WO2018065656A1

WO2018065656A1 - Sensor for sweat

Info

Publication number: WO2018065656A1
Application number: PCT/ES2017/070656
Authority: WO
Inventors: Antoni Baldi Coll; César FERNÁNDEZ SÁNCHEZ; Emma Paola CÓRCOLES PERPIÑAN
Original assignee: Consejo Superior De Investigaciones Científicas (Csic)
Priority date: 2016-10-07
Filing date: 2017-10-09
Publication date: 2018-04-12

Abstract

The present invention relates to a sensor (1) for measuring pH and the concentration of different analytes in a sweat sample from an individual. More specifically, the sensor (1) can be worn by an individual to carry out measurements continually, and comprises a support (2) that can be connected to fluid-transporting means (3) for transporting and analysing the sweat by means of at least a first ion-selective transistor (13), a second ion-selective transistor (14) and an electrochemical sensor (15), which are in contact with various parts of the fluid-transporting means (3).

Description

OBJECT OF THE INVENTION

The object of the present invention is a sensor for measuring the pH and measuring the concentration of various analytes in a sweat sample of an individual. More specifically, this sensor is intended to be carried by the individual, so that the sensor is in contact with the user's skin to absorb sweat and measure the pH and concentration of analytes such as glucose, lactic acid, alanine, or electrolytes such as chlorides, sodium or potassium, BACKGROUND OF THE INVENTION

Currently, there is a significant increase of! number of individuals who do sports, or who have diseases that must be monitored, and that require systems or methods that determine biological parameters of their body.

These systems and methods are mainly based on the analysis of exhaled gases, or the analysis of chemical components in blood, to establish e! physical state of! individual. Recently, new devices or methods have appeared to determine these biological parameters in a non-invasive way, through! sweat of the individual

An example of these devices is shown in US2015057515A1 which describes a device that collects and analyzes a plurality of sweat samples that have previously been collected over a discrete period of time to allow the analysis of biomarkers in blood in relation to time. To perform the analysis of the biomarkers, it uses a single sensor selected from a chemical condenser, a chemical field effect transistor, an ion selective electrode, a sensor based on electrical-chemical impedance spectroscopy and an electrochemical potentiostat. Despite this, when this device uses sensors potentiometric, includes a reference electrode (for example Ag / AgCI) to perform the measurements. This Ag / AgCI electrode exhibits interference with the sample chlorides if it does not have an internal solution of constant concentration. Additionally, even if it has said internal solution, said internal solution is difficult to integrate into the device itself, as well as to ensure that it lasts a long measurement time without being contaminated. Additionally, the device described in this document is limited to the direct measurement of biomarkers.

Another example of these devices is reflected in WO2007146047A1 which discloses patches, systems and methods for measuring glucose in sweat. A preferred embodiment of this document is a patch to monitor the concentration of sweat glucose in real time. To do this, it uses glucose oxidase (GOx) as a reagent. More specifically, this patch collects the sweat by storing it in a reservoir using a permeable membrane, and measures the concentration of peroxide obtained from the reaction of the glucose with the GOx. Despite this, this device requires valves and pumps to perform the different operations that comprise a measurement, such as for the addition of GOx solution in the chamber where the detection electrodes are. Valves and pumps are difficult elements to integrate into a small space, and therefore increase both the manufacturing complexity of the device and the price of obtaining and selling.

On the other hand, the amperometric measurement by means of the detection of hydrogen peroxide resulting from the enzymatic reaction of glucose oxidase is problematic because it is carried out at a high potential to which other species present in sweat can also be oxidized and produce interference.

In addition, the use of a mediator such as ferrocyanide or ferrocene is recommended for glucose measurement; Most glucose meters on the market use mediators, since it greatly reduces the potential for glucose detection. The mediator cannot be immobilized since, once it has reacted, it has to be diffused to the working electrode in order to be detected.

