Sensors and Materials, Vol. 13, No. 3 (2001) 155-167
MYUTokyo
S &M 0439

Gingival Crevicular Fluid [GCF]­
Collecting Device for Analyzing Microvolume
Sample Solutions
Masaki Yamaguchi, Yukiko Fukushi, Katsuya Yamazaki1
and Masashi Kobayashi 1
Toyama University,Toyama 930-8555, Japan
1Toyama Medical and Pharmaceutical University,Toyama 930-0194, Japan(Received October 7, 2000; accepted February 5, 2001)
Key words:tus

gingival crevicular, noninvasive, blood glucose, testing tape, capillary, diabetic melli­

The authors aim to develop noninvasive methods of measuring blood glucose levels by
analyzing body fluids collected painlessly. In this report, we describe the manufacturing ofa gingival crevicular fluid (GCF)-collecting device with a highly sensitive glucose testingtape that causes no pain for the subjects and has proven its usefulness and medical safety.
The testing tape must be adequately colored for measurement. This study clarified thefollowing: 1. The structure of a GCF-collecting device that can analyze very small
volumes of GCF has been devised, and the quantity collected can be adjusted by varying
the size of the testing tape. 2. Two types of GCF-collecting device are proposed which canbe used even in cases of periodontitis. The GCF-collecting devices can be used asnoninvasive devices in dolorimetry. 3. The manufacturing procedure for the glucose
testing tape has been clarified with N, N-diethyl-p-phenylendiamine sulfate selected as achromogen. The advantage of this procedure is that no materials with toxic or carcinogenic
properties are used.

1.

Introduction

It is essential for patients with diabetes mellitus to maintain their blood glucose levelwithin a normal range. To achieve this goal, many diabetic patients carry a portable self­
measuring glucose analyzer to withdraw their own blood to examine their glucose level.
With this they can determine the appropriate level of exercise, insulin dose or restricted

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food intake for maintaining their health. Thus, the development of a noninvasive methodof measuring the blood glucose level is an urgent necessity, and putting such a method into
practical use will not only enable us to reduce or eliminate the physical and mental stressand pain suffered by patients with diabetes mellitus, but will also allow us to utilize themethod widely for the screening of patients who are at high risk of developing a diabetic
condition.
We have continuously been focusing on the development of noninvasive methods ofmeasuring the blood glucose level by analyzing body fluids such as painlessly collectedsaliva.c1,2J We are the first investigators worldwide to recognize the importance ofdeveloping a noninvasive blood glucose analyzer that can measure blood glucose levels bydetermining the glucose concentrations in the fluid slowly secreted from gingival crev­
ices.<3l We are currently attempting to develop a noninvasive blood glucose analyzer and to
put it into practical use.
There is a V-shaped crevice surrounding the teeth between the teeth and the gingiva(the gum). This crevice is called the gingival crevice or gingival pocket and is present
around all teeth. The depth of the crevice within the clinically normal range is generally
between 0.5 and 2.0 mm. When the gum becomes infected or injured because of an oraldisease such as periodontitis or gingivitis, the crevice becomes deeper in proportion to theseverity of the inflammation in the area. Gingival crevicular fluid (GCF) is an extracellular
fluid secreted from the epithelia of the gingival crevice, whose presence was first reported
by Brill et al. in 1958.<4l The components of GCF are mainly derived from plasma, and its
flow rate is 2 to 3 µl per hour per tooth. Accordingly, particularly in the United States andEurope, much attention is currently being paid. to the attempt to establish methods ofmeasuring the concentrations of specific substances contained in GCF and to apply this tononinvasive screening for disease and to biological determinations in vivo.<5l
To develop a working system for GCF analysis, however, a device which can not only
collect a very small volume (only a few microliters) of the secreted GCF but which can also
determine the concentrations of the secreted substance before evaporation and drying of
the samples needs to be devised. Unfortunately, such a device, which can quantify specificsubstances contained in a very small volume of body fluid, has not yet been developed.
Thus, most previous studies regarding GCF have mainly focused on the secreted volumesof GCF. In this report, we discuss the following topics as a first step in the development of
noninvasive methods for the measurement of blood glucose level.
1. The structural features of the GCF-collecting device for the analysis of microvolume
samples and a quantitative analyzing test.
2. Diagnostic test for pain (dolorimetry) in using the GCF-collecting device in 20
subjects.
3. Evaluation of the blood glucose testing tape, which is used for the determination of
glucose concentrations and exhibits high biological safety.

