U.S. patent application number 17/668181 was filed with the patent office on 2022-08-25 for potentiometric wearable sweat sensor.
The applicant listed for this patent is The Johns Hopkins University. Invention is credited to Dong-Hoon Choi, Garry R. Cutting, Peter Searson.
Application Number | 20220265180 17/668181 |
Document ID | / |
Family ID | |
Filed Date | 2022-08-25 |
United States Patent
Application |
20220265180 |
Kind Code |
A1 |
Choi; Dong-Hoon ; et
al. |
August 25, 2022 |
POTENTIOMETRIC WEARABLE SWEAT SENSOR
Abstract
A potentiometric sensor that includes a housing and working
electrode is provided. The housing includes a reference electrode,
a first hydrogel that contains a reference solution, and a salt
bridge. The sensor is wearable and can be used for continuous
on-body sweat measurements.
Inventors: |
Choi; Dong-Hoon; (Baltimore,
MD) ; Searson; Peter; (Baltimore, MD) ;
Cutting; Garry R.; (Baltimore, MD) |
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Applicant: |
Name |
City |
State |
Country |
Type |
The Johns Hopkins University |
Baltimore |
MD |
US |
|
|
Appl. No.: |
17/668181 |
Filed: |
February 9, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16099176 |
Nov 5, 2018 |
11272868 |
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PCT/US17/31031 |
May 4, 2017 |
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17668181 |
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62332949 |
May 6, 2016 |
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International
Class: |
A61B 5/145 20060101
A61B005/145; G01N 27/403 20060101 G01N027/403; A61B 5/0537 20060101
A61B005/0537; A61B 5/1477 20060101 A61B005/1477; A61B 5/00 20060101
A61B005/00; G01N 27/416 20060101 G01N027/416 |
Claims
1. A method of measuring an ion concentration comprising: placing a
potentiometric sensor on the skin of a subject, wherein the
potentiometric sensor comprises: a reference electrode; a first
hydrogel containing a reference solution; a salt bridge; and a
working electrode, wherein the salt bridge is in direct contact
with the skin; generating sweat under the salt bridge, wherein the
ions in the sweat form an ionic circuit between the reference
electrode and the working electrode; and measuring a potential
difference proportional to the ion concentration, thereby measuring
the ion concentration.
2. The method of claim 1, wherein the salt bridge comprises a
second hydrogel.
3. The method of claim 1, wherein measuring the ion concentration
is continuous.
4. The method of claim 1, wherein the ion is selected from the
group consisting of chloride, potassium and sodium.
5. The method of claim 4, wherein the ion is chloride.
6. The method of claim 5, further comprising diagnosing whether the
subject has cystic fibrosis (CF) based upon the chloride ion
concentration.
7. The method of claim 5, further comprising assessing an intensity
of a workout based upon the chloride ion concentration.
8. A method of manufacturing a potentiometric sensor comprising:
forming a through hole in a substrate; forming a working electrode
on a first side of the substrate; forming a reference electrode on
a second side of the substrate surface opposite to the first side
of the substrate; forming a salt bridge within the through hole;
forming a reference solution hydrogel on the reference electrode
and the salt bridge; and forming an encapsulating layer on the
reference solution hydrogel.
9. The method of claim 8, wherein the through hole is formed by a
laser drilling process or a molding process.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 16/099,176 filed Nov. 5, 2018, now pending;
which is a 35 USC .sctn. 371 National Stage application of
International Application No. PCT/2017/031031 filed May 4, 2017,
now expired; which claims the benefit under 35 USC .sctn. 119(e) to
U.S. Application Ser. No. 62/332,949 filed May 6, 2016, now
expired. The disclosure of each of the prior applications is
considered part of and is incorporated by reference in the
disclosure of this application.
BACKGROUND OF THE INVENTION
Field of the Invention
[0002] The present invention relates to a potentiometric sensor for
measuring the concentration of chloride ions in sweat.
Background Information
[0003] Potentiometric sensing is a method developed to measure the
concentration of an ion in solution. This method measures the
electrical potential difference between a reference and a working
electrode (FIG. 1). The potential of the reference electrode should
be constant. The potential of the working electrode is dependent on
the concentration of the ion in solution. In order to detect
chloride ions, a Ag/AgCl electrode is used as the working
electrode. The measured potential is converted to a concentration
by measuring the potential for a range of solutions of known
concentration. This calibration curve is then used to determine the
concentration of an unknown test solution.
[0004] Sweat chloride is a biomarker for cystic fibrosis (CF) and
for electrolyte loss during exercise. The chloride ion
concentration in the sweat of CF patients is typically 60 to 150
mM, much higher than in healthy individuals (typically 10-40 mM),
and hence sweat chloride testing is the most widely used assay for
diagnosis of CF. The assay involves collection of a sweat sample
and analysis by coulometric titration, manual titration, or an ion
selective electrode. Chloride is the most abundant ion in sweat,
and hence is also a potential biomarker for electrolyte loss. The
successful development of a wearable sweat chloride sensor can
reduce the cost and time for laboratory-based sweat testing for CF
patients, and can provide real-time information for healthy
individuals during exercise.
[0005] The development of wearable sensors to measure biomarkers in
sweat is recognized as a major technological challenge. There are
three main candidate technologies for wearable sweat chloride
sensors: titration devices, conductivity measurements, and
potentiometric sensors. Wearable titration sensors have been
reported, but require subsequent analysis on a separate instrument.
Wearable conductivity sensors are readily miniaturized, but are not
chloride specific. Potentiometric measurements rely on the
relationship between the ion concentration and the electrochemical
potential of an electrode. This is a well-established analytical
technique that can be readily miniaturized. Chloride ion detection
relies on the equilibrium between chloride ions and silver chloride
(AgCl(s)+e-Cl-(aq)+Ag(s)), and can be measured using silver
chloride electrodes that are widely employed in electrophysiology
and analytical chemistry. There are relatively few examples of
wearable sodium and potassium ion sweat sensors that use ion
selective membranes, and wearable potentiometric chloride
sensors.