As mentioned above, in the literature you can find devices that can measure the concentration of ions in sweat. More specifically, these devices are based on the use of at least one electrode. selective and of an Ag / AgCi reference electrode to measure the concentration of ions in the sweat with the intention of, for example, detecting signs of dehydration in individuals during physical exercise. Despite this, it is known that devices using Ag / AgCl reference electrodes are not accurate because they suffer from interference due to variations in chloride concentration. Because of this, some of these devices comprise an additional structure on the Ag / AgCl electrode that is formed by depositing a hydrogel containing a known concentration of salts so that this electrode behaves as a reference electrode with internal electrolyte. This hydrogel makes the device an impractical solution to manufacture and with a very limited life time, since the hydrogel can be dried or the salts can be depleted. Furthermore, it is known that the hydrogel introduces interference due to the potential of Donan that is established between the interior and exterior of said material, and which depends on the concentration of salts in the contained solution. Therefore, it is necessary to develop new sensors for the detection of anaiites and pH that solve the aforementioned technical problems.

DESCRIPTION OF THE INVENTION The present invention relates to a portable sensor by an individual for continuously measuring a pH and a concentration of anaiites in the sweat of an individual, wherein the sensor comprises a support linkable with fluid transport means for transport and analyze sweat. More specifically, the means of transporting fluids include:

"A first microcanai with a first entry and a first exit, where the first entry is intended to understand sweat,

»A second microcanai with a second input and a second output, where the second input is intended to comprise a reference composition, ® a third microcanai with a third input and a third output, where the third input is linked to the first and second exit, and

»An absorbent pad linked to the third outlet and intended to produce the movement of sweat through the first microcanai, and the reference composition through the second microcanai; and intended to mix sweat with the reference composition along the third microcanai. While the support includes:

® a first ion selective transistor intended to contact the first microchannel to perform a first pH measurement of! sweat,

"A second ion selective transistor intended to contact the second microchannel to perform a second measurement comprising the pH value of the reference composition, and

"An electrochemical sensor intended to contact the third microchannel to perform a third measurement comprising the value of the concentration of at least one product obtained from the reaction between sweat and the reference composition, and

«A processing unit linked with the electrochemical sensor and with the first and second transistor, where from the first and second measurement the processing unit differentially calculates the pH of the sweat, and from the third measurement the unit of processed calculates e! analyte concentration value of! sweat.

In the present invention, "sweat pH" is preferably understood as the concentration of hydrogen ions in sweat. In the present invention, "sweat analytes" are understood to be those compounds that are detected, such as glucose, lactate or α-anine. The concentration of each of the analytes in sweat is directly related to the concentration of the products of the reactions of! sweat with the reference composition. Preferably, each microchannel has a width between 0.5 mm to 4 mm, a thickness of 0.01 to 0.5 mm and a length between 0.5 mm to 100 mm and the material that it constitutes has a porosity between 10% to 90%.

Optionally, this support comprises a memory unit to store the value of! pH of! sweat and the value of sweat analyte concentration.

Additionally, the support comprises a data transmission unit linked to the processing unit to establish communication with an external device that represents and / or stores e! pH value of sweat and the value of the concentration of sweat analytes. Preferably, the external device is a tablet, computer or smartphone,

Optionally, the support comprises a display unit to represent the pH value of sweat and the concentration of sweat analytes.

Preferably, the support comprises a printed circuit board which in turn comprises the first and second ion selective transistor, the electrochemical sensor, the data transmission unit and the processing unit. Additionally, the support comprises a first fastening means linked to the printed circuit to removably fix the sensor to a part of the individual's body, where when the sensor is in use the first entry is in contact with the skin of the user allowing the first entry to understand the sweat of the individual. Likewise, the support incorporates a second fastening means linked to the printed circuit to removably fix the means of transporting fluids to the printed circuit.

Preferably, the first, the second and the third microchannel form a single disposable body and are made of a material with absorbent properties that make both the sweat and the reference composition transported by capillarity from the first and the second inlet to the third output, and that in the third microchannel the sweat and the reference composition are mixed by diffusion.

The material with absorbent properties is selected from paper, cellulose, nitroceiuous, vinylidene polyfluoride, polyisulfone, nylon, and a combination of the above.

Preferably, the reference composition comprises:

a) hydrolytically active salts selected from sodium phosphate and sodium chloride, and

b) at least one enzyme reagent selected from the list consisting of glucose oxidase, peroxidase, lactate oxidase, peroxidase, aianine dehydrogenase, diaphorase, nicofinamide adenine dinucieotide (NAD ⁺ ) and a combination of the above, and

c) at least one mediator selected from the list consisting of ferrocene or hexaminorutenium (III).

