Method for Measuring Average Concentrations of Cortisol and Glucose Using Earwax

Abstract

A method for measuring glucose and cortisol levels in earwax, wherein the measured cortisol and glucose levels are interpreted as average cortisol and glucose levels; and a safe, practical, reliable, and hygienic medical device for self-extracting earwax.

Description

[0001] Background of the present invention

[0002] A. Field of the invention

[0003] This patent relates to a method for measuring cortisol and glucose concentrations, namely using earwax to measure the average levels of cortisol and glucose.

[0004] B. Description of related literature

[0005] The first problem

[0006] Lack of a reliable and harmless method to measure the average level of glucose

[0007] Chronic diseases are the leading cause of death globally (71%), and diabetes ranks fourth (WHO, 2018). Additionally, according to the same report by the World Health Organization, 650 million adults have another chronic disease, such as obesity. Even more shockingly, the proportion of overweight adults over 18 years old is as high as 39%. Moreover, this additional chronic disease is gradually showing an upward trend. During the period from 1975 to 2016, the global prevalence of obesity almost tripled (NCD-RisC etc., 2017).

[0008] These two prevalent diseases are closely related. Nearly 90% of the most common type of diabetes, or type II diabetes, is associated with overweight (Wu etc., 2014). Additionally, these diseases usually have the same metabolic changes, namely a gradual increase in glucose. Therefore, the diagnosis of diabetes requires testing glucose levels. On the other hand, subjects with elevated glucose levels but not within the diabetes range are also 4.5% more likely to be obese (Meigs etc., 1998).

[0009] Currently, the measurement of "short-term" glucose samples (such as serum) has significant limitations in evaluating the average glucose level. This is because glucose levels vary greatly during the day, and some stress events that occur during the day (Dagogo-Jack, 2010), smoking (Frati etc., 1996), hypertension (Modan etc., 1985), body mass index (BMI) (Hiller etc., 1988), and physical activity (Allen etc., 2009) also affect glucose levels.

[0010] To obtain accurate glucose test results, there are currently some standardized glucose measurement methods, such as fasting before measurement or testing postprandial glucose concentration. However, such measurement methods impose requirements and restrictions on patients and often cannot accurately reflect the average level of glucose, which is crucial for the long-term glucose control of diabetic patients. In fact, the results detected by these methods are often lower than the mean values of fasting serum glucose (FSG) or postprandial serum glucose (PSG) (Peter et al., 2006).

[0011] Glycated hemoglobin (HbA1c) is a form of hemoglobin that is positively correlated with both fasting and postprandial glucose indices, so it is usually used as an indicator to reflect the long-term average level of glucose (Monnier et al., 2006; Bonora et al., 2001; Rohlfing et al., 2002), and is also considered the most standard method to reflect the average glucose concentration. However, compared with diabetic patients, the correlation between postprandial glucose or fasting glucose levels and HbA1c in healthy individuals is weaker (van′t Riet et al., 2010). Studies have shown that in the general population, the correlation between PSG and HbA1c is 0.33, and the correlation between FSG and HbA1c is 0.46, while these correlations are 0.71 and 0.79 respectively in diabetic patients (van′t Riet et al., 2010). These results undermine the effectiveness of HbA1c in screening tests (Dagogo-Jack, 2010). On the other hand, in healthy individuals and diabetic patients with poor glucose control, the association between fasting glucose level and HbA1C is stronger than that between postprandial glucose level and HbA1c (Monnier et al., 2006). This situation means that the HbA1c test index of diabetic patients with abnormal diet may be within a reasonable range. This shortcoming limits the ability to use the HbA1c index to closely monitor the average glucose level of these patients. Therefore, a more accurate method to reflect the average glucose concentration should comprehensively balance postprandial and fasting glucose levels. HbA1c can reflect the average glucose concentration in the last three months, but it is most affected by the plasma glucose concentration in the last month (75%) (Leow, 2016; Mortensen & Válund, 1988; Tahara & Shima, 1993). In addition, HbAlc cannot provide glucose test information in a shorter period, for example, tracking early changes after blood glucose control, and this period requires relatively strict metabolic control (Goldstein et al., 2004; J.H. Kim et al., 2012; Koenig et al., 1976).

[0012] In addition, this method has other limitations. On the one hand, it is affected by some variables. For example, aging can change the concentration of HbA1c (Dagogo-Jack, 2010). On the other hand, some common diseases, such as anemia (Sundaram et al., 2007) and some hemoglobinopathies (up to 7%) (Weatherall, 2011), may also affect its level. Even long working hours can lead to an increase in HbA1c levels (Azami et al., 2018). In addition, its detection is expensive, and often the experimental detection conditions do not allow (Sacks, 2011). Finally, this method actually indirectly estimates the mean glucose level because it is only a related protein rather than sugar itself. Therefore, some scholars even doubt its effectiveness as a diagnostic indicator for diabetes and prediabetes (Dagogo-Jack, 2010).

[0013] Recently, glycated albumin has been used as an indicator for measuring the mid-term stage of 2 to 4 weeks. However, some sociodemographic variables, such as age, body mass index (BMI) (Miyashita et al., 2007), or diseases that affect albumin metabolism, such as thyroid dysfunction, nephrotic syndrome, or cirrhosis (KJ Kim & Lee, 2012), make the determination of glycated albumin somewhat unstable in clinical practice (Huh et al., 2014). In addition, so far, its effectiveness as a diagnostic method is not clear.

[0014] It should also be noted that all these samples, including the measurement of glucose, HbA1c, or glycated albumin, are obtained from blood samples, which means they are costly because they require skilled technicians, such as nurses, to operate. Moreover, they may have some side effects, such as bleeding and infection, which are even more common and complex in patients with metabolic diseases, such as diabetes. However, despite the above disadvantages, glucose level measurement is still the most needed laboratory test in primary care centers in some countries / regions (Salinas et al., 2014; Zunic, 2012), and it also ranks third in the laboratory test costs of the health system (Zunic, 2012). HbA1c is also one of the most demanded laboratory tests, and it is even considered that the demand for HbA1c is still underestimated (Salinas et al., 2012). Therefore, we need to develop a better sample that can not only directly reflect the average glucose concentration at different times but also be more economical and harmless.

[0015] The second problem

[0016] Lack of reliable and practical methods for measuring the average concentration of cortisol

[0017] Major depressive disorder (MDD) is another chronic prevalent disease, yet its clinical diagnosis is not very reliable due to the heterogeneity of its symptoms (Lieblich et al., 2015). Therefore, research has been dedicated to developing an accurate biomarker to improve the consistency of its diagnosis. Among them, cortisol levels have been the most popular biomarker because it is the most common neurobiological change in this syndrome (Pariante, 2009). However, due to the reactivity of cortisol secretion, the results of this hormone are very different. It not only has a very obvious circadian rhythm (Bhagwagar, 2003; Bhagwagar et al., 2005), but is also affected by several typical variables. For example, food intake (Gibson & Checkley, 1999), nicotine (Steptoe & Ussher, 2006), physical exercise (Hill et al., 2008) and stress levels (Kirschbaum et al., 1993; Sharpley, 2012) can all affect its short-term levels. This situation means that most current biological specimens (such as plasma or serum) are not the most suitable specimens to reflect the average cortisol concentration, which is a key indicator to describe the state changes of different types of MDD.

[0018] Not long ago, hair samples were started to be used as an indicator to measure the average cortisol concentration level (Dettenborn et al., 2012). Studies have found that hair can provide an indicator to measure the average cortisol concentration because it can accumulate the hormone but is not affected by complex factors like short-term specimens (Short et al., 2016a). However, it also has some limitations. Most studies on its effectiveness have been conducted by comparing hair cortisol concentration (HCC) with single or several cortisol specimens without considering the nocturnal cortisol levels. This limitation can explain why most correlation coefficients between hair and one or more short-term saliva specimens have been moderate so far (D′Anna-Hernandez et al., 2011; Sauvé et al., 2007; van Holland et al., 2012; Xie et al., 2011). In fact, few successful studies have confirmed the effectiveness of hair. An ideal study should correlate HCC with continuous cortisol levels, or cortisol levels measured during the day and at night. Those studies that correlated HCC with 24-hour urine collection showed diverse results. Sauvé et al. (2007) found only a moderate correlation between HCC and 24h urinary cortisol collection, while the study by Short et al. (2016b) showed no significant correlation.

[0019] Finally, hair may not reflect the average level of cortisol concentration as well as expected. For example, it is unclear whether sweat glands are also involved in the internal transport of a certain amount of cortisol in the hair (Sharpley, 2012). However, it is certain that sebaceous glands transport cortisol into the hair and are thus innervated by a network of nerve fibers (Okumura, 1967). In addition, acute factors can also affect hair growth. There is a body of evidence suggesting that neurohormones and neurotransmitters released during stress can significantly affect the hair growth cycle (Paus et al., 2006, 1997; Botchkarev, 2003). Moreover, steroids are deposited in the hair follicles, and thus future hair loss also depends on local metabolic variables that reflect the hair growth cycle (Terao & Katayama, 2016). These variables may explain why hair cortisol treatment protocols recommend excising keratinized tissue from the posterior part of the scalp, as less hair growth variability is observed in this area (Pragst & Balikova, 2006). This may imply that although acute cortisol mixing variables may not affect HCC, their local covariates may do so. Therefore, it is reasonable to assume that hair does not accurately reflect the long-term systemic level of cortisol but provides an indicator for local observation.

[0020] Hair specimens also encounter some practical problems that hinder their widespread clinical use. The areas with less hair growth variability are also the areas of the posterior vertex that are most affected when people start to experience hair loss. In fact, this type of hair loss (type IV) affects 40% of men over 40 years old and 10% of women over 40 years old (Hamilton, 1951), not including a large number of people who cannot provide samples because their hair has not reached the minimum length (at least one centimeter to reflect the cortisol concentration of the previous month). A recent study showed that up to 30% of participants were unable or unwilling to provide this sample for various reasons, including aesthetic reasons (Fischer et al., 2016). More importantly, accurately cutting off 1 mm of hair seems to be an impossible task. However, being able to distinguish the average cortisol levels between weeks is very important for doctors. The antidepressant effect usually starts to show its effect after 3 weeks of treatment rather than 4 weeks (Tanum & Malt, 1996). This means that the antidepressant effect related to long-term cortisol levels cannot be accurately described by using hair samples. In fact, different from non-keratinized tissues, the analysis time of hair is relatively long. In fact, analyzing a single saliva sample may take 4 hours and 20 minutes, but analyzing a hair sample takes more than 30 hours. This difference means that the working time has increased by eight times. Therefore, this is a very inefficient process. This situation can explain why its cost is 44.3% higher than that of analyzing short-term cortisol specimens such as saliva (Bristow, 2017). All of the above factors hinder its widespread clinical application.

