3.5. Applications
The proposed PADs fabricated by wax dipping were investigated
for their applicability for the detection of glucose and protein
in real samples based on colorimetric assays. Detail of the size and
shape of an iron mould used in the experiments display in Fig. 5A.
A 5 L volume of 10×
BCG working reagent or glucose reagent
was spotted onto the separate detection zones of the PAD device.
After the reagents were allowed to dry for 10 min at room temperature,
the bottom end of the PAD device was dipped for 1 min
into either a standard solution of glucose of varying concentration
(0–1000 mg dL−1), a standard solution of BSA of varying concentration
(0–10 g dL−1) or a sample solution.
Then, the colour intensity was allowed to develop from colourless
to a strong blue or yellow for the protein and glucose reactions,
respectively. Images of the colorimetric reaction of protein and glucose
are shown in Fig. 5B. To measure the colour intensity, Adobe
Photoshop CS2 was used to convert the images from RGB into grey
scale format before analysis. A calibration curve was plotted for
glucose or protein concentration versus colour intensity. The calibration
curves are shown in Fig. 6A and B. The linear range for
the glucose assay was 0–500 mg dL−1 (r2 = 0.989) and 0–6 g dL−1
for the protein assay (r2 = 0.990). The range of glucose concentrations
that is linear for the assay in our proposed method is broader
than for the conventional method based on the glucose oxidase
spectrophotometric method, in which the linear range of GLUCOSE
liquicolor is only up to 400 mg dL−1. Similarly, the assay range for
protein detection in our method is also better than the conventional
BCG method, which is only linear up to 5 g dL−1. Both results
demonstrate that the wax dipping method for PAD fabrication
can be potentially applied for detection of glucose and protein
Fig. 5. Paper based microfluidic device fabricated by the wax dipping method used
for colorimetric applications: (A) detail of the size and shape of an iron mould and
(B) paper device after fabrication by the wax dipping method (top) and after use for
the detection of protein and glucose (bottom).
simultaneously at levels that are clinically significant. Subsequently,
the PAD was utilised for quantitative detection of glucose
and protein in real samples, and the results were compared to
the conventional methods. Two samples of control serum with
different levels of protein and glucose were tested by the wax
dipped PAD and conventional methods. The comparison of the
results demonstrated that both methods were rather similar. When
analysed by a paired t-test, no significant difference was found
between the two methods at a 95% confidence interval. In addition,
the PAD was also used to measure the glucose level from
real plasma samples, and the results were compared with both
conventional enzymatic glucose oxidase and Accu-Check point-ofcare-
testing methods. The analysis of the results using one-way
ANOVA demonstrated that the concentration of glucose in the
plasma as determined by the three methods was not significantly
different (p > 0.05) (Table 1). It can therefore be concluded that