http://dx.doi.org/10.5935/0103-5053.

20160164
J. Braz. Chem. Soc., Vol. 28, No. 1, 197-201, 2017.

Short Report
Printed in Brazil - ©2017 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00

Colorimetric Detection of Glucose in Biological Fluids Using Toner-Based

Microzone Plates

Paula B. M. Silva,a Karoliny A. Oliveiraa and Wendell K. T. Coltro*,a,b

a
Instituto de Química, Universidade Federal de Goiás, 74001-970 Goiânia-GO, Brazil
b
Instituto Nacional de Ciência e Tecnologia de Bioanalítica, INCTBio, 13083-861 Campinas-SP, Brazil

This report describes the use of toner-based microzone plates for quantitative determination of
glucose in artificial urine and human serum samples using colorimetric detection. The proposed
approach has exhibited a linear response for glucose concentration levels from 0 to 10 mmol L-1
with determination coefficient (R2) of 0.996. The achieved values for sensitivity and limit of
detection were 5.1 AU (mmol L-1)-1 and 0.6 mmol L-1, respectively. The glucose analysis in artificial
serum samples revealed error lower than 5% in comparison with certified values provided by the
supplier. Lastly, the proposed approach has also provided suitable accuracy (92-105%) for glucose
measurements in artificial urine samples.

Keywords: bioanalytic, clinical assay, disposable device, point-of-care testing, portable

instrumentation

Introduction substrate. In 2003, do Lago et al.16 proposed the direct

fabrication of microfluidic devices using a laser printer for
In the last years, the miniaturization of analytical the deposition of toner layers on polyester films followed
systems has received considerable attention due to their by a thermal lamination for sealing of channels at low
attractive advantages including low reagent and sample temperature. Since this pioneering report, laser printing
consumption, portability, minimal waste generation, short technology (LPT) has been used to produce microfluidic
time analysis, and high-throughput capability.1-7 Standard toner-based analytical devices (µTADs) at very low cost. As
photolithography is one of the main microfabrication previously reviewed,13 examples of toner-based devices for
technologies allowing the fabrication of microfluidic electrophoretic separations, deoxyribonucleic acid (DNA)
channels on different platforms including glass, silicon extraction and analysis, polymerase chain reaction (PCR)
and quartz. Although this technology provides excellent amplification, passive micromixers, protein concentrator
resolution, it is laborious, expensive and time consuming.8,9 and electrospray tips have been successfully reported.
Furthermore, cleanroom facilities and sophisticated Recent publications have also demonstrated the use of
instrumentation are required thereby limiting the access to LPT to produce tunable hydrophobic valves17 as well as
few research groups. For this reason, simpler, cheaper and polyester-toner devices for microdroplets generation18 and
faster technologies have been developed to promote the rapid reciprocating mixers.19 Additional examples regarded to the
spread of miniaturized analytical systems worldwide.10-12 indirect use of toner for the fabrication of electroanalytical
Among the alternative fabrication methodologies, toner- devices,20-25 microchip free-flow electrophoresis,26 polymer
based techniques offer simplicity and low instrumental microfluidic devices27-29 and integrated microfluidic cell
requirements, thus making possible their implementation devices have been also reported.30
in any laboratory or research center.13,14 Due to the advantages associated with the fabrication
Toner was firstly explored for microfabrication in 2001, simplicity and low cost per device, some authors have
when Tan et al.15 suggested the use of a photocopying extended the use of LPT to produce toner-based devices for
machine to create a high-relief master for prototyping of clinical assays involving the use of portable equipment like
microfluidic devices in poly(dimetilsyloxane) (PDMS) cell phone camera, scanner, digital camera and handheld
optical microscope for colorimetric measurements. The
*e-mail: wendell@ufg.br association of toner-based devices and colorimetric
198 Colorimetric Detection of Glucose in Biological Fluids Using Toner-Based Microzone Plates J. Braz. Chem. Soc.