[0021] Some variables also affect the cortisol levels in hair. For example, gender may change HCC. Multiple studies have shown that men are more likely to increase HCC than women (Garcia-Leon et al., 2018; Vanaelst et al., 2012). In addition, although the "washout" or cortisol removal effect caused by external factors (such as ultraviolet radiation or the use of cleaning products) in the part less than four centimeters from the hair root has been excluded (Dettenborn et al., 2010), it is not clear whether other removal effects (in other words, the part of the specimen that has just grown out of the scalp) will also reduce its level. In addition, although significant differences in hair levels are related to this step of capillary cortisol analysis, hair research is not consistent with specific cortisol extraction methods. When the specimen is crushed instead of cut into small pieces, up to 3.5 times more cortisol can be extracted (Davenport et al., 2006). Finally, it is not clear whether certain beauty treatments (such as hair dyeing) will also affect HCC (Manenschijn et al., 2011; Sauvé et al., 2007).

[0022] The third problem:

[0023] Lack of efficient and safe devices for cleaning the external ear canal

[0024] Unfortunately, so far, no earwax self-cleaning device has been as effective as the traditional clinical methods for cleaning the external ear. This limitation means that, although earwax samples have potential uses in measuring long-term levels of glucose and cortisol, their widespread clinical application seems unrealistic. Because so far, only qualified doctors can perform the tests safely, using clinical methods to extract samples is very expensive.

[0025] In addition, although cleaning the external ear is not a medical indication, millions of people have this dangerous habit every day. Cotton swabs are the most common method of self-cleaning the ears (Khan et al., 2017), and at the same time, they are also the main risk factors for several external ear diseases, such as abnormal earwax and bleeding (Ahmed et al., 2014; Nussinovitch et al., 2004).

[0026] Given its potential serious side effects, the popularity of cotton swabs may not only be related to their ease of use. First of all, these devices may be addictive. The fibers around the external ear canal are sensitive, and the stimulation of them will trigger a sense of pleasure in the body. However, long-term use of these devices will trigger a vicious cycle, that is, the "itch-scratch cycle", and this vicious cycle will continue. Therefore, the popularity of cotton swabs can not only explain the general increase in the sense of itching, but also the reason for their abuse (Mochizuki et al., 2014; Pata et al., 2003). Therefore, considering their huge success in the global commercial market at present, a simple warning about this potential side effect may not be sufficient as a measure to reduce their huge demand. For example, the sales of only one brand in the United States in 2005 were 189.3 million US dollars, and increased to 208.4 million US dollars in 2014. A recent market study found that during the period from 2011 to 2017, the growth rate of their sales was 30% in the United States, 32% in China, and 26% in Europe, and the expected growth rates in the next five years are 20%, 24% and 19% respectively (Hexa Reports, 2017). Therefore, an efficient and safe alternative device needs to be developed to replace the high-risk cotton swabs.

[0027] Unfortunately, so far, this has not been achieved. Currently, several commercially available ear cleaning products are ineffective or have little effect. Comprehensive and systematic evaluations have shown that although some earwax decomposition solutions (such as solutions containing mineral oil) may have certain effects, it is not clear which of them causes this decomposition. In addition, so far, no device (mechanical or electronic) has been as effective as extraction by an expert or using a syringe (such as the Reiner-Alexander syringe) (Clegg et al., 2010)

[0028] The first solution

[0029] Cerumen reflects the average levels of glucose and cortisol

[0030] Few biological specimens can reflect the average levels of glucose and cortisol. Adipose tissue may be one of them because of its property of accumulating substances (Szymczak & Milewicz, 1998). However, it seems highly unrealistic to perform a biopsy on a patient's tissue as it is not only dangerous but also more expensive than collecting a blood sample. However, another more accessible biological specimen can also reflect these levels: cerumen. Cerumen is an oily secretion mainly composed of lipids (Inaba et al., 1987). It is secreted by the apocrine and sebaceous glands around the ear canal (Montagna, 1955). This secretion can accurately reflect the average levels of glucose and cortisol because it is not affected by local and acute effects, as the concentration of glucose and cortisol is not influenced by nerve fibers or local metabolic variables. In fact, unlike the sebaceous glands of hair follicles, the apocrine and sebaceous glands of the ear are not innervated by nerves (Bende, 1981).

[0031] Bees also produce wax, and the role of the honeycomb also indicates that cerumen may have more advantages than blood samples. On the one hand, bees can store (accumulate) their sugar (honey) in the honeycomb (Fratini et al., 2016), and on the other hand, since the honeycomb has antibacterial properties, it will not be consumed by microorganisms (Ghanem, 2011). The wax produced by the human body also has this property (Stoeckelhuber et al., 2006). Therefore, this indicates that cerumen can not only accumulate glucose and cortisol for a long time but also be protected from epidermal flora. This feature may mean that if there is a self-sampling cerumen device, i.e., a device that can collect cerumen samples from the patient's home without the need for collection under special storage or transportation conditions like blood samples.

[0032] Cortisol and glucose are two highly reactive substances. Measuring their long-term levels is crucial as they are altered in epidemics. However, so far, current biological specimens can only measure their short-term levels; even if some samples can be accumulated to measure the levels of cortisol and glucose over a longer period, their widespread clinical application is unrealistic and expensive. Cerumen can measure these substances over a long term and is a feasible and reliable biological specimen. However, it is not clear whether this unique specimen can accumulate these reactive substances over a long term.

[0033] Therefore, the applicant has developed an analytical method for detecting glucose and cortisol in cerumen samples.

[0034] The method used in the present invention has been systematically evaluated. Preliminary studies have also confirmed the efficiency and reliability of earwax analysis and determined whether glucose and cortisol can be detected in this new type of sample. Finally, the effectiveness of various types of sponges in removing artificial wax from pigskin was also tested.

[0035] Results: Cortisol levels have not been measured in earwax before. Cortisol and glucose were detected in this oily secretion. The time required to analyze cortisol in earwax is much less than that required to analyze cortisol in hair. Cellulose sponges with special abrasiveness and absorbency are the most effective method for removing artificial wax from pigskin.

[0036] Conclusion: Earwax may be the most accurate and effective sample for measuring long-term cortisol and glucose levels. Cellulose sponges are an effective, economical, and safe extraction material.

[0037] The second solution

[0038] Design a safe and reliable ear self-cleaning device

[0039] Due to the above problems, the applicant has developed a medical device that provides an effective, safe, and hygienic method for self-extracting earwax.

[0040] In addition, the earwax self-sampling device of the present invention can provide a suitable sample for analyzing glucose and cortisol levels using this biological sample. Summary of the invention

[0041] Therefore, the main object of the present invention is to provide a method for measuring glucose and cortisol levels in earwax.

[0042] Another main object of the present invention is to develop a new medical device for reliable, effective, safe, and hygienic self-extraction of earwax.

[0043] The following description details the method of the present invention for using earwax to measure glucose and cortisol levels, as well as the designed medical device, and their various advantages are obvious to those skilled in the art.

[0044] Brief description of the example figures

[0045] Figure 1 Left side view of the first embodiment of the medical device of the present invention, showing the tip cross-section (with sponge).

[0046] Figure 2 Perspective view of the tip of the medical device of the present invention (without sponge)

[0047] Figure 3 Top view of the tip of the medical device of the present invention (without sponge)

[0048] Figure 4 Left side view of the second embodiment of the medical device of the present invention, showing the tip cross-section (with sponge)

[0049] Figure 5 Illustration of the correlation results between baseline - EGC and FSG

[0050] Figure 6 Illustration of the correlation results between baseline - HbA1c and FSG

[0051] Figure 7 Illustration of the correlation results between tracking EGC and PSG

[0052] Figure 8 Illustration of the correlation results between tracking HbA1c and PSG

[0053] Figure 9 Illustration of the correlation results between baseline EGC and the average blood glucose level

[0054] Figure 10 Illustration of the correlation results between baseline HbA1c and the average blood glucose level

[0055] Figure 11 Illustration of the correlation results between tracking EGC and the average blood glucose level

[0056] Figure 12 Illustration of the correlation results between tracking HbA1c and the average blood glucose level

[0057] Figure 13 Illustration of the correlation results between HCC and ECC

[0058] Detailed description of the present invention

[0059] This description will describe the method of the present invention for measuring long - term glucose and cortisol levels in earwax according to preferred embodiments. In the most general embodiment, the method of the present invention includes:

[0060] Extracting an earwax sample from the ear by any suitable method. The minimum amount of earwax required for measuring the average levels of cortisol and glucose is 0.8 milligrams;

[0061] Preparing the earwax sample by measurement means or methods for measuring the levels of cortisol and glucose.

[0062] Determining cortisol and glucose using any known means or methods, where the levels of cortisol and glucose are interpreted as the average levels of cortisol and glucose.

[0063] The extraction of the earwax sample can be carried out in a conventional manner, such as using a Reiner - Alexander syringe or by using any suitable extraction device.

[0064] The preparation of the samples can be carried out in different ways, depending on the measurement methods of cortisol and glucose used. The samples can contain pure and dry earwax. Their glucose and cortisol analysis can be carried out in different ways, such as using immunohistochemical reactions or ELISA.

[0065] In a first specific embodiment of the present invention, earwax is obtained from the outer ear using a Reiner-Alexander syringe.

[0066] In such a specific embodiment, the preparation of the samples and the measurement of cortisol and glucose levels are carried out as follows:

[0067] Sample preparation

[0068] Cortisol extraction

[0069] a) Dry the earwax sample with N 2 steam until all water has evaporated from the sample. This step can also be accomplished using freeze-drying means.

[0070] b) Weigh the dried earwax sample to standardize the weight of cortisol by dry weight. Standardization means adjusting the measured weight to a standard ratio so that the data can be compared;

[0071] c) Homogenize the dried sample with 1 ml of phosphate-buffered saline (PBS) solution to obtain a solution of earwax in PBS. The amount of phosphate-buffered saline (PBS) solution can be 10 times the weight of the earwax. For example, 100 g of earwax uses 1000 μl of phosphate-buffered saline (PBS) solution. In addition, any hydrophilic solvent, such as physiological saline, can be used.