Preferably, the product of the reaction between the sweat and the reference composition comprises the reaction of the sweat with the enzymatic reagents and / or the mediators and is hydrogen peroxide, alanine dehydrogenase, diaphorase, ferricinium cation, or hexaminorutenium cation (II).

This reference composition is comprised in dehydrated form in an absorbent container that is in contact with the second inlet, so that when the sensor is in use, sweat travels from the first inlet to the second inlet, so that The reference composition dissolves in the sweat and when the second microchannel is completely wet the reference composition is wetted and a concentration gradient of the reference composition is established that travels by diffusion to the second outlet and then is dragged by the sweat flow to the absorbent pad.

Alternatively, the reference composition is comprised in liquid form in a container, where this container is in contact with the second inlet, so that when the sensor is in use, the container releases the reference composition, which moves from the second entrance to the absorbent pad.

Optionally, the first inlet comprises a patch with a first layer of material with absorbent properties to contact the individual's skin, a second layer of adhesive material to fix the first layer to the individual's skin and a third layer of insulating material to cover The second layer

Preferably, the first and second ion selective transistor measure sweat pH by measuring the changes in a threshold voltage of the transistor caused by variations in the concentration of hydrogen ions present in the sweat and in contact with the dielectric of the door. Preferably, the second ion selective transistor is in contact with the hydrolytically active salts of the reference composition that are in a stable concentration.

More specifically, the first ion selective transistor is a measurement ion selective transistor (ISFET) and the second reference ion selective transistor (REFET) to obtain a differential measurement of! Sweat pH and obtain highly reliable measurements.

It should be noted that hydrogen peroxide is a product of the reaction of sweat glucose with glucose oxidase, that the ferricinium cation is the product of the reaction of hydrogen peroxide generated in the previous reaction with peroxidase and that hexaminorutenium ( II) is the product of the reactions between alanine and alanine dehydrogenase in the presence of nicotinamide adenine dinucleotide in oxidized form NAD ⁺ and its coupled reaction with diaphorase.

More specifically, these reactions are as follows:

Glucose:

GOx (glucose oxidase)

Glucose + 0 ₂ + H ₂ 0> Gluconic acid + H ₂ 0 ₂

HRP (peroxidase)

H ₂ 0 + ₂ Ferrocene> H ₂ 0 + ₂ Ferricinium ⁺

+

H ₂ 0 ₂ + ₂ Ferrocene> H ₂ 0 + ₂ Ferricinic Lactic acid:

Lox (lactate oxidase)

lactate + 0 ₂ > pyruvate + H ₂ 0 ₂

HRP (peroxidase)

H ₂ 0 + ₂ Ferrocene> H ₂ 0 + ₂ Ferricinium ⁺

To the girl:

AIDH (Alanine Dehydrogenase)

L-Alanine + Η, Ο + NAD ⁺ > pyruvate + NH, + NADH

DP (diaphorase)

NADH + hexaminorutenium (III)> NAD ⁺ + hexaminorutenium (II) hexaminorutenium (II)> hexaminorutenium (III) + e The measures of lactic acid, chlorides and alanine are indicative of low blood oxygen levels, dehydration stages and muscle damage, respectively.

Optionally, the fluid transport means comprise several second microchannels, so that each second microchannel carries a different reference composition, for example one of these second microchannels would be intended for hydrolytically active salts, another for enzymes, and another for ios mediators In this way, the dimensions of each second microchannel can be optimized and flow rate controlled.

Optionally, enzymes are immobilized in the third channel, to reduce the use of enzymes.

In this way a new high sensitivity sensor is obtained that allows to measure the pH of the sweat continuously and the concentration of sweat analytes continuously for a prolonged period of time that can reach several hours, unlike the state devices of the technique that are usually designed for a single use or for discrete measurements over time. Additionally, this new sensor has a better sensitivity due to the use of two ion selective transistors, where one measures the pH of sweat and the other measures the pH of the reference composition that has a constant pH. In this way, not only the sensitivity is improved but also the problems derived from using Ag / AgCI electrodes in the presence of a high content and variability of sweat chlorides are avoided, which makes it difficult to obtain reliable results.

The division of the sensor into two attachable parts, one that contains means for transporting fluids and that is disposable, and another, non-disposable, that contains the electrochemical and electronic support and devices, makes frequent use of the sensor more economically affordable.