[0006] Two publications describing miniaturized chloride sweat
sensors are described (see below). Gonzalo-Ruiz et al. reports
measurements immediately after inducing sweat in subjects but does
not report measurements as a function of time. Lynch et al. reports
only measurements in a test solution; neither reference reports any
on-body measurements. Furthermore, neither reference reports the
time response of its device.
[0007] Gonzalo-Ruiz's apparatus is used to sense chloride ions in
sweat. The apparatus includes a screen-printed Ag/AgCl electrode
covered by pHEMA hydrogel matrix containing KCl (the hydrogel
matrix was used as a reservoir for the reference solution) as the
reference electrode, a screen-printed Ag/AgCl electrode as the
working electrode, and two other electrodes (cathode and anode) for
sweat generation (FIGS. 2A-2B).
[0008] Lynch's apparatus is used to sense chloride, potassium, and
sodium ions in a sweat sample. The apparatus is not wearable. The
components for chloride ion sensing include a Ag/AgCl electrode
covered by a hydrogel containing the reference solution as the
reference electrode and a Ag/AgCl electrode as the working
electrode (FIG. 3).
[0009] In current designs of wearable sweat sensors (chloride ions
as well as other ions) the reference electrode is covered with a
gel containing the reference solution. Transport of ions between
the reference solution and test solution (sweat) results in changes
in the potential of the working electrode and results in
measurement error. As a result, these devices cannot be used for
continuous on-body measurements. Thus, there is a need for sweat
sensors that are suitable for continuous on-body measurements.
SUMMARY OF THE INVENTION
[0010] The present invention is based on the discovery that a sweat
chloride sensor integrated with a salt bridge minimizes
equilibration and enables stable measurements over extended periods
of time.
[0011] One embodiment of the present invention is to provide a
potentiometric sensor that includes a housing and a working
electrode. The housing includes a reference electrode, a first
hydrogel that contains a reference solution, and a salt bridge.
[0012] In another embodiment, the salt bridge includes a second
hydrogel.
[0013] In another embodiment, the first and second hydrogels are
the same.
[0014] In another embodiment, the hydrogel is agarose.
[0015] In another embodiment, the housing includes
polydimethylsiloxane (PDMS).
[0016] In another embodiment, the reference and working electrodes
are Ag/AgCl electrodes.
[0017] In another embodiment, the reference solution includes 1M
KCl.
[0018] In another embodiment, the salt bridge includes an ion
selective polymer.
[0019] In another embodiment, the ion selective polymer is Nafion
or polydiallyldimethylammonium chloride (polyDADMAC).
[0020] In another embodiment, the sensor monitors the concentration
of an ion in sweat.
[0021] In another embodiment, the sensor is wearable.
[0022] In another embodiment, the ion is selected the ion can be
chloride, potassium or sodium.
[0023] In another embodiment, the sensor is used to monitor
chloride ion concentration in a cystic fibrosis (CF) subject.
[0024] In another embodiment, the sensor is used to monitor
chloride ion concentration as a function of workout intensity.
[0025] Another embodiment of the present invention is to provide a
method of measuring an ion concentration in sweat. The method
includes a step of placing a potentiometric sensor on the skin of a
subject. The potentiometric sensor includes a reference electrode,
a first hydrogel containing a reference solution, a salt bridge,
and a working electrode. The salt bridge is in direct contact with
the skin. Another step includes generating sweat under the salt
bridge. The ions in the sweat form an ionic circuit between the
reference electrode and the working electrode. A third step
includes measuring a potential difference proportional to the ion
concentration in the sweat, thereby measuring the ion
concentration.
[0026] In another embodiment, the salt bridge used in the method
includes a second hydrogel.
[0027] In another embodiment, the step of measuring the ion
concentration is continuous.
[0028] In another embodiment, the ion being measured can be
chloride, potassium or sodium.
[0029] In another embodiment, the ion being measure is
chloride.
[0030] In another embodiment, the method includes the step of
diagnosing whether the subject has cystic fibrosis (CF) based upon
the chloride ion concentration.
[0031] In another embodiment, the method includes assessing an
intensity of a workout based upon the chloride ion
concentration.
[0032] Other aspects and advantages of the invention will be
apparent from the following description and the appended
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1. Potentiometric measurement.
[0034] FIGS. 2A-2B. Potentiometric sensor for measurement of
chloride ions in sweat (FIG. 2A) Photograph of fabricated sweat
sensor (FIG. 2B) Schematic view of working electrode and reference
electrode. Note that the gel containing the reference solution
reservoir has a large contact area with the test solution. This
results in rapid transport between the reference solution and test
solution, which also results in a change in the potential of the
reference electrode.
[0035] FIG. 3. Sensor array to detect chloride, potassium, and
sodium ions in sweat.
[0036] FIG. 4A-4B. (FIG. 4A) Proposed sweat sensor with a salt
bridge, (FIG. 4B) salt bridge geometry.
[0037] FIGS. 5A-5C. Parametric studies (FIG. 5A) A schematic view
of a test device for parametric studies, and (FIGS. 5B-5C) measured
output voltage as a function of a time according to (FIG. 5A) the
length of the salt bridge (L=0.9, 2.8, 4.8, and 10.4 mm, D=635
.mu.m) and the diameter of the salt bridge (D=100 mm, 150, 380, and
635 .mu.m).
[0038] FIGS. 6A-6B. (FIG. 6A) A test device adopting an ion
selective polymer poly(diallyldimethylammonium chloride) (pDADMAC)
for the salt bridge, (FIG. 6B) Measured output voltage.
[0039] FIGS. 7A-7G. Fabrication process of the prototype sweat
sensor.
[0040] FIGS. 8A-8C. Photographs of the fabricated sweat sensor.
[0041] FIGS. 9A-9C. Simultaneous measurement of output voltage and
chloride concentration measured at: (FIG. 9A) forearm, (FIG. 9B)
chest, and (FIG. 9C) back.
[0042] FIGS. 10A-10C. Comparison between the sweat sensor described
in this ROI and a sweat sensor fabricated to mimic the devices
described in Gonzalo-Ruiz et al. 2009 and Lynch et al. 2002. (FIG.
10A) Design of sweat sensor used to mimic the devices in
Gonzalo-Ruiz et al. 2009 and Lynch et al. 2002, (FIG. 10B)
photograph of an on-body test, and (FIG. 10C) chloride
concentrations measured by the device described in this ROI and the
device fabricated to mimic the sensors reported in Gonzalo-Ruiz et
al. 2009 and Lynch et al. 2002.
