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DEVELOPMENT OF HEMATOCRIT MONITORING SENSOR


USING SCREEN PRINTED CARBON ELECTRODE

Seung-Ro Lee1and Chong-Min Kyung2


1
Center for Integrated Smart Sensors, Korea Advanced Institute of Science and Technology,
Daejeon, Republic of Korea
2
Department of Electrical Engineering, Korea Advanced Institute of Science and Technology,
Daejeon, Republic of Korea

ABSTRACT RBCs have the greatest weight and are forced to the bottom
Hematocrit (HCT) is actually a calculated value of the tube. The WBCs and platelets form a thin layer,
obtained from modern automated hematology analyzers. It called the buffy coat, between the RBCs and the plasma,
is the product of the mean cell volume (MCV) and the red and the liquid plasma rises to the top. The height of the red
blood cell (RBC) count, both of which are directly cell column is measured as a percent of the total blood
measured by the analyzer. Therefore, if there are any column. The higher the column of red cells, the higher the
inaccuracies in measurement of the MCV or RBC count, hematocrit. Most commonly, the hematocrit is measured
the HCT will reflect those inaccuracies. In this study we indirectly by an automated blood cell counter. It is
have demonstrated the feasibility of electrochemical important to recognize that different results may be
methods for determination of hematocrit level in whole obtained when different measurement principles are used.
blood samples. The sensitivity of the hematocrit biosensor For example, the microhematocrit tube method will give
was 1.01A/% and the average coefficient of variation was slightly higher results than the electronic methods when
5.04%, respectively. RBCs of abnormal shape are present because more plasma
is trapped between the cells.
KEYWORDS The hematocrit is typically measured from a blood
Capillary blood, Hematocrit (Ht or HCT), White sample by an automated machine that makes several other
Blood Cells (WBCs), Red Blood Cells (RBCs), Plasma, measurements at the same time [2, 3]. Hematocrit is a
Hemoglobin (Hb or Hgb), Anemia, Disposable biosensor, parameter that reflects the fractional volume of a blood
POCT (Point-of -care testing). sample that is occupied by packed red blood cells (RBCs)
[4, 5]. Normal values vary with age and sex. Some
representative ranges are [6, 7]:
INTRODUCTION
The hematocrit is used to screen for anemia, or is at birth: 42-60%
measured on a person to determine the extent of anemia. six to 12 months: 33-40%
An anemic person has fewer or smaller than normal red adult males: 42-52%
blood cells. A low hematocrit, combined with other adult females: 35-47%
abnormal blood tests, confirms the diagnosis. The
hematocrit is decreased in a variety of common conditions When a tube of blood is centrifuged, the red cells will
including chronic and recent acute blood loss, some be packed into the bottom of the tube. The proportion of
cancers, kidney and liver diseases, malnutrition, vitamin B red cells to the total blood volume can be visually
12 and folic acid deficiencies, iron deficiency, pregnancy, measured, as shown in Fig. 1.
systemic lupus erythematosus, rheumatoid arthritis and
peptic ulcer disease. An elevated hematocrit is most often
associated with severe burns, diarrhea, shock, Ad dison's
disease, and dehydration, which is a decreased amount of
water in the tissues. These conditions reduce the volume of
plasma water causing a relative increase in RBCs, which
concentrates the RBCs, called hemoconcentration. An
elevated hematocrit may also be caused by an absolute
increase in blood cells, called polycythemia. This may be
secondary to a decreased amount of oxygen, called
hypoxia, or the result of a proliferation of blood forming
cells in the bone marrow (polycythemia vera).[1]
Blood is made up of red blood cells, white blood cells
(WBCs), platelets, and plasma. A decrease in the number
or size of red cells also decreases the amount of space they
occupy, resulting in a lower hematocrit. The hematocrit Figure 1: Photographs of the laboratory tabletop
may be measured manually by centrifugation. A thin centrifuge. The glass capillary tube containing blood is
capillary tube called a microhematocrit tube is filled with mounted on a centrifuge, and the Hematocrit is determined
blood and sealed at the bottom. The tube is centrifuged at by measuring the ratio of the length of accumulated RBCs
10,000 RPM (revolutions per minute) for five minutes. The to the total length of the blood sample.

