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 Journal of The Electrochemical Society, 2022 169 027515

Multifunctional Periodontal Probes and Their Handheld

Electronic System for Simultaneous Temperature, pH, and Depth
Measurements
Nicole L. Ritzert,*,z Asha Rani,a Tae-Jun Ko, Jeffrey J. Kim,b and Shin Ae Kimz
Department of Innovation and Technology Research, ADA Science & Research Institute, Gaithersburg, Maryland 20879
United States of America

Microscale temperature and pH sensors, contained within an area of 125 μm × 750 μm, were fabricated on disposable 3D printed
periodontal probe tips (0.5-mm diameter) with Marquis color-coded calibration marks using microfabrication and electrochemical
methods. The probe tips were inserted in a reusable, wireless handheld electronic system that records the temperature and pH of
specific oral sites such as a gingival sulcus. The linear response and precision of each sensor were tested in solutions of varying
temperature and pH representing the physiological range of the oral cavity. The temperature and pH sensors showed linear response
from 25 °C to 60 °C (±0.1 °C) and pH 4.0 to pH 8.0 (±0.1 pH unit), respectively. A calibration function performed by the system
software increased precision between probes tenfold for temperature sensors (deviation range: 0.69 °C to –0.07 °C) and twofold for
pH sensors (deviation range: 0.07 to 0.04). Wireless communication along with compatible software provides portability and
facilitates chairside use. Simultaneous measurement of temperature and pH along with gingival sulcus depth may detect early signs
of inflammation that current methods, such as probing and radiography, may not identify, thus minimizing potential tissue damage
caused by periodontal disease.
© 2022 The Author(s). Published on behalf of The Electrochemical Society by IOP Publishing Limited. This is an open access
article distributed under the terms of the Creative Commons Attribution 4.0 License (CC BY, http://creativecommons.org/licenses/
by/4.0/), which permits unrestricted reuse of the work in any medium, provided the original work is properly cited. [DOI: 10.1149/
1945-7111/ac50e4]

Manuscript submitted October 13, 2021; revised manuscript received January 6, 2022. Published February 17, 2022. This paper is
part of the JES Focus Issue on Women in Electrochemistry.
Supplementary material for this article is available online

Periodontal disease involves inflammation of the periodontium, the activity14–16 in addition to specific biomarkers for periodontal
tissues including the gingiva, connective tissue, and alveolar bone that disease.9,17–19 Periodontal probes containing sensors have detected
surround and support teeth.1–3 A recent National Health and Nutrition bacteria as well as measured the temperature or pH of the gingival
Survey (NHANES) reports that 47.2 %, or 64.7 million, of U.S. adults sulcus. For example, relative sulfide concentration in the gingival sulcus
suffer from periodontitis,2 which involves tissue loss;3–5 in addition, was used as an indicator of gram-negative bacterial activity.20 However,
according to the Global Burden of Disease Study, at least 10 % of the many bacteria do not produce volatile sulfur compounds, which creates
global population has severe periodontitis.5 Timely treatment is needed the potential of undetected infection. Although a periodontal tempera-
to minimize tissue and tooth loss, as periodontitis may compromise oral ture probe for detecting small (±0.1 °C) temperature differences caused
function (e.g., chewing and speaking) and thus negatively impact the by inflammation at localized periodontal sites was proposed,21 the
overall quality of life. Moreover, periodontal diseases are associated temperature change was represented by a two-color diode. Thus,
with other inflammatory factors and conditions, such as smoking, temperature change over time could not be recorded. Miniaturized
diabetes, heart disease, respiratory disease, and a few cancer types.6 pH sensors showed that periodontitis22–24 and peri-implantitis23–26
This association with several risk factors further accentuates the increased the pH of the gingival or peri-implant sulcus by up to two
importance of early and accurate detection of periodontal disease for pH units; however, these sensors required separate electrodes with
managing oral and systemic health. connection wires as well as independent assessment of gingival health
Severity of periodontal disease is currently assessed using methods (e.g., gingival or plaque index score). Previous periodontal probes
such as visual-tactile examination, radiographs, and probing which rely contained sensors were wired to a large main control unit, making
on visual cues of inflammation and/or tissue loss.7–10 For example, simultaneous probing and checking of measured values difficult. In
inflammation of the gingiva (i.e. gingivitis) is defined by the American addition, measuring simultaneously multiple parameters would improve
Academy of Periodontology (AAP) and the European Federation of early detection of inflammation.
Periodontology (EFP) as bleeding on probing of ⩾10 % sites in the This study focuses on developing sensors and their handheld
mouth.4 Probe depth also plays a role in determining the stage of electronic system for simultaneously measuring site-specific tempera-
periodontal disease.4 Several types of periodontal probes have been ture, pH, and probe depth for assessing periodontal health. Using
used, and the modifications in design have focused mainly on microfabrication and electrodeposition processes, we fabricated tem-
improving the accuracy of probing depth.8,9,11,12 However, probing perature and pH sensors on the end of disposable periodontal probe tips
accuracy relies on the experience of the examiner and the minimal with dimensions and features similar to commercially available probes.
amount of visual tissue loss or gain detectable. Radiographic bone loss These probe tips can be inserted in a reusable handheld body system
only detects if loss and lesions that have already occurred and may not containing electronics and software capable of signal processing, power
detect early stages of periodontal disease.13 Therefore, probing and control, display, and wireless data transfer. The entire probe system,
radiographs are of limited utility to detect initial stages of inflammation. including the disposable probe and body system, is designed to be
Clinical indicators, such as temperature and pH, associated with portable and easy to use chairside. We describe the design and in vitro
inflammation may provide quantifiable information to detect disease characterization of the new periodontal probe-based sensors, their
handheld electronic system, and their potential to detect inflammation
at specific gingival sites.
*Electrochemical Society Member.
a
Present address: Science Systems and Applications, Inc., Lanham, Maryland 20706 Experimental
USA.
b
Present address: Naval Postgraduate Dental School, US Navy, Bethesda, Maryland
Reagents.—Agar (ash 2.5 % to 4.5 %), graphite rods (3-mm
20889, USA. diameter, 99.995 %), and iron(III) chloride hexahydrate
z
E-mail: [email protected]; [email protected] (FeCl3·6H2O, 98.0 %–102 %) were purchased from Sigma-Aldrich
 Journal of The Electrochemical Society, 2022 169 027515