[0043] FIGS. 11A-11B. Microduct.RTM. sweat collection system (FIG.
11A) Webster sweat inducer, and (FIG. 11B) Macroduct collector.
(Macroduct sweat collection system)
[0044] FIG. 12. An illustration of potentiometric sensor
components.
[0045] FIGS. 13A-13F. Fabrication of a thin film, wearable,
potentiometric sweat chloride sensor. (FIG. 13A) Laser drilling of
a 30 .mu.m hole that defines the salt bridge. (FIGS. 13B-13C)
E-beam evaporation of a 10 nm Cr adhesion layer and 300 nm Ag film.
(FIG. 13D) Chloridization of the Ag layer in 50 mM FeCl.sub.3
solution. (FIG. 13E) The reference solution gel is introduced into
the salt bridge and on top of the substrate. (FIG. 13F) The
reference solution gel is sealed using a UV curable resin.
[0046] FIGS. 14A-14F. Thin film potentiometric chloride sensor with
integrated salt bridge. (FIG. 14A) Schematic illustration of the
sensor. (FIG. 14B) Top side (R.E.--reference electrode). (FIG. 14C)
Bottom side (W. E.--working electrode). (FIG. 14D) Cross-section
view of the sensor. (FIG. 14E) Optical image of the sensor after
fabrication. (FIG. 14F) A sensor attached on the forearm using a
transparent adhesive bandage.
[0047] FIGS. 15A-15C. (FIG. 15A) Electrical connector for the sweat
sensor. (FIG. 15B) Attaching the connector to the sensor. (FIG.
15C) The sensor assembled with the connector.
[0048] FIGS. 16A-16D. Sensor calibration and reproducibility. (FIG.
16A) Schematic illustration of the calibration procedure. All
devices were calibrated three times following this procedure. (FIG.
16B) Sensor voltage versus time for three trials of a single
device. (FIG. 16C) Calibration curve for multiple sensors (N=6).
Data represent mean.+-.SD (red dots: measured voltages, solid line:
calibration curve obtained by the linear regression). Inset shows
sensor voltage versus activity of the test solution (red dots:
sensor voltages taking the junction potential into account, solid
line: Nernst equation). (FIG. 16D) Measurement accuracy (.zeta.)
due to variation among multiple sensors. The measurement accuracy
was obtained by substituting the measured voltage (V) into the
calibration curve (inset).
[0049] FIG. 17. Temperature dependence of sensor voltage.
Calibration curves were measured at 22 (black circles) and
32.degree. C. (blue squares). The sensor voltage at 32.degree. C.
predicted by the Nernst equation and the voltage at 22.degree. C.
(red triangles) are very close to the measured values (blue
squares).
[0050] FIG. 18A-18B. Sensor measurement in 20 .mu.l of 10 mM NaCl.
(FIG. 18A) Voltage and (FIG. 18B) concentration for a sensor with a
30 .mu.m diameter salt bridge over 2 hours.
[0051] FIG. 19A-19D. The role of the salt bridge in sensor
performance. (FIG. 19A) Schematic illustration of measurement
set-up. (FIG. 19B) Measured output voltage versus time for sensors
with salt bridge diameters of 30 .mu.m, 500 .mu.m, or 1 (N=3).
(FIG. 19C) Chloride concentration versus time (N=3). (D) Chloride
ion concentration for sensors with a 30 .mu.m diameter salt bridge
measured over 12 hours.
[0052] FIGS. 20A-20C. Measurement accuracy. (FIG. 20A) Procedure
for testing measurement accuracy. (FIG. 20B) Concentration obtained
from the sensor versus known test solution concentration. As a
guide, the dotted lines show .+-.5 mM. (C) Chloride concentration
obtained from the sensor for a test solution concentration of 150
mM over repeated measurements. The dotted line represents 150.+-.5
mM. The inset shows the sensor voltage and the solid line
represents voltage corresponding to 150 mM.
[0053] FIGS. 21A-21C. Dose response curve. (FIG. 21A) Measurement
set-up. The test solution was initially 100 mL of DI water. 1 mL of
1 M NaCl solution was added to the water every single minute. (FIG.
21B) Dose response curve. (FIG. 21C) Measured concentration and
calculated test solution concentration of the test solution.
[0054] FIGS. 22A-22F. On-body tests for a healthy individual while
exercising. (FIG. 22A) Exercise profile for constant load test.
(FIG. 22B) Representative sweat chloride measurement. (FIG. 22C)
Sweat chloride concentrations at t=30, 45 and 60 min (N=3). Bars
represent mean.+-.SE. (FIG. 22D) Exercise profile for graded load
test. (FIG. 22E) Measured sweat chloride concentration. (FIG. 22F)
Sweat chloride concentration at t=30, 45, and 60 mins (N=3). Bars
represent mean.+-.SE (* p<0.05).
DETAILED DESCRIPTION OF THE INVENTION
[0055] Other aspects and advantages of the invention will be
apparent from the following description.
[0056] A challenge in developing wearable potentiometric sensors is
that equilibration between the reference solution and the test
solution over time results in a measurement error. In recent work,
the critical role of the salt bridge in determining the sensor
performance has been reported. An analytical model to assess the
rate of equilibration between the reference and test solutions, and
hence to predict the measurement error as a function of salt bridge
geometry was used. The model was validated by a series of
parametric studies, which allowed the design of rules for specific
applications. The present invention uses these criteria to design
and optimize a wearable thin film chloride sweat sensor. Building
on previous work, the present invention makes the following key
advances: (1) a fabrication process to integrate the salt bridge
into a thin film sensor is described; (2) the reliability and
reproducibility of the sensor is described; and (3) and in vivo
results from in vivo testing which indicate that sweat chloride
concentration is dependent on exercise intensity are presented. The
device is fabricated on a plastic substrate and can be easily and
comfortably worn on the body using a commercial adhesive bandage.
The device shows reliable performance over 12 hours. The accuracy
of the device is evaluated over the concentration range of about 10
to 150 mM, and the calibration curve and dose response of the
fabricated devices are also presented. Finally, the concentration
changes during an exercise with graded exercise load are
presented.
