRED 2L standard curve, mid-range.

Different reagent baseline.

[035] FIG. 22 and 23 are graphs depicting an illustration showing heating curves for heating reagent samples for about 5 minutes.

[036] FIG. 24 is a graph depicting a comparison of the sensitivity of the FRED 2S reagent to available gas phase aldehydes.

[037] FIG. 25 is a graph depicting the same data presented using RGB color space. [038] FIG. 26 is a graph depicting that the reagent appears stable at moderately high temperatures for 24 hours. In the graph, H is the color reading, the reagent was heated for 24 hours at 50 C (about 125 degrees F) and then its response to propanaldehyde assessed.

[039] FIG. 27 is a graph depicting that the reagent maintains stability over time during refrigerated storage as evidenced by consistent performance in terms of propanaldehyde sensitivity over time, as shown.

[040] FIG. 28 is a screen shot of color assessment using photoshop, as examples of what can be accomplished using cellular or other camera methodology to send a sample electronically to a lab, and then what the lab does to perform the assay.

[041] FIG. 29 is a screen shot of color assessment using photoshop, as examples of what can be accomplished using cellular or other camera methodology to send a sample electronically to a lab, and then what the lab does to perform the assay.

[042] FIG. 30 is a screen shot of color assessment using photoshop, as examples of what can be accomplished using cellular or other camera methodology to send a sample electronically to a lab, and then what the lab does to perform the assay.

DETAILED DESCRIPTION

[043] The following description is exemplary in nature and is not intended to limit the scope, applicability, or configuration of the invention in any way. Various changes to the described embodiment may be made in the function and arrangement of the elements described herein without departing from the scope of the invention.

[044] As used in this application and in the claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. Additionally, the term "includes" means "comprises." Further, the terms "coupled" and "associated" generally mean electrically, electromagnetically, and/or physically (e.g. , mechanically or chemically) coupled or linked and does not exclude the presence of intermediate elements between the coupled or associated items absent specific contrary language. [045] Although the operations of exemplary embodiments of the disclosed method may be described in a particular, sequential order for convenient presentation, it should be understood that disclosed embodiments can encompass an order of operations other than the particular, sequential order disclosed. For example, operations described sequentially may in some cases be rearranged or performed concurrently. Further, descriptions and disclosures provided in association with one particular embodiment are not limited to that embodiment, and may be applied to any embodiment disclosed.

[046] Moreover, for the sake of simplicity, the attached figures may not show the various ways (readily discernable, based on this disclosure, by one of ordinary skill in the art) in which the disclosed system, method, and apparatus can be used in combination with other systems, methods, and apparatuses. Additionally, the description sometimes uses terms such as "produce" and "provide" to describe the disclosed method. These terms are high-level abstractions of the actual operations that can be performed. The actual operations that correspond to these terms can vary depending on the particular implementation and are, based on this disclosure, readily discernible by one of ordinary skill in the art.

[047] The systems and methods described below relate to non-invasive testing systems and methods of using such systems to identify the presence of various substances in the exhaled breath of a test subject. The substances can either be detected directly in the exhaled breath or in a condensate thereof. As discussed in more detail below, a testing system generally includes a breath collection device, a reagent contained in the breath collection device that exhibits a colorimetric reaction when exposed to a substance present in exhaled breath, and a measurement device that is capable of quantifying the colorimetric reaction resulting from the interaction of the substance in the exhaled breath with the reagent. In some embodiments, reagents and other components of the systems described herein are referred to as FRED reagents, tubes, etc. The term FRED simply refers to a shortened form of "Free Radical Enzymatic Device" and is not intended to be limiting in any way. For example, a FRED tube can be used with various reagents and to detect elements— it is not limited only to devices or reagents that are configured to detect free radicals.

[048] The section headings used herein are for organizational purposes only, and are not to be construed as limiting the subject matter disclosed.

[049] Aldehydes in human breath

[050] Unbalanced oxidative processes in the body cause production of volatile aldehydes as cells are injured. These aldehydes are exhaled and can be measured in human breath to serve as a rapid assessment of oxidative stress.

[051] Oxidation reactions are normal and necessary— even defining— components of aerobic life. Generation of oxygen free radicals occurs when one component of the complex system of cellular metabolism is not in harmony with the rest, when the immune system is responding to certain threats, and as a result of toxic exposures. There are elegant interactive detoxification mechanism present in tissues that— apart from stress and illness— prevent acute severe tissue oxidation and destruction.

However, even in healthy people these mechanisms are insufficient to completely prohibit the gradual chronic tissue injury caused by oxidation of cellular

components— the aging process. As an unfortunate twist, the detoxification methods for free radicals get less functional as people age, in part because they have been attacked by free radicals themselves.

[052] Oxidation of cellular components results when free radicals escape detoxification methods. Hydroxyl radical, superoxide, bleach, peroxynitrite and other strong oxidants react entirely indiscriminately on tissues, and thereby directly attack proteins and the structural lipids of cellular membranes (in a self-sustaining chain-reaction process of lipid peroxidation) with resulting formation of carbonyls (double bonds between carbon and oxygen). The membranes need to heal, and do so as well as they can, until the cell gradually loses its integrity, loses flexibility and function, fails, and dies.

[053] FIG. 1 illustrates a structure of an aldehyde. When cellular membrane lipid molecules are oxidized, the resulting carbonyl is in the form of an aldehyde moiety (shown in FIG. 1 with 'R' indicating a saturated or unsaturated carbon chain).

Unlike protein carbonyls or oxidative injury to DNA, the lipid oxidation aldehyde products are volatile, and are cleared to a great extent in the breath, making them readily measurable. For example, acetaldehyde is transferred from blood to breath 15 times as extensively as ethanol.

[054] Note that when human cell membrane lipids suffer oxidation to form lipid aldehydes, it is not an enzymatically regulated, controlled or favorable response, but rather is considered a harmful stress that occurs because of failure of control (failure of homeostasis). Indeed, this process is the prototypical "oxidative stress".

[055] The lipid aldehydes formed have various characteristics depending on the length and structure of the attached carbon chain ('R'). As noted, most of these aldehydes are highly volatile, and some are reasonably water-soluble. All are fairly reactive molecules themselves. The most common aldehydes are toxic—

formaldehyde and acetaldehyde both have unpleasant pharmacologic effects and are carcinogens. Others aldehydes are also well-known carcinogens, such as acrolein and crotonaldehyde. All the lipid aldehydes tend to be strongly scented or odorous. Some are pleasant and mostly harmless (the scent of cinnamon results from an aldehyde). But in contrast, some smell like one might imagine death to smell.

Indeed, aldehydes can be measured and may be the determinant of when olive oil becomes rancid. Malondialdehyde is the most studied of all the breath aldehydes because there is a readily available research assay for it.