(St. Louis, MO, USA). Alfa Aesar (Tewksbury, MA, USA) was the followed by room temperature stabilization for 24 h. To pattern the
source of potassium nitrate (KNO3, 99.0 % min), iridium(IV) metal layers, we implemented a lift-off process using
chloride (IrCl4, 99.9 5%, Ir 56.5 % min), sulfuric acid (H2SO4, a positive photoresist (SPR220-3.0, Dow, Midland, MI, USA) as
99.9999 %, 92 % min), phosphoric acid (H3PO4, 85 % aqueous the sacrificial material. After defining photoresist patterns with
solution), boric acid (H3BO3, 99.99 %), and sodium hydroxide 320 mJ cm−2 exposure on the stabilized polyimide bottom film, we
(NaOH, ⩾ 98 %). Hydrogen peroxide (H2O2, 30 % (w/w) aqueous used e-beam evaporation to sequentially deposit Ti/Pt (20 nm/250 nm;
solution) and potassium chloride (KCl, ACS reagent) were acquired Fig. 1a-ⅲ) for the temperature sensor and Ti/Au (20 nm/300 nm;
from Taylor Scientific (St. Louis, MO, USA). Glacial acetic acid Fig. 1a-ⅳ) for the pH sensing electrodes, interconnection lines, and
(CH3COOH, 99.7 %, ACS reagent) was from Avantor, Radnor, PA, contact pads. To remove the sacrificial photoresist, we sequentially
USA). Ultrapure water (18 MΩ·cm), referred to DW, for preparing immersed the wafer in acetone and then isopropanol alcohol (IPA) for
solutions and cleaning glassware was from an EASYpure UV system a few minutes followed by DW rinse. After metallization, a top
(Barnstead-Thermolyne, Dubuque, IA, USA) or Picopure 3 system insulating polyimide layer was spin-coated, patterned, and cured
(Hydro Service and Supplies, Durham, NC, USA). under the same conditions as the bottom polyimide film (Fig. 1a-ⅴ).
Finally, to release the probes from the silicon wafer, we immersed the
Fabrication of disposable probes.—The microfabrication of wafer in buffered oxide etchant (BOE 6:1), which selectively etches
polyimide-based flexible probes is presented in Fig. 1. Using an the silicon oxide sacrificial layer (Fig. 1a-ⅵ). After rinsing the
electron beam (e-beam) evaporator (Infinity 22, Denton Vacuum, released probes with DW and drying them, we examined the surface
Moorestown, NJ, USA), we deposited 200-nm thick silicon oxide and metal patterns using an optical microscope (L200, Nikon, Tokyo,
(SiO2) to be used as a sacrificial layer to release the sensors from the Japan), a field emission scanning electron microscope (FE-SEM; Ultra
100-mm silicon wafer support (Fig. 1a-ⅰ). For generating a 5-μm 60, Carl Zeiss, Oberkochen, Germany), and profilometer (Dektak XT,
thick polyimide bottom film on the sacrificial layer, we spin-coated Bruker, Billerica, MA, USA).
photo-definable polyimide (HD-8820, HD MicroSystems, To protect both the delicate probe materials and oral tissues,
Wilmington, DE, USA) at 3000 rpm for 60 s and then patterned frames were made using biocompatible resin (VeroWhite and
the probe geometry using photolithography with 350 mJ cm−2 g-line VeroBlack) 3D polyjet printing (R&D technologies, North
exposure (MA6/BA6, Suss MicroTec, Garching, Germany). After Kingstown, RI, USA) with 16 μm resolution. Polyimide probes
patterning the bottom polyimide film, we cured the film for 2 h at were bonded to the frame using biocompatible adhesive (EPO-
275 °C in a nitrogen oven (Fig. 1a-ⅱ). To reduce thermal stress in the TECK MED-301, Epoxy Technology, Billerica, MA, USA) as
polyimide film, we adjusted temperature ramping and cooling rates shown at the bottom of Fig. 1b. Because the resin has relatively
to 1.5 °C min−1 using the programmable oven (Heratherm, Thermo high flexural strength (11,000 psi to 16,000 psi) and both the resin
Fisher Scientific, Waltham, MA, USA) under a nitrogen atmosphere, and adhesive are biomedical grade, the probes are highly resistant to

Figure 1. Fabrication of flexible periodontal probe including temperature and pH sensors at the end of the probe. (a) Each step shows the cross-sectional view of
sensors (S), an interconnection line (IL), and a contact pad (CP) of the probe. (b) Optical image of the developed flexible probe (top) and the probe bonded to the
frame (bottom). The dotted lines mark the location of the cross section of the sensors, interconnection line, and contact pad in (a). FE-SEM images of (c) the
iridium oxide film and (d) the silver/silver chloride electrodeposited on the pH sensors. Scale bars indicate (b) 7 mm and (c), (d) 1 μm.
 Journal of The Electrochemical Society, 2022 169 027515

