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Correspondence: Dr. Anders Wolff, MIC – Department of Micro and PCR