[056] As cells attempt to heal themselves from oxidative injury, the free-radical- formed lipid aldehydes are released into the surrounding liquid, and emerge into the blood circulation, where they are soon carried to the lungs. The volatility of lipid aldehydes leads to these compounds being preferentially released from the blood and into human exhaled breath. Many aldehydes have been identified in breath, including the smallest (formaldehyde) and large multi-carbon aldehydes. Although there can certainly be significant oxidative activities in the lung tissue itself (e.g., in acute exacerbations of asthma) aldehydes in the breath have been shown to be systemically sourced, for example, in a variety of different cancers (including breast and prostate cancer) and not just when the cancer is in the lung itself.

[057] The aldehyde group on these free-radical attacked cell membrane lipids (e.g., as depicted above in FIG. 1) is measurable in exhaled breath using large machinery such as gas chromatography/mass spectroscopy as well as somewhat simpler chemical methods. Measurement of breath aldehydes can be useful in delineating oxidative stress occurring in disease states such as ranging and different as cancer and lung disease, as well as baseline formation of lipid aldehydes, which occur chronically during the process of aging.

[058] In some embodiments described herein, a dry Schiff reagent is provided to replace liquid phase Schiff reagents for aqueous aldehyde assessment. This reagent can be incorporated into a small, filtered plastic straw or other breath collection device. Exhaled breath of a person can be delivered to the straw or other breath collection device so that the reagent can capture the aldehydes in the breath. A color change by the reagent can be assigned a numerical value that correlates to the concentration of aldehyde in the breath. This process can be relatively rapid. In some embodiments, the test results can be available in just a few minutes after the breath sample is provided. Such tests can also be relatively inexpensive, with no massive and expensive laboratory machinery as had been required in the past.

[059] The methods and systems disclosed herein provide a profoundly enhanced sensitivity for aldehydes by greatly eliminating noise that has confounded most every other breath assay technology. In particular, the methods and systems described herein can be configured to be sensitive in a low part per billion (PPB) range, and reaches up to 1500 PPB or more. As the human breath aldehyde range is generally between 100 and 500 PPB, the methods and systems described herein are well-suited for human measurement. In addition, the method and systems described herein can also detect other analytes with new and/or improved sensitivity, including, for example, malondialdehyde and the known aldehyde carcinogen, acrolein as well as the other major volatile lipid aldehydes. [060] Many advantages can be provided by the methods and systems for measuring oxidative stress described herein. For example, these methods and systems improve the ability to identify the effects of health improvement interventions in people who already have good health. This is particularly useful because augmenting the duration and quality of life requires more than just fighting disease, it requires battling the normal process of aging that occurs in even the healthiest people. In addition, the uniquely sensitive methods and systems described herein provide information that can help mankind— and the individual person— identify the interventions most likely to be beneficial in combating chronic oxidative

deterioration of aging.

[061] Aldehydes in disease

[062] Oxidative stress has been poorly addressed by the academic medical community. It is frequently mentioned and respected as a critical component of disease, yet academics have failed to well study it or try to resolve it, perhaps in part because of certain biases inherent in the academic medical mindset. These biases include a strong tendency to lump various people into disease categories into which they may fit only poorly, and then attempt to use non-specific therapies on all the people in these artificial groups, with poor or no measurements of the immediate effects of the anti-oxidant regimen being applied, and little effort to adjust the regimen or the dosing based on biochemical efficacy in the individual. The academic tendency has been away from personalizing diagnosis and therapy, and this tendency has prohibited effective research. Academic medical research has provided few, and at most moderately effective therapies for oxidative stress— which is a highly individual process. However, there has been research that is increasingly reaching the mainstream academic that cannot be ignored, and the attention and funding by the National Institutes for Health is clearly following.

Personalized medicine is clearly on the rise, and people will be demanding the tools to measure their own health status, instead of relying on the results of big studies involving other people in order to determine their nutritional and other interventions. The MD world is beginning to get the message, but lags behind the nutrition and supplement fields.

[063] The military has been interested in oxidative stress longer than many other government organizations. Diseases related to high altitude and exercise therefore have received attention. Acute Mountain Sickness (intracranial and intrapulmonary pressure changes that occur in people moving rapidly to high altitude) is a disease in which the careful harmonization of normal aerobic oxygen metabolism is thrown askew. Breath malondialdehyde increases as soldiers climb to altitude and decline upon return to base camp. There is a direct correlation of breath malondialdehyde with the development of acute mountain sickness symptoms. Malondialdehyde in exhaled breath correlates with the degree of Acute Mountain Sickness (see FIGS. 2 and 3), and is affected by exertion in Marines working at moderately high altitude.

[064] Likewise, environmental toxicology research has received funding, and work in this field reveals malondialdehyde in breath to be correlated both which Cobalt and Chromium exposure FIGS. 4 and 5. Exhaled malondialdehyde (as an assessment of oxidative injury) correlates to exhaled levels of cobalt and chromium and Tungsten (not shown). This heavy metal toxin and aldehyde relationship is likewise found to be relevant in cigarette smokers.

[065] And as a more immediately relevant factor for most people, exhaled aldehydes (again in the form of the most studied— malondialdehyde) correlate to particulate air pollution exposure during exercise, supporting a role for caution in individuals who exercise outdoors in cities or along highways (FIG. 6). As shown in FIG. 6, breath malondialdehyde increases after exercise in conditions of exposure to particulate air pollution. This serves as an example of the desire to personalize the measurements and interventions to individual people and their situations.

[066] Also of relevance to individuals is the effect of acute highly strenuous exercise on oxidative stress. It is not commonly known that the efforts the body undertakes to control oxidative stress are overwhelmed during significant exertion. This has been measured using assays for serum aldehydes called the TBARS assay (FIG. 7) and is also being studied in the breath. FIG. 7 illustrates blood

malondialdehyde, as assessed by TBARS post-exercise. It has been suggested that this sort of acute strenuous exercise, although causing oxidative stress in the short term, does activate enzymatic anti-oxidant pathways more chronically, and is thus potentially beneficial to the overall health of individuals, by keeping their antioxidant pathways turned on day and night. Indeed, this positive effect of exercise on decreasing oxidative injury has been specifically identified during pulmonary rehabilitation in patients with chronic obstructive pulmonary disease, in which urinary aldehydes were used as the outcome measure.

[067] Additionally, elite triathletes who undertake a special three month breath holding training curriculum to improve tolerance to hypoxemia reveal a significant decrease in breath hold-related oxidative stress, as measured by blood aldehydes. Multiple antioxidant vitamins used together (Vit C, Vit E, and beta-carotene) lowered aldehydes in blood in subjects at rest, but failed to affect post-exercise blood aldehydes.

[068] Asbestosis and silicosis reveal high levels of aldehydes in exhaled breath, and indeed the breath aldehydes are better than aldehydes found in other body fluids in terms of distinguishing health from disease. Thus, the amount of breath that can be exposed to a reagent is substantially more than the amount of blood, thus improving accuracy and sensitivity of tests for volatile compounds that tend to move into the breath compartment from the body fluid compartment rapidly. Accordingly, patients with lung cancer can also be discriminated on the basis of breath aldehydes.