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2.

Gingival Crevicular Fluid Collecting Device

2.1 Collection and analysis

GCF is secreted at less than 1 µl per hour, and the liquid usually dries up completely inabout one min at room temperature. With a GCF-collecting device it must be possible tocollect the GCF in about 30 s and to analyze the concentration before evaporation. The
GCF-collecting device and built-in glucose testing tape must satisfy at least four conditions
as follows:
1. The device must have a structure such that the collected sample solution is auto­
matically absorbed in a highly sensitive glucose testing tape. Before the concentration
changes due to evaporation of the particle sample, the testing tape must be adequately
colored for measurement.
2. The collecting device must be able to be inserted into the gingival crevice with awidth of 0.1 mm without pain.
3. The quantity of the sample solution is to be determined by the structure of thecollecting device.
4. The safety of the materials used for the device must be confirmed in order for it to
be inserted orally.

2.2 GCF-collecting device

We manufactured a GCF-collecting device that has a capillary drain and built-in highsensitivity glucose testing tape as shown in Fig. 1. Capillary action is used to pump GCF
automatically. Three sheets of film are laminated to form the capillary drain. The middle
film has a drain and the plane films on each side function as covers. The drain leads

Measuring aperture( <P 1.6mm)

Plane film
(Polyester)

Glucose testing tape
( <P 2.2mm,Thickness:0.4mm}'
Surfactant

I«
Fig. 1. Structure of gingival crevicular fluid (GCF) collecting device.

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samples to a built-in testing tape between the plane films. Since the plastic film is water
repellent, a swfactant (sucrose fatty acid ester, DK ester, Daiichi Kogyo Seiyaku Co., Ltd)
diluted to 5% in ethanol is applied to the inside face of the two plane films. This sucrose
fatty acid ester is in a food additive, so organic and environmental safety are fully
confirmed by the manufacturer. Since the collecting device is to be placed in the mouth of
the subjects, it is desirable that it be designed to be disposable. Therefore, only low-cost
polyester film (Teijin. Co. Ltd.) is used for these plastic films. To enable insertion into agingival crevice with a width of about 0.1 mm, the thickness of the film is selected to be 30µm, and the three layers are contacted by an ultrasonic welding method.
Figure 2 shows a standard device (a) and a device for the elderly (b). These two devices
cover a range of depths associated with periodontitis and also corresponding to the increaseof gingival crevicular depth due to aging. Figure 3 shows the relation between the water­
holding volume and the diameter of the GCF-collecting device with built-in testing tape.
The experimental results shown are the average of five measurements wiLh glucose
standard liquid used as a sample solution. The difference in the water-holding volume
between the standard device and the device for the elderly corresponds to the length ofthese capillary tubes. The water-holding volume is proportional to the diameter of thetesting tape, and for the purposes of calibration, the variation (CV) in the capillary is a
maximum of 7%.

3.

Dolorimetry

To test the pain during insertion, dolorimetry is perf ormed on twenty normal subjects
using these GCF-collecting devices. (Fig. 4) Each subject inserts the device twice in theupper and lower front teeth and in the bicuspids at random. None of the subjects
complained of excruciating pain. More than 85% using the standard device and 70% using
the device for the elderly collected GCF without pain. (Table 1) This result corresponds tothe physiological viewpoint that, although tactile points exist around the gingival crevice,
there are no pain points. In a previous experiment where the device was inserted by adentist, GCF could be collected without pain in more than 90% of subjects.(3) The results
suggest that these devices are usable for collecting GCF, and that the pain can be decreased
by increasing the degree of skill of the user.

4.

Glucose Testing Tape

4.1 Manufacturing method

A glucose testing tape used for measuring the glucose concentration in GCF requires
the following characteristics:
1. The sensitivity of detection of a testing tape compared with ones currrently available
on the market (for example, ureal testing tape) must be improved by two digits. The
required accuracy is 0.5 mg/dl.

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37.5

1.2

·o·-·-------·-·(a) Standard devicecf>2.2(b) Devicefor the ElderlyFig. 2. Comparison of the two kinds of GCF-collecting devices (values in mm).