[0072] d) Divide the solution obtained in step c) into a first solution portion and a second solution portion, and add each solution portion to its respective test tube;

[0073] e) Add 0.5 ml of ether to the first solution portion to obtain a PBS earwax solution mixed with 0.5 mg of ether; the ratio between the two substances is 1:1. However, other suitable substances of the same kind can also be used.

[0074] f) Stir the test tube containing the solution of step e) for at least one minute, and after resuspension, add 0.5 mg of ether to the resulting solution, with a ratio of 1:1 to PBS;

[0075] g) Cool the mixed solution obtained in step g) at a temperature of -18 to -21 °C (preferably -20 °C) for at least two hours to ensure that the liquid part is frozen and does not contaminate the organic part. This step can extract those compounds that are significantly dissolved in ether, such as cortisol. This is because although the ether part remains liquid at -20 °C, the phosphate part freezes;

[0076] h) Extract the compounds that are significantly soluble in ether from the cooled solution;

[0077] i) Dry the remaining liquid solution by the N 2 displacement method. However, other methods, such as evaporation, can also be used.

[0078] j) Store the dried part obtained in step i) at -80 °C for further use;

[0079] k) Add 300 pg of cortisol to the second solution part to obtain a PBS earwax solution mixed with cortisol;

[0080] l) Add 0.5 ml of ether to the solution obtained in step k) to determine the amount of purified cortisol as a means of evaluating the extraction efficiency;

[0081] m) Stir the test tube containing the solution obtained in step I) for at least one minute. After resuspension, add 0.5 mg of ether to the obtained solution, with a ratio of 1:1 to PBS;

[0082] n) Cool the mixed solution obtained in step m) at a temperature of -18 to -21 °C (preferably at -20 °C) for at least two hours to ensure that the liquid part is frozen and does not contaminate the organic part;

[0083] o) Extract the compounds soluble in ether from the cooled solution;

[0084] p) Dry the remaining liquid solution again by the displacement method with N 2 ;

[0085] q) Store the dried part obtained in step i) at -80 °C for further use;

[0086] r) Dissolve 0.5 ml of 300 pg / ml purified cortisol solution in PBS with a pH of 7 in a third test tube (without the homogenized solution). This step aims to obtain the efficiency of extracting cortisol from earwax.

[0087] s) Perform the same steps as for the first solution part and the second solution part to extract cortisol from them.

[0088] Cortisol Quantification

[0089] According to the manufacturer's instructions (Enzo Life Sciences, Farmingdale, NY), ELISA technology was used to quantify the concentration of cortisol in cerumen samples, and the quantification method was as follows:

[0090] a) The extracted samples were reconstituted using the buffer provided by the manufacturer. This method uses colorimetric competitive ELISA technology to quantitatively analyze cortisol. Specifically, the buffer was added to the extracted samples, and the resulting solution was allowed to stand for 20 minutes, or other time ranges. 20 minutes is sufficient for the solution to rehydrate and make it easier for the buffer to be resuspended. Then, the solution was stirred within 1 minute to homogenize it. During this process, the buffer provides a stable solution because it can maintain the pH value within a certain range whether an alkali or an acid is added.

[0091] b) The standard curve of an enzyme-linked immunosorbent assay (ELISA) reader (NovoStar) was used to measure the total amount of cortisol in the samples;

[0092] c) Fluorescence technology was used to normalize the dry weight of cerumen, where the fluorometer was excited in the range of 530 - 570 nm and read in the range of 590 - 600 nm. Although multiple variables such as age, gender, different medical conditions, and stress levels may affect the cortisol level in cerumen, to avoid confounding the detection results of cortisol by covariates, cholesterol levels were used for comparison in the experiment because cholesterol levels are not affected by the above variables.

[0093] Glucose quantification

[0094] A portion of the cerumen solution previously dissolved in PBS for measuring cortisol levels was used to measure glucose levels. The quantification method of glucose was as follows:

[0095] a) According to the manufacturer's instructions, Kit SERA-PAK PLUS (Bayer HealthCare) technology was used to measure the glucose level in the dissolved cerumen solution. The glucose absorption was quantified in triplicate at 505 nm. The average value of its absorption was used to obtain the glucose concentration (mg / dl), and the total amount of glucose dissolved in the solution was calculated based on the initial weight of the normalized sample.

[0096] In the second specific embodiment of the present invention, cerumen was obtained using the extraction device of the present invention, and the extraction device includes:

[0097] A handle (1) having a first end (2) and a second end (3), and the second end (3) has connecting means, which may include a thread (4) in a preferred embodiment of the present invention;

[0098] Detachable head (tip), including a base (5) and an elongated sponge holder (6) hanging down from the upper part of the base and extending longitudinally, wherein the lower part of the base includes a threaded housing (7) with internal threads for accommodating the threads (4) of the handle (1), and wherein the sponge holder (6) has a star-shaped cross-section;

[0099] An elongated sponge (8), having a central longitudinal housing (not shown) with a star-shaped cross-section for accommodating the sponge holder (6) of the base (5).

[0100] The handle (1) and the base (5) can use any suitable coupling device, such as a snap joint (9).

[0101] The sponge (8) is preferably made of cellulose and adhered to the sponge holder (6) using a non-allergenic adhesive.

[0102] As mentioned above, the cross-section of the sponge holder (6) is star-shaped, which can improve the efficiency of earwax extraction when rubbing the sponge (8) inside the ear. However, the cross-section of the sponge holder can be any suitable shape.

[0103] The base (5) is wider than the handle (1) and is used as a safety brake to prevent the tip from being inserted into the ear canal.

[0104] The handle (1) is characterized by a rotationally symmetric shape, which facilitates the user to rub the sponge by rotating the handle.

[0105] The sponge (8) is wrapped and sealed in a wet state to keep it soft. The wetting agent used is magnesium chloride (MgCl 2 )), which can act as an antibacterial agent to prevent the growth of microorganisms during the shelf life. Magnesium chloride can not only prevent the sponge from breeding microorganisms, but also help to extract earwax. In addition, it is also used to treat dermatitis (Zhai et al., 1999), which is the most common side effect of using cotton swabs (Ahmed et al., 2014). Other known antimicrobial agents can also be used.

[0106] Insert the tip with the sponge (8) into the ear and rotate the sponge (8) in the ear canal for about 30 to 60 seconds to obtain earwax.

[0107] In the second embodiment of the present invention, sample preparation is carried out by adding 500 μl of PBS buffer solution to a 5 ml test tube. Separate the sponge from the plastic holder and then place it in the tube. After the sponge has absorbed all the solution, squeeze and absorb it repeatedly for 2 minutes (or adjust as needed), squeeze the sponge until it is dry and then take it out of the test tube. Subsequently, through N 2Replace the obtained solution by drying and resuspend the obtained content in 500 μl of ultrapure water. Keep the obtained solution in the test tube at 4 °C until further use, or it can also be cooled within other suitable temperature ranges. It is obvious to those skilled in the art that other amounts of PBS can also be used in this process, and the ratio between its weight and volume is usually 1:2, which is also the reason for using 500 μl here.

[0108] In this second specific embodiment, according to the manufacturer's instructions, measure the cortisol level by ELISA technology and measure the glucose level in the dissolved earwax solution by using the Kit SERA-PAK PLUS (Bayer HealthCare) kit. Other suitable known methods can also be used, such as liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0109] Pilot study

[0110] Earwax removal

[0111] Clean the ears of the volunteers using a Reiner-Alexander syringe, because so far, this is the unique safe method for effectively removing earwax in the outer ear (Clegg et al., 2010). This syringe is a traditional method for otolaryngologists to remove abnormal earwax. Before cleaning both ears, use an otoscope to examine the external auditory canal to exclude the presence of outer ear lesions, such as abnormal earwax or tympanic membrane perforation. Briefly, slowly inject water at 37 degrees Celsius into the external auditory canal using the Reiner-Alexander syringe. The injection process will produce a slight sense of compression because the warm water will wash away the earwax. Collect the drained water and the extracted earwax in a kidney-shaped basin.

[0112] Analysis of cortisol and glucose using earwax samples

[0113] Extract cortisol according to the first specific embodiment of the present invention and perform cortisol and glucose quantification according to the first specific embodiment of the present invention.

[0114] The following pilot study was conducted in a group of healthy participants to standardize the cortisol and glucose extraction protocols for earwax samples.

[0115] Results of measuring glucose and cortisol using earwax samples (Table II.1)

[0116] Table II.1. Pilot study - Cortisol and glucose analysis of samples from five volunteers (Volunteers 0 - 4)

[0117]

[0118] Effectiveness of the cortisol extraction and quantification protocol in earwax samples compared with blood samples

[0119] As mentioned above, to control the extraction efficiency of cortisol, 0.5 ml of 300 pg / ml purified cortisol solution was dissolved in PBS at pH 7, and the same extraction protocol as for the earwax samples was performed. The results showed that the average cortisol concentration was 281.48 ± 5.16 pg. / ml, corresponding to the average of the cortisol concentrations in three test tubes (289.2 pg / ml, 273.42 pg / ml, and 281.6 pg / ml). This result showed an extraction efficiency of 93.8 ± 1.72%, indicating the high efficiency of the cortisol extraction protocol using earwax samples.

[0120] The effectiveness of the extraction process can also be evaluated in another way, that is, by adding 300 pg of purified cortisol to the homogenized solution of dissolved earwax before adding ether (see Methods). This step was carried out to determine whether the earwax components interfere with the purification of cortisol. The results are shown in Table 2, which shows that the average recovery rate of purified cortisol obtained from the earwax homogenate was 99.3%, which also confirmed the high efficiency of using earwax samples to measure cortisol levels.

[0121] Table II.2: Recovery rate of cortisol levels in earwax samples

[0122]

[0123] This process was also evaluated by using three serum samples from a previous project as a control group (Participants 0 - 2) (Table 3). It is certain that the protocol for measuring cortisol levels using earwax is indeed reliable because when the same serum samples were measured for cortisol levels using the current earwax protocol, the results were almost the same (compare Column 3 and Column 4 in Table 3)

[0124] Table II.3: Serum cortisol levels of three participants (control group)

[0125]

[0126] Finally, it can be seen from comparing Table II.4 and Table II.5 that the analysis of cortisol in earwax using a Reiner - Alexander syringe is faster than measuring cortisol in hair.