[069] Asthma and chronic obstructive pulmonary disease can be monitored using breath assays for aldehydes. Breath aldehyde testing is sensitive enough to be able to discriminate asthmatic children's variable exposure to air pollution.

[070] Cigarette smokers reveal higher levels of aldehydes in breath, and

interestingly, have been reported to have lower levels of hydrogen peroxide (a prototypical oxidant). Given the variable antioxidant defenses from person to person, the important issue is whether the body is being damaged or not, and that is best revealed by measurements of the damage itself as revealed by aldehydes, as opposed to measurement of the damaging compounds (peroxide). It has been shown that smoking cessation decreases malondialdehyde in the blood. As another example of the benefits of measuring aldehydes (evidence of injury) instead of the oxidants themselves, patients with Community Acquired Pneumonia (CAP) who are smokers have a higher exhaled breath aldehyde level than non-smokers, but a lower exhaled hydrogen peroxide level. This attests to the several chemical pathways of oxidation, but the ubiquitous nature of the aldehyde signal when cellular oxidation occurs.

[071] Obstructive sleep apnea causes free-radical oxidative stress as well.

Malondialdehyde is elevated and antioxidant defenses are altered in children who have obstructive sleep apnea, and normalize after adenotonsillectomy.

[072] That more effective antioxidant regimens are needed is supported by the failure of vitamin E supplementation to alter blood and breath aldehydes in patients with congestive heart failure.

[073] Most of the volatile contributors to exhaled breath reflect systemic processes— as exemplified by carbon dioxide in the breath being formed throughout the body. Although many of the studies in the literature that use breath aldehyde assessment are focused on pulmonary conditions, this is a simple reflection of the interest of those medical researchers who study breath tests— primarily the lung physicians. However, aldehydes are sufficiently volatile, and are formed in every tissue undergoing oxidative stress, that the breath aldehydes measured are now thought by most to indeed reflect systemic oxidative insults primarily. This is in contrast to non-volatile constituents in exhaled breath such as cytokines and salts, which clearly reflect lung processes.

[074] The methods and systems disclosed herein provide improved sensitivity for breath aldehydes to the point where measurement of them has relevance for even people with low aldehydes at baseline. In some embodiments, a dry powder reagent is provided that lessens noise and increases responsiveness to a broader range of aldehydes present in breath. In addition, results can be provided from a breath test using the dry powder reagent in a relatively shortly time. In some embodiments, the results are provided within 15 minutes, more preferably, less than 10 minutes, and, even more preferably, less than 7 minutes (e.g., about 6 minutes).

[075] In some of the examples described herein, propanal is used as a standard test gas because it is a mid-sized aldehyde that is a known component of breath, and provides a very robust test-bed when working in a PPB assay range.

[076] Linearity of the assay down to zero, and batch-to-batch reproducibility is excellent. Two batches made 5 days apart reveal substantially similar standard curves (range 0-800 PPB) (see, e.g., FIG. 8). FIG. 8 illustrates two standard curves for propanaldehyde (propanal) using the reagent technology and colorimetric measurement systems described herein. The curves from two separate batches of reagent are nearly identical, have excellent linearity, attesting to the robustness of the assay methodology. Note— human breath range is generally 100-500 PPB, at least in health, making this assay particularly useful. The regression lines for these standard curves are in the 95% or higher range, which is clear support for the robustness of the assay methodology.

[077] Other exhaled breath constituents such as nitrogen, oxygen, carbon dioxide, and water do not react with the reagent, reducing the occurrence of noise. High levels of acetone alter the reagent in a different and recognizable manner than the reaction with aldehydes and is not likely to be a confounder but requires some investigation in diabetics. Preferably, a controlled volume of exhaled breath is delivered to the reagent. The controlled volume can be, for example, about 2 liters.

[078] The human range identified with this technology has been determined in small studies to be from approximately 100 PPB to 500 PPB in subjects not acutely ill. The methods and systems described herein operate effectively in that range. Note that the technology can identify a summation of multiple aldehydes in the breath, and not just malondialdehyde, providing broad applicability. Back-to-back reproducibility in human testing has shown coefficients of variation of 10% and lower.

[079] The reagents described herein have been tested with specific available gas- phase aldehyde standards including propanal, acetaldehyde and butanal with similar responsiveness. The reagents also react readily with the vapor phase of

formaldehyde, malondialdehyde, and acrolein.

[080] Aldehydes are formed not only from oxidation of lipids, but also from dehydrogenation of alcohols. Because methanol (convertable to formaldehyde) and isopropyl (convertable to propanal) are toxic to ingest, ethanol is the only relevant dietary alcohol for which we may need to control. Alcohol ingestion prior to testing may confound the assay, particularly in certain subjects who have aberrant versions of the aldehyde dehydrogenase enzyme (common in Asians). The people who have this aberrant enzyme accumulate acetaldehyde in the blood that causes facial flushing and illness upon consumption of ethanol. Such factors should be considered in reviewing a test result if applicable.

[081] The methods and systems described herein also require smaller volumes of reagent in the sampling tube than conventional systems, making it substantially easier for the patient to breathe through the sampling tube. In some embodiments, the systems require 2.5 liters of exhaled air or less, and more preferably 2.0 liters of exhaled air or less. Accordingly, a sufficient sample can generally be accomplished with one breath for patients whose lung capacity has not been adversely influenced by lung disease or other conditions. Children can perform the tests with two breaths.

[082] Using the methods and systems described herein, aldehydes, which are formed during oxidative insult to cells and released into the blood and then into the breath, can be readily measured. The disclosed methods and systems can be provided to be sensitive to below 100 PPB, making these methods particularly effective within the human breath range of 100-500 ppb. In addition, the reagents described herein exhibit robust reproducibility of manufacture and assay

characteristics, and are responsive to a broad range of relevant breath aldehydes. [083] In some embodiments, the reagents can be heated before colorimetric reading. For example, in one embodiment, a 5-minute heating step can be provided. Such methods and systems provide both a much improved human use experience and a broader assessment of relevant aldehydes that both indicate oxidative stress and that can be toxic themselves. Given the degree of accuracy and sensitivity of the methods and systems described herein, the methods and systems described herein should be the evaluative method of choice as a biomarker to determine efficacy of nutritional and lifestyle interventions related to oxidative stress not only in ill people, but in healthy individuals as well.

[084] In various embodiments, the methods and systems described herein provide at least some of the follow improvements and advantages, making these methods particularly help for point-of-care services. Specifically:

1. Numerous difficulties in creating a dry Schiff reagent were recognized and eliminated and/or greatly reduced, including a notable loss of Schiff during heating/drying; variability in source dye; sensitivity to a variety of non-aldehyde components in breath; unacceptable sensitivity to aldehydes; lack of sensitivity to lower molecular weight aldehydes.

2. The dry Schiff reagents described herein are sensitive to a variety of aldehydes including low molecular weight aldehydes (tested in gas phase in a human breath simulation) in the range that is found in actual human breath.