physical stress as well as biological and toxicological reactions To determine the response of the pH sensors to changes in pH,
during use. we measured the voltage, Emeasured, between the pH sensing and
probe reference electrodes:
Electrodeposition of iridium oxide and silver/silver chloride to
form pH sensors.—After the fabricated flexible probes were bonded E measured = EIrOx − EAg AgCl probe [1]
on 3D printed frames, iridium oxide (IrOx) and silver/silver chloride
(Ag/AgClprobe) were electrodeposited sequentially on the Au pH Voltage was measured and recorded by the SP-300 potentiostat in a
sensing and probe reference electrodes, respectively, as shown in series of buffer solutions32 with 40 mmol l−1 H3PO4, 40 mmol l−1
Figs. 1a-ⅶi and 1a-ix. Both electrodes were 25 μm × 750 μm (area CH3COOH, 40 mmol l−1 H3BO3, 137 mmol l−1 NaCl, and
of 1.9 × 10–4 cm2). Electrodeposition was performed with a model 3 mmol l−1 KCl contained in a polystyrene 48-well plate at room
SP-300 potentiostat (Bio-Logic, Knoxville, TN, USA) with an ultra- temperature (20 °C to 22 °C). pH values were adjusted to values
low current electrometer. Electrolyte solutions were contained in a between 4.0 and 8.0 in 1.0 increments using 1.0 mol l−1 NaOH.
10-ml beaker or 5-mm diameter glass dish, serving as the electro- To determine the effect of temperature on the potential of IrOx
chemical cell, in a Faraday cage. All glassware was immersed in 3 and Ag/AgClprobe, Ag/AgClwire was prepared by immersing 1-mm
mol l−1 nitric acid for at least 30 min and then rinsed with copious diameter Ag wire (99.99 %, Goodfellow, Coraopolis, PA, USA) in
amounts of water before use. Auxiliary and external reference 50 mmol l−1 FeCl3 for 1 min to 2 min. Voltage was measured
electrodes were a graphite rod and Ag∣AgCl∣1 mol l−1 KCl, between IrOx or Ag/AgClwire and the Ag∣AgCl∣1 mol l−1 KCl
respectively. This reference electrode, referred to as Ag/AgClref, reference electrode in 10 ml of each buffer solution immersed in a
was placed in a salt bridge containing 30 g l−1 agar and 0.2 mol l−1 water bath at room temperature or adjusted to 37 °C or 52 °C using a
KNO3. The working electrode lead of the potentiostat was connected hotplate.
to either the pH sensing or reference electrode on the probe.
Electrical contact was made to the sensor electrodes by inserting Handheld body system configuration.—The handheld body
the probe contact pads in a custom printed circuit board (PCB) with system incorporates wireless communication for mobility and ease
a USB connector. Only the end of the probe containing the sensing of handling. Inside the plastic housing, a signal processing circuit
elements was immersed in solution. Immediately before electro- board enabled signal processing, data display and storage, and
deposition, electrodes were cleaned electrochemically in 0.5 mol l−1 external data transfer via Bluetooth communication system
H2SO4 by stepping the applied potential between +1.50 V and (Fig. 2a). By using an ultra-low-power system-on-chip (SoC;
–0.40 V vs Ag/AgClref, poising for 5 s at each potential, for at least nRF51822, Nordic Semiconductor, Trondheim, Norway), we can
30 cycles and then rinsing with water. program direct interaction of various analog and digital peripherals
IrOx films were formed first, followed by Ag/AgClprobe. IrOx for sensor output, bias voltage check, battery monitoring, and
was electrodeposited from a solution of 4 mmol l−1 IrCl4, 0.3 % onboard commercial temperature sensor output in parallel. For
(w/w) H2O2, and 40 mmol l−1 oxalic acid with pH adjusted to 10.5 controlling the organic light-emitting diode (OLED) display module,
using potassium carbonate (K2CO3); this solution was kept covered we used the inter-integrated circuit block. For storing calibrated
in the dark at room temperature for at least three days until the color measurements from the temperature and pH sensors, we used flash
turned pale violet and then stored at 4 °C.27,28 A potential of memory. On the housing, the body system contains an OLED
+0.65 V vs Ag/AgClref was applied to the probe pH sensing display unit, a power switch, three input buttons (measurement,
electrode for 10 min or 15 min to form the IrOx film. Thickness of calibration, and mode buttons), an external port (USB micro-B 2.0)
the IrOx was estimated by performing cyclic voltammetry of the for battery charging & UART parameter setting, a probe connector
films in 0.1 mol l−1 H2SO4.29,30 After depositing IrOx, Ag was (USB-C 3.0 plug type), and a ventilation opening for an onboard
electrodeposited on the probe reference electrode (Ag/AgClprobe) commercial temperature sensor (Fig. 2c).
using Techni Silver CylessII solution (Technic, Cranson, RI, USA).
A current density of 13 mA cm−2 or 50 mA cm−2 was applied for
1 min. Thickness of the Ag was estimated by integrating the charge Temperature and pH calibration and temperature compensa-
passed while oxidizing Ag films at +0.35 V vs Ag/AgClref. Ag was tion.—To minimize measurement errors due to variations in the
redeposited after oxidation. A layer of AgCl was then formed by fabrication process, calibration should be performed each time the
immersing the tip of the probe in 50 mmol l−1 FeCl3 for 1 min and disposable probe is replaced. The temperature and pH sensors were
rinsing with water. calibrated using our body system following the procedure outlined in
Fig. 2b. When the power of the body system is turned on with the
Equipment and testing performance of sensors.—Once the probe connected, the system is initialized and then displays mode
probes were bonded to the frame, we tested the performance of options. By clicking the “mode” button, the user can select between
the temperature sensors using a four-wire measurement circuit to “Temperature calibration” and “Measurement” modes. In
increase the accuracy.31 The Au lead resistance does not affect the “Temperature calibration” mode, the system reads an initial resis-
voltage measurement and thus allows a highly linear correlation tance of the temperature sensor and room temperature from an
between the Pt resistance and temperature. To confirm the correla- onboard commercial temperature sensor (STS35-DIS, Sensirion AG,
tion, we applied a constant current of 0.5 mA to the circuit using a Zurich, Switzerland). Then, the initial resistance of the temperature
current source (Keithley 6221, Keithley Instruments, Cleveland, OH, sensor on the probe is calibrated to the value of the measured room
USA) and monitored the voltage with a digital multimeter (2480 R, temperature based on the preloaded temperature coefficient of
Data Precision, Wakefield, MA, USA), a SourceMeter (2611B, resistance. After the one-point “Temperature calibration,” a beeping
Keithley Instrument, Cleveland, OH, USA) or an oscilloscope sound occurs, indicating the start of “pH calibration.” For “pH
(MDO3024, Tektronix, Beaverton, OR, USA) in a temperature- calibration,” the user can use two standard buffer solutions with pH
controlled environment (i.e., DW-filled 5-ml beaker). The tempera- 4 and 7 to determine the change in voltage, Emeasured, with pH (i.e.,
ture was increased in 5 °C steps over the range of 25 °C to 60 °C the slope in a plot of Emeasured vs pH). By sequentially immersing the
with 1 °C precision (Super-Nuova Stirring Hotplate, Barnstead probe in the standard buffers following the display instruction,
Thermolyne, Ramsey, MN, USA). To account for any temperature Emeasured is measured for each pH, and the measured values are fit
difference between the hotplate and DW in the beaker, we used a with a linear regression plot to determine the slope. The following
Fluke (Everett, WA, USA) model 52 K/J thermometer to monitor the equation was used to adjust for temperature effects on Emeasured,
temperature of the DW in real time. assuming a constant chloride concentration:
 Journal of The Electrochemical Society, 2022 169 027515