[0127] Table II.4: Time required for analyzing cortisol and glucose in earwax samples collected with a Reiner - Alexander syringe

[0128]

[0129] Table II.5: Time required for analyzing cortisol in hair and related costs (relative to obtaining the same parameters from saliva samples)

[0130]

[0131]

[0132] However, it can also be seen from Table II.4 that when using a Reiner-Alexander syringe, the collected earwax samples need to be dried before being used for analyzing glucose and cortisol levels, which increases the total time required for analyzing these samples. Therefore, the device of the present invention provides a mechanism that can collect earwax without injecting any solution.

[0133] Conclusion

[0134] Preliminary studies have shown that glucose and cortisol levels can be detected in earwax samples. The time required to analyze cortisol in earwax is significantly less than the time required to analyze cortisol in hair. In addition, the study also found that the most suitable sponge for removing artificial earwax is made of cellulose.

[0135] Validity study

[0136] Method

[0137] The subjects were mainly recruited from the staff and student volunteers of the Universidad Católica del Norte (UCN) in Coquimbo, Chile. All subjects were evaluated by the same clinical researcher. The sample included thirty-seven healthy participants, 20 of whom were female, with an average age of 29.9 years and an average BMI of 25.6 kg / m 2 .

[0138] All subjects were recruited between July 6 and August 3, 2018, during the southern hemisphere winter. Previous studies have found that seasons can change the triglyceride composition of earwax (Cipriani et al., 1990). Due to the different earwax characteristics in terms of composition and quantity, Asians and people with intellectual disabilities were excluded respectively (Cipriani et al., 1990; Crandell & Roeser, 1993). The subjects did not report a history of medical diseases in the current or previous month, including ear diseases (such as abnormal earwax, tympanic membrane perforation, or otitis media) and metabolic diseases (such as diabetes, glucose, or lactose intolerance). The participants had not used any medications for at least one month. According to the definition of DMS-III (Pichot, 1986), if the subjects reported using illicit drugs in the past month or being under severe stress, they would also be excluded.

[0139] The validation study had two interviews, each one month apart, at baseline (day = 1) and follow-up (day = 30). During the baseline assessment, the researcher conducted a comprehensive clinical interview with the subject to rule out any medical conditions such as ear diseases, metabolic diseases, or psychiatric disorders. Socio-demographic data were also recorded during the assessment. Once the subject was included in the study, the ears were cleaned using a Reiner-Alexander syringe, as this is, to date, the unique safe method for effectively removing earwax from the outer ear (Clegg et al., 2010), and it is also the traditional method frequently used by otolaryngologists to remove abnormal earwax. The subjects were instructed to avoid using cotton swabs or any ear cleaning method during the follow-up period, which allowed the researchers to collect standardized earwax secretions after thirty days (at the follow-up assessment). Approximately 3 - 8 mg of earwax is the secretion volume for about 4 weeks.

[0140] Comparable earwax secretion amounts between the left and right ears (Cipriani et al., 1986) provided case controls rather than the possibility of a cross-sectional study. Thus, at the follow-up assessment, the left ear was cleaned using a Reiner-Alexander syringe [control group], and the right ear was cleaned using the extraction device of the present invention. Some environmental factors were self-assessed using the Hassles scale (Kanner & Coyne, 1981), such as the most common daily environmental disturbances (their frequency and severity); some sudden factors in the subjects' lives in the previous month (such as major life events) were evaluated using the Recent Life Changes Questionnaire (RLCQ; Miller & Rahe, 1997). The participants also evaluated their stress perception in the previous month using the Perceived Stress Scale (PSS; Cohen, 1994). All psychological test tools were verified Spanish versions. Finally, a standardized satisfaction survey was conducted to evaluate the participants' experience using the device of the present invention. This evaluation was scored and summed based on a 5-point scale of the attitude scale construction technique (Likert scale) (Spector, 1985). The survey also recorded some categorical and persistent variables, such as the participants' knowledge of or previous use of cotton swabs. Some anthropometric data, such as weight, height, body mass index (BMI), and waist circumference, were also recorded in detail during the visit.

[0141] Overall results: Socio-demographic data, anthropometric data, and self-administered questionnaire results

[0142] Results:

[0143] Table III.1: Socio-demographic and anthropometric data

[0144]

[0145]

[0146]

[0147]

[0148] Table III.3: Results of the self - management questionnaire

[0149]

[0150]

[0151] Overall, it can be seen from Tables III.1, III.2 and III.3 that the subjects consisted of a fairly homogeneous young sample, mainly composed of women (54.1%). In terms of anthropometric variables, they were also a healthy group of people. However, compared with samples from other Chilean control groups, this group of participants faced an increasing number and more severe troubles and life events (Herane - Vives et al., 2018). This also explains why their stress perception was higher than that of other healthy Latin samples (Cohen, 1994).

[0152] Evaluate the extraction device of the present invention

[0153] Background : So far, there has not been a safe self - cleaning device that is as useful as the traditional clinical method of removing earwax. Such a self - cleaning device can replace the dangerous cotton swabs. In this study, we evaluated an original and safe self - sampling earwax device (Trears), namely their reliability, effectiveness and user experience.

[0154] Method : The weights of 37 right - ear earwax samples collected using Trears were compared with the weights of the same number of left - ear earwax samples collected using the traditional clinical method (Reiner - Alexander syringe). These samples represented the earwax secretion in the past month. The experience of the subjects using this earwax self - cleaning device was also evaluated using a standardized satisfaction survey.

[0155] Results : Compared with the Reiner - Alexander syringe, the effect of removing earwax using Trears was significantly better (p < 0.001). There was no significant difference in the reliability of the two methods. The Trears tip with a humidity of 50% (105.1 μl) was more effective in removing earwax than that with a humidity of 12.5% (30 μl) (p < 0.05). The subjects thought that using Trears was safer and more comfortable than using cotton swabs.

[0156] Conclusion: In healthy individuals, using Trears is a more convenient, reliable, and effective method for self-cleaning the external ear canal. It can also replace the currently widely used but high-risk cotton swabs.

[0157] Preliminary research results confirmed that a special abrasive and absorbent sponge can easily remove artificial wax from animal skin (Herane-Vives and Benohr, 2018). However, its effectiveness has not been tested in earwax. This case-control study tested the effectiveness and safety of an earwax extraction device (Trears) using this type of sponge in healthy participants.

[0158] Earwax samples

[0159] A clinical research assistant for this study was trained in the use of the Reiner-Alexander syringe by an otolaryngology specialist on May 30, 2018. Before cleaning both ears, an otoscope was used to examine the external ear canal to exclude the presence of external ear diseases, such as abnormal earwax or tympanic membrane perforation. Briefly, water at 37 degrees Celsius was slowly injected into the external ear canal using the Reiner-Alexander syringe. The injection process causes a mild sensation as the warm water from the syringe flushes out the earwax. The drained water and the extracted earwax were collected in a kidney-shaped basin. During the follow-up period, the participants self-cleaned their right ear using Trears according to the manufacturer's instructions ( Figure 1 ).

[0160] Figure 1 : Instructions for using Trears.

[0161] Ear cleaning device

[0162] Warning

[0163]

[0164]

[0165] Warning

[0166] Do not reuse or wash the disposable tips. The cleaning instructions are only for single use (one per ear canal). Reusing the tips may cause infection or damage to the ear. If needed, Trears can be cleaned with warm soapy water TM and dried thoroughly before storage.

[0167] · Earwax is produced near the entrance of the external ear canal.

[0168] Do not insert Trears TM beyond the depth allowed by the safety brake. Using Trears beyond the safety brake range TMIt may damage the eardrum and hearing structures, resulting in permanent hearing loss and / or tinnitus / pain / dizziness.

[0169] · Abnormal earwax should be removed by a medical professional.

[0170] · Please follow the instructions step by step to practice safe use of this product. Misuse of Trears TM may cause serious injury. Please read all instructions carefully before use.

[0171] Using TREARS TM Before, please read this instruction carefully

[0172] Ear cleaning device

[0173] Instructions for use

[0174] Please follow the following instructions to clean your ears safely and effectively.

[0175] Preparation

[0176] 1. Wash your hands.

[0177] 2. Remove Trears TM device and a sealed disposable cleaning tip from the housing, then place it on a clean surface.

[0178] Warning: If the cleaning tip is not adequately sealed, do not use or reuse it.

[0179] 3. Remove a cleaning tip from the package and insert it into the Trears TM device, then rotate it clockwise until it clicks.

[0180] Cleaning

[0181] 4. Carefully and gently insert Trears TM (connected with a disposable cleaning tip) into your ear. Do not force the cleaning tip into the ear canal.

[0182] Note: The device has a safety brake. Do not insert the device into the ear canal beyond the range allowed by the brake.

[0183] 5. Clean your ear by wiping the cleaning tip on the entire inner wall of the ear for about 30 to 60 seconds.

[0184] Due to the safety brake of Trears TM you can clean your ear safely.

[0185] Ear cleaning device

[0186] Instructions for use

[0187] Please follow the following instructions to clean your ears safely and effectively.

[0188] Post-treatment

[0189] 6. Remove the cleaning head from Trears TM and rotate it counterclockwise.

[0190] 7. Discard the used cleaning head into any household bin. The cleaning head is biodegradable.

[0191] Warning: Do not flush down the toilet.

[0192] 8. Repeat the same steps for the other ear.

[0193] 9. Store the Trears TM device and the sealed cleaning head in a dry place.

[0194] The sponge on the disposable cleaning head has been pre-wetted with magnesium chloride (MgCl 2 ), so it has different humidity levels. The magnesium oil used during the study was a 31% aqueous solution of magnesium chloride, which can also be used for massage, skin regeneration and care. Due to its high magnesium content, the solution has a smooth and moisturizing fluid texture. It does not contain any oils itself, but has a smooth feeling like that of oils. Each milliliter of magnesium oil contains approximately 103 mg of magnesium element. Four earwax samples were labeled, weighed and stored at low temperature. All earwax samples were labeled, weighed and stored at 4 degrees Celsius.

[0195] Statistical analysis

[0196] The normality of the data was examined using the Kolmogorov-Smirnov statistical test and graphical methods (including histograms). All samples were normally distributed (all p > 0.05), except for the left ear samples during follow-up (p = 0.03). Therefore, we used the repeated t-test to compare the amount of earwax extraction from the left and right ears at baseline, and the amount of earwax extraction at baseline and during follow-up; the Wilcoxon paired signed-rank test to compare the amount of earwax extraction from the left and right ears during follow-up; and linear regression analysis to determine the association between the amount of earwax extracted by Tears and different biological variables, or the association between the same amount and the questions in the customer satisfaction survey. The significance level was set at p less than or equal to 0.05 (two-tailed).