[0057] To overcome problems discussed in the Background of the
Invention section, a salt bridge between the reference solution and
the working electrode and test solution was introduced. The present
invention describes a sweat sensor to reliably measure the
concentration of chloride ions in sweat. By introducing a salt
bridge, ion transport between the reference solution and test
solution is very slow and hence the potential of the reference
electrode remains constant for extended times. Therefore, reliable
on-body measurements can be made over time.
[0058] FIG. 12 illustrates the potentiometric sensor 1 components.
The potentiometric sensor includes a housing 3 and working
electrode 11. The housing includes a reference electrode 5, a first
hydrogel that contains a reference solution 7, and a salt bridge
9.
[0059] As shown in FIG. 4A, the proposed sweat sensor in this
invention consists of: (1) a reference electrode (a Ag/AgCl
electrode, (2) a hydrogel containing a reference solution, (3) a
salt bridge filled with the hydrogel and designed to minimize ion
transport between the reference solution and test solution, (4) a
working electrode (a Ag/AgCl electrode), and (5) an integrated
package to optimize sweat measurements.
[0060] On generation of sweat under the sensor, the ionic circuit
between the working electrode and the reference electrode is
completed, resulting in a potential difference that is related to
the chloride ion concentration in sweat. Using a calibration curve
determined for the sensor prior to use, the potential difference
can be related directly to a chloride concentration.
[0061] Transport of ions between the reference solution and the
test solution (sweat) is dependent on the salt bridge geometry, and
the materials used in the sensor. The design of the sensor has been
optimized to minimize ion transport and maximize the time of
measurement. Designs using an ion selective polymer for the salt
bridge have been tested.
Parametric Studies
[0062] To optimize the geometry of the salt bridge, parametric
studies have been performed.
[0063] FIG. 5A shows a schematic view of a test device for
parametric studies. The test device has two chambers (a reference
and a test chamber) and the chambers are connected by a salt
bridge. The reference and test chambers are filled with a reference
solution of 1M KCl and a test solution of 10 mM NaCl, respectively.
Numerous devices with different salt bridge geometries were tested,
and then their voltage drift were compared.
[0064] FIGS. 5B-5C show the results. As the salt bridge is longer,
the voltage drift decreases (FIG. 5B). Also, the voltage drift is
reduced as the salt bridge diameter decreases (FIG. 5C).
[0065] In another embodiment, the use of ion selective polymers in
the salt bridge to reduce ion transport and improve measurement
stability was explored. For example, polymers such as Nafion and
polyDADMAC (polydiallyldimethylammonium chloride) have been tested
(FIGS. 6A-6B). In preliminary experiments, devices with ion
selective polymers require several hours for stabilization. After
the output voltage is stabilized, the test device has a constant
output voltage for at least 20 hours.
Prototype Sweat Sensor and On-Body Tests
[0066] One example of the fabrication process is described in FIGS.
7A-7G.
[0067] Step 1. Fabrication of the polydimethylsiloxane (PDMS)
housing for the reference electrode chamber (FIG. 7A). The PDMS
housing that contains the reference solution and the wire reference
electrode is formed by a casting process. A metal wire is inserted
into a mold that is then filled with PDMS. The metal wire serves as
a template to form the hole in the PDMS housing into which the wire
electrode is inserted. The PDMS is then cured at 75.degree. C. for
1 hour. The wire is then removed and the PDMS housing removed from
the mold. The wire diameter is dependent on the diameter of the
reference electrode wire to be used, but is typically 635 .mu.m.
The thickness of the PDMS housing is about 5 mm, but can be
decreased to make the sensor dimensions smaller.
[0068] After removal of the PDMS housing from the mold, a hole
punch is used to form the reference solution chamber. An additional
hole is punched in the housing that is later used to seal the
reference electrode wire into the housing. The diameter of the
reference electrode chamber is typically about 5 mm, but can also
be smaller to reduce the size of the sensor.
[0069] Step 2. Fabrication of the PDMS base with the salt bridge
channel (FIG. 7A).
[0070] The PDMS base that contains the salt bridge channel is also
formed by a similar casting process using a metal wire template.
Typically a metal wire of 100 .mu.m in diameter is used to form a
cylindrical channel in PDMS. The PDMS with the template wire still
in place are removed from the mold. Next the template wire is
removed from the PDMS. The PDMS is then cut into slices with a
thickness that defines the length of the salt bridge. Each slice
forms the base of a sensor with a hole that will be the salt bridge
channel. Typically the thickness of the PDMS base is 3 mm. The wire
diameter defines the diameter if the salt bridge channel and can be
changed by using a different diameter template wire. The length of
the salt bridge is dependent on the thickness of the slice cut from
the PDMS block.
[0071] Step 3. The PDMS housing with the reference electrode
chamber and PDMS base with the salt bridge channel are plasma
bonded (FIG. 7B). A reference electrode (Ag/AgCl electrode) wire is
inserted into the hole for the reference electrode (FIG. 7C).
[0072] Step 4. The second hole in the PDMS housing is then filled
with PDMS to fix the reference electrode wire into the housing. The
PDMS is then cured at 75.degree. C. for 1 hour (FIG. 7D).
[0073] Step 5. The reference electrode chamber is filled with a
hydrogel containing the reference solution. In many experiments, 1
M KCl in agarose was used (4% w/v ration agarose gel, 1 M KCl
solution: agarose gel=20 ml: 0.8 g). Vacuum is applied to the
chamber through the salt bridge channel to fill the salt bridge
with the hydrogel (FIG. 7E). The gel in the reference electrode
chamber containing the reference solution and the salt bridge are
the same. The concentration of the reference solution and the
composition of the gel in the reference chamber can be changed
depending on the requirements.
[0074] Step 6. The top of the reference electrode chamber is sealed
with PDMS. The device is placed in an oven at 45.degree. C. for 5
hours to cure the PDMS on the top of the housing. The PDMS cap
prevents evaporation of the reference solution in the reference
chamber (FIG. 7F).
[0075] Step 7. Working electrode. A hole is formed in the housing
for the working electrode using a 1 mm hole punch. A Ag/AgCl wire
electrode is inserted into the hole (FIG. 7G).