DESCRIPTION OF THE DRAWINGS

To complement the description that is being made and in order to help a better understanding of the characteristics of the invention, according to an example A preferred embodiment of the same is accompanied as an integral part of said description, a set of drawings where, for illustrative and non-limiting purposes, the following has been represented: Figure 1.- Shows a schematic view of a preferred embodiment of the sensor.

Figure 2 - Shows a schematic view of the sensor holder.

Figure 3.- Shows a graph of a first example of measurement using the sensor.

Figure 4.- Shows a graph of a second example of measurement using the sensor.

Figure 5.- Shows a graph of a third example of measurement using the sensor. PREFERRED EMBODIMENT OF THE INVENTION

In a preferred embodiment of the invention, the sensor (1) is used to continuously measure a pH and a concentration of various analytes in sweat, of an individual while performing sports.

This sensor (1) is designed to be carried, or adjusted, in accessories, and / or clothing, thus keeping in contact with the individual's skin to continuously measure the pH and concentration of certain individual sweat analytes, and thus produce an early diagnosis of possible health adversities.

More specifically, as shown in Figure 1, the sensor (1) comprises a support (2) linked to fluid transport means (3) for transporting and analyzing sweat. This support (2) comprises a first fastening means (4), as well as a bracelet, to fix, removably, the sensor to an individual's wrist, as well as a second fastening means (5), to fix, of removable form, the means of transport of fluids (3) to the sensor (1), because said means of transport of fluids (3) are disposable and are composed of a strip of paper in a substantially Y-shaped form. It should be noted that both parts are easily aligned and snapped with 4 posts in the corresponding holes of a PCB,

The support contains some fasteners that ensure good contact and alignment of the paper! With the chips in the reader.

More specifically, the means for transporting fluids (3) comprise: a first microcanny (6), a second microcanny (7), a third microcanny (8) and an absorbent pad (9). The first microcanai (6) comprises a first inlet and a first outlet, where the first inlet is linked with a patch (10) intended to be fixed on the individual's skin to contact and absorb sweat. Thus, when the sensor (1) is in use, sweat travels from the first inlet to the absorbent pad (9). More specifically, the second microcanai (7) comprises a second inlet and a second outlet, where the second inlet is linked to a container (11), as well as a "blister", which in turn contains a reference composition with enzymatic reagents in liquid form, and the second outlet is linked to the first outlet of the first microcanny (8). Thus, when the sensor (1) is in use, the individual presses the container (11) that releases the reference composition and this moves from the second inlet to the absorbent pad (9).

The third microcanai (8) comprises a third inlet and a third outlet, where the third inlet is linked to the first and second outlets, and the third outlet is linked to the absorbent pad (9). Thus, when the sensor (1) is in use, the absorbent pad (9) causes the movement of sweat through the first microcannet, and the reference composition through the second microcanny; and said sweat and said reference composition are mixed by diffusion along the third microcanai. More specifically, the absorbent pad (9) continuously maintains the pumping by capillarity of the sweat and of the reference composition.

Preferably, the reference composition comprises hydrolytically active salts, glucose oxidase between 8 and 640U / mi, peroxidase between 64 and 2580 U / mi and ferrocene between 2.5 and 10 mM. Thus, the reference composition is a pH 7 buffer that also contains enzymes and mediators, preferably redox mediators, which directs the biorecognition and transduction of biochemical species. In this preferred embodiment these enzymes and mediators react with sweat glucose, obtaining a complete chemical reaction resulting from the total sweat mixture with the reference composition as follows:

GOx (glucose oxidase)

Glucose + G ₂ + H ₂ 0> Gluconic acid + H ₂ 0 ₂

HRP (peroxidase)

H ₂ 0 + ₂ Ferrocene> H ₂ 0 + ₂ Ferricinium ⁺

+

H ₂ 0 ₂ + ₂ Ferrocene> H ₂ 0 + ₂ Ferricinium

Thus, it is obtained that the ratio between ferricinium, hydrogen peroxide and glucose is 2: 1: 1, and therefore the sensor (1) can monitor the glucose level of the individual.

To monitor said pH and glucose level, as shown in Figure 2, the support (2) comprises a printed circuit board (12) which in turn comprises a first and a second ion selective transistor (13 , 14), an electrochemical sensor (15) with three electrodes, and a data transmission unit (17), all linked to a processing unit (16).