3. There is minimal to no water noise with the reagents described herein.

4. There is minimal to no C02 noise with the reagents described herein.

5. The reagents described herein provide beneficial marketability features, including point-of-care testing and results, or lab-based assays.

6. The reagents described herein are generally inexpensive to manufacture with very inexpensive ingredients as described in more detail below. 7. The tube designs described herein ensure that the spectroscopic readings are not adversely affected by the tube and plastic.

8. The reagents described herein have exhibited sufficient stability.

9. Coefficient of variation in humans has been found to be relatively low.

For example, in some embodiments, variation has been found to be less than about 7 %.

10. The measure of human intrasubject CV compared to intersubject range is adequate (e.g., less than 0.5).

11. In early human studies, there is an attractive correlation between results obtained using the systems and methods described herein and human Body Mass Index.

[085] Example 1 - Dry Schiff Reagent

INGREDIENTS:

sodium metabisulfite (MBS)

pararosanilin hydrochloride (PRHC1) in DDI H20

INSTRUCTIONS

Combine the PRHC1 and the MBS solutions in a 1:20 v/v ratio

Let solutions react to completion

Filter solution if needed

Add silica gel

Lyophilize without heat until reagent is completely dry.

Once reagent is dry, transfer to a glass container with < 20mL of head space.

Note: avoid exposure to atmosphere.

This reagent can identify atmospheric aldehydes, and must be protected from them for best results. [086] Examples of lyophilized reagent are shown in FIGS. 9A and 9B. The appearance is slightly pink; however, if desired, this can be converted to a white color by means of certain post-manufacture interventions (which can be minimal). It may be preferably to maintain the illustrated appearance, however, to avoid reducing the level of reproducibility of the reagent to the manufacturing process. Other dyes can be provided to make the color change on breath exposure more "exciting".

These post-manufacture interventions can be added on to a manufacturing protocol easily and at a little relative cost.

[087] 40 mgs of reagent is placed in a FRED tube, which is preferably protected from ambient air/ambient aldehydes. In some embodiments, the reagent is placed in the tube under argon or otherwise sealed.

[088] Example 2 - Methods of using reagents.

[089] To obtain improved aldehyde sensitivity in the human breath range, the systems described herein can be used in the following manner.

1. Subject blows through a tube for 4 liters. 2 or 3 liters (or less) can be acceptable; however, modestly more accuracy and sensitivity may be able to be obtained with higher exposure volumes.

2. After breath exposure, the exposed reagent can be heated to 75 degrees C for 5 to 20 minutes (longer or shorter can also be accomplished, and different temperatures can be used.

3. Addition of acid (300 microliters) to the reagent.

4. Immediately followed by measure of the absorbance at 572 nm of the colored fluid that is formed by the acid reacting with the reagent.

5. A number (FRED 2S score) is created. [090] Example 3 - Assay:

[091] The reaction speed is partially dependent on aldehyde concentration;

therefore, in one embodiment that uses the FRED 2S system it can be beneficial for the patient to blow 4L of breath through the tube as opposed to 2L. However, in other embodiments (including, for example, the "FRED 2 Long" embodiments described herein) 2L can be sufficient.

[092] The patient can blow 2L of breath, pause, and preferably shake the tube to agitate the reagents (actually exposing initially unexposed surfaces of the reagent). The patient can then continue to blow through the tube for an additional 2L. This may be stressful for the patients that have limiting respiratory conditions (asthma, COPD, etc.) but is advantageous for the sensitivity of the reagent.

[093] As noted above, a 3 liter breath or less can also be acceptable for the FRED 2S system or for other systems. However, it should be noted that there does not appear to be a need to perform the 4 liter exposure all at once. Therefore, a rest break of up to several minutes can be acceptable, if needed. The assay has also been tested successfully down to 2 liters of breath exposure. For subjects that have high aldehyde levels, breath exposures can be acceptable at even lower volumes.

[094] After the tubes have been exposed to human breath, they can be heated at 75°C for five minutes (although other heating times and temperatures are

acceptable). After heating, the assay continues with addition of acid and

spectrophotometric reading or a photograph can be taken and color effects determined using graphic image software or text stream assessment.

[095] Example 4 - Bench assay

1. ) Have patients exhale 2 liters into a FRED tube in a vertical position; remove the tube from mouth, shake it, and then exhale an additional 2L through the tube. This can be done with 2 liters at once, or several breaths that add up to 2 liters.

2. ) Remove the top piece from the FRED tube and transfer the exposed reagent into a 1.5mL microcentrifuge tube. 3. ) Immediately transfer the tube to a microcentrifuge tube heater.

4. ) Heat at 75°C for 20 minutes, no shaking required.

5. ) Prepare a 96 well microplate and a reader set to read absorbance at 572nm and a pathlength correction for 175uL.

6. ) Once the plate and reader are ready, set the plate in the reader with the plate exposed

7. ) Open the 1.5 mL microcentrifuge tube, pipette in 300uL of H₃PO₄ (1M).

8. ) Immediately close the tube and shake for 2-3 seconds, allowing all of the reagent to be exposed to the acid.

9. ) IMMEDIATELY after the silica has settled to the bottom of the microcentrifuge tube, leaving the liquid at the top— a process that happens spontaneously over 10-20 seconds— carefully pipette off 175uL of the supernatant into a well of the plate. Be sure to LEAVE SILICA in the microcentrifuge tube (pipetting must be carefully done). The silica will be discarded. Comments: It is preferable that each sample of reagent is exposed to the acid for the same amount of time.

10) Measured Absorbance at 572 nm is then compared to standards. [096] Example 5 - Production assay

1) Have patients exhale 2 liters into a FRED 2 tube in a vertical position; remove the tube from mouth, shake it, and then exhale an additional 2L through the tube.

2) Place FRED 2 in REVELAR 2 Warmer attachment auxiliary unit (see FIG. 10) for 20 minutes. In some embodiments, the time can be less than 20 minutes, and preferably, less than 10 minutes, and even more preferably, about 5 minutes or less.

3) Remove the tube from the heater and squeeze the upper portion to fracture the glass vial, releasing onto the reagent 300uL of H₃PO₄ (1M) (phosphoric acid) that is within the glass vial. (Note, this is not a particular harsh or concentrated acid, but care should be taken to assure it won't spill into machine or onto skin.)

4) Shake the FRED 2S tube to mix the liquid with the powder reagent. This step can be automated.

5) Place the tube in the REVELAR reader.

6) REVELAR uses an internal spectrophotometer to read absorption of the liquid at about 572 nm. Alternatively, an RGB sensing chip and LED light source can be provided, or a CCD/CMOS chip can be used to take a digital image of the sample.

7) REVELAR provides this absorption or color assessment information on the screen. The score can reflect a range, called the FRED SCORE. In some embodiments, the range can be between about 400 to 800; however other ranges can be provided to present the user with, for example, a "grade" (A through F) or color scale (Green through Red) or any other type of scale that marketing thinks is appropriate.