Alternative Methods of Fabrication
[0076] Other methods for fabrication and other configurations have
been developed. For example, in one embodiment, planar Ag/AgCl
electrodes are used.
[0077] FIGS. 8A-8C show photographs of the fabricated sweat sensor.
The device can be easily attached to the body using a wrist band
from a fitness device (e.g., Fitbit, (FIG. 8B) or a commercial band
aid (FIG. 8C).
[0078] On-body tests to monitor chloride concentration in sweat
were performed. Three human subjects participated in these tests
and did a constant-load exercise on an exercise bike for an hour.
The devices were attached at various locations including forearm,
chest, and back. The output voltage of the sensor was measured
during the test by DAQ (data acquisition) systems and LABVIEW.
[0079] FIGS. 9A-9C show output voltage and chloride concentration
measured simultaneously at three different locations. Once the
subject begins sweating a stable signal is measured.
[0080] The chloride concentrations measured during the exercise was
in the range 5-40 mM, typical of a normal individual. Ten on-body
tests with 14 sensors were performed, and the measured chloride
concentrations are in the normal range (Table 1.)
TABLE-US-00001 TABLE 1 On-body test results (Subject 1: male, age =
35, height = 196 cm, weight = 100 kg, Subject 2: female, age = 19,
height = 170 cm, weight = 60 kg, Subject 3: age = 28, height = 155
cm, weight = 64.4 kg). Test Subject Position Wearing Perspiration
Cl-- 1 Subject 1 Forearm Fitbit band <5 min 25 mM 2 Subject 1
Forearm Fitbit band <5 min 20 mM 3 Subject 1 Forearm Fitbit band
10 min 25 mM 4 Subject 1 Forearm Fitbit band 12 min 38 mM 5 Subject
1 Forearm Fitbit band <5 min 20 mM Band aid <5 min 26 mM 6
Subject 1 Forearm Fitbit band 12 min 11 mM Band aid 12 min 12 mM 7
Subject 2 Forearm Fitbit band 17 min 10 mM 8 Subject 3 Forearm
Fitbit band 8 min 20 mM 9 Subject 3 Forearm Band aid 20 min 4 mM
Chest Band aid 9 min 8 mM Back Band aid 13 min 40 mM 10 Subject 3
Chest Band aid 19 min 8 mM Back Band aid 22 min 5 mM
[0081] In this invention, to minimize a measurement error caused by
transport of ions between the reference solution and the test
solution (sweat), the salt bridge with an optimized geometry is
adopted. To verify that the salt bridge can minimize the
measurement error, the chloride concentration was compared to that
obtained from a device fabricated to mimic the design in
Gonzalo-Ruiz et al. 2009 and Lynch et al. 2002. FIG. 10A shows a
schematic illustration of a device typical of those described in
Gonzalo-Ruiz et al. 2009 and Lynch et al. 2002. Note that a similar
design has been used to fabricate devices to measure sodium ions,
ammonium ions (Gonzalo-Ruiz et al. 2009; Rose et al. 2015;
Guinovart et al. 2013; Bandodkar et al. 2014). Calibration curves
were obtained for both devices using known concentrations between
10 mM-100 mM. Both of the sensors were attached to the forearm with
a band aid strip (FIG. 10B), and the output voltages were measured
at the same time.
[0082] FIG. 10C shows the chloride concentration measured by the
device described in this ROI and a device fabricated to mimic that
described in Gonzalo-Ruiz et al. 2009 and Lynch et al. 2002. The
concentration measured by the sensor designed to mimic the device
described in Gonzalo-Ruiz et al. 2009 and Lynch et al. 2002 is much
higher than that measured by the proposed device, which is caused
by ion transport between the reference solution and the test
solution (sweat). Since the chloride concentration of the reference
solution (1 M KCl) is much higher than the sweat chloride
concentration, the apparent sweat concentration increases.
Sensor Fabrication
[0083] The sensor is fabricated on a 125 .mu.m thick PET
(Polyethylene terephthalate, Melinex.RTM. ST) film (FIGS. 13A-13F).
First, the salt bridge was defined by laser drilling a 30 .mu.m in
diameter hole in the PET film (FIG. 13A). To assess the role of
salt bridge diameter on performance, sensors were also fabricated
with salt bridge diameters of 500 .mu.m and 1 mm. Next, a 10 nm Cr
adhesion layer and a 300 nm Ag film were patterned on the top and
bottom of the PET film by e-beam evaporation using shadow masks
(FIGS. 13B-13C). Ag/AgCl electrodes were formed by incubating the
patterned Ag electrodes in 25 .mu.L of 50 mM FeCl.sub.3
(Sigma-Aldrich) solution for 3 min (FIG. 13D). The Ag/AgCl
electrodes on the top and bottom side serve as the reference and
working electrodes, respectively. Next, approximately 200 .mu.L of
a 4 w/v % agarose gel (Invitrogen) containing 1 M KCl (Sigma
Aldrich) solution was injected onto the reference electrode and the
salt bridge hole (FIG. 13E). In this step, vacuum was applied to
the hole to ensure complete filling with the gel. Finally, the gel
was covered by a UV curable resin (Addison Clear Wave Coating, AC A
1450) (FIG. 13F). The resin was introduced on the gel using a
syringe, and then cured by UV exposure. The wearable potentiometric
sweat sensor was fabricated on a plastic PET substrate. The
fabrication process is relatively simple and cost-effective, and
involves laser drilling, e-beam evaporation and UV exposure
processes (FIGS. 13A-13F). To connect the sweat sensor to the data
acquisition board (USB-6363, National Instruments) to measure the
output voltage, an electrical connector was manufactured using 3D
printing and machining (FIGS. 15A-15C). The connector consists of
two electrodes with screw terminals and a plastic housing (FIGS.
15A-15C).