Q :Device for the elderly D :Standard device

4...--------..-----------,

2.5

3

Diameter of testing tape (mm)Fig. 3. Comparison of water-holding volume with diameter of testing tape of the GCF-collecting
device.

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Pain points

Fig. 4. Method of dolorimetry.
Table 1
Dolorimetry of GCF-collecting devices.
Sensation

Standard device

Device for the elderly

No pain
Some pain
Excruciating pain

140
20
0

118
42
0

(87.5%)(12.5%)(0%)(73.8%)(26.2%)(0%)

2. The testing tape must be made using only materials of which the biocompatible
safety has been confirmed by experiment. The materials should be neither toxic nor
carcinogenic.
The procedure for manufacturing the glucose testing tape, which used N, N-diethyl-p­
phenylenediamine sulfate (DPD sulfate) as a chromogen, is shown in Fig. 5.
1. A surfactant, 0.05 g of sodium dodecyl sulfate (CH2 (CH2)11OSO3Na, Wako Pure
Chemical Industries, Ltd.), and a stabilizer for various chemicals, 0.5 g of sodium alginate,are dissolved in 30 ml of distilled water. This is solution A.
2. Glucose oxidase (GOD, ECl .1.3.4, 220 units/mg, Wako Pure Chemical Industries,Ltd.) and peroxidase (POD, ECl.11.1.7, 100 units/mg, Wako Pure Chemical Industries,Ltd.) are dissolved in 10 ml of distilled water. This is solution B.
3. Solution C-1 is made from 1.44 g of gelatin dissolved in 30 ml of distilled water at
40°C, after which 30 ml of neutral phosphoric acid buffer solution (pH6.86) is added tokeep it warm. Solution C-2 is made from 1.44 g gelatin dissolved in 60 ml of boric acidbuffer solution (pH9.18) at 40 °C. A chromogen, DPD sulfate, is added to C-1 and C-2.
Table 2 shows six types of buffer solutions made using various solvents and soluteconcentrations.

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Buffer solution (40-S0'C)

Distilled water
Sodium alginate

r- Gelatin

Sodium dodecyl
sulfateMixing, Dissolution
A Distilled water
B
Rapid mixing

GOD,
POD

t

Dissolution
e
Ko p

.

f

4

C-1, C-2

0'C N,N-di thyl-pe
phenylenediamine

D-1, D-2
Dipping with filter papers � Dry for 2 h at 35 ° C in drying oven

Dissolution

Fig. 5. Manufacturing procedure for glucose testing tape.
Table 2
Combinations of various concentrations of chromogen and solvent.
Concentration ofN,N-diethyl-pphenylenediamine
sulfate (g/dl)

Solvent for
N,N-diethyl-pphenylenediamine
sulfateType 1
2
3

0.125
0.250
0.375

Diluted neutral
phosphoric acidbuffer solution(pH6.86)

6.00
4.25
3.50

Type4
5
6

0.250
0.375
0.500

Borate standard
solution (pH9.18)

8.25
7.50
5.50

pH

4. Solution D-1 is made by mixing solutions A, Band C-1. Solution D�2 is made bymixing solutions A, Band C-2.
5. Filter papers are dipped in the solution D (D-1, D-2) for 60 s, then dried for about 2
h at 35°C in a drying oven.
Generally, as the coloring characteristics of DPD sulfate change depending on thebuffer solution, the gradient and linearity (correlation coefficient) of the coloring charac­
teristics are calculated to detemrine the difference due to using different solvents. Six types

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of buffer solutions are selected as shown in Table 2. The final pH of the buffer solution
decreases in proportion to the concentration of chromogen.
The reflectance of these six glucose testing tapes is measured with a spectrophotometer(U-3010, Hitachi, Ltd.) using a glucose standard solution. The reflectance changes
depending on whether the paper is dry or wet. Using the wet condition as standard, therelative reflectance can be calculated using the following equation:
R=

Rmes

(1)

Ro ,

where R0 is the reflectance measured with zero mg/dl glucose standard solution, andRmes is the reflectance at each glucose concentration.
The optical density, OD, is obtained from this relative reflectance by the equation:
OD=-logR.