[097] Note: preferably, each sample of reagent is exposed to the acid for the same amount of time.

[098] Under some circumstances, each batch of reagent may be slightly different. In such cases, it can be desirable to quality check each batch using a gas

standardization system. A "universal" standard curve can be provided for every batch of reagent that was properly produced. The slopes of the standard curves are very similar among various batches of reagents, however the intercepts may vary slightly. A change in intercept from batch to batch will lead to an offset in measurements when comparing one standard measured by two batches. This can be corrected with an addition or subtraction step (a batch correction factor). This correction may not be needed if the batch to batch consistency is acceptable.

However, if desired and/or needed, a correction factor can be added.

[099] A programmable calibration or correction factor can be reasonably automated to the individual batch or such a correction factor can be incorporated in the central laboratory for the remote LDT version of the assay. This can involve one of two very simple techniques. First, in a preferred embodiment, REVELAR 2 can be configured to read a bar code or other indicator on the tube that represents a number from 1 to 10 that indicates within 10% the relevant correction factor.

Alternatively, the user could enter such a number manually.

[0100] Second, a blank/unexposed measurement can be made as a comparator. This is not necessarily simply placing an unexposed tube through the assay, although it COULD be). NOTE— this correction factor step may not be necessary, however, it can be added if desired to improve consistency of measurements and/or if reagent production batches vary more than a certain amount.

[0101] Utility and characteristics of assays

[0102] With the larger variety of propionaldehyde gas concentrations that are commercially available, we were able to test the accuracy and reproducibility of the current reagent to propionaldehyde in detail, and this is considered our primary test gas. We focused on examining the lower ranges (200-600ppb) for FRED 2S that are more applicable to what we would/should see from human breath (subsequent figures).

[0103] With the new FRED 2S reagent, we received much improved signals, as is revealed in FIG. 11. There is a clear and significant difference between means at 0, 1.7 and 4.6 ppm as well as no overlap among these data. (Note: This is a somewhat different spectrophotometer measurement, so different Y axis then subsequent figures.)

[0104] Effect of moisture/water on assays

[0105] Noise caused by variable humidity absorbed during breath exposure can interfere with readings, significantly limits the results obtained by a reader.

Accordingly, it is desirable to eliminate or reduce the effect that water or moisture has on a test result.

[0106] The reagents described herein, including the FRED 2S reagent, are not significantly affected by water. For example, to determine the effects of moisture, the FRED 2S was examined using our bench gas exposure system and relevant vapor phase water exposures as well.

[0107] Reagent was tested with different volumes of humidified gas. Four liters of humidified artificial breath gas were delivered to four tubes containing reagent. Six liters of the same humidified gas were delivered to four additional tubes. These tubes were then subjected to the FRED 2S assay (heated for 20 minutes at 75°C, treated with acid, and their absorbances read in the spec as per the methodology described above). Using t-testing, it was found that there was no significant difference (p=0.567) between the mean absorbance values for each set of four tubes exposed to different volumes of humidified gas, however there was a indeed a significant increase (p=0.021) in the mean weight of each tube confirming that there was indeed greater water absorption during the longer exposure. Regression of water tube weight increase (water added) to absorbance revealed an insignificant R- square of 0.02, indicating no association between water added and absorbance. This indicates that increased water being delivered to the reagent did not affect the absorbance values (or FRED 2s scores).

[0108] Effect of CQ2 on assays

[0109] C02 was not found to have a significant effect on the absorbance value of the reagent. The sensitivity of the reagent to humidified 100% C02 and humidified breath gas (5% C02, 21% N2, 74% 02) was tested to ensure that the reagent is accurately measuring exhaled aldehydes and not a component of breath. 100% C02 did not increase absorbance of the reagent when compared to an identical flow of breath gas.

[0110] Effect of gas Aldehydes on assays

[0111] A brief comparison of various gas aldehydes (Figures 12A-D) was performed to ensure that the reagent reacts to aldehydes other than the main test gas (propionaldehyde). In this experiment, each gas phase aldehyde was delivered to the reagent in concentrations of both 500ppb and lOOOppb, which is in the human exhaled breath range. The reagent batch used in this test had been exposed to atmospheric air in the lab for an extended period and had absorbed ambient aldehydes, giving a somewhat higher baseline absorbance. As noted above, atmospheric exposure is preferably avoided in the production/commercial system. The FRED 2S reagent reacts to different aldehydes although we were not able to detect nonanal below 1000 ppb using the FRED 2S system, at least with this overly atmosphere-exposed batch. The FRED 2 Long assay, discussed below, was able to detect nonanal.

[0112] Additional description of the FRED 2S reagent characteristics.

[0113] FIG. 13A illustrates a FRED 2S standard curve in an aldehyde range relevant to human breath. The axis are noted in reverse, because this is to serve as a standard curve, and for actual breath samples, the input would be absorbance, and the output variable is PPB (or some other score). As shown in FIG. 13A, the reagent is sensitive to aldehydes in a range that is applicable to human breath (0 - 600ppb) and that there is only slight overlap with significant differentiation among the means at each concentration of aldehyde.

[0114] Storage of Reagent (Pre Exposure)

[0115] We have tested the reagent under several conditions in order to establish the optimal storage method. There are a few variables that should be controlled, if possible.

[0116] The reagent can be stored at room temperature (approx. 25°C) or at any temperature below that. It is unadvisable to expose the reagent to high heat.

Heating may cause a non-permanent color shift. Though the color shift is non- permanent, it should be avoided to ensure that it does not cause "noise" during the assay process. We recognize that shipping may expose reagent to higher

temperatures if not shipped in a cool container (which is easy to do).

[0117] It is also desirable that the reagent be kept under air-tight conditions. Any bench reagent storage container should preferably be sealed with parafilm.

Additionally, it is beneficial to reduce the amount of headspace in the storage container. Packing under argon or other gases can also be desirable. [0118] However, even if the reagent is inadvertently exposed to room air aldehydes, it can still function effectively as evidenced by FIG. 13B (showing an

atmospherically exposed reagent sensitivity to relevant aldehyde concentrations) with a notably high and variable baseline absorbance.

[0119] To clarify, note that even when the reagent has been undesirably exposed to room air aldehydes, (as evidenced, for example, by the high and more variable baseline absorbance in the above figure), the reagent can still exhibit good sensitivity to propanal. That is, the reagent doesn't go 'bad' when it turns pinker with room air exposure. However, because a reagent effectively measures the room air aldehydes after exposure, it is preferable that the reagent not be exposed excessively to room air prior to use. Also, if possible, manufacturing should be performed in an aldehyde free setting with minimal exposure to room air. In addition, the reagent powder is preferably loaded into tubes, which are then sealed from atmosphere (argon filled wrappers). The room air aldehyde absorbance takes place over 30 minutes, so brief exposures are insignificant unless ambient aldehyde levels are high.