Figure 2. Configuration of the handheld body system. (a) Block diagram illustrating internal circuit components inside the body system. (b) Flow chart of
calibrating process for temperature and pH sensors. (c) Handheld body system exterior design of top view and side view. The body system includes two USB
connectors, one power switch, three selection buttons, and a color display.

kept until the next calibration. If “No” is selected, the system goes
E measured = a·T ·pH + b + c·T [2] back to the “mode selection” to repeat the calibration without
overwriting the numbers. Otherwise, the previously calibrated values
T is the buffer temperature, a is a constant relating changes in pH can be used by selecting the “Measurement” mode. In the
and temperature to the IrOx voltage, b is the voltage at pH 0.0, and c “Measurement” mode, the system reads and calculates the tempera-
is a constant related to changes in the Ag/AgClprobe voltage with ture and the pH values based on the most recent calibration numbers.
temperature. Thus, the only effects on the voltage are temperature The measured temperature and pH values are displayed on the
and pH. This equation was derived from the temperature dependence display unit, and up to 100 (≈128 KB) measurements are simulta-
of Ag/AgCl33,34 and IrOx.34,35 For these studies, c was initially— neously stored in the body system.
0.00086 V K−1.34 With a and b calculated in calibration mode, our
system compensated for the change in pH value with temperature.
Results
The temperature compensation equation is updated as the pH
calibration proceeds. After finishing the “pH calibration,” another Sensor and probe materials.—Initially, sensors were fabricated
beep sounds, and the display screen on the handheld body system using silicon as the structural substrate, but we found flexible
asks a question about whether the calibration data is going to be used polyimide substrates were easier to handle without breaking than
or not. If “Yes” is selected, the calibrated data for the temperature silicon probes which were too brittle (Figs. 3a–3c). Polymers such as
and the pH sensors are overwritten on the flash memory. The data is polyimide and polyethylene terephthalate are commonly used in
 Journal of The Electrochemical Society, 2022 169 027515

Figure 3. Multifunctional periodontal probe system. (a) Bright-field microscopic image of a Pt temperature sensor (Temp sensor) covered by top insulation layer
and two Au electrodes (Electrodes) for pH sensing. This corresponds to the fabrication steps in Figs. 1a–1v. (b) Bright-field microscopic image of sensors after
electrodeposition. pH sensing electrode with IrOx film (left) and reference electrode with Ag film (right). This figure corresponds to the fabrication steps in
Figs. 1a–1ix before AgCl formation. (c) 18 microfabricated probes on a silicon wafer before releasing, corresponding to the fabrication steps in Figs. 1a–1v.
(d) Microfabricated probe before (left) and after (right) bonding probe tip to a frame (right). (e) Prototype of the handheld multifunctional periodontal probe
system consisting of a disposable probe tip inserted in a reusable body system (right). (f) Easy connection using USB-C connector between the probe tip and the
body system. (g) Probing on a standard typodont with soft gums. Scale bars indicate (a,b) 100 μm and (d) 10 mm.

biosensor and implantable devices due to their high flexibility, patients, the handheld body system is reusable and easily connects to
electrical resistivity, thermal and chemical stability, and the probe tip with a USB-C connector (Fig. 3f). During device
biocompatibility.31,36–40 We selected platinum (Pt) for the tempera- measurement operations, electrical signals from the sensors on the
ture sensor due to its high linear correlation between electrical probe are transferred to the body system through interconnection
resistance and temperature (Fig. 3a). Gold (Au) was used as the leads and contact pads. The body system contains a signal proces-
interconnection line and pad material due to its biocompatibility and sing circuit board, a display unit, a power switch, three input buttons,
electrical and chemical stability (Fig. 3b). Titanium (Ti) provided a rechargeable batterie, an external port, and a wireless communica-
adhesion between Pt or Au and polyimide. tion module. In addition, a calibration function can compensate for
For the pH sensing electrode, iridium oxide (IrOx) was used due variations in the fabrication process.
to its (electro)chemical stability, particularly in complex media, as
well as ease of fabrication on microscale electrodes.28,30,41–47 The Fabricated probe design and features.—The tip of a periodontal
potential of the IrOx film is sensitive to proton concentration, as probe must fit inside the periodontal pocket. The tip has a shape of a
shown in the following formula:34,35 long cone with a diameter of less than 600 μm and rounded ends to
avoid tissue damage during measurement.48 Therefore, the tempera-
1 1 ture and pH sensors were integrated with a probe tip with a 500 μm
IrO 2 + H + + e− ⇌ I2 O3 + H2 O [3]
2 2 diameter, and features are similar to those of commercially available
periodontal probes. Two different colors of printing resin were used
According to the Nernst equation, the potential of the IrOx film, in making the frame to show the depth of probe penetration (Figs. 3d
EIrOx, depends linearly on pH:35 and 3g). The color markings are 3 mm, 5 mm, 7 mm, and 10 mm
RT RT from the end of the probe with highlights in two regions like
0 0
EIrOx = E IrOx + 2.303 log c H + = E IrOx − 2.303 ·pH [ 4] Marquis color-coded probes.
nF nF At the end of the probe, a temperature sensor, pH sensing
0 electrode, and reference electrode were successfully fabricated
where EIrOx is the standard potential of IrOx, R is the molar gas
within a 125 μm × 750 μm area as shown in Figs. 3a and 3b. To
constant, T is temperature, n is number of electrons, F is the Faraday
minimize self-heating and power consumption during temperature
constant, and pH is −log c H +. Thus, the expected slope of a plot of sensing, Pt resistance was maximized using a long-serpentine design
EIrOx vs pH is −58 mV pH−1 unit at room temperature (21 °C). (10 vertical lines) with 4-μm width and 8-μm pitch. The corners
Silver/silver chloride was chosen as the probe reference electrode were rounded to increase the current density. Two Au electrodes for
(Ag/AgClprobe) because Ag/AgCl is low-cost, fabricated readily, the pH sensing and reference electrodes were designed and
nontoxic, and nonpolarizable. Importantly, the potential of the fabricated as two rectangles (25 μm × 750 μm) on either side of
Ag/AgClprobe is independent of pH34 so that any change in the temperature sensor (Fig. 3a) to maximize the area of these
Emeasured (Eq. 1) is attributed to EIrOx. electrodes on the narrow probe design. Using appropriate photo-
masks, additional sensors could be integrated for simultaneous
Handheld multifunctional periodontal probe system configura- sensing of physiological or biological parameters along with the
tion.—The multifunctional periodontal probe system consists of a temperature and pH sensors without changing the size or shape of
probe tip (Fig. 3d) and a handheld body system (Fig. 3e) for the probe.
monitoring periodontal health in a site-specific manner. The probe To make the pH sensor, IrOx and Ag/AgClprobe were electro-
tip includes a microfabricated probe to sense multiple signals and a deposited sequentially on their respective Au electrodes (Fig. 3b).
probe frame to mechanically support and protect the probe. The We found that electrodeposition generated thicker Ag and IrOx films
microfabricated sensors (Figs. 3a–3c) detect temperature and pH with a higher success rate than sputter deposition, as determined
using transduction of electrical signals. While the probe tip is using FE-SEM and profilometer images (not shown). Cyclic
designed for one-time usage to minimize contamination between voltammetry (CV) of Au in the electrodeposition solution revealed
 Journal of The Electrochemical Society, 2022 169 027515