[0197] Results

[0198] Tables III.1, III.2, and III.3 provide detailed sociodemographic data, anthropometric data, and self-administered questionnaire results, respectively. Most participants found the use of Trears to be very comfortable, effective, and safe. They also described that, compared to cotton swabs, using Trears was more effective, safer, and more pleasant. Although only 14.3% of people were willing to purchase the product, most still indicated that they might consider using the product (60.7%) (Table V.1).

[0199] Although there was no difference in the amount of baseline earwax samples extracted from the left and right ears, much more earwax was extracted using Trears than using the Reiner-Alexander syringe (p < 0.001) (Table IV.2). After cleaning with the baseline Reiner-Alexander syringe, the production of earwax in both ears increased significantly (both p < 0.05). The amount of earwax extracted from the left ear during follow-up was also significantly higher than that of the baseline right earwax sample (p < 0.05).

[0200] Although different top pin thicknesses did not show any difference in the amount of earwax extracted, sponges with a humidity of 50% or 105.1 μl MgCl 2 (Table IV.3) extracted more earwax than sponges with 12.5% (p < 0.05) (Table IV.4). No biological or psychological variables changed the amount of earwax secretion (all p > 0.05) (Table IV.5). Although different variants of Trears did not change the participants' evaluation of the Trears usage experience, when the subjects faced more or more severe troubles, they considered its effectiveness mediocre and worse than cotton swabs (Table IV.6).

[0201] Charts:

[0202] Table IV.1: Satisfaction Survey of Trears

[0203]

[0204]

[0205] Table IV.2: Comparison of Earwax Extraction Using the Reiner-Alexander Syringe and Trears Device

[0206]

[0207] Table IV.3: Trears Humidity

[0208]

[0209]

[0210] Table IV.5:

[0211] Linear regression models between cerumen extracted by Trears and some biological and psychological variables

[0212]

[0213]

[0214]

[0215] Discussion

[0216] The study found that after baseline cleaning of both ears with a Reiner - Alexander syringe, the amount of cerumen produced increased significantly. Trears removed more cerumen compared to the clinical method (Reiner - Alexander syringe). Most participants found using Trears comfortable, effective, and safe. They also reported that it was more effective, safe, and comfortable than using cotton swabs. Although only 14.3% of people were willing to buy the product, most participants said they might consider using this option (60.7%).

[0217] It is worth noting that during the follow - up period, regardless of the extraction method used, the amount of cerumen increased significantly rather than decreased. This may be due to insufficient cerumen after baseline external ear cleaning with a Reiner - Alexander syringe, causing the ear glands to increase cerumen secretion as a compensatory mechanism. The design of our research protocol also took this difference into account. We instructed participants to avoid cleaning their ears in subsequent stages to extract a standardized amount of cerumen to trace the cerumen produced in that month. This likelihood was also strengthened after observing that 35.7% of the participants were heavy cotton swab users and another 28.6% used them at least occasionally.

[0218] In addition to warm water, the Reiner - Alexander syringe can also be used to extract more cerumen. Cipriani et al. (1986) used an extraction method of injecting a solution of alcohol / ether 3:1 v / v (2.02 ± 0.22 mg / week). This method extracted more cerumen (2.25 ± 0.18 mg / week) than the conventional extraction method. The results of Cipriani et al. (1986) were also confirmed in this experiment - after comparing the baseline results, it was found that there was no volume change in cerumen related to the ear side. However, the baseline amount of cerumen does not represent the cerumen secretion amount after one month, because in healthy mixed - race populations, Trears extracted eight times more cerumen than the syringe.

[0219] Although highly inadvisable, the use of cotton swabs will continue to increase. Therefore, there is a current need for a safe alternative. Unfortunately, all current self-cleaning products or devices are ineffective or have limited effectiveness compared to using clinical methods such as the Reiner-Alexander syringe. However, Trears is not only as useful as traditional earwax removal methods but is also capable of extracting more earwax than clinical methods. The cleaning head material of Trears is a special abrasive and absorbent honeycomb sponge that has previously shown utility in removing artificial liquid wax. In addition, a mineral oil has been added to the cleaning head of Trears, which has shown some effect on removing earwax. We found that adding a higher concentration (50%) of a mineral oil (such as magnesium chloride) increased the extraction rate of Trears compared to using a relatively dry cleaning head (concentration of 12.5%). Future Trears devices will reach this humidity level. In addition, the cumulative effect of the cleaning head plus mineral oil can explain these important research results.

[0220] Measuring mean glucose concentration using earwax

[0221] Background: Elevated mean blood glucose concentration is associated with many epidemics and chronic diseases. Currently, there is no test that can accurately reflect its long-term level, is reasonably priced, convenient, and harmless. Earwax may meet these characteristics. The applicant correlated fasting and postprandial blood glucose levels with baseline and follow-up samples of earwax and glycated hemoglobin (HbA1c).

[0222] Method: Thirty-seven healthy participants provided two right ear earwax samples and two serum samples, which were collected one month apart. Baseline measurements were taken after an 8-hour fast, and follow-up samples were collected after ingestion of a standard meal. The baseline earwax glucose concentration (EGC) represents the average concentration of fasting and postprandial glucose levels for a previously unknown period, while the subsequent EGC represents the average of the same data for the current month. Both HbA1C samples represent the retrospective average glucose concentration between one and three months. The mean blood glucose was calculated using the average of their respective baseline and follow-up data. The baseline level and follow-up level of each sample were compared. The effects of several covariates were studied in these samples, including the correlation of fasting serum glucose [FSG] with their respective baseline EGC and HbA1c samples, and the correlation of postprandial serum glucose [PSG] with their respective follow-up EGC and HbA1c samples. The mean blood glucose level was correlated with all of the above HbA1c and EGC samples. Different blood glucose levels were predicted using baseline and follow-up EGC and HbA1c samples.

[0223] Results: All the tracked concentrations were higher than their respective baseline concentrations. The earwax samples were not affected by any covariates. Although all the associations between EGC and blood glucose levels showed strong positive correlations (all R > 0.60; p < 0.001), the associations of HbA1c with different blood glucose levels all showed moderate or weak correlations (all R < 0.50; 0.10 < p < 0.01). Baseline EGC predicted a significant increase in mean blood glucose levels (all p < 0.001).

[0224] Conclusion: EGC reflects blood glucose levels more accurately than HbA1c. Earwax is more stable than blood glucose and HbA1c in measuring glucose concentration. The EGC results indicate that earwax can better represent long-term average glucose levels.

[0225] Although other studies have also measured glucose levels in earwax samples (Masuda et al., 1978; Shichjo & Masuda, 1979), and even glucose levels in diabetic patients (Khasanov and Popova, 1984), it remains unknown whether the EGC found in previous studies can accurately represent long-term blood glucose levels. This is why in this study, we measured the levels of EGC, blood glucose, and HbA1c in samples from healthy participants during fasting and after a standardized meal, and compared the baseline levels and tracked levels of each sample. This project also studied the effects of several covariates on glucose levels. Fasting serum glucose [FSG] was correlated with its baseline EGC and HbA1c, and postprandial serum glucose [PSG] was also correlated with its tracked EGC and HbA1c levels. Mean glucose levels were correlated with all the above HbA1c and EGC samples. Different blood glucose levels were predicted using baseline and tracked EGC and HbA1c samples.

[0226] Research hypotheses:

[0227] 1) All the concentrations of the tracked tests are more important than their respective benchmark concentrations;

[0228] 2) Earwax is more stable than blood glucose and HbA1c in reflecting blood glucose concentration;

[0229] 3) All the associations between EGC and different blood glucose levels are stronger than the associations between HbA1c and the same blood glucose measurements, and

[0230] 4) Baseline EGC can predict a drastic increase in mean blood glucose levels.

[0231] Methods:

[0232] Thirty-seven healthy participants provided two right ear wax and two serum samples, which were collected one month apart. Baseline measures were taken after an 8-hour fast, and follow-up samples were collected after ingestion of a standard meal. Although the retrospective period of ear wax glucose accumulation is unclear, the follow-up EGC covered glucose accumulation for the current test month. Both HbA1C samples represented retrospective average glucose concentrations between one and three months. The average blood glucose level was calculated using the mean between its baseline and post-follow-up blood glucose levels. The baseline and follow-up levels of each sample were compared, and the effects of several covariates were studied in these samples. Fasting serum glucose [FSG] was correlated with its baseline EGC and HbA1c, and the same was true for postprandial serum glucose [PSG] with its follow-up EGC and HbA1c. The average blood glucose level was correlated with all of the above HbA1c and EGC metrics.

[0233] Ear wax samples:

[0234] Right ear wax baseline samples were collected after an 8-hour fast. Right ear wax follow-up samples were collected two hours after the subjects had their meal (a standard 236-ml liquid meal). All samples were labeled and stored at 4 degrees Celsius.

[0235] Ear wax was sampled using a Reiner-Alexander syringe.

[0236] Ear wax samples obtained by ear irrigation were dried using the N 2 displacement method. Briefly, 50 ml was aliquoted into four test tubes, and each tube was inserted with a sleeve connected to an N 2 gas cylinder, and a constant temperature bath was used to maintain a constant temperature of 25 °C. When the nitrogen gas flow was turned on, it would displace the evaporated H 2 O, thus drying the sample. Once the base sample was dried (as was the case with the left sample obtained on day 30), the weight of the dried ear wax sample was obtained by subtracting the weight of the empty test tube (previously weighed). Finally, 125 μl PBS was added to each test tube, the contents of each tube were resuspended, and the samples were combined into a 5-ml test tube. The resulting sample was resuspended in 500 μl PBS and stored at 4 °C until use.

[0237] Ear wax samples collected using Trears

[0238] Using Ear wax samples obtained using the instrument were processed by rinsing the sponge cleaning head with 500 μl of PBS for 2 minutes. Since the extraction mechanism of Trears is drying, N 2The drying time is much less. In fact, contrary to the Reiner-Alexander syringe, Trears does not inject water.