[0120] If the reagent is stored under the proper conditions it has the potential to sustain its reactivity indefinitely. Under laboratory conditions we have used the same batch of reagent for several months. We believe that the reagent will maintain a consistent sensitivity to aldehydes for at least 6 months, and probably significantly longer.

[0121] Assay heating time

[0122] In some embodiments, during the FRED 2S process the exposed reagent is heated to about 75 degrees C for about five minutes before being assayed. Table 1 and FIG. 14 illustrate such embodiments. FIG. 14 illustrates a graphical

presentation of potential utility of 5 minute heating time, as opposed to 20 minute heating time, for a FRED 2S assay. However, it should be understood that heating times and temperatures can be adjusted as desired to improve performance. Table 1. Potential utility of 5 minute heating time during FRED 2S assay

5 min heat @ 75 °C

ppb Abs. Mean Std. Dev. CV

0 581

0 735

0 649

0 683

662 64.54972 0.097507

600 776

600 777

600 793

600 795 782 9.539392 0.012199

[0123] Color change of reagent

[0124] The F2S reagent will change colors when exposed to aldehydes. The reagent starts out pink and will transform to a darker pink/purple color the intensity of which is relative to the amount of aldehyde being delivered. This color change is measured at 572 nm. Only a modest color change occurs upon breath exposure, but this is greatly enhanced by heating the reagent to 75 degrees C for 5 minutes, which greatly accelerates the Schiff reaction. Alternatively, the exposed reagent can just be left at room temperature for 24 hours, and the heating step can be eliminated.

[0125] FIG. 15 illustrates an appearance of FRED 2S reagent after heating, with the left tube being humidified 0 ppb artificial breath gas and the right tube being humidified 1000 ppb propanal. FIG. 15 illustrates the difference in the color of the FRED 2S reagent after undergoing the FRED 2S assay (heating). The

microcentrifuge tube on the left has been exposed only to humidified zero gas, and the right hand tube was exposed to 1 PPM humidified propanal. This is the color differentiation that the spectrophotometer is analyzing. As shown in FIG. 15, the change is readily distinguishable even to the human eye.

[0126] Human range data

[0127] Experiments with human subjects using the FRED 2S reagent have provided encouraging and interesting results. Tests show individuals will normally range from 0-600 ppb of exhaled aldehyde (FIG. 16 and 17). FIG. 16 illustrates FRED 2S raw absorbance score for 11 subjects (back to back assays) and FIG. 17 illustrates FRED 2S human subject scores (converted to PPB using propanal as standard). We have found an average intrasubject coefficient of variation (CV) of 0.066 (6.6%) and a Hunt quotient of 0.459 (11 subjects).

[0128] Additionally, we see that aldehyde concentration is correlated with body mass index BMI and estimated body fat % (FIGS. 18A and 18B). FIG. 18A illustrates a FRED 2S mean raw score (abundance) vs. Body Mass Index in 7 subjects, and FIG. 18B illustrates FRED 2S raw score (absorbance) vs. Body Mass Index in 7 subjects. All replicates presented as opposed to simply the means.

Accordingly, there is an apparent association with other clinical indicators of health such as BMI.

[0129] Example 6 - FRED 2 Long

[0130] "Long acting" test can also be provided. FRED 2 LONG (F2L) is an embodiment that includes the same reagent as in F2S, but provides a longer exposure period. In this example, the breath exposure can be about 2 Liters or less; however, larger volumes can be used as well if desired. After breathing, the sample tube is then processed 24 hours later by addition of the acid with immediate absorption measurement exactly as described for F2S. Thus, the 20 minute heating step of F2S is effectively replaced with a 24 hour room temperature step for F2L. Otherwise, the tests are substantially identical (except that, as noted above, 2 L of breath can be used for F2L). [0131] The longer reaction time provides enhanced sensitivity to aldehydes (Figures) even without the heating step. However, the long acting reagent can have a greater degree of variability than the short acting reagent. This may be, at least in part, due to the lower volume breath sample. Additionally, variability can be introduced by lengthening the period between measurements (e.g., from 24 hours to several weeks after exposure). This may result in part from a failure to keep the samples completely free from room air contamination. Therefore, it is desirable to know the date of the sample to facilitate accounting for such changes such as in the case where patients to mail their samples to a testing center, which would lead to a large variety in assay times between each sample. There is little overlap between gas exposures, however there is also increased variability within each set as compared to F2S.

[0132] FIG. 19 illustrates a FRED 2L low PPB standard curve, FIG. 20 illustrates a FRED 2L extended standard curve, and FIG. 21 illustrates a FRED 2L standard curve, mid-range with a different reagent baseline.

[0133] iPhone application-based system

[0134] The pictures above of the FRED 2S reagent were taken in relatively uncontrolled conditions with an iPhone, which provides images with readily interpretable colors. It is very reasonable to consider that such pictures, of an exposed reagent net to a color calibrator, could be obtained with an iPhone app, when an exposed reagent tube is placed into a Pulse-supplied box with an intrinsic color calibrator (a pink square). The app then sends the picture to a server, which measures the color of, for example, 100 pixels using appropriate color-reading software (e.g., Photoshop) and averages them, compares to the calibrator(s), and adjusts if necessary based on the batch number (also supplied by the picture of the exposed tube). All this can be done automatically. Indeed, the assessment can be performed by picking up the color codes of the pixels in the image data stream, without the additional step of converting to Photoshop. [0135] Difficulties can be experienced fine-tuning the 800 ppb control gas. This is caused by the very low flow in one bottle required to obtain this concentration. Even with very high end regulators, it is very hard to get this just right. Note that these difficulties do not reflect a defect of the reagent, but of the standards.

[0136] FIGS. 22 and 23 show heating curves for heating reagent samples for about 5 minutes. A comparison of the sensitivity of the FRED 2S reagent to available gas phase aldehydes is shown in FIG. 24. Note that the reagent is more sensitive to propanaldehyde than nonanal, butanal and acetaldehyde. Also note that the reagent generally is more sensitive based on water solubility of the aldehyde

(prop>butane>nonanal). Acetaldehyde is an exception to the water solubility consideration as a determiner of reagent reactivity. These tests are limited by the commercial availability of aldehyde tests gases. FIG. 25 illustrates the same data presented using RGB color space. Note that these data were obtained by taking a picture using an iPhone 3G.

[0137] The reagent appears stable at moderately high temperatures for 24 hours. FIG. 26 shows a graph with H as the color reading. The reagent was heated for 24 hours at 50 C (about 125 degrees F) and then its response to propanaldehyde assessed.

[0138] The reagent maintains stability over time during refrigerated storage as evidenced by consistent performance in terms of propanaldehyde sensitivity over time, as shown in FIG. 27.

[0139] Screen shots of color assessment using photoshop, as examples of what can be accomplished using cellular or other camera methodology to send a sample electronically to a lab, and then what the lab does to perform the assay. FIGS. 28-30 show three images of test tubes of exposed reagent with pixels selected from a grid. The color space determination of those pixels, and a process by which these color space values are averaged for a final complete valuation. One skilled in the art can see that this can be performed directly from the data stream as well, without translation through photoshop. Many more, or ALL the relevantly colored pixels can be incorporated into the image assessment.