(2)

The calibration curves between the optical density, OD, and the glucose level of thesesix testing tapes have been measured. Figure 6 shows the correlation between the gradient

Q: gradientcorrelation coefficient

6:

0.03

<::s

0.02

.2

,,

0.01

,,

0.9

0
0

0

0.2

0.4

0.8
0.6

Concentration of chromogen (g/dl)
Fig. 6. Measured results of coloring characteristics of testing tape as a function of chromogen
concentration. (wavelength: 530 nm)

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163

and the correlation coefficient of these calibration curves. From the results, the following
become clear:
1. The sensitivity of the reciprocal of absorbance (reflectance) of the glucose testingtape shows a maximum at a wavelength of about 530 nm. The gradients of these buffer
solutions are measured at this wavelength, and it is found that the gradient increases inproportion to the final pH of the solution.
2. The correlation coefficient increases in proportion to the decrease in the final pH. In
this evaluation, the best sensitivity is obtained when the solute concentration is 0.375 g/dlof DPD sulfate (chromogen) and when a mixed solution of diluted neutral phosphoric acidbuffer solution is used as the solvent.
3. In all cases, the correlation coefficient increases in proportion to the density of DPD
sulfate.

4.2 Calibration curve and time-course changes of testing tape

The glucose testing tape is made using 0.375 g/dl of chromogen and a diluted neutral
phosphoric acid buffer solution. Figure 7 shows the calibration curve of the absorbanceconverted from the reflectance. When the glucose concentration is more than 5 mg/dl, thereflectance saturates. The correlation coefficients in the ranges from 0 to 5 mg/dl and from0 to 10 mg/dl are 1.00 (calibration curve A) and 0.99 (calibration curve B), respectively.
The coefficient of variation (CV) obtained from each concentration for both calibration
curves shows a maximum of 12 % . Figure 8 shows the changes over a period of time in a

- : 0-10 mg/di (B)-----: 0-5 mg/di (A)
0.3

A

.£

I

)'/

0.2

/

0.1

0
0

5

10

Glucose level (mg/di)
Fig. 7.

Calibration curve of testing tape measured using the spectrometer.

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7
6

5

Q)
Q)

3
2

1
0

0

5

10

day

15

Fig. 8. Time-course changes of testing tape with 5 mg/dl of glucose standard solution.

standard solution of 5 mg/dl glucose calculated using calibration curve B. In the first fourdays the glucose levels changed by 22%. Thereafter, they varied by± 10% for 5 mg/dl forabout seven days. Based on these results, it is desirable to use this glucose testing tape after
four days aging.

4.3 Calibration curve of GCF-collecting device

Figure 9 shows the results of the calibration curve obtained using a GCF-collecting
device. This curve is measured using a manufactured optical measuring system in therange from Oto 10 mg/dl. In this measurement, a green LED (E1L51-3G, Toyoda Gosei
Co., Ltd.) irradiates the glucose testing tape, and the reflectance is measured by aphotodiode (S2506-02, Hamamatsu Photonics., K.K.). For comparison, a calibration
curve measured by the spectrophotometer is also shown. The gradient of the spectropho­
tometer is 2.32 x 10-2 and the coefficient of variation (CV) is 12 % maximum. On the other
hand, the gradient of the optical measuring system is 2.07 x 10-2 and the CV is 9%
maximum. Both con-elation coefficients are more than 0.98. If the condensing character­istics of the green LED can be improved further, it may be possible to improve the accuracyof the measurement. In conclusion, with the GCF-collecting device the necessary preci­sion for measuring glucose concentration can be achieved.

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165

0 : Spectrophotometer

D: Optical measuring system
§
·rn

·0a

0.2

0.1

Glucose level (mg/dl)
Fig. 9.

5.

Calibration curve of GCF-collecting device measured by an optical measuring system.

Conclusions

We manufactured a GCF-collecting device with a highly sensitive glucose testing tapethat causes no pain to the subjects and proved its usefulness and medical safety. The
following results became clear:
1. The structure of a GCF-collecting device that can analyze very small volumes of
GCF is devised, and the quantity collected can be adjusted by the shape of its capillarydrain and the size of the testing tape.
2. Two types of GCF-collecting device are proposed which can be used even in casesof periodontitis. Based on results from dolorimetry, the GCF-collecting devices can beused as noninvasive devices.
3. The manufacturing procedure for the glucose testing tape is clarified with N, N­
diethyl-p-phenylendiamine sulfate selected as a chromogen. The advantage of this proce­dure is that no materials with toxic or carcinogenic properties are used. From themeasurements, the coefficient of variation of this GCF-collecting device in the range 0 10
mg/dl is a maximum of 9%.
In the future, we aim to establish methods for noninvasive measurement for blood
glucose levels by performing bench tests using these GCF-collecting devices.