Calibration of the Sweat Chloride Sensor
[0084] Calibration of the sensors was performed prior to all
measurements. All devices were calibrated in the following way: (1)
the working electrode was rinsed in running deionized (DI) water
for 40 s, (2) 100 .mu.L of 10 mM NaCl (Fisher Scientific) solution
was placed on the working electrode of the sensor using a
micropipette, (3) the sensor voltage was measured and recorded for
3 minutes, (4) steps 1-3 were repeated with 50 and 100 mM NaCl
solutions, (5) the sensor voltage for each solution was determined
by averaging the recorded voltages over last 1 min, and (6) using a
linear least squares fit (V-log C), the relationship between the
measured voltage and the concentration of the test solution was
established. All calibrations were performed at room temperature.
The output voltage of the sensors was measured and recorded by a
data acquisition (DAQ) system (USB-6363, National Instruments) and
Labview software (National Instrument).
Role of the Salt Bridge on Sensor Performance
[0085] To assess the influence of the salt bridge on performance,
sensors were fabricated with 30 .mu.m, 500 .mu.m, and 1 mm diameter
holes and their output voltages measured for 2 hours in contact
with 110 .mu.L of 10 mM NaCl solution. To prevent evaporation of
the test solution, the sensor was mounted in a custom chamber, and
the edges of the sensor sealed with Kapton tape (Uline). The
chamber was fabricated from a 2.2 mm thick PDMS
(polydimethylsiloxane, Sylgard 184, Dow Corning) film with an 8 mm
diameter hole. The PDMS film was prepared by a conventional curing
process (75.degree. C. in a convection oven for 1 hour), and the
hole was punched manually. A glass slide was then oxygen plasma
bonded to one side of the PDMS film.
Device Accuracy
[0086] To assess device accuracy, the chloride concentration
determined from the sensor measurement was compared to the known
values of the test solutions in the concentration range 10-150 mM.
Each test solution was prepared independently, and not from a
diluted stock solution. The procedure was as follows: (1) a sensor
was calibrated as described in Section 2.2., (2) 100 .mu.L of a
test solution was introduced onto the working electrode using a
micropipette, (3) the sensor voltage was measured for 5 min. and
the average sensor voltage determined for the last 1 minute, (4)
the sensor working electrode was rinsed for 40 s using DI water and
the measurement repeated with a new test solution. The measured
voltages were converted to concentration values using the
calibration curve, and the measured concentration compared to the
concentration of the test solution.
Dose Response Curves
[0087] To assess the sensor performance in real time, dose response
curves were obtained in the following way. A sensor was partially
immersed into 100 mL of DI water so that the working electrode was
completely submerged. Then, 1 mL of 1 M KCl solution was added to
the solution every minute, and the output voltage was continuously
recorded. To ensure good mixing, the test solution was agitated
using a stirring bar.
On-Body Tests
[0088] On-body tests with a healthy subject while exercising on a
stationary bike were performed in compliance with a protocol
approved by the institutional review board (IRB) at Johns Hopkins
University (HIRB00004232). Tests were performed at a constant load
or with three incrementally increasing loads. The sensor was
attached to the middle of the flexor aspect of the forearm with a
commercial adhesive bandage (Nexcare, Tegaderm.TM.). Before
attaching the sensor, the area on the forearm was swabbed with
alcohol and DI water. Prior to the test, the subject was asked to
spin on a stationary bike at 45 W for 10 min as a warm-up. For the
constant load test, the subject was asked to spin on the exercise
bike at 100 W for one hour. During the test, the sensor voltage was
continuously monitored. For the graded load test, the subject was
asked to spin sequentially at 100, 125, and 150 W for 30 min, 15
min and 15 min, respectively. Each test was performed three times
on different days. Each sensor was calibrated at room temperature
(22.degree. C.) before each test. The skin temperature during
on-body tests was typically around 32.degree. C., and the
differences in sensor voltage between 22.degree. C. and 32.degree.
C. (V.sub.32.degree. C.-V.sub.22.degree. C.) were verified to be in
agreement with the values predicted by the Nernst equation.
Therefore, all calibration curves were recorded at room temperature
and adjusted for skin temperature using the Nernst equation. A
paired-sample Students' t-test was performed to check the
dependency of the sweat chloride concentration on the exercise load
(* indicates p<0.05).
Influence of Temperature on Sensor Calibration
[0089] The voltage of a potentiometric sensor is dependent on
temperature. According to the Nernst equation, the voltage of a
potentiometric chloride sensor is given by:
V = - 2.303 .times. RT F .times. log .times. a Cl - sweat a Cl -
ref ##EQU00001##
where R is the gas constant, F is Faraday's constant, T is
temperature, a.sub.Cl.sub.-.sup.sweat is the activity of chloride
ion in sweat and a.sub.Cl.sub.-.sup.ref is the activity of chloride
in the reference solution. The mean ionic activity coefficients
(.gamma..+-.) for 10, 50, 100 NaCl are 0.903, 0.822, and 0.779,
respectively. The mean ionic activity coefficient for 1 M KCl
solution is 0.604.
[0090] The sensors in this study were pre-calibrated at room
temperature (22.degree. C.), however, the skin temperature during
on-body was about 32.degree. C. To assess the influence of
temperature on sensor voltage, calibration curves at 32.degree. C.
were measured (FIG. 17). The difference in sensor voltage between
22.degree. C. and 32.degree. C. (.DELTA.V=V.sub.32.degree.
C.-V.sub.22.degree. C.) were 4.3, 2.2 and 2.0 mV in 10, 50, and 100
mM NaCl solution, respectively. The differences predicted by the
Nernst equation are 3.7, 2.4, and 1.8 mV, in 10, 50, and 100 mM
NaCl solution. Therefore, the small measured shift in the sensor
voltage between room temperature and skin temperature is very close
to the values predicted by the Nernst equation. Therefore, for the
on-body tests, calibration curves were recorded at room temperature
and adjusted for skin temperature using the Nernst equation.
Thin Film Sweat Chloride Sensor
[0091] The wearable potentiometric sensor consists of planar
Ag/AgCl reference and working electrodes located on opposite sides
of a PET film and connected by a laser drilled hole that defines
the salt bridge (FIG. 14A). The reference chamber consists of an
agarose gel containing 1 M KCl and sealed with a UV curable resin
to prevent evaporation of the reference solution in the hydrogel
(FIG. 14B). The working electrode is formed on the bottom side of
the PET film and directly contacts the skin when attached to the
body (FIG. 14C). The main role of the salt bridge is to provide an
ionic path between the reference and test solutions (FIG. 14D),
however, the salt bridge geometry also determines the rate of
equilibration between the reference and test solutions, and hence
determines the time over which accurate measurements can be made.