increasing current, particularly two couples near –0.15 V and environment (between 25 °C and 45 °C) independent of probing
+0.25 V vs Ag/AgClref upon cycling, likely due to IrOx precipita- locations or periodontal health.50,51
tion on the Au surface (Fig. S1 (available online at stacks.iop.org/ The temperature coefficient of resistance (TCR) was calculated
JES/169/027515/mmedia)).30 Onset of oxygen evolution, which between the measured temperature range (25 °C to 60 °C) using the
induces precipitation of IrOx on the Au surface,30,41 appeared near following equation:
+0.6 V vs Ag/AgClref. An applied potential of +0.65 V vs
Ag/AgClref generated continuous, blue-green IrOx films28,30,43 R2 − R1 R 60 C − R25 C
TCR = = ° ° (°C−1) [5]
within 10 min to 15 min (Fig. S2). Potentials negative of +0.65 V R1 × (T2 − T1 ) R25 C × (60 − 25)°C
°
produced little to no IrOx whereas those positive of +0.65 V cracked
the film or expanded it over the insulation surrounding the electrode. Based on the equation above, an average TCR value of (3.86 ± 0.18)
Estimated thickness of the IrOx films was between 200 nm and × 10−3 °C−1 (mean ± SD) from 8 probes was comparable to the
600 nm, assuming a surface concentration of 7.8 × 107 mol cm−2 TCR of pure annealed Pt wire (3.92 × 10−3 °C−1). The TCR
μm−1 for IrOx;29,30 the number of moles per unit area was variance of 18 temperature sensors from a single batch was below
determined by integrating the charge in the oxidation wave near 2 %. Last, the response time of the temperature sensors was tested.
+1.0 V vs Ag/AgClref during CV in 0.1 mol l−1 H2SO4 (Fig. S3). The response time was measured as the time required to reach
This thickness was similar to that of electrodeposited films in 63.2 % or 90 % of the final temperature after immersing a
previous studies.30,43 IrOx films cracked after CV, so these temperature sensor in heated water (60 °C) at room temperature
electrodes were not used for pH measurements. During Ag electro- (22 °C). The response times for heating to 63.2 % and 90 % of the
deposition on Au, the measured potential was typically –1.0 V vs temperature change (from 22 °C to 60 °C) were 0.18 ± 0.04 s and
Ag/AgClref (Fig. S4). Thickness of the bright, smooth Ag films was 0.27 ± 0.08 s, respectively. Therefore, a temperature sensor stored at
between 0.8 μm and 1.0 μm, determined by integrating the charge room temperature is capable of measuring the temperature of a
passed while oxidizing the Ag film (Fig. S5), assuming 100% current patient’s periodontal pocket within 0.3 s. Based on the high linearity,
efficiency and an Ag density of 10.5 g cm−3.49 A matte brown film reproducible TCR measurements, and fast response time, we
appeared after forming AgCl on the Ag. Chemical oxidation with conclude that the microfabricated temperature sensors are suitable
FeCl3 provided more uniform films than electrochemical oxidation for accurate temperature monitoring in the oral environment.
in chloride solutions.
From a single 100-mm silicon wafer, 18 probes were developed pH sensor performance.—Voltage, Emeasured, between the pH
as shown in Fig. 3c. Supporting structures with 8-mm width held sensing and reference electrodes (Eq. 1) was measured in buffer
all probes with 200-μm wide small notches to minimize probe solutions within an oral physiological pH range23–26,52 of 3.0 to 8.0.
loss during release from the wafer at the end of microfabrication As shown in Fig. 5, the voltage decreased as the pH increased, as
(Fig. 1a-ⅵ). The notch width held each probe tightly without loss as expected (Eq. 4). Voltage values were considered stable when they
well as decreased probe damage during individual detachment from fluctuated by less than ±2 mV during a single measurement.30,42,43 Only
the supporting structures. After probes were separated, they were solutions of pH 4.0 to 8.0 were used for analysis because the voltage
bonded to the probe frame with biocompatible adhesive to form a fluctuated by more than ±2 mV after the probe was immersed in
complete probe tip (Fig. 1a-vii). Each probe tip was connected to a solutions with pH less than 4.0 for several minutes. The difference
lab-designed PCB, and its sensitivity to temperature and pH changes between Emeasured and the stable voltage was less than 10 % of the
were tested. Based on measurements from multiple batches, the stable voltage within 5 s for pH 4.0 to 7.0 and 30 s for pH 8.0. These
fabrication yield exceeded 94 %. Additionally, the optimized probe response times agree well with previous studies using electrodeposited
design and mechanical flexibility achieved in this study allowed easy IrOx.28,41–43,46 Longer response times at higher pH were also reported
adherence on the probe frame without physical damage to either by Marzourk et al.,28 although they attributed this observation to
sensors or the polyimide film. Based on a preliminary physical diffusion through the protective film. The effect in the current study
endurance test, probing over 100 times on a standard typodont with could be due to slower proton diffusion in IrOx films near neutral pH.30
simulated soft gums showed no physical or functional damage to the The Emeasured values were plotted vs pH to generate a calibration
probe and temperature sensors (Fig. 3g). Therefore, the probe is plot at three time periods, viz., 5 s, 30 s, and stable voltage, for each
expected to be robust enough for localized measurement of pH sensor (Fig. 5 and Table I). Intervals were determined at the 95%
temperature and pH inside periodontal pockets. confidence level for at least three repetitions.53 The Emeasured vs pH
plot showed high correlation (R2 > 0.99) for each time period, and
Temperature sensor performance.—To analyze the temperature error generally decreased with time, indicated by decreasing CI. The
sensing capability of the microfabricated probes, we tested the slope ranged from –72.0 mV pH−1 to –81.9 mV pH−1 for four
correlation between the surrounding temperature change in DW and probes. Both the magnitude and range of the slope correspond well
its consequent resistance change from randomly selected 8 probes with reported electrodeposited IrOx films,28,30,35,43–45 although some
from 4 batches. The four-wire temperature circuit measured the studies reported lower slope magnitudes around –60 mV
resistance change of the temperature sensor with ±0.1 °C precision, pH−1.28,42,46 The magnitude of the slope was larger than the
similar to that previously reported,24 at a randomly selected expected value of 58 mV pH−1 (Eq. 4), which may be due to
temperature between 25 °C and 45 °C. The average Pt resistance contributions from a hydrated oxide produced during electrodeposi-
values from 8 sensors were linearly correlated to the temperature tion, for example:54
from 25 °C to 60 °C as shown in Fig. 4a. As expected, each sensor
showed a small variation at each temperature between batches, but 3 + 1 3
[IrO 2 (OH)2 ·2H2 O]2 − + H + e− ⇌ [Ir2O 3(OH)3·3H2 O]3 − + H2 O
the linearity in resistance response was similar. The resistance 2 2 2
change to temperature was independent of solution types such as [6]
DW and phosphate-buffered saline (PBS) shown in Fig. 4b.
Therefore, we expect the sensor to monitor sufficiently the intraoral According to the corresponding Nernst equation:

0 RT 0 RT 3 0 RT
EIrOx = E IrOx + 2.303 log c H + 3 / 2 = E IrOx + 2.303 · log c H + = E IrOx − 3.455 ·pH [7]
nF nF 2 nF
 Journal of The Electrochemical Society, 2022 169 027515

Figure 4. Linear resistance changes of microfabricated Pt temperature sensors depending on temperature change between 25 °C and 60 °C. (a) Averaged
resistance plot with linear regression (dotted line; R2 > 99 %; n = 8). Error bars represent the standard deviation (SD). (b) Examples of resistance of a sensor
measured by three different solutions. DW is distilled water and PBS is phosphate-buffered saline, pH = 7.4.

Figure 5. (a) Voltage (Emeasured) between iridium oxide (IrOx) and silver/silver chloride (Ag/AgClprobe) as a function of time in buffers of varying pH containing
40 mmol l−1 H3PO4, 40 mmol l−1 CH3COOH, 40 mmol l−1 H3BO3, 137 mmol l−1 NaCl, and 3 mmol l−1 KCl. (b) Emeasured as a function of pH for data in
Fig. 4a for three sampling times. Error bars represent a 95 % confidence interval for at least three repetitions.

Table I. Slope and y-intercept from the plot of voltage (Emeasured) vs pH for four pH sensors at room temperature (20 °C to 22 °C) for different
elapsed times after immersion in each buffer solution. Stable slope and y-intercept were determined from plots where voltage fluctuated by less than
±2 mV at each pH. Intervals were determined at the 95 % confidence level for at least three repetitions.

Sensor A B C D
−1
stable slope (mV pH ) –81.9 ± 0.8 –80.3 ± 0.9 –72.0 ± 1.6 –74.0 ± 0.6
stable y-intercept (mV) 769 ± 4 747 ± 7 617 ± 14 662 ± 3
slope, 5 s (mV pH−1) –81.1 ± 2.4 –80.0 ± 4.1 –71.8 ± 3.6 –72.5 ± 1.2
y-intercept, 5 s (mV) 770 ± 1 758 ± 27 623 ± 30 665 ± 15
slope, 30 s (mV pH−1) –81.9 ± 0.6 –79.9 ± 0.6 –72.0 ± 2.1 –73.9 ± 2.8
y-intercept, 30 s (mV) 772 ± 1 750 ± 4 620 ± 20 665 ± 12

the slope of a plot of Emeasured vs pH for the hydrated IrOx would be for three repetitions on a single sensor, which is more than reported
higher in magnitude than for an anhydrous film. values of ±2.5 mV,28 ±5 mV,43 and ±0.05 mV42 but still less than the
The y-intercept, which represents the voltage at pH 0.0, differed smallest reported difference (+0.2 in pH) between sites without and
by ≈150 mV among the pH sensors. Previous studies have also with periodontal pockets.23,52
shown that the y-intercept can vary by a considerable amount The potential of Ag/AgClwire and IrOx changed by (+0.31 ±
between electrodes, even after aging, cycling the potential, or 0.09) mV °C−1 and (–0.61 ± 0.23) mV °C−1, respectively, between
specific storage conditions,30,41,44 indicating that each pH sensor 21 °C and 52 °C, based on data from three Ag/AgClwire and three
will need to be calibrated before use. Differences could be due to IrOx samples at pH 6.7. The reported value is ≈ –1 mV °C−1 for
varying ratios of Ir oxidation states between IrOx films.54 The Ag/AgCl34 and IrOx35 at pH 7. The magnitude of the slope in a plot
Emeasured values at one pH were within ±8 mV (≈±0.1 of pH value) of EIrOx vs pH changed by 0.1 mV pH−1 C−1, which was less than
 Journal of The Electrochemical Society, 2022 169 027515