The first ion selective transistor (13) is intended to contact the first microchannel (8) to perform a first measurement comprising the pH value of sweat, while the second ion selective transistor (14) is intended to contact the second microchannel (7) to perform a second measurement comprising the pH value of the reference composition. Thus, the first ion selective transistor (13) is a selective measurement ion transistor (ISFET), while the second ion selective transistor (14) is a selective ion reference transistor (REFET), producing results highly reliable

Alternatively, the first ion selective transistor (13) is an ISFET with a selective membrane that allows to detect other ions, other than hydrogen ions, such as chlorides, sodium, potassium or calcium. Alternatively, the support (2) comprises a plurality of first ion selective transistors (13) so that the concentration of several ions in the first microchannel (6) can be measured simultaneously. It should be noted that the polarization of the gate of the first and the second ion selective transistor (13, 14) is performed with a common electrode that is in contact with the sweat mixture and reference composition in the liquid state, for example one of the electrochemical sensor electrodes (15). More specifically, the electrochemical sensor (15) is intended to contact the third microchannel (8) to perform a third measurement of the concentration of products selected from hydrogen peroxide and the ferricinium cation.

On the other hand, from the first and second measurement, the processing unit (18) calculates differentially the pH of sweat, and from the third measurement, the processing unit (16) calculates the glucose concentration value .

The data transmission unit (17) forms a wireless network, preferably bluetooth type, with an external device such as a Tablet, computer or smartphone, not shown, to represent and / or store the pH value of sweat and the value of the concentration of products selected from hydrogen peroxide, ferricinium cation, and hexaminorutenium (II) cation.

It should be noted that support (2) comprises a power supply system that feeds: the first and second ion selective transistor (13, 14), the electrochemical sensor (15), the data transmission unit (17), and the processing unit (16). Additionally, the support (2) comprises an electrical regulation unit, linked to said power supply system that provides adequate potentials for complete chemical reactions to occur.

Measurement Examples

By means of this preferred embodiment, a series of measures have been carried out to demonstrate the correct operation of the sensor (1), as well as to verify that it can take measurements greater than one hour. It should be noted that these measures have been carried out considering that an individual when exercising produces on average an amount of Ι μί / ΓϊΐΙη / στη ² , thus the first, second and third microcanai (6, 7, 8) must have 2 mm wide and 4 cm long to generate a flow of 2 pL / min in this way the patch (10) has an area of at least 2 cm ² . It should be noted that the first, the second and the third microcanna! (6, 7, 8) are made on Whatman No.1 paper.

A first measurement example is shown in Figure 3 and continuously represents the difference in power over time! between the first and the second measurement (mV) over a period of 3000 s.

A second example of measurement is shown in Figure 4 and continuously represents over time the relationship between the current (mA) of the third measurement and the concentration of sweat glucose (mM) over a period of 12000 s.

A third example of measurement is shown in Figure 5 and continuously represents the difference in power over time! between the first and the second measurement (mV) for different sweat pH values, and the current (A) of the third measurement for different sweat glucose concentrations (mM) over a period of 5000 s.

In another preferred embodiment, not shown, the reference composition is dehydrated in an absorbent container, which is in contact with the second inlet, so that when the sensor is in use, sweat travels from the first inlet to reaching the second inlet, so that the reference composition dissolves in sweat and when the second microcanai is completely wet a diffusion concentration gradient of the reference composition is established, which travels to the second outlet and then is dragged by the flow of sweat to the absorbent pad.

Claims

1 .- Sensor (1) portable by an individual to continuously measure the pH and the concentration of analytes in an individual's sweat, where the sensor (1) comprises a support (2) and fluid transport means (3 ) to transport and analyze the sweat, and linkable with the support (2), the sensor (1) being characterized in that:

»Means for transporting fluids (3) include:

or a first microchannel (6) with a first input and a first output, where the first input is intended to understand sweat,

or at least a second microchannel {7} with a second input and a second output, where the second input is intended to comprise a reference composition,

or a third microchannel (8) with a third input and a third output, where the third input is linked to the first and second output, and

or an absorbent pad (9) linked to the third outlet and intended to produce the movement of sweat through the first microchannel, and the reference composition through the second microchannel; and intended to mix sweat with the reference composition along the third microchannel, and