[0140] As described herein, the color change in the reacted reagent can be collected with a dedicated spectrophotometer, or a cell phone camera, or a digital camera, or a CMOS or CCD chip in a special device, or it can be read by the human eye compared to color chip standards (visual analog scale). In some embodiments, one of more color chips can be incorporated into the measurement system that will allow for standardization/comparison of color readings or different cameras, cell phone cameras, CCD, CMOS chips.

[0141] Tubes with one or more standardized stable colors can be incorporated with the system to be used to calibrate the instrument on site, or to identify calibration factors that will require adjustments as a central server if the sample is sent digitally to the central lab.

[0142] The sample's reagent manufacturing lot number can be read using bar code or equivalent technology and transmitted to the reading machine or remote laboratory server.

[0143] The device can use an unexposed breath tube as a standard to determine if the reagent is likely to still be functional

[0144] In some embodiments, laboratory samples can be delivered by email, digital stream, text stream, image stream, or other electronic means.

[0145] As described in more detail herein, a dry Schiff reagent can be provided that is stable, consistent, and that has good reproducibility. In some embodiments, the reagent is formed using lyophilization instead of heated drying to combine an aqueous phase Schiff reagent with a material such as silica beads which allows for the gas being blown through the reagent to have lots of surface area for contact, and prevents the dry reagent from completely impeding gas flow.

[0146] In other embodiments, iPhone apps, software applications, or other mobile technology that now exists or can be envisioned can be used to provide colorimetric processing to complete an assay. Alternatively, such technology can be used to send test information (e.g., a color image) via text or other standard modality to a central laboratory server which can then send the information back to the user.

[0147] In other embodiments, one or more color controls can be provided to assist the process. For example, a white box with or without embedded/incorporated colored paint chips (or other stable color presentation method) can be used along with a digital camera, iPhone, Droid or other such device. After exposure to the gas sample, the reagent can be placed in the color control and a picture can be taken.

[0148] The methods and systems described herein are not limited to exhaled breath, but can be used for other purposes. For example, the methods and systems can be used for environmental assessments. Additionally, it can be used for headspace assessment above wine vats or in connection with foods being prepared for human or animal consumption.

[0149] An algorithm using RGB or any other color space can be made that takes the raw data and presents a score that can be numerical (continuous or discrete), or categorical. Such a score may, for example, be presented as a "propanaldehyde equivalent score", meaning that the subject has the equivalent amount of aldehydes in their breath as would read out with a, for example, 400 ppb propanal exposure.

[0150] The reagent breath exposure times can be varied (shortened, decreased volume) if necessary for people who have very high aldehyde levels.

[0151] The breath maneuver can modified to discriminate alveolar release of aldehydes from airway release of aldehydes into the exhaled air stream.

[0152] The system can be used for measuring ambient aldehydes before breath assessment to ensure that ambient levels do not significantly contaminate the human readings.

[0153] The tube material can affect the reading using various imaging technologies. Accordingly, optimal materials can be selected depending on the particular reader being used. For example, in some embodiments, the tube can be formed of polypropylene. [0154] In one preferred embodiment, a subject breathes through a 3.5 inch hollow tube in which a powdered reagent (e.g., a dry Schiff reagent mixed with fine beads such as silica) is held. The reagent can be protected from room air by being enclosed in a glass vial, and the glass vial can be fractured within the hollow tube by, for example, squeezing the hollow tube at the level of said glass vial between fingers. This action frees the reagent powder, which is then contained in the tube by two filtered ends of the hollow tube. The exhales a fixed volume through one end of the tube to expose the reagent to the breath gas. The reagent can then be heated within the hollow tube for a predetermined time (such as 5 minutes) at a

predetermined temperature (such as 75 degrees), after which an optional additional reagent (comprising an acid such as phosphoric acid, sulfuric acid, or hydrochloric acid) can be added, if desired, to enhance the reaction. Then, the color of the resultant product can be measured or analyzed using a visual analog scale or a colorimetric sensing device such as a spectrophotometer or camera (including but not limited to a CMOS or CCD device) which may be incorporated into a cell phone or other portable technology. Optionally, this color data can be sent to a remote human or computerized processer for analysis of the color changes, interpretation of the color of the reagent by assessing pixel color space data from the image data stream and comparing to known standards or by using a visual analog scale comparing the color of reagent to standardized colored paint chips. The analyte concentration or quantity can be reported back to the user through electronic communication, electronic display or other means.

[0155] Non-exhaustive list of examples of use of the technology

1. A woman wants to see how fast she is aging relative to her peers, in terms of a real biologic measurement. She gets together with her friends and they all measure their breath aldehyde concentrations and learn that her regular exercise and conscientious diet and nutritional supplements are associated with a lower production of aldehydes, which are formed from oxidative stress to lipid membranes. She continues with her healthy regimen, and other friends are thereby encouraged to do the same. A man ingests a large amount of fatty foods and is curious if there is immediate evidence of injury to his body by so doing or whether he will just have to wait. He measures his breath aldehydes and learns that there is immediate oxidative stress causes by eating the fatty foods.

A doctor wishes to know if a diabetic patient has high levels of oxidative stress evident to help determine how many antioxidants to start on the patient, and which antioxidant might work best for this individual patient. He/she measures the breath aldehydes of her patient over time and compares to an available large normative database, and then recommends nutritional interventions and other lifestyle modifications aimed at diminishing the oxidative stress if appropriate.

A pharmaceutical company wishes to have a biomarker for oxidative stress to help obtain information about whether their new therapy may work or not in human systems. They incorporate breath aldehyde assessment into their large double blind placebo controlled studies as an easily useful biomarker of cellular oxidation.

A neonatologist wants to know whether supplemental oxygen given to a premature baby, combined with various other interventions, is causing oxidative injury to the lungs or body. She measures breath aldehydes over time as one potential marker of such injury, and if there is sufficient data to support altering interventions, does so.

A company is trying to make a beverage that embodies the notion of the fountain of youth, and is trying to slow down aging. They consider oxidative stress to be a key component of aging. They use breath aldehydes as a biomarker to help them identify good candidates in their hunt for the fountain of youth.

A smoker wants to know whether he might have injury to his body that he is not aware of, and performs breath aldehyde measurements over time to help determine this.

A smoker wants to learn if he is in the 20% of smokers that will suffer aggressive loss of lung function by smoking. He has learned of new data that implicates the inability to deal with oxidative stress as one component of the risk determination. Working with his doctor in a holistic fashion, he incorporates aldehyde measurements in the breath into his personal assessment.

A patient is frustrated by the lack of personalizable data that she can obtain about antioxidants and her health. She wants to know which antioxidant regimen is likely to work best for her. She develops a system in which she tries each of several nutritional supplements over time, monitoring her breath aldehydes and her general feeling of wellbeing over time, and uses this to help determine which regimen seems most likely to be effective for her body. A food preparation company has a bad reputation that its food causes oxidative injury. It knows otherwise, but cannot convince people because of the momentum of the accusation. It performs a careful study using several biomarkers including breath aldehydes to determine that their food is not causing particular oxidative stress.