the reported value of 0.2 mV pH−1 °C−1.35 The difference between programmed on the SoC using the voltage measured simultaneously
measured and reported values may be due to differences in ionic with the commercial or the fabricated temperature sensors.
strength and/or chloride concentration. Regardless of discrepancies The integrated body system provided reliable readings with
between measured and reported values, the effect of temperature on calibration. Figure 6 shows the temperature and pH values with
potential needs to be considered when determining pH using the and without calibration. When measuring temperature after calibra-
sensors in this study. For example, a change in temperature between tion, the temperature difference between a control thermometer and
room temperature (21 °C) and average subgingival temperature our periodontal probe system was negligible (mean ± SD, −0.07 °C
(35 °C) would produce ≈14 mV difference in Emeasured and thus an ± 0.06 °C). However, the differences between the preset temperature
error in pH of ≈0.2. This error is at the threshold of distinguishing value and the measured temperature of the uncalibrated sensor
differences in pH between healthy and diseased sites, indicating a were approximately tenfold higher comparable to calibrated sensors
need for temperature compensated pH values. (0.69 °C ± 0.11 °C). pH measurements were also verified in pH 4.0
and pH 7.0 buffer solutions. After performing pH calibration, the
Handheld body system performance.—After the sensors passed measurement error, calculated as (measured pH- buffer pH)/(buffer
the performance evaluation (i.e., linear response over range 25 °C to pH) × 100 %, was ≈1 % on average from 10 measurements.
60 °C with ±0.1 °C precision and pH 4.0 to pH 8.0 with ±0.1 pH unit However, pH measurements without calibration, the measurement
precision), the probe tip was connected to a reusable handheld body error increased to ≈3 %. The difference in pH among replicates from
system through a USB-C connector (Fig. 3f). As shown in Fig. 3e, the same probe was 0.04 ± 0.05 and 0.07 ± 0.48 with and without
the main body system is integrated into a plastic housing (150 mm calibration, respectively. These results confirm that the calibration
length × 30 mm width × 16 mm height) similar in size to a handheld function compensates well for any variation in the fabrication of the
oral thermometer, and the overall weight of the system was 50.6 g. temperature and pH sensors.
The entire system is designed to be operated with one hand. The
design and components of the body system as proof-of-concept are Wireless data transfer and software.—The body system includes
illustrated in Fig. 2c. wireless communication for portability and compatible software for
The body system provides intuitive operation for calibration, facilitating clinical use as shown in Fig. 7. The wireless commu-
measurement, and mode selection by clicking buttons following the nication uses the Bluetooth low-energy module (nRF51822, Nordic
displayed message. By connecting and removing the disposable Semiconductor) to minimize battery use. To enable low-power
probe tip through a general USB connector, the body system can wireless communication with a 2.4 GHz radiofrequency, we used a
be operated without additional alignment and training as well as 2.4 GHz ISM band working antenna (RFANT5220110A2T, Walsin
reduce the risk of cross-contamination between patients. A dispo- Technology, Taipei, Taiwan). The speed of initial device connection
sable barrier sleeve similar to a digital X-ray sensor sleeve may and data transfers were 250 ms and 225 ms per data log, respec-
provide additional protection. The power supply uses a rechargeable tively. Up to 100 measured temperature and pH values can be saved.
lithium-polymer battery that can run for up to 10 h. In addition, the All saved values were transferred to external devices using a custom
body system has a low battery alert on the display with a sound alert Android software, as shown in Fig. S6, and the transferred data were
to prevent unexpected system shutdown during the patient examina- saved in external devices as a text file. Data can be transferred to
tion if the battery level drops below the preset level. electronic devices for data storage and downstream analyses without
manual connection or additional support (Fig. 7). Furthermore, the
Temperature and pH measurements with handheld body transferred data can be linked to the dental chart software in the
system.—For temperature sensing, the body system used a four- future to be used as auxiliary data for periodontal disease diagnosis
wire measurement circuit similar to the sensor testing scheme. Two based on multi-layered analysis. All data transfers will be the Health
wires of the four-wire circuit were connected to a constant current Insurance Portability and Accountability Act (HIPAA) compliant.
source (0.5 mA), and the other two wires were connected to a high-
resolution analog-to-digital converter (ADC; ADS1115, Texas
Discussion
Instruments, Dallas, USA) to read the change in voltage with
106.38 Hz sampling rate. Due to the high input impedance of the In this study, we developed a multifunctional periodontal probe
ADC, the body system can measure Pt resistance without the system which includes a handheld body system with a disposable
influence of lead resistance. Because the voltage across the Pt probe tip containing microscale temperature and pH sensors. Our
temperature sensor was measured simultaneously with a constant data indicate that the microfabricated sensors can measure potential
current applied, any variation in resistance due to self-heating of the temperature and pH differences between healthy and inflamed
temperature sensor can cause an error in the reading. To minimize periodontal sites in a timely manner. Combined pH and probe depth
any self-heating error, we designed the sensing mechanism to only measurements may support the diagnosis of periodontal disease
apply current through the temperature sensor when the measurement activity,3 indicated by previous studies reporting increased pH upon
button is activated with the sub-milliampere current. formation of periodontal pockets whereas gingivitis generates little
For low-power pH sensing, the body system integrated a or no noticeable pH change.24,55 However, one paper showed that
configurable analog front-end chip (AFE; LMP91200, Texas gingivitis could increase pH by +0.2 after seven days although little
Instruments, Dallas, USA) designed for two-electrode sensors. The change is detected initially.23 Thus, by measuring pH changes over
pH sensing circuit with AFE, ADC, and SoC functionally included a time, inflammation caused by gingivitis may be initially undetect-
buffer amplifier and temperature compensation. Because any bias able whereas that caused by early stages of periodontitis could be
current between the working and reference electrodes introduces an detected.
offset voltage error in the system, a buffer amplifier with high input Our handheld body system includes wireless communication for
impedance and very low input bias current (max. ±125 fA at 25 °C) high mobility and compatible software to enable chairside use.56–58
was implemented in the pH sensing circuit. To reduce the errors In addition, the microfabricated probe is built with biocompatible
between replicate measurements, as shown in Fig. 5a, the pH value materials and shows a constant TCR value throughout testing
was determined as an average value from 10 sets of 25 sampled without polymer film damage. In previous studies, we tested the
values when the standard deviation (SD) of 10 sets converged within biocompatibility of similar materials in primary mouse epithelial
a preset value (e.g., SD < 0.07). If the SD of the first 10 sets was tongue cells and found no cytotoxicity.31 The probe tips are
larger than the preset, another (eleventh) set would be adopted disposable to minimize cross-contamination between patients. pH
instead of the first set of measurements. The SD comparison process and temperature of saliva were measured in previous studies using
for pH sensing could be repeated for up to 2 min. Temperature and mouthguards;50,51,59,60 however, this data represents processes in the
pH calibration and temperature compensation functions were also entire oral cavity. By integrating microscale sensors at the end of a
 Journal of The Electrochemical Society, 2022 169 027515