The sensor was fabricated by conventional thin film deposition
processes (FIG. 14E) and the sensor was easily attached to the body
using an adhesive bandage (FIG. 14F). For all measurements the
sensor was connected to a data acquisition (DAQ) system (FIGS.
15A-15C).
Calibration and Reproducibility
[0092] Devices were calibrated in test solutions of 10, 50, and 100
mM NaCl (FIG. 16A), spanning the range of healthy individuals and
individuals with cystic fibrosis. A typical result from three
sequential trials of a single sensor shows stable and reproducible
values with voltage variations of less than 1 mV for each
concentration (FIG. 16B). The average calibration curve (V vs. log
C) obtained from multiple sensors (N=6) shows a slope of
52.8.+-.0.7 mV decade-1 (FIG. 16C).
[0093] To compare the slopes of the calibration curves to the
Nernst equation, the activity coefficients need to be taken into
account. The activity coefficients are significantly less than 1.0
at the concentrations reported here: =0.903 for 10 mM NaCl, =0.822
for 50 mM NaCl, =0.779 for 100 mM NaCl), and =0.604 for 1 M KCl
(reference solution). In addition, for the sensor configuration
described here, the junction potential lowers the measured
potential by about 2.0 mV. Replotting the sensor voltage versus
activity and taking into account the junction potential, the slope
of the calibration curves is 58.5 mV, identical to the theoretical
value of 58.5 V predicted by the Nernst equation at 22.degree. C.
(FIG. 16C inset).
[0094] The deviation in sensor voltage from the average values
(FIG. 16C) corresponds to an average variation in chloride
concentration of less than 1 mM in 10 mM NaCl solution and about 6
mM in 100 mM NaCl solution (FIG. 16D). The maximum variation of 2.5
mV in 100 mM NaCl solution, corresponds to an error of 10 mM. The
relatively small variation between sensors (FIG. 16D) indicates
that each sensor does not need to be calibrated prior to use,
depending on the desired accuracy of the measurement, significantly
reducing time and cost.
Junction Potential
[0095] A junction potential V.sub.junction is developed at the
interface between liquids with different ion compositions. The
junction potential between the test solution and the salt bridge
which has the same composition as the reference solution, was
calculated from the Henderson equation:
V junction = V TS - V SB = RT F .times. i = 1 N z i .times. u i ( a
i TS - a i SB ) i = 1 N z i 2 .times. u i ( a i TS - a i SB )
.times. ln .function. ( i = 1 N z i 2 .times. u i .times. a i TS i
= 1 N z i 2 .times. u i .times. a i SB ) ##EQU00002##
where V.sub.TS is the potential of the test solution, V.sub.SB is
the potential of the salt bridge (reference solution), R is the gas
constant, T is temperature, F is Faraday's constant, z, is the
valency of ion i, u.sub.i is mobility, and a.sub.i is activity. N
is total number of ions in all solutions and the superscripts of TS
and SB refer to test solution and salt bridge, respectively. The
relative mobility u.sub.CL-/u.sub.K+=1.036 and
u.sub.Na+/u.sub.K++=0.677 at 22.degree. C.
[0096] The calculated junction potentials at 22.degree. C. in 10,
50 and 100 mM NaCl test solutions and 1 M KCl reference solution
are -1.8, -0.5 and 0.3 mV, respectively. The calculated junction
potentials at 32.degree. C. in 10, 50 and 100 mM NaCl test
solutions and 1 M KCl reference solution are -1.7, -0.4 and 0.3 mV,
respectively. The sensor potential
V=V.sub.Nernst+V.sub.junction.
The Role of the Salt Bridge on Sensor Performance
[0097] To assess the role of the salt bridge on performance,
sensors were fabricated with salt bridge diameters of 30 .mu.m, 500
.mu.m or 1 mm. The length of the salt bridge is defined by the
thickness of the PET substrate (125 .mu.m). The sensors were
mounted in a holder with 110 .mu.L of 10 mM NaCl (FIG. 19A). For
sensors with a 30 .mu.m diameter salt bridge, the sensor voltage
remained stable over 2 hours, but decreased rapidly with salt
bridge diameters of 500 .mu.m and 1 mm (FIG. 19B). The output
voltages were converted to chloride ion concentrations using the
calibration curve for each sensor (FIG. 19B). The measured
concentration for sensors with a 30 .mu.m diameter salt bridge
remains very close to the concentration of the test solution (10
mM) over the 2 hour duration. In contrast, the sensors with 500
.mu.m and 1 mm diameter salt bridges show a rapid increase in
apparent chloride ion concentration over the same period. For a 500
.mu.m salt bridge, the apparent concentration is about 64 mM after
2 hours.
[0098] The changes in sensor voltage and the corresponding chloride
ion concentration are due to equilibration between the reference
and test solutions. The concentration of the reference solution (1
M) is much larger than the test solution (10 mM), and hence the
changes are dominated by the concentration change in the test
solution. Since the concentration change is dependent on the volume
of the test solution, the equilibration problem is more significant
when the test (sweat) volume is very small. These results
illustrate the important role of the salt bridge in potentiometric
chloride sweat sensors.
[0099] To test the long-term performance of sensors with a 30 .mu.m
diameter salt bridge, the sensor output over 12 hours with 110
.mu.L of the test solution was recorded (FIG. 19D). The measured
chloride ion concentration increased to 14 mM over 12 hours, a
drift rate of about 0.3 mM h.sup.-1. The relevance of these results
to on-body tests and the sweat volume under the sensor are
discussed in "Sweat volume under the sensor and concentration drift
rate (Q)".
Sweat Volume Under the Sensor and Concentration Drift Rate (Q)
[0100] The groves in skin have a depth of about 40 .mu.m and the
androgenic (body) hair on forearm is about 30 .mu.m in diameter.