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Acknowledgements
Prut of this research is supported from the FY 1998 Proposal-Based Immediate-Effect
R & D Promotion Program of the New Energy and Industrial Technology Development
Organization in Japan, which was entitled "Development of Noninvasive Measurement
Blood Glucose Using Gingival Crevicular Fluid."

References

2
3

4
5

M. Yamaguchi, M. Mitsumori and Y. Kano: IEEE Engineering in Medicine and Biology
Magazine 17 (1998) 59.
M. Yamaguchi, K. Kataoka, Y. Kano, N. Takai, Y. Yoshida and G. Egusa: Saliva Glucose
Testing Tape Used for Noninvasively Monitoring Blood Glucose, 21th Annual International
Conference of the Engineering in Medicine and Biology Society, No. 587 (1999) p. 840.
M. Yamaguchi, S. Yamada, M. Okubo and Y. Yoshida: Development of Noninvasively
Measuring Blood Glucose Using Gingival Crevicular Fluid, R&D Report for FY 1998 Proposed­
Based brunediate-Effect R&D Program of New Energy and Industrial Technology Development
Organization (NEDO) in Japan (2000) 8. [in Japanese]
N. Brill and B. Krasse: Acta Odont. Scandinav. 16 (1958) 233.
0. Kjellman: Swed. Dent. J. 63 (1970) 11.

Masaki Yamaguchi was born in Nagoya, Japan, in 1963.

He received the B.S. and M.S. degrees in engineering from
Shinshu University, Japan, in 1985 and 1987. He joined the
Research Laboratory of Brother Industries, LTD., Japan, in
1987. He received the doctoral degree in engineering from
Shinshu University in 1994. He started an assistant profes­
sor at Tokyo University of Agriculture and Technology
since 1995. He is ctmently working as an associate profes­
sor in faculty of engineering at Toyama University since1999. His primary research interests focus on the develop­
ment of noninvasive medical sensors and welfare apparatuses. His noninvasive measurement approaches include
blood glucose, human stress, and other clinical analytical items. In 2001 he received
Nikkei BP Technology Awards in Medicine/Biotechnology Section from Nikkei Business
Publications, Inc. for his achievements in the study of noninvasively measuring blood
glucose via gingival crevicular fluid. He is a member of the IEEE EMBS, the Institute ofElectrical Engineers of Japan (IEEJ), the Japan Society of Medical Electronics andBiological Engineering (JME), and the Japan Diabetes Society.

Sensors and Materials, Vol. 13, No. 3 (2001)

167

Yukiko Fukushi was born in Hokkaido, Japan,in 1977. She
received the B.S. degree in engineering from Toyama Uni­
versity, Japan, in 2000. She is a master's course student of
the Graduate School of Science and Engineering at the
Toyama University. Her current research interest is devel­
opment of measuring blood glucose levels via gingival crev­
icular fluid-collecting device. She is a member of the Japan
Diabetes Society.

Katsuya Yamazaki received his M.D. in 1986 and his Ph.D.
degree in medical science from Toyama Medical and Phar­maceutical University, Toyama, Japan, in 1991. Since 1994,
he has been working at the Department of Internal Medicine,
Toyama Medical and Pharmaceutical University. His re­search interests include non-invasive blood glucose mea­
surement, and disease management of diabetes using com­puter systems.

Masashi Kobayashi received his M.D. in 1967. He worked
at the University of St. Louis and Kansas University Medical
Center from 1970 to 1976 and studied at the Stanford Uni­
versity, the Department of Endocrinology, Gerontology andMetabolism, from 1976 to 1978. He was a professor at the
Department of Interrnal Medicine, Toyama Medical andPharmaceutical University, in Japan. He is now a vice­president of the Toyama Medical and Pharmaceutical Uni­
versity and a director of the university hospital. His researchinterests include disease management of type 2 diabetes andinsulin resistance.