A pharmaceutical company wishes to perform a phase 4 study of a blockbuster drug that lowers cholesterol. They wish to assess antioxidant effects of their drug, which if effective, they will use in their marketing program. They study breath aldehydes in a carefully controlled and planned study.

A mountain guide or the military branch wishes to have some clue as to who might be at highest risk of suffering from Acute Mountain Sickness. They measure breath aldehydes in subjects before and after climbing to altitude. NASA wants to understand the effect of space flight on human oxidative biology. The perform aldehyde breath tests using a reagent in a breath tube, and the astronauts send a picture of the reagent post exposure to NASA laboratory for image processing to determine oxidative stress.

A professional basketball team wishes to determine if their oxidative metabolism is improved by using nasal strips (Breathe-right™ strips), so they perform a study using breath aldehydes as a marker of oxidative stress, comparing athletes who wear the strips with those who do not, or comparing oxidative stress through aldehyde measurement during one game with strips, and one without the nasal strips.

15. A Dentist or dental device company wishes to learn how much systemic oxidative injury to important tissue occurs during teeth whitening. They use breath aldehyde measurements to modify and improve their product to avoid undesired systemic injury.

[0156] In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:

1. A method for creating a reagent to sense analyte(s) within a gas sample, comprising:

providing a reagent that changes color when exposed to the analyte of interest; and

exposing the reagent to the analyte of interest,

wherein the reagent is mixed with a material chosen to allow easy passage of gas flow through and over the reagent to increase the exposure of reagent to the analyte of interest.

2. The method of claim 1, wherein the reagent is initially made in aqueous phase.

3. The method of claim 1, wherein the material chosen to allow easy passage of gas flow through and over the reagent is chosen from one or more of the following: silica bead, sand, polystyrene, or other such materials.

4. The material of claim 3, wherein the size of the particles is optimized to allow for sufficient gas flow through the reagent.

5. The method of claim 1, wherein an aqueous phase (liquid) reagent is mixed with material chosen to allow for easy passage of gas flow through the reagent, the mixture being accomplished by direct addition.

6. The method of claim 1, wherein an aqueous phase (liquid) reagent is mixed with material chosen to allow for easy passage of gas flow through the reagent, the mixture being accomplished by direct addition, and then lyophilization or freeze-drying to remove the water from the reagent.

7. The method of claim 1, wherein the reagent is an aldehyde-sensitive material.

8. The material of claim 7, wherein the reagent is a Schiff reagent.

9. The material of claim 8, wherein the reagent includes a combination of sulfuric acid or metabisulfite and one or more of the following: rosanilin, pararosanilin, fuschin, basic fuschin, in proportions that optimize aldehyde sensitivity for the given application.

10. The method of claim 1, wherein the reagent is placed in a device appropriate for capturing the sample of interest.

11. The device of claim 10, wherein the device is a tube designed for collection of exhaled breath.

12. The device of claim 11, further comprising a tube in which breath is passed through, one-or more reagents for breath constituents to react with, and optional filters.

13. The device of claim 12, wherein there is one or more additional reagents, powders or fluids that can be added prior to or after assay is performed.

14. The device of claim 13 wherein the additional reagent(s) are contained within a separate compartment within the tube.

15. The device of claim 14, wherein the separate compartment is a vial made of glass or other appropriate material that is placed within the tube.

16. The device of claim 12, wherein the additional reagent is an acid.

17. The device of claim 16, wherein the acid is chosen from among any one or combination of the following: any concentration of Sulfuric acid, phosphoric acid, hydrochloric acid, water.

18. The method of claim 1, wherein the reagent is heated for a predetermined period of time prior to or during assay.

19. The method of claim 18, wherein the heating time is between 1 second and 24 hours.

20. The method of claim 19, wherein the heating time is 5 minutes.

21. The method of claim 18, wherein the heating temperature is between 25 and 500 degrees C.

22. The method of claim 21, wherein the heating temperature is 75 degrees C.

23. A method for transmitting a sample to a laboratory comprising: capturing or reacting an analyte or analytes of interest with a reagent that changes color, hue or light absorption characteristics upon exposure to said analyte(s), taking a picture of the altered reagent before and after, or just after, the reaction; and

transmitting that picture or the data representing that picture to a

computational device or human operator at a remote laboratory over wireless, internet, telephone wires, cell phone signals or other communication technology.

24. The method of claim 23, wherein the picture is taken by a CCD or CMOS device.

25. The device of claim 24, wherein the CCD or CMOS chip records image data using any of several well-known color spaces, such as RGB, CMYK, or other color space systems.

26. The method of claim 23, wherein a computer or human operator then analyzes the color change, hue, or light absorption characteristics compared to baseline and/or compared to known standards to provide an analysis of the quantity or concentration of the analyte of interest.

27. The method of claim 25, wherein individual components of a color space that serves as the means of measuring color or light absorption characteristics are measured, or retrieved from the data stream, are individually recorded and a mathematical algorithm applied that allows for determination of analyte

concentration, quantity, or exposure.

28. The method of claim 26, wherein the results of the laboratory assay are transmitted back to the original site of sample collection using wireless, internet, telephone wires, cell phone signals or other communication technology.

29. The method of claim 23, wherein the reagent is contained within a material that is most conducive to photographic assessment, such as any clear or partially translucent material, for example polypropylene.

30. A method and device for determining aldehydes in exhaled breath, comprising:

providing a 3.5 inch hollow tube in which a powdered reagent (comprising a dry Schiff reagent mixed with fine beads such as silica) has been optionally kept protected from room air by being enclosed in a glass vial;

fracturing the glass vial within the hollow tube by squeezing the hollow tube at the level of said glass vial between fingers, freeing the reagent powder to be held between two filtered ends of the hollow tube;

receiving a fixed volume of breath through the tube to expose the breath gas to the reagent; and

heating the reagent within the hollow tube for a predetermined time (such as 5 minutes) at a predetermined temperature (such as 75 degrees).

31. The method of claim 30, further comprising:

adding an optional additional reagent (consisting of an acid such as phosphoric acid, sulfuric acid, or hydrochloric acid) to enhance the reaction; and measuring the color of the resultant product using a visual analog scale or a colorimetric sensing device such as a spectrophotometer or camera (including but not limited to a CMOS or CCD device).

32. The method of claim 31, wherein the camera or spectrophotometer is incorporated into a cell phone or other portable technology and images and/or color data is sent to a remote human or computerized processer for analysis of the color changes.

33. The method of claim 32, further comprising interpreting the color of the reagent by assessing pixel color space data from the image data stream and comparing to known standards or by using a visual analog scale comparing the color of reagent to standardized colored paint chips; and

reporting the analyte concentration or quantity back to the user through electronic communication, electronic display or other means.