144
A. S. Cerda-Kipper and S. Hosseini
Electrochemical affinity sensors have considerable potential in developing
portable analytical devices for detecting analytes such as blood glycated hemoglobin
(HbA1c) (Liu et al. 2012). An integrated microfluidic system was developed to
achieve automation of the whole aptamer–antibody sandwich assay to perform the
measurements of HbA1c (Table 6.1) (Tankova et al. 2012). The aptamer was implemented as a main ligand to capture HbA1c or Hb in the blood samples and to facilitate the accuracy and cost reduction. The incorporation of CL detection scheme
provided a higher sensitivity for the microfluidic system. For incorporation of CL to
the device, Hb or HbA1c specific aptamers were used to coat the magnetic beads.
Furthermore, Hb- or HbA1c specific biotinylated aptamers were combined with
streptavidin-coated magnetic beads in saline-sodium citrate (SSC) buffer. The beads
were then blocked with BSA to minimize non-specific binding in whole blood analysis. When the capturing of Hb or HbA1c in the blood samples were accomplished,
the unbound fractions were washed away. Subsequently, acridinium ester-labeled
Hb or HbA1c antibodies were added to particularly bind with the captured Hb or
HbA1c molecules. Lastly, CL was produced through the addition of signal reagents
of H 2 O 2 as well as NaOH and a luminometer was used for its measurement. The
process of aptamer–antibody sandwich assay on magnetic beads within the integrated
microfluidic system is shown in Fig. 6.6a.
The chip design is shown in Fig. 6.6b. The authors developed a tri-layer microfluidic chip, consisted of two PDMS layers, comprising a thick-film and a thin-film
used as an air channel layer and a liquid channel layer, respectively on top of a glass
substrate. The major micro-components, such as normally-closed valves, a transport
unit (a closed chamber), a waste chamber and five open chambers were integrated
into this device. The microfluidic chip used a vacuum pump and an air compressor
controlled via electromagnetic valves (EMVs) to automatically trigger the fluid transport for performing the assay. PDMS employed to fabricate the microfluidic chip,
was created by the use of a CNC machining procedure to construct a master mold
accompanied by a PDMS replica-molding process. Initially, the inverse microstructures were etched on PMMA, and after the CNC machining procedure the master
mold was further polished. Lastly, to form the inverse microfluidic structures, the
PDMS replica was fabricated on the PMMA master mold. For the casting process of
the PDMS, the elastomer and the curing agent were combined with a weight ratio of
10:1 and a vacuum pumping process was used to remove the air bubbles. Ultimately,
one glass plate and the two layers of PDMS were bonded together via utilization of
an oxygen plasma treatment.
For the assay procedure (Fig. 6.6b), either Hb- or HbA1c-specific aptamers were
used to pre-coat the magnetic-bead conjugates, that were then loaded into the sample
chamber and combined with freeze-thawed whole blood. By manipulation of the
transportation unit and the microvalve, the binding between the Hb or HbA1c in
the blood sample and the aptamer took place in an incubation process for 10 min.
Subsequently, an external magnet was utilized for the purpose of collecting the targetaptamer-bead complexes, whilst the nonbinding substances and the supernatant were
washed away through activation of the micropump which instead directed a phosphate
buffer over the waste outlet. Afterwards, to form sandwich-like structures with the
A. S. Cerda-Kipper and S. Hosseini
Electrochemical affinity sensors have considerable potential in developing
portable analytical devices for detecting analytes such as blood glycated hemoglobin
(HbA1c) (Liu et al. 2012). An integrated microfluidic system was developed to
achieve automation of the whole aptamer–antibody sandwich assay to perform the
measurements of HbA1c (Table 6.1) (Tankova et al. 2012). The aptamer was implemented as a main ligand to capture HbA1c or Hb in the blood samples and to facilitate the accuracy and cost reduction. The incorporation of CL detection scheme
provided a higher sensitivity for the microfluidic system. For incorporation of CL to
the device, Hb or HbA1c specific aptamers were used to coat the magnetic beads.
Furthermore, Hb- or HbA1c specific biotinylated aptamers were combined with
streptavidin-coated magnetic beads in saline-sodium citrate (SSC) buffer. The beads
were then blocked with BSA to minimize non-specific binding in whole blood analysis. When the capturing of Hb or HbA1c in the blood samples were accomplished,
the unbound fractions were washed away. Subsequently, acridinium ester-labeled
Hb or HbA1c antibodies were added to particularly bind with the captured Hb or
HbA1c molecules. Lastly, CL was produced through the addition of signal reagents
of H 2 O 2 as well as NaOH and a luminometer was used for its measurement. The
process of aptamer–antibody sandwich assay on magnetic beads within the integrated
microfluidic system is shown in Fig. 6.6a.
The chip design is shown in Fig. 6.6b. The authors developed a tri-layer microfluidic chip, consisted of two PDMS layers, comprising a thick-film and a thin-film
used as an air channel layer and a liquid channel layer, respectively on top of a glass
substrate. The major micro-components, such as normally-closed valves, a transport
unit (a closed chamber), a waste chamber and five open chambers were integrated
into this device. The microfluidic chip used a vacuum pump and an air compressor
controlled via electromagnetic valves (EMVs) to automatically trigger the fluid transport for performing the assay. PDMS employed to fabricate the microfluidic chip,
was created by the use of a CNC machining procedure to construct a master mold
accompanied by a PDMS replica-molding process. Initially, the inverse microstructures were etched on PMMA, and after the CNC machining procedure the master
mold was further polished. Lastly, to form the inverse microfluidic structures, the
PDMS replica was fabricated on the PMMA master mold. For the casting process of
the PDMS, the elastomer and the curing agent were combined with a weight ratio of
10:1 and a vacuum pumping process was used to remove the air bubbles. Ultimately,
one glass plate and the two layers of PDMS were bonded together via utilization of
an oxygen plasma treatment.
For the assay procedure (Fig. 6.6b), either Hb- or HbA1c-specific aptamers were
used to pre-coat the magnetic-bead conjugates, that were then loaded into the sample
chamber and combined with freeze-thawed whole blood. By manipulation of the
transportation unit and the microvalve, the binding between the Hb or HbA1c in
the blood sample and the aptamer took place in an incubation process for 10 min.
Subsequently, an external magnet was utilized for the purpose of collecting the targetaptamer-bead complexes, whilst the nonbinding substances and the supernatant were
washed away through activation of the micropump which instead directed a phosphate
buffer over the waste outlet. Afterwards, to form sandwich-like structures with the