detection is quite attractive for point-of-care (POC) microplate consisted in 33 wells arranged into 11 columns
testing due to operational simplicity and low instrumental with 3 wells each, as depicted in Figure 1.
requirements. Furthermore, the mentioned electronic
devices for image capture are globally affordable and
their use do not require extensive training. This platform
has been successfully used for capillary-driven flow
based clinical assays31 and dengue diagnostics through
enzyme-linked immunosorbent assay (ELISA) on printed
zones.32 When compared to capillary-driven toner-based
microfluidic devices, the laser printing of microzone array Figure 1. Layout of the microplate containing 33 wells arranged into
on transparency films is based on a single printing step,32 11 columns with 3 wells each.
while the fabrication of multilayer toner-based microfluidic
devices require the laser cut of an intermediary transparency Colorimetric detection
film, an alignment step and, finally, a thermal lamination
to promote the channel sealing.31 Colorimetric detection was performed using the
In this current report, we describe the use of toner- scanner mode of a Deskjet multifunction printer (Hewlett-
based microzone plates for glucose analysis in biological Packard, model F4280), with 600-dpi resolution. The
fluids using colorimetric measurements. Glucose has been images were converted to 8-bit grayscale and analyzed in
selected as a target analyte because of its clinical importance Corel Photo‑Paint software. The arithmetic mean intensity
for diagnosis and monitoring of diabetes.33 Colorimetric of each pixel was used to measure the color intensity.
measurements were performed on printed toner zones
filled with cellulose paste. Glucose concentration levels Glucose assay
were quantitatively determined in artificial serum and
urine samples. Prior to the colorimetric assays, 15 µL-aliquots
of a cellulose paste made of cellulose powder and
Experimental water (1:4 m/v) were added to the zones and allowed to
dry at room temperature. Afterwards, 10 µL of 0.6 mol L-1
Materials and chemicals potassium iodide solution and 10 µL of a solution
containing HRP and glucose oxidase enzymes (1:5)
Cellulose powder, glucose oxidase (181 U mg -1), prepared in 100 mmol L-1 phosphate buffer (pH 6) were
D-glucose, horseradish peroxidase (HRP) (73 U mg-1), manually pipetted inside each microzone. The microplate
potassium iodide, sodium monohydrogen phosphate, was kept at room temperature during 30 minutes to dry.
and sodium dihydrogen phosphate were purchased from Finally, 10 µL of standard or artificial sample solution were
Sigma‑Aldrich Co. (Saint Louis, MO, USA). Artificial added into the microzones.
serum samples level I (lot No. SCNO 13041) and level II
(lot No. SCPA 12081) were acquired from Doles Reagents Biological samples
(Goiania, GO, Brazil). Transparency films (Filipaper Pro
100-µ model) and toner cartridge CB540A were obtained Artificial samples of human urine and serum were used
from Filiperson (Rio de Janeiro, RJ, Brazil) and Hewlett- to demonstrate the analytical and clinical feasibility the
Packard (Palo Alto, CA, USA), respectively. All chemicals proposed approach. Artificial urine solution was prepared
were used without any purification. according to the procedure described by Martinez et al.34
The solution was prepared with 1.1, 2.0, 25, 70, 2.5, 90,
Fabrication of toner-based microzone plates 10, 2.0, 7.0, 7.0 and 25 mmol L-1 of lactic acid, citric
acid, sodium bicarbonate, urea, calcium chloride, sodium
Toner-based microzone plates were fabricated according chloride, sodium sulfate, magnesium sulfate, potassium
to the procedure reported by our group.32 Microplates monohydrogen phosphate, potassium dihydrogen
were drawn in Corel Draw software version 13.0 and phosphate and ammonium chloride, respectively. The
then printed on a polyester film surface using a laser final volume was adjusted to 200 mL with ultrapure water
printer with 1200‑dpi resolution (model LaserJet 1102w, and the pH was adjusted to 6.0 using hydrochloric acid
Hewlett-Packard). The toner provided a hydrophobic (1.0 mol L-1). Artificial serum samples levels I and II were
barrier restricting the area for bioassays. The layout of the obtained from Doles Reagents (Goiânia, GO, Brazil).
Vol. 28, No. 1, 2017 Silva et al. 199

