Elektronika 2010-11.pdf - Instytut Systemów Elektronicznych ...

Fig. 4. Portable real-time PCR DNA analyzer: a) LOC on author’s
finger, b) view of the docking station ready to work
Rys. 4. Przenośny analizator DNA PCR czasu rzeczywistego:
a) mikrosystem typu lab-on-chip w porównaniu do monety
10-centowej, b) widok stacji dokującej gotowej do pracy
a)
Fluorescence intensity [au]
a)
b)
b)
100
Normalized fluorescence intensity
[au]
80
60
40
20
0
100 140 180 220 260 300 340 380 420
Time [s]
0,6
0,5
0,4
0,3
0,2
0,1
0
Fluorescence signal
Temperature profile
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
PCR cycle
Fig. 5. Real-time PCR of Campylobacter j. DNA: a) fluorescence
intensity change following PCR temperature profiling, b) S-curve
– like bar graph describing kinetics of DNA amplification
Rys. 5. PCR czasu rzeczywistego DNA bakterii Campylobacter j.:
a) zmiany intensywności fluorescencji w czasie cykli temperaturowych
reakcji PCR, b) krzywa typu S opisująca kinetykę namnażania
materiału genetycznego
cialized PCR temperature controller connected to the computer.
The holder with ready to use LOC is positioned in the
docking station (15 × 5 × 7 cm 3 ) in the way ensuring laser light
introduction into PCR microchamber and fluorescence light
collection [10]. Than, PCR temperature profiling and fluorescence
signal acquisition are started. The laser light does not
illuminate whole PCR microchamber but it does only in part
of the chamber which corresponds to a laser light distribution
cone. Therefore, the fluorescence is induced and emitted from
a volume in picoliter range.
View of the chip and hand-held docking station with positioned
chip holder just before start of the PCR process
is shown on Fig. 4.
The pre-validation tests of LOC – based system for detection
of Campylobacter j. were carried out with 48 chicken
fecal samples [9]. All the steps – from sample preparation to
final result – were performed in the single chip with 2,5 μl volume
of reagents. The ratio of PCR efficiencies between onchip
and on-tube (reference) was up to 300%. The sensitivity
of on-chip PCR was determined as 0,7–7 ng/ml of template
DNA – similar result were obtained for on-tube amplification.
The LOC real-time PCR process took 30 min – at least 4 times
shorter than PCR on-tube.
An example of the fluorescence intensity change following
temperature profiling during PCR process is shown on Fig. 5a.
S-curve graph of the real-time PCR process has been compiled
on the base of average fluorescence intensity during
extension step of each PCR cycle (Fig. 5b). Obtained S-curve
is comparable to the characteristic achieved by the use of the
chip observed under epifluorescence microscope equipped
with PMT.
Carried out pre-validation tests confirmed usefulness
of the developed optical instrumentation, as well as the whole
LOC-based system for real-time PCR detection of Campylobacter
j.
Summary and conclusions
Presented here idea and technical realization of novel fluorescence
readout has been successfully tested and applied
in various LOC-based instrumentation. The main advantage
of presented here solution is a ratio of the price of optical readout
components to the sensitivity of the whole unit. It has been
obtained by application of low-cost components and “intelligent”
software for conditioning of collected data. As the result, sensitivity
of the novel system is comparable to sophisticated “large
scale” solutions with 100-times lower consumables costs. The
novel image sensor – based fluorescence detection enables development
of point-of-care devices with advanced fluorescence
detection with sensitivity level comparable to standard laboratory
equipment. Currently, there are works on further development of
the CCD image sensor – based fluorescence detection method
and instrumentation toward multiwavelength detection. Preliminary
works indicated also that developed optical instrumentation
can be successfully used also in portable cocaine detector
for monitoring of professional driver’s cocaine abuse [11],
in LOC-based gel electrophoresis DNA analyzer or microcytometer
for optical characterization of bio- samples [12].
The works were/are financed by 6. FP OPTOLABCARD, 7 FP.
LABONFOIL and Statutory Grants of PWr. Author would like
to thank to J. Dziuban, A. Górecka-Drzazga, P. Knapkiewicz,
P. Szczepańska and W. Kubicki from Wrocław University of Technology,
J. Koszur and B. Latecki from Institute of Electron Technology
in Warsaw, D. D. Bang from Danish Technical University and J.
M. Ruano-Lopez from Ikerlan in Spain for fruitful co-operation.
Elektronika 11/2010 35