Figure 6. Measured (a) temperature and (b) pH values with and without calibration. (a) The temperature difference between the preset value and measured value
was calculated from 31 different temperature points. (b) Measured pH values in pH 4.0 (grey) and pH 7.0 (red) buffers with and without calibration (n = 10).
Each graph has scatter plots (left) and box and whisker plots (right).

Figure 7. Conceptual schematic for future application in a dental clinic. The developed multifunctional periodontal probe system measures the physiological and
biological parameters, (temperature and pH in the current version) of the periodontal pocket in real time, transmits the measured values wirelessly, and stores and
analyzes data according to the tooth position.

periodontal probe, localized sites such as a specific periodontal have been used to collect GCF and then measure biomarkers. In one
pocket can be probed to locate potentially infected periodontal sites example, GCF samples were sent to a laboratory to analyze the
precisely and map individual teeth. Both the frame and probe are potential of several proteins in assessing the treatment of periodontal
developed with a flexible polymer which makes the probe system disease.19 Direct measurement of biomarkers using chairside tests at
suitable for natural teeth as well as titanium dental implants, specific sites can decrease analysis time and minimize errors in
providing for peri-implant disease detection. sample collecting and storing. However, current chairside tests rely
One potential limitation of this technology is that intraoral on qualitative colorimetric detection or measure one biomarker at a
temperature and pH measurements may be susceptible to food intake time.20,73,74 Building upon our platform for temperature and pH
and residues,61 time of day,50 smoking habits,62 tooth location,21 sensing, in the future, our system could provide quantitative,
gender22 and other dental conditions such as root caries.63,64 However, simultaneous measurements of a variety of biomarkers.
our system is designed to record data over time, so changes in an Future studies also include determining the effect of electrolyte
individual patient’s baseline values could be tracked to help control for composition and protein adsorption on measured pH as well as
these factors. Although smoking is a well-known modifiable risk factor improving the probe design and materials. For example, we expect
of periodontitis,65 studies have shown that smokers exhibit less the potential of Ag/AgClprobe to vary with chloride concentration, which
inflammation, indicated by fewer bleeding sites and incidence of may be minimized by coating the sensor with an antifouling
gingivitis, than non-smokers at early disease stages,66,67 thus possibly membrane.75 Such coatings may also mechanically protect the surface
compromising early detection of periodontal disease based on inflam- of the sensors during contact with gingival tissue.28,76 In addition,
mation. Other potential complications include possible variations in multiple microscale sensors could be integrated along the probe tip to
individual inflammation response to microbial infection or dental provide information about the vertical depth. Furthermore, a current
plaque68,69 although these relationships are still under investigation. level and safety circuit of the integrated system will be refined to meet
Considering these challenges, the probe tip is designed so that the medical-grade electrical safety standards.
additional sensors can be incorporated to improve the accuracy of
diagnosis and prognosis of periodontal disease. Many biomarkers,
Conclusions
such as proteins, bacteria and their products, ions, volatile com-
pounds, and genotypic/phenotypic markers in the GCF, have been Using a combination of microfabrication and electrodeposition
identified for potential use in periodontal disease diagnosis and techniques, microscale temperature and pH sensors were integrated
prognosis.17,18,55,70,71 Measuring biomarkers from specific gingival at the end of the probe tip with similar shape, size, and markings as
sites could provide specific and accurate information for current commercially available periodontal probes. These sensors may
periodontal disease activity and risk factors.72 Indeed, test strips detect inflammation caused by periodontal disease because the
 Journal of The Electrochemical Society, 2022 169 027515

sensors exhibited a linear response to physiologically relevant 25. D. Karpavicius, M. Stasikelyte, N. Baseviciene, U. Sakalauskaite, S. Ratkute, and
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This research was funded by ADA Foundation grant number 43. I. A. Ges, B. L. Ivanov, D. K. Schaffer, E. A. Lima, A. A. Werdich, and F.
97700140. Microfabrication was performed in the Center for J. Baudenbacher, Biosens. Bioelectron., 21, 248 (2005).
Nanoscale Science and Technology (CNST) located at the National 44. E. E.-D. M. El-Giar and D. O. Wipf, J. Electroanal. Chem., 609, 147 (2007).
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authors would like to thank the CNST staffs, Todoc engieers, Erin 46. H. Khani and D. O. Wipf, J. Electrochem. Soc., 166, B673 (2019).
Claussen (ADA Science & Research Institute), Prerna Gopal (ADA 47. Z. Zhou, D. Pan, C. Wang, H. Han, H. Wei, and F. Pan, J. Electrochem. Soc., 168,
Science & Research Institute), Gerry Stafford (NIST), and Tom Moffat 097501 (2021).
48. International Organization for Standardization, (2012), Dentistry — Periodontal
(NIST) for their technical support and helpful discussions. probes — Part 1: General requirements, ISO 21672-1, https://www.iso.org/
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ORCID 49. W. M. Haynes, CRC Handbook of Chemistry and Physics (CRC Press, Boca Raton,
FL) p. 4 (2011).
Nicole L. Ritzert https://orcid.org/0000-0001-8680-6299 50. J. E. Choi, K. M. Lyons, M. C. McLean, and N. J. Waddell, BDJ Open, 2, 16008 (2016).
Shin Ae Kim https://orcid.org/0000-0002-6729-8097 51. J. E. Choi, K. M. Lyons, J. A. Kieser, and N. J. Waddell, BDJ Open, 3, 17015
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