Standard, artificial and diluted solutions were

prepared using ultrapure water processed through a water
purification system (Direct-Q® 3, Millipore, Darmstadt,
Germany) with resistivity equal to 18.2 MΩ cm. Recovery
experiments were carried out to study the accuracy of
the proposed methodology for glucose analysis. For this
Figure 2. Optical micrograph showing colorimetric assays for glucose at
purpose, artificial urine samples were spiked with glucose concentration levels ranging from 0 (column 1) to 10 mmol L-1 (column 11)
standard solutions at three concentration levels (2, 4 and with 1 mmol L-1 increments.
6 mmol L-1).
as previously optimized.37 The resulting color intensity
Results and Discussion was taken into consideration to establish a relationship
with the analytical concentration. Figure 3 presents
The toner layer printed on polyester surface provides an a good correlation between the color intensities and
effective hydrophobic barrier for chemical or biochemical glucose concentrations. The analytical curve varied from
assays performed on zones or wells defined in white color 0 to 10 mmol L-1 exhibited a great relationship with a
during the laser printing step. In this area, the printer coefficient determination (R2) equal to 0.996. The limit of
does not deposit toner allowing the production of zones detection (LOD) and analytical sensitivity achieved were
according to desirable dimensions. The limitation of this 0.6 mmol L-1 and 5.1 AU (mmol L-1)-1, respectively. The
technology makes reference to the toner layer thickness, LOD value was calculated based on the color intensity
which depends on the laser printer resolution. According related to three times the standard deviation of the control
to a previous report, the printed zones are defined with a zone and the angular coefficient of the analytical curve.
depth of ca. 5 µm (equivalent to the toner layer thickness).32 The LOD achieved on printed zones is better than the
When compared to wax printing technology,35 recognized value previously reported using capillary-driven toner-
as one of the simplest methods to produce paper-based based microfluidic devices.31 The reason is associated
analytical devices, the laser printing allows the fabrication with the poor color homogeneity inside detection zones
of microzone plates in a single printing step using simpler on toner-based microfluidic devices. The poor uniformity
instrumentation. In this current report, zones were filled is caused due to the weak interaction between enzymes
with a cellulose paste to ensure the retention of the and/or colorimetric indicators with the substrate surface
added reagents and to provide a better color distribution allowing their loading to zone borders, thus resulting in
on the whole zone. Moreover, this strategy improved the formation of a color gradient. In addition, the found
the repeatability of colorimetric measurements through LOD is similar to the value obtained using microfluidic
digital images captured with a scanner and prevented the cloth-based analytical devices (µCADs)38 and slightly
interference of external light commonly observed in the higher than the LOD’s achieved in representative articles
drop formed inside microzones.32 on microfluidic paper-based analytical devices34,36,39-42
Glucose colorimetric assay was then carried out using and µCADs coupled with electrochemiluminescence and
toner-based microzones filled with cellulose paste. The colorimetric detectors.43,44
reaction is based on glucose conversion to gluconic acid The precision of the colorimetric measurements
and hydrogen peroxide by glucose oxidase (GOx) enzyme was investigated aiming the intra and inter-microplate
in the presence of HRP, which catalyzes the reduction comparisons. For this purpose, a standard glucose solution
of hydrogen peroxide and the oxidation of iodide to was added inside microzones and analyzed under similar
molecular iodine. These sequential reactions promote a experimental conditions during five consecutive weeks. The
color change from colorless to brown.34,36 Figure 2 displays relative standard deviation (RSD) values recorded for the
an optical micrograph showing developed color for glucose intraplate measurements (n = 3) ranged from 0.4 to 2.0%.
concentration levels from 0 (column 1) to 10 mmol L-1 On the other hand, the RSD found for the colorimetric
(column 11). As it can be seen, the higher the concentration, response in an interplate comparison (n = 5) was 6.1%.
the stronger the color intensity. As referenced by Dungchai et al.,36 the normal glucose
In order to obtain quantitative information from levels in serum and urine are 2.5-5.3 and 0.1-0.8 mmol L-1,
scanned images, the pixel intensity values were respectively. In addition, concentration levels above
determined using a graphic software which allowed the 7.0 mmol L-1 in serum and 1.4 mmol L-1 in urine samples
glucose analysis in biological fluids. For this purpose, may be indicative of diabetes mellitus or other disorder.34
images were scanned after a reaction time of 15 min, Based on the analytical performance of the proposed hybrid
200 Colorimetric Detection of Glucose in Biological Fluids Using Toner-Based Microzone Plates J. Braz. Chem. Soc.

toner-cellulose zones, the LOD, the linear concentration Conclusions

range and the inter and intraplates comparisons achieved
make the use of this disposable platform possible for In summary, printed microzones have been successfully
clinical studies using biological samples. used for colorimetric analysis of glucose in artificial
biological samples. Toner-based microzones were printed
on polyester film and filled with a cellulose paste. This
strategy ensured acceptable analytical performance for
clinical applications. The achieved LOD (0.6 mmol L-1) as
well as the linear concentration range (0‑10 mmol L-1) and
the acceptable accuracy (92-105%) make this technology
quite attractive for diagnosis of diabetes or other disorder
associated with glucose concentration levels. The
advantages related to the use of polyester-toner devices (low
cost, ease of fabrication and low instrumental requirements)
in association with colorimetric measurements through
portable electronic devices can be useful for POC testing.
Future advances may enable the use of a color scale to allow
the direct and visual analysis of glucose concentrations.
Figure 3. Calibration curve for glucose assay. The symbol and the error
bars mean the average and standard deviation values (n = 3), respectively. This can result in an important tool for clinical screening.
The linear regression equation obtained was y = –0.46 + 5.10x.
Acknowledgments
Biological samples
This project has been supported by Conselho Nacional
The clinical feasibility of the toner-based platform was de Desenvolvimento Científico e Tecnológico (CNPq) grant
demonstrated using artificial serum and urine samples. No. 448089/2014-9 and Fundação de Amparo a Pesquisa
Firstly, artificial serum samples (levels I and II) with do Estado de Goiás (FAPEG). The authors gratefully
certified concentration levels were analyzed according acknowledge the scholarships granted from Coordenação
to the procedure described earlier. While serum sample de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
level I was prepared without dilution, the serum to KAO and from CNPq to PBMS as well as the researcher
sample level II was diluted in water (1:2, v/v). Based fellowship granted from CNPq to WKTC (grant No.
on the analytical curve shown in Figure 3, the glucose 311744/2013-3).
concentrations found in both serum samples were
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