978-1-4673-5983-2/13/$31.00 2013 IEEE 2165 Transducers 2013, Barcelona, SPAIN, 16-20 June 2013
However, the centrifugation does not easily allow the
continuous measurement of hematocrit, and the analysis is
relatively time-consuming, automated machinery are
expensive, bulky, and not generally portable.
Electrochemical sensors based on an electrode
modified with immobilized enzyme have been reported
previously [8]. A disposable amperometric biosensor to
detect blood glucose has also been developed. Various
biosensor electrode fabrication techniques have been
reported in the literature, including rolling, screen printing
[9, 10] and sputter deposition techniques [11]. Typically, a
biosensor is based on a screen printed electrode. A
carbon-paste electrode (CPE) is made from amixture of
conducting graphite powder and a pasting liquid. These
Figure 3: (a) Photographs of the potable hematocrit meter.
electrodes are simple to make, and offer an easily
(b) Photographs of the hematocrit bio-sensor strip.
renewable surface for electron exchange. These electrodes
are widely used, mainly for voltammetric measurements.
Carbon paste-based sensors [12, 13, 14, 15, 16, 17, 18, 19,
EXPERIMENTAL
20, 21, 22, 23, 24, 25, 26, 27, 28 and 29] Ledru et al., 2006;
In this study, electrochemical measurements to
Corgier et al., 2007; Piermarini et al., 2007; Tangkuaram et
estimate the newly developed hematocrit sensor strips,
al., 2007; Honeychurch et al., 2007; Djellouli et al., 2007)
which consist of a two electrode system, were performed
are also applicable in coulometry (both amperometry and
using an electrochemical analyzer. The equipment
potentiometry).
employed included a cyclic voltammeter, an amperometer
(WonATech Co., Ltd), and WPCIPG software version 1.0
FABRICATION installed on a personal computer. All structural materials of
In this paper presents a hematocrit sensor strip, which the sensor chip and reagents were commercially available:
was developed using a screen printed carbon electrodes analytical reagent grade potassium hexacyanoferrate (II)
(SPCE), as shown in Fig. 2. (i.e. ferricyanide) with 99.9 % purity. Triton X-100 wetting
agent was purchased from Sigma Aldrich. Deionized (D.I.)
water was used throughout the experiments to prepare the
samples and buffers solutions. A 0.1Mphosphate buffer
solution (PBS) was freshly prepared by mixing solutions of
KH2PO4 and K2HPO4. The pH of the prepared solution was
7.03. Then, 0.4 of the electron transfer mediator was also
immobilized onto the sensing area using the dispensing
method. The hematocrit biosensor can be measured after
drying for 24 h at room temperature. The heparin mixed
blood sample was prepared daily, and stored overnight to
reach mutarotational equilibrium before use. All
electrochemical experiments were carried out in a cell
Figure 2: Schematic representation of the hematocrit containing 0.1M phosphate buffer solution at room
bio-sensor screen-printed carbon electrodes temperature (252) using screen printed carbon as the
working and reference electrodes. In this paper, we used
The Hematocrit biosensor consisted of a bottom, a amperometry techniques to study the electrochemical
middle, and upper plate on to the 180m thick PET properties of the immobilized mediator solution on screen
(polyethylene terephthalate) substrate. The fabrication printed carbon electrodes for biosensor applications.
process of the sensor is as follows: Potassium hexacyanoferrate (II) can be immobilized by
(a) The working and reference electrodes are formed simple adsorption on a screen printed carbon sensing
on the fabricated bottom plate. (b) Then the bottom plate is electrode. In addition, the ferro-ferri cyanide redox
combined with the middle plate. (c) Hydrophilic solution coupling electrochemical reaction [25] was used as the
(Triton X 100 + Ruthenium) is deposited on the channel of sensing mechanism for this hematocrit biosensor. The
the combined structure (formed bottom plate + middle ferro-ferri redox reaction can occur at a screen printed
plate). (d) Holes to form the inlet and outlet are fabricated carbon electrode surface (sensing area), which produces an
in the upper plate. The hematocrit biosensor is oxidation and a reduction current from the hematocrit
accomplished by a combination between (c) and (d). concentration. In this work, potassium ferrocyanide was
The hematocrit biosensor is comprised of a screen used as the mediator [26, 27]. Potassium ferricyanide had
printed carbon electrode structure on polyethylene an electronic mediator function, and received electrons to
terephthalate (PET) substrates. The fabricated electrode become potassium ferrocyanide. Ultimately, the electric
has an electrical resistance (500). Fig. 3 shows a current (e) was detected by the screen printed carbon
photograph of the hematocrit meter and SPCE biosensor electrode, and potassium ferrocyanide changed to
strips, respectively. potassium ferricyanide.