Assuming the gap between the sensor and skin is 30 .mu.m, the sweat
volume (v) under the sensor (the sensing area is 2.5 cm by 2.5 cm)
is about 20 .mu.L. To estimate the maximum measurement error during
on-body tests, the sensor voltage in a test solution volume of 20
.mu.L of 10 mM NaCl for 2 hours was recorded. The concentration
increase was about 2.2 mM for 2 hours (FIGS. 18A-18B),
corresponding to a concentration drift rate Q of about 1.1 mM
h.sup.-1 which is 3.7 times larger than Q value of 0.3 mM h.sup.-1
at v=110 pt. In practice, with a continuous sweat generation rate,
sweat will flow out of the region under the sensor onto the skin. A
typical sweat generation rate of 1 .mu.L cm.sup.-2 min.sup.-1 would
result in 750 .mu.L sweat under the sensor (2.5 cm.times.2.5 cm) in
2 hours, much larger than the 20 .mu.L in this test. Therefore, the
concentration drift rate during the 2 hour on-body tests is
expected to be negligible for the salt bridge geometry and sensor
design employed here.
Measurement Accuracy and Dose Response
[0101] To further evaluate measurement accuracy and sensor
performance, test solutions over the concentration range 10-150 mM
were measured (FIG. 20A). One hundred .mu.L of the test solution
was placed on the working electrode of the sensor using
micropipette, and the concentration of the test solution obtained
from the sensor voltage and calibration curve. The concentration
determined from the sensor and the known concentration of the test
solution are in excellent agreement with a Pearson correlation
coefficient of 0.9968 (FIG. 20B). The measurement accuracy is 0.13
mM in 10 mM solution and increases to 4.7 mM in 150 mM solution.
The accuracy decreases as the concentration of the test solution
increases since the output voltage is inversely proportional to log
C.
[0102] To assess reproducibility, the chloride ion concentration
was measured repeatedly in 150 mM solution (FIG. 20C) following
sensor calibration. The average sensor voltage was 47.2.+-.1.2 mV
(mean.+-.standard deviation) and from the calibration curve an
average variation of 5.3.+-.8 mM is obtained. As described above,
for low chloride ion concentrations, variations in sensor voltage
can be ignored. For example, for a 10 mM test solution, a 5 mV
variation in sensor voltage corresponds to a concentration
difference of 2 mM (for a 53 mV decade.sup.-1 calibration curve).
However, as the concentration of the test solution increases, the
measurement error caused by the sensor voltage variation cannot be
ignored. For example, the difference between the measured and
calibration voltages in the sixth trial in 150 mM solution (FIG.
20C) is only 3 mV, but the concentration measurement error is 25
mM.
[0103] To assess the sensor response time, dose response curves
were recorded. 1 mL of 1 M NaCl solution was pipetted every minute
into 100 mL of DI water, and the output voltage of the sensor was
recorded as a function of time (FIG. 21A). From the dose response
curves (FIG. 21B) a response time constant of about 2 s at all
concentrations was obtained. The concentration obtained from the
sensor is in good agreement with the calculated concentration (FIG.
21C), and the variation was below 5 mM.
[0104] To assess sensor performance, trials were performed with a
healthy subject while exercising on a stationary bike. Two types of
tests were performed. In the first set of trials, the subject was
requested to spin at constant power (100 W) for 60 minutes (FIG.
22A). In the second set of trials, the subject was requested to
increase the power after 30 minutes and again after 45 minutes
(FIG. 22D). In general, the onset of sweating occurred after 15-20
minutes, at which point a stable chloride ion concentration was
measured (FIG. 22B, FIG. 22E).
[0105] At constant exercise load, the measured chloride
concentrations were in the normal range for healthy individuals
(10-40 mM). To assess changes in the sweat concentration during the
test, values at 30 (C.sub.1), 45 (C.sub.2), and 60 minutes
(C.sub.3) were compared (FIG. 22C). There was no statistical
difference in sweat chloride as a function of time.
[0106] To assess the role of exercise intensity on sweat chloride
concentration, a graded exercise load test was performed (FIG.
22D). In contrast to exercise at constant load, the sweat
concentration increased in response to an increase in exercise
intensity (FIG. 22E, FIG. 22F). The chloride concentration in sweat
is known to increase with increasing sweat rate when the sweat rate
exceeds the absorption rate of the sweat gland (Dill et al. 1966;
Emrich et al. 1968). Therefore, the increase in sweat chloride with
exercise intensity is likely due to an increase in sweat rate.
[0107] In sum, a thin film, potentiometric sweat chloride sensor
with integrated salt bridge was fabricated and tested. The salt
bridge minimizes equilibration between the reference solution and
sweat sample and enables stable measurements over extended periods
of time. The sensor showed a very small concentration drift (<4
mM) over 12 hours even though the volume of the test solution was
only 110 .mu.L. The measurement variation was less than 2 mM at low
chloride ion concentration (10 mM) and 5 mM at high concentration
(150 mM), spanning the range for healthy individuals and CF
patients, typically 10-150 mM, and hence the device could be used
as a diagnostic tool for CF. In on-body tests, the sweat chloride
concentration in healthy individuals was shown to be dependent on
exercise intensity, indicating that the sensor has a potential for
a fitness monitoring applications.
Applications
[0108] This sensor can be used as a wearable sensor to monitor
sweat concentration of CF (cystic fibrosis) patients.
[0109] One current technology, the Macroduct.RTM. Sweat Collection
System (ELITech Group) includes (1) Webster sweat inducer that
administers pilocarpine using iontophoresis (FIG. 11A), and (2)
Macroduct collector to collect the sweat induced by iontophoresis
(FIG. 11B). The collected sweat is then sent to a lab for analysis.
Macroduct.RTM. Sweat Collection System is commonly used in a
hospital to collect sweat for CF diagnosis. The proposed sensor
could be used to replace the Macroduct collector (FIG. 11B) to
enable measurement of the chloride concentration in the clinic,
without having to send the sample to the lab for analysis.
[0110] This wearable chloride sensor could also be used to measure
electrolyte balance during workouts and hence could be integrated
into a fitness sensor.
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[0142] Although the present invention has been described in terms
of specific exemplary embodiments and examples, it will be
appreciated that the embodiments disclosed herein are for
illustrative purposes only and various modifications and
alterations might be made by those skilled in the art without
departing from the spirit and scope of the invention as set forth
in the following claims.
* * * * *