* the support (2) includes:

or a first ion selective transistor (13) intended to contact the first microchannel (6) to perform a first measurement of sweat pH, or a second ion selective transistor (14) intended to contact the second microchannel (6) to perform a second measurement comprising the pH value of the reference composition, and an electrochemical sensor (15) intended to contact the third microchannel (8) to perform a third means comprising the concentration of at least one product obtained from the reaction between sweat and the reference composition, and

or a processing unit (16) linked with the electrochemical sensor (15) and with the first and second transistor (13, 14), where from the first and second measurement the processing unit (16) calculates differentially the Sweat pH, and from the third measurement the unit Processing (16) calculates the concentration value of sweat analytes.

2 - Sensor according to claim 1, characterized in that the support (2) comprises a memory unit for storing the pH value of sweat and the value of the sweat analyte concentration.

3. - Sensor according to claims 1 or 2, characterized in that the support comprises a data transmission unit (17) linked to the processing unit (16) to establish communication with an external device that represents and / or stores the or sweat pH value and the sweat analyte concentration value.

4. - Sensor according to claim 3, characterized in that the external device is a Tabiet, computer or smartphone.

5. Sensor according to claim 1, characterized in that the support (2) comprises a display unit to represent the pH value of sweat and the concentration of sweat analytes.

6. - Sensor according to claim 3, characterized in that the support (2) comprises a 0 printed circuit board, which in turn comprises the first and second ion selective transistor (13, 14), the electrochemical sensor (15) , the data transmission unit (17) and the processing unit (16),

7. - Sensor according to claim 6, characterized in that the support (2) comprises a first fastening means (4) linked to the printed circuit to removably fix the sensor (1) to a part of the individual's body , where when the sensor (1) is in use the first entry is in contact with the user's skin allowing the first entry to comprise a continuous sweat sample of the individual.

0

8. - Sensor according to claim 6, characterized in that the support (2) comprises a second fastening means (5) linked to the printed circuit for removably fixing the fluid transport means (3) to the printed circuit . 5

9. Sensor according to claim 1, characterized in that the first, the second and the Third microchannel (6, 7, 8) form a single disposable body and is made of a material with absorbent properties.

10. Sensor according to claim 9, characterized in that the material with absorbent properties is selected from at least one of the following materials: paper, cellulose, nitrocellulose, vinylidene polyfluoride, polyisulfone, nylon, and a combination of the above.

1 1 Sensor according to claim 1, characterized in that the reference composition comprises:

b) at least one enzyme reagent selected from the list consisting of: glucose oxidase, lactate oxidase, peroxidase, alanine dehydrogenase, diaphorase, nicotinamide adenine dinucieotide (NAD ⁺ ) and a combination of ios above and

c) at least one mediator selected from the list consisting of ferrocene, or hexaminorutenium (iii).

12. Sensor according to claim 1, characterized in that the product of the reaction between sweat and enzymatic reagents and / or mediators is hydrogen peroxide, ferricinium cation, or hexaminoruthenium cation (II).

13. - Sensor according to claim 1, characterized in that the reference composition is dehydrated in an absorbent container, where this absorbent container is in contact with the second inlet, so that when the sensor (1) is in use , the sweat travels from the first inlet to the second inlet, so that the reference composition dissolves in the sweat and when the second microchannel (7) is completely wet a concentration gradient is established by diffusion of the composition reference that moves to the second exit and then is dragged by sweat to the absorbent pad (9).

14. - Sensor according to claim 1, characterized in that the reference composition is comprised in liquid form in a container (1 1), wherein this container (1 1) is in contact with the second inlet, so that when the sensor (1) is in use, the container (1 1) releases the reference composition that travels from the second inlet to the absorbent pad (9).

15. - Sensor according to claim 1, characterized in that the first inlet comprises a patch (10) with a first layer of material with absorbent properties to contact the skin of the individual, a second layer of material of adhesive material to fix the first layer to the skin of the individual and a third layer of insulating material to cover the second layer.

16. - A sensor according to claim 1, characterized in that the fluid transport means (3) comprise several second microcanies (7), each of which carries a different reference composition.

17. Sensor according to claim 16, characterized in that it comprises three second microcanaies (7), wherein one of these second microcanaies (7) is intended for hydrolytically active salts, another for enzymes, and another for mediators.