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Figure 4: Amperometric profiles of the hematocrit
bio- sensor showing the change in the response current of Figure 6: Characteristics of the linear correlation
each hematocrit level with time. Working and reference coefficient. Calibration curve: R2=0.9703, y=-3E-08x+6E
electrodes are SPCE. Applied potential is +1.5 V. -0 6

As shown in Fig. 4, chrono amperometric profiles of Fig. 5 shows the characteristic measurement of the
the hematocrit biosensor showing the change in the fabricated disposable hematocrit biosensor, which was
response current of each hematocrit level with time. repeated 50 times, and indicates that the disposable SPCE
Repeatability of the current responses for each hematocrit biosensor has good linearity and considerable
levels, 0(plasma), 30, 47, 75 and 100% were prepared prior reproducibility. Fig. 6 shows the correlation between the
to conducting electrochemical measurements, as shown in current value detected by the newly developed bio-sensor
Fig. 5. The currenttime response of screen printed carbon strip and the hematocrit level of whole blood. The screen
electrode to each hematocrit concentration was also printed carbon electrode achieved a steady state current in
studied by amperometry in whole blood samples at a fixed less than 30 s. And the linear slope is y=-3E-08x+6E (%)
potential of 1.5 volt (V). The sensor presents a fast current with a correlation coefficient of R2=0.9703. The sensitivity
response to hematocrit concentration and reached to hematocrit concentration is ~0.3 A10%-1 with a lowest
steady-state within 10s. detection limit of 0 %. The sensor exhibits excellent
linearity in the wide range from 0 to 100 %.

Table 1: Repeatability evaluation of different hematocrit


levels
HCT level
0% 30% 47% 75% 100% Total
(%), N=50
Average 6.17 5.74 0.51 4.45 3.41

(A) E-06 E-06 E-06 E-06 E-06
Relative
standard 3.85 2.09 3.12 1.31 2.14

deviation E-07 E-07 E-07 E-07 E-07
(A)
Coefficient
of
6.24 3.64 6.12 2.94 6.26 5.04
variation
(%)

Figure 5: Comparison of hematocrit determinations Table 1 show that a linear relation exists in the blood
by the electrochemical method. Repeatability of the current hematocrit level from 0 to 100 %, and the coefficient of
responses for each hematocrit levels, 0, 30, 47, 75 and variation in each hematocrit level was under 5.04 %.
100 % were prepared prior to conducting electrochemical
measurements. RESULTS AND DISSCUSSION
The scope of this study is to establish standard
Along with the addition of hematocrit %, the oxidation procedures of hematocrit bio-sensor fabrication with main
current decreases linearly and the corresponding focuses on low production cost and dependable
calibration plot is shown in Fig. 5 and Fig.6. Fig. 5 reproducibility. The hematocrit is a test that measures the
compares results by the electrochemical and micro percentage of blood that is comprised of red blood cells. A
hematocrit methods. Fig. 5 shows the precision of the disposable hematocrit bio-sensor was successfully
electrochemical hematocrit method. Measurements must developed in this research using screen printing technique.
be made with fresh blood samples, because sodium ions The fabricated hematocrit bio-sensor was tested using
equilibrate across the erythrocyte membrane with time, relevant electrochemical methods such as cyclic
resulting in low hematocrit. voltammetry and chrono-amperometry methods. The

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biosensor responded linearly to whole blood samples with 2000.
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Diagnostic Tests, 4th ed. Upper Saddle River, NJ: * Correspondence to: Ph.D. Seung-Ro Lee, Center for
Prentice Hall, 2001. Integrated Smart Sensors/KAIST, Tel: +82-42-350-8704,
[7] Kjeldsberg, Carl R. Practical Diagnosis of Fax: +82-42-350-8700, E-mail address: leesr@kaist.ac.kr
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