[0239] Specifically, 500 μl of the buffer solution PBS was added to a 5-ml tube; then it was placed in a 5-ml test tube. The sponge was separated from its plastic support and introduced into the tube. After the sponge had absorbed all the solution, it was squeezed repeatedly and absorbed for 2 minutes, and then the sponge was squeezed dry and removed from the test tube. Subsequently, the resulting solution was dried by the N 2 displacement method, and the contents were resuspended in 500 μl of ultrapure water. The solution in the test tube was stored at 4 °C until use.

[0240] Serum samples

[0241] Fasting blood samples were drawn (antecubital vein, 3-cc syringe, and blood collection tubes without anticoagulants). All serum samples were collected in the morning. One HbA1c and blood glucose sample [FSG] were collected in the morning at the baseline visit. Participants had been instructed to avoid eating or drinking for eight hours before the assessment. Another HbA1c and blood glucose sample [PSG] were also collected during the follow-up. These follow-up samples were collected after the ingestion of a standard liquid meal. 236 ml of the liquid meal contained 23.6% protein, 44.8% carbohydrates, 28.8% fat, 28.8% fat, 1% fiber, and 1.1 ml of 236 ml% β-hydroxy-β-butyric acid butyl ester. The mean glucose concentration in serum was estimated from the mean between fasting and postprandial blood glucose levels.

[0242] Serum glucose analysis:

[0243] Preparation of serum samples. Blood samples obtained under fasting and postprandial conditions were stored at 4 °C for 24 hours to facilitate serum clotting and separation, and then centrifuged at 1000 x g for 20 minutes at 4 °C. The precipitate was collected with a 1-ml syringe into 2-ml labeled plastic tubes. Once the serum was obtained, they were stored at -20 °C until use.

[0244] Quantification of glucose extracted from cerumen and serum:

[0245] Glucose was quantified by enzymatic oxidation assay and quantification of oxidized glucose label in 96-well plates according to the instructions given by the enzyme supplier (BioVision Inc., Milpitas, CA, USA). The standard curve was 0, 2, 4, 6, 8, and 10 nmol glucose standards per well. 50 μl of the standard, the aqueous part, and a 1:25 serum dilution were added to the remaining wells together with 50 μl of a glucose quantification mixture containing glucose assay buffer, 2 μl of a glucose probe, and 2 μl of an enzymatic glucose mixture. After measuring the absorbance at 570 nm in a microplate reader (NovoStar), the mixture was immediately reacted for 30 minutes at 37 °C in the dark. The absorbance of the standard curve was adjusted to a linear equation, and the glucose content was calculated by interpolation within the adjusted curve.

[0246] Quantification of glycosylated hemoglobin in serum samples:

[0247] The content of glycated hemoglobin (HbA1c) was quantified using a sandwich ELISA method. According to the instructions of its supplier (Abbexa Ltd., Cambridge, UK), the standard curve used was 0.30125, 6.25, 12.5, 25, 50, 100, and 200 ng / ml HbA1c standards. The standards and 100 μl of the standards, as well as undiluted serum, were added to the wells of a plate coated with an HbA1c antibody, and allowed to react with shaking at 37 °C for 60 minutes. After discarding the contents and washing twice with the washing solution, 100 μl of an HbA1c detection antibody conjugated with biotin was added, and then reacted with shaking at 37 °C for 30 minutes. After discarding the contents and washing five times with the washing buffer, 90 μl of TMB medium was added to each well and reacted at 37 °C in the dark for 20 minutes. Finally, 50 μl of the solution was added to each well, and the absorbance at 450 nm was quantified in a microplate reader (NovoStar). The absorbance of the standard curve was fitted to a straight line, and the absorbance of the sample was interpolated into the curve.

[0248] Statistical analysis

[0249] The normality of the data was checked using statistical tests and graphical methods. All other values were normally distributed (all p > 0.05). Therefore, repeated t-tests were used to compare the baseline and follow-up blood glucose levels of different samples. Linear regression analysis was used to determine the associations between glucose concentration and different biological and psychological variables, and EGC and HbA1c specimens were used to predict different blood glucose levels. Pearson correlations were used to determine the associations between baseline and follow-up EGC and different blood glucose levels, or between baseline and follow-up HbA1c and different blood glucose levels. Cohen's correlation criteria were used: low when r = 0.1 - 0.3, moderate when r = 0.3 - 0.5, and high when r = 0.5 - 1.0 (J Cohen, 2013). The time required to analyze each sample was also recorded. The significance level was set at p ≤ 0.05 (two-tailed).

[0250] Results

[0251] Tables III.1, III.2, and III.3 provide detailed sociodemographic and anthropometric data, as well as the results of the self-management questionnaire, respectively. Using Trears significantly reduced the time required to analyze earwax compared to the time required using Reiner-Alexander syringes (compare Tables II.4 and V.1). All follow-up concentrations were higher than their respective baseline concentrations (Table V.2). Earwax samples were more stable than HbA1c or blood glucose levels as their glucose concentrations were not affected by any covariates (Table V.3). Age had a direct effect on baseline HbA1c samples. Additionally, follow-up HbA1c and PSG levels increased with increasing years of education; smoking decreased FSG and PSG levels (Table V.3). All associations between EGC and blood glucose levels showed strong positive correlations (all R > 0.60; p < 0.001), while HbA1c showed moderate or weak correlations (all R < 0.50; 0.10 < p < 0.01)( Figure 5 , 6, 7, and 8). The association between follow-up HbA1c and FSG was stronger (R = 0.48, p < 0.001) compared to the association between baseline HbA1c and FSG (R = 0.43, p < 0.001)( Figure 6 and 8 ). Both of these EGC associations were stronger than the HbA1c associations. However, the follow-up EGC samples also showed a stronger association with PSG (R = 0.90, p < 0.001) compared to the baseline EGC samples with FSG levels (R = 71 p < 0.001)( Figure 5 ). The mean blood glucose level also showed a stronger association with earwax rather than HbA1c specimens (see Figure 7 , Figure 9 , 10and 11, 12). Among them, the postprandial earwax samples also showed the strongest association with the average blood glucose level (R = 0.84, p < 0.001)( Figure 11 ).

[0252] Table V.1: Time for analyzing different samples

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260] Discussion

[0261] The study found that all the tracked concentrations using blood glucose, HbA1c, and EGC specimens were significantly higher than their respective baseline concentrations, thus confirming that the study was properly conducted. Earwax is more stable than HbA1c and blood glucose samples because its glucose level is not affected by any covariates. Although all the associations between EGC and blood glucose levels showed strong positive correlation coefficients (all R > 0.60; p < 0.001), the HbA1c associations between the two blood glucose levels only showed moderate or weak correlations (all R < 0.50; 0.10 < p < 0.01). Tracking both EGC and HbA1c measures showed the strongest correlations with PSG, with the correlations between tracked EGC and PSG being (R = 0.90, p < 0.001 and R = 0.48, p < 0.01) respectively. However, the largest increases in predicting blood glucose levels were achieved through baseline EGC and HbA1c (β = 13.2, p < 0.001; β = 11.7, p < 0.01) respectively.

[0262] Previous studies have confirmed that in healthy populations, the association between HbA1c and fasting or postprandial blood glucose levels is very small (van t Riet et al., 2010). This result may be due to moderate reasons for the samples, or because HbA1c usually shows a stronger association in populations with elevated glucose levels (such as diabetic patients) (van t Riet et al., 2010).

[0263] It must be emphasized that baseline EGCs cannot be fully comparable to their follow-up samples because they represent different periods of glucose accumulation in earwax. Indeed, the study design only allowed us to standardize the earwax secretion volume of follow-up samples after baseline ear cleaning. Intense physical activity or certain stress events prior to this may temporarily increase baseline EGC samples. In this sense, baseline EGCs may represent several accumulations of long-term fasting and postprandial blood glucose levels. However, not only baseline-HbA1c, but also baseline-EGC can predict the maximum increase in the average blood glucose level, which also indicates that EGC better represents the average glucose concentration rather than fasting or postprandial blood glucose concentration. It is well known that HbA1c is mainly affected by FSG rather than PSG because people fast for longer periods during the day than they eat (Monnier et al., 2006). Baseline EGC may also be mainly affected by fasting blood glucose levels because its glucose concentration is lower than that of its follow-up earwax samples (p < 0.01).

[0264] Earwax is undoubtedly better than HbA1c at reflecting glucose levels. Not only because all correlations between EGC and blood glucose levels are stronger than the correlation coefficients observed between HbA1c and blood glucose levels, but also because stronger correlations are shown between follow-up EGC and PSG (R = 0.90; p < 0.001) than between baseline EGC and FSG (R = 0.71; p < 0.001). Therefore, the correlations between baseline EGC during the fasting period and different blood glucose levels may be more significant than those observed between follow-up EGC and different blood glucose levels.

[0265] Previous studies have shown that HbA1c levels are affected by age. HbA1c is also affected by educational level, and the corresponding type of work is likely to explain this. Studies have shown that these jobs that require a higher level of education are associated with an increase in working hours (Uehata, 1991), and working hours in turn are also associated with an increase in HbA1c (Azami et al., 2018). We also verified previous results that showed that smoking reduces FSG and PSG. In contrast, earwax is also a more stable specimen because its cortisol level is not affected by any covariates. Our study results indicate that the next step should be to start testing EGC in patients with diabetes and obesity.

[0266] Some previous studies have used the area under the curve rather than the mean between fasting and postprandial glucose levels to estimate mean glucose concentration (Avignon et al., 1997). However, the mean between fasting and postprandial blood glucose levels has also been shown to be a very accurate indicator. In fact, Svendson and his team found that in 18 type 1 diabetic patients, the mean glucose level obtained from approximately 2 to 300 measurements per individual was almost perfectly correlated with HbA1c (R = 0.96) (Aaby Svendsen et al., 1982). Ozmen et al. found that the mean blood glucose level caused by fasting and postprandial plasma glucose levels was also closely correlated with HbA1c in type 2 diabetic patients (Ozmen et al., 2006). Recently, the mean between postprandial and fasting blood glucose levels in women with gestational diabetes has also been used (Koren et al., 2016). Therefore, this index may be more relevant to the mean blood glucose level in healthy individuals, as their 24-hour blood glucose level variation is less than that of diabetic patients (Praet et al., 2006).

[0267] Inter-individual differences in the ability of subjects to absorb different dietary components may also affect their blood glucose levels (Freckmann et al., 2007). This is why some studies use the glucose tolerance test after ingestion of 75 grams of glucose rather than measuring postprandial levels after a standard meal (Ensure). This meal contains various nutrients such as protein, lipids, and glucose, and their absorption rates may vary, which ultimately affects the level of PSG. However, we used a widely used test to provide a liquid meal that is easily absorbed. In addition, participants with allergies to certain foods, such as lactose intolerance, were excluded.

[0268] Regarding the difference in blood glucose levels between plasma and serum, some studies report that plasma glucose is higher than serum glucose, while other studies have found no difference. However, it is not recommended to diagnose diabetes by measuring glucose in serum (American Diabetes Association, 2010). However, we did not use FSG or PSG levels for any diagnosis because we recruited healthy subjects and used different samples to investigate their blood glucose levels.

[0269] In summary, earwax reflects the mean glucose level better than blood glucose and HbA1c samples because it is more stable and its glucose concentration can better predict the mean glucose concentration rather than its post-fasting or postprandial levels.

[0270] Measurement of cortisol levels using earwax samples

[0271] Background: The diagnosis of depression is considered to be not entirely reliable. This may be due to the substantial heterogeneity of the syndrome. An accurate biomarker could improve the consistency of this diagnosis. Cortisol levels are commonly measured in the diagnosis of depression because it is a common neurobiological alteration under this syndrome. However, due to the secretion characteristics of reactive cortisol, the cortisol results using short-term samples vary greatly. These samples are not suitable for reflecting the average cortisol concentration because some acute factors affect its cortisol level. It has been found that hair cortisol concentration (HCC) reflects the long-term cortisol system level because it accumulates hormones over a long period. However, its widespread use seems unrealistic. In addition, it is not clear whether certain acute effects actually alter HCC. Ear wax cortisol concentration (ECC) may be a more convenient and accurate sample for reflecting the average cortisol concentration.

[0272] Method: The ears of 37 healthy subjects were cleaned during baseline sampling. One month later, the ECC was analyzed from the right ear of the subjects. During the follow-up period, the subjects also provided 1 cm of hair, retrospectively representing the accumulation of hair cortisol in the past month. The ECC and HCC were compared and correlated with each other.

[0273] Results: The ECC was significantly greater than the HCC (P < 0.001). The ECC and HCC showed a significant moderate positive correlation (R = 0.39; p = 0.03). Although the HCC of males was higher than that of females (p < 0.001), the ECC was not affected by gender.

[0274] Conclusion: The ECC may be another sample that can accurately reflect the average cortisol concentration. Compared with hair, ear wax accumulates a higher concentration of cortisol. Ear wax is also a more stable sample because its cortisol level is not affected by any covariates. Finally, the time required to analyze the ECC is much less than the time required to analyze the HCC.

[0275] Objectives and Hypotheses:

[0276] Although we recently found that this hormone could be detected in this secretion, it is not clear whether its content represents the average cortisol concentration. This is why in this study, we correlated the cortisol concentration (ECC) and HCC in ear wax samples from 37 healthy subjects. We hypothesized that: 1) the time required to analyze the ECC is less than the time required to analyze the HCC, 2) the cortisol content in the ECC is more than that in the HCC, 3) there is a positive correlation between the ECC and HCC, and 4) common short-term and hair cortisol covariates do not affect the ECC.

[0277] Methods:

[0278] We compared and correlated cortisol levels obtained from swabbed cerumen samples and 1 cm hair samples obtained during the same visit. Both samples represent the cortisol accumulation over the last month. The effects of several covariates were studied in these samples. The time required for the analysis of ECC was also recorded.

[0279] Cerumen samples obtained using Trears:

[0280] (See instructions Figure 1 )

[0281] Cerumen cortisol analysis:

[0282] Purifying cortisol in cerumen samples : After obtaining the cerumen samples by the device, the samples resuspended in 500 μl PBS were homogenized with a 1 ml syringe. Then, 500 μl of diethyl ether was added, and each sample was vortexed for 1 minute and then placed at -20 °C for 2 hours. Thereafter, the liquid part (organic part) of each sample was transferred to a new 5 ml test tube and appropriately labeled and dried using the above N 2 displacement method. After drying, the samples were resuspended in 500 μl PBS, and the cortisol levels were quantified. On the other hand, the aqueous solution remaining after extraction with diethyl ether was used to determine the glucose level in the cerumen.

[0283] Determining cortisol in cerumen samples:Cortisol was quantified using ELISA according to the instructions provided by its supplier (Enzo Life Sciences, Farmingdale, NY, USA). The cortisol content was quantified by competitive colorimetric ELISA technology using a standard curve of 0, 156, 313, 625, 1250, 2500, 5000, and 10,000 pg / ml of cortisol standards. 100 μl of the standard solution, organic sample portion, and serum diluent were added to the wells of a plate coated with anti-mouse antibody. In addition to the above solutions, 50 μl of cortisol blue conjugate covalently bound to alkaline phosphatase and 50 μl of mouse monoclonal antibody against cortisol were added to all wells. After adding the antibody, it was agitated and incubated for 2 hours to allow the cortisol present in the sample / standard to compete with the cortisol of the conjugate and the anti-hormone antibody. Due to the interaction of this antibody with the antibody, it will remain bound to the well and adhere to the well wall. After 2 hours, the wells were thoroughly washed, and 200 μl of p-nitrophenyl phosphate (pNpp) was added to each well and incubated for 1 hour without agitation. This step causes the p-nitrophenyl phosphate to be converted by an enzyme-mediated reaction mediated by alkaline phosphatase covalently linked to cortisol in p-nitrophenol, and its coloring is inversely proportional to the amount of cortisol present. Finally, 50 μl of the solution for terminating the enzymatic reaction was added to each well. The plate was read at 405 nm in an ELISA reader (NovoStar), the absorbance of the standard curve was adjusted to a 4-point logarithmic curve, and the absorbance of the sample was interpolated into this curve to obtain the concentration of the sample (pg / ml). The concentration was multiplied by the microliters of the sample (500 μl) and then divided by the weight of the dried sample to obtain the concentration of cerumen in mg / mg. The concentration was multiplied by the dilution factor to calculate the concentration in serum.

[0284] Hair samples

[0285] A trained clinician collected hair samples from all subjects. The presence and frequency of biological contaminants or procedures that may affect hair cortisol levels were measured, including cosmetic treatments (dyeing, bleaching, permanent straightening or curling) and the frequency of shampooing. Hair samples were collected from the vertex of the posterior part of the head and cut as close to the scalp as possible with clean scissors. For this study, four bundles of hair were taken from different positions at the posterior vertex, and each bundle of hair was about the thickness of a 1-cm rubber band. In the laboratory, 1-cm-long hair was cut from the root of each hair bundle, which approximately represents the hair growth in the past month and is equivalent to a 1-month retrospective assessment of cortisol. The total weight of the 1-cm portion selected from each hair bundle is approximately equal to 25 - 50 mg of hair. After collection, the hair samples will be stored in a sealed container at room temperature in a light-free environment.

[0286] Hair cortisol analysis

[0287] Before analysis, hair samples were washed in 1 ml of isopropanol to remove external contaminants, then the isopropanol was removed from the vial and the hair was dried in a clean air environment for 48 hours. After complete drying, five ceramic balls were added to each tube and the hair samples were ground into powder using an MPbio Fast Prep (MP Biomedicals, LLC). To extract cortisol, 1.75 ml of methanol was added to each sample and the samples were incubated for 20 hours while constantly rotating the samples.

[0288] The hair, methanol, and ceramic balls were poured into polypropylene tubes (Sarstedt AG&Co, Germany) that separated the ceramic balls from the rest of the mixture. The tubes were centrifuged at 3000 RCF to separate the hair and methanol, and then 1.25 ml of the clear methanol supernatant was poured into 2 ml polypropylene cryotubes. The methanol was then removed using a vacuum centrifuge (Scan Speed 40, Labgene) and the tubes were frozen at -80 °C until cortisol ELISA was required. Cortisol levels were measured using a commercially available competitive ELISA (Salimetrics LLC, USA). The samples were thawed and reconstituted with 0.125 ml of Salimetrics cortisol assay diluent, and then the samples were assayed according to the manufacturer's protocol. Results were presented as picograms of cortisol per milligram of hair. All hair samples were analyzed in the Biomarker Analysis Laboratory at Anglia Ruskin University, Cambridge, UK (www.anglia.ac.uk) (Albermann & Musshoff, 2012).

[0289] Statistical analysis

[0290] Statistical tests and graphical methods (e.g., histograms) were used to examine the normality of the data; the ECC and HCC values were normally distributed. Therefore, we used a paired t-test for paired samples to compare ECC and HCC. Pearson correlation was used to determine the association between HCC and ECC. Cohen's correlation criteria were used: low when r = 0.1 - 0.3, moderate when r = 0.3 - 0.5, and high when r = 0.5 - 1.0 (J Cohen, 2013). Linear regression analysis was used to determine the association between glucose concentration and different biological and psychological variables. The time required to analyze ECC was also recorded. The significance level was set at p ≤ 0.05 (two-tailed).

[0291] Results

[0292] Tables III.1, III.2, and III.3 list the detailed results of the sociodemographic, anthropometric, and self - administered questionnaires, respectively. The time required to analyze ECC using Trears is half the time required to analyze ECC using the Reiner - Alexander syringe (compare Tables II.4 and VI.1). The time required to analyze ECC using Trears is four times less than the time required to analyze HCC (Table VI.1). ECC is significantly more than HCC (Table VI.2). Although HCC is elevated in men compared to women (P < 0.001) (Table VI.3), gender does not affect ECC. ECC is moderately positively correlated with HCC (R = 0.39, p = 0.003)( Figure 13 )

[0293] Table VI.1: Time required to analyze ECC

[0294]

[0295]

[0296]

[0297]

[0298] Please see Table 13.

[0299] Discussion:

[0300] Extracting ECC using Trears is significantly more efficient than using the Reiner - Alexander syringe. When compared to the time required to analyze HCC, its performance is even better. The cortisol concentration in ear wax is significantly higher than that in hair. Also, compared to hair, ear wax is a more stable sample for reflecting cortisol levels because it has no covariates. In fact, we have also confirmed the conclusion of previous studies that HCC is elevated in men compared to women (Garcia - Leon et al., 2018; Vanaelst et al., 2012).

[0301] Hair is another specimen that can accumulate cortisol concentration, and it has a significant positive correlation with specimens that have been proven to reflect long - term cortisol levels. This correlation is strengthened after finding that the new sample is not affected by any acute factors. The correlation between ECC and 24 - hour urine collection or continuous cortisol levels may also increase. There is some evidence that HCC is affected by some acute factors, such as sweat (Sharpley, 2012) and nerve fibers (Okumura, 1967).

[0302] The cortisol concentration in cerumen accumulates higher than that in hair. Cortisol is indirectly transported to the hair shaft by an unclear multi-compartment model. However, cortisol is directly secreted from the sebaceous glands into the external auditory canal through a simple single-compartment model. A potential limitation is related to the comparison between ECC and HCC. Although the same sample of subjects was used in this study, their results are not strictly comparable because the analysis of these samples was conducted in two different laboratories. Clark et al. (1998) showed that in the control group undergoing the standard adrenocorticotropic hormone test, the significant deviation ratio between five different immunoassays was as high as 1.2. (Jeremy Cohen et al., 2006). However, we found that the ECC / HCC ratio was as high as 14.3. Despite the large deviation, ELISA technology was used in both laboratories. Therefore, it is impossible to find this difference by chance. Future studies may correlate ECC with nail cortisol levels, which is another specimen that may accumulate cortisol levels over a long term (Izawa et al., 2015). Ultimately, ECC should be measured in patients with depression.

[0303] Conclusion:

[0304] For healthy people, cerumen may be a more economical, convenient, and effective method of self-cleaning the outer ear. This device can also replace the risky use of cotton swabs. Cerumen samples can accurately reflect the average levels of cortisol and glucose. Common acute effects do not affect the levels of glucose and cortisol in cerumen.

Claims

1. A method for measuring the levels of glucose and cortisol in earwax, comprising the following steps: extracting an earwax sample from the ear by any suitable method; preparing the earwax sample according to a measurement method to measure the levels of cortisol and glucose; measuring cortisol and glucose using any known means or method, wherein the levels of cortisol and glucose are interpreted as long-term average cortisol and glucose levels, wherein: the extraction of earwax is carried out by an extraction device comprising the following parts: a handle having a first and a second end, the second end having a coupling device: a detachable sponge tip, which includes a base and an elongated member that directly hangs from the upper part of the base and extends longitudinally, wherein the lower part of the base has a portion for coupling the handle, and wherein the elongated member has a star-shaped cross-section; an elongated sponge, which has a central longitudinal housing for accommodating the elongated part of the base; wherein earwax is obtained by inserting the tip with the sponge into the ear and rotating the sponge within the ear canal, wherein: the preparation of the sample is carried out as follows: a) drying the earwax sample until all moisture has evaporated from the sample, and this step is completed using freeze-drying; b) weighing the dried earwax sample to standardize the amount of cortisol by dry weight, and normalization means adjusting the measured weight to a standard ratio so that data can be compared; c) homogenizing the dried sample with 1 ml of phosphate buffered saline PBS to obtain a PBS solution of earwax, and the volume of the phosphate buffered saline PBS solution is 10 times the weight of the earwax by 10, and in addition, any hydrophilic solvent can be used; d) dividing the solution obtained in step c) into a first solution portion and a second solution portion, and adding each solution portion to a test tube separately; e) adding a solvent to the first solution portion in a ratio of 1:1 between PBS and the first solution portion to obtain a mixed solution of earwax in PBS and the solvent; f) stirring the test tube containing the solution obtained in step e) for at least one minute to mix it thoroughly, and adding 0.5 mg of diethyl ether after resuspension, with a ratio of 1:1 to PBS; g) cooling the mixed solution obtained in step g) at a temperature of -18 to -21 °C for at least two hours to ensure that the liquid part is frozen and does not contaminate the organic part, and this step extracts those compounds that are clearly dissolved in diethyl ether, and this step is taken because although the diethyl ether part remains liquid at -20 °C, the phosphate part is frozen; h) extracting the compounds that are clearly soluble in diethyl ether from the cooled solution; i) drying the remaining liquid solution; j) storing the dried part obtained in step i) at -80 °C for further use; k) adding 300 pg of cortisol to the second solution portion to obtain a solution of earwax mixed with cortisol in PBS; I) adding 0.5 ml of solvent to the solution obtained in step k) to quantify the amount of purified cortisol; m) stirring the test tube containing the solution obtained in step I) for at least one minute, and adding 0.5 mg of ether to the obtained solution after resuspension, with a ratio of 1:1 to PBS; n) Cool the mixed solution obtained in step m) at a temperature of -18 to -21 °C for at least two hours, ensuring that the liquid part is frozen and does not contaminate the organic part; o) Extract the compounds that are clearly soluble in diethyl ether from the cooled solution; p) Dry the remaining liquid solution; q) Store the dried part obtained in step i) at a temperature between -20 and -90 °C for further use; r) Dissolve 0.5 ml of a 300 pg / ml purified cortisol solution in PBS with a pH between 6.8 and 7.

2. This step is carried out to enhance the efficiency of the cortisol earwax extraction protocol. s) Perform the same procedure on the second solution part as on the first solution part to extract cortisol therefrom; The measurement of cortisol is carried out by the following method: a) Reconstitute the extracted sample using the buffer assay provided by the manufacturer, which uses a colorimetric competitive ELISA technique. Cortisol is quantified by adding buffer to the extracted sample to obtain a solution and allowing it to stand and stir; b) Measure the total amount of cortisol in the sample using a standard curve - microplate reader NovoStar for measuring cortisol; c) Normalize the dry weight of the earwax using fluorescence techniques, where the fluorometer is excited in the range of 530 - 570 nm and reads in the emission range of 590 - 600 nm. Since multiple variables such as age, gender, different medical conditions, and stress levels affect the cortisol level in earwax, while the cholesterol level is not affected by the above variables, the cholesterol level is used to avoid confounding of the cortisol results by these covariates. Follow the manufacturer's instructions to measure the glucose content in the dissolved earwax solution using a kit, where the glucose absorption is quantified in triplicate at 505 nm, and the glucose concentration in mg / dl is obtained using its absorption average. Calculate the total amount of glucose in the solution based on the initial weight of the sample after the standardization process.

2. The method for measuring the glucose and cortisol levels in earwax according to claim 1, wherein 100 g of earwax uses 1000 ul of phosphate buffered saline (PBS) solution.

3. The method for measuring the glucose and cortisol levels in earwax according to claim 1, wherein the hydrophilic solvent is physiological saline.

4. The method for measuring the glucose and cortisol levels in earwax according to claim 1, wherein the compound is cortisol.

5. The method for measuring the glucose and cortisol levels in earwax according to claim 1, wherein in step g), the mixed solution obtained in step g) is cooled at a temperature of -20 °C for at least two hours to ensure that the liquid part is frozen and does not contaminate the organic part.

6. The method for measuring the glucose and cortisol levels in earwax according to claim 1, wherein in step n), the mixed solution obtained in step m) is cooled at a temperature of -20 °C for at least two hours to ensure that the liquid part is frozen and does not contaminate the organic part.

7. The method for measuring the levels of glucose and cortisol in earwax according to claim 1, wherein in step a), N 2 steam is used to dry the earwax at ambient temperature.

8. The method for measuring the glucose and cortisol levels in earwax according to claim 1, wherein the earwax is dried by using a lyophilization method in step a).

9. The method for measuring glucose and cortisol levels in earwax according to claim 1, in step e), the solvent comprises diethyl ether.

10. The method for measuring glucose and cortisol levels in earwax according to claim 1, in step i), by means of N 2 Steam dries the remaining liquid solution at ambient temperature.

11. The method for measuring glucose and cortisol levels in earwax according to claim 1, in step I), the solvent comprises diethyl ether.

12. The method for measuring the levels of glucose and cortisol in earwax according to claim 1, in step p), using N at ambient temperature 2 to steam-dry the remaining liquid solution.

13. The method for measuring glucose and cortisol levels in earwax according to claim 1, in step a) of cortisol measurement, the solution is allowed to stand for 20 minutes.

14. The method for measuring glucose and cortisol levels in earwax according to claim 1, in step a) of cortisol measurement, the solution is stirred within 1 minute.

15. The method for measuring glucose and cortisol levels in earwax according to claim 1: The sample preparation is carried out by adding PBS buffer solution into a test tube; separating the sponge from its plastic support and introducing it into the tube; after the sponge has absorbed all the solution, squeezing the sponge repeatedly to absorb; squeezing the sponge dry and taking it out of the test tube; drying the obtained solution; resuspending the obtained content in ultrapure water, and storing the obtained solution until use; In the dissolved earwax solution, the cortisol level is measured by ELISA technique, and the glucose level is measured by using the said kit, and the operations are all carried out according to the manufacturer's instructions.

16. The method for measuring glucose and cortisol levels in earwax according to claim 15, in the sample preparation, the ratio between the weight of the sponge and the volume of PBS is 1:

2.

17. The method for measuring glucose and cortisol levels in earwax according to claim 15, in the preparation of the sample, the resulting solution is dried by N 2 displacement method.

18. The method for measuring glucose and cortisol levels in earwax according to claim 15, in the sample preparation, the obtained solution is stored at 4°C.

19. The method for measuring glucose and cortisol levels in earwax according to claim 1, the extraction device further has the following features: The coupling device of the handle comprises a thread; The coupling device of the base comprises a housing which has an internal thread corresponding to the thread of the handle.

20. The method for measuring glucose and cortisol levels in earwax according to claim 1, the extraction device further has the following features: The elongated member has a star-shaped cross-section, which improves the extraction efficiency of earwax when rubbing the sponge in the ear; The longitudinal housing located at the center has a star-shaped cross-section for accommodating the elongated member of the base.

21. The method for measuring glucose and cortisol levels in earwax according to claim 1, the extraction device further has the following features: The sponge is made of cellulose.

22. The method for measuring glucose and cortisol levels in earwax according to claim 1, the extraction device further has the following features: The sponge is adhered to the elongated member using a non-allergenic glue.

23. The method for measuring glucose and cortisol levels in earwax according to claim 1, the extraction device further has the following features: The base is wider than the handle, acting as a safety brake, which prevents the tip from being inserted into the ear canal.

24. The method for measuring glucose and cortisol levels in earwax according to claim 1, the extraction device further has the following features: The sponge is packaged and sealed under wet conditions, and a wetting agent containing magnesium chloride MgCl2 is used to make it soft.

25. The method for measuring the levels of glucose and cortisol in earwax according to claim 1, wherein the extraction device further has the following characteristics: Insert the tip with a sponge into the ear and rotate the sponge in the ear canal for 30 to 60 seconds to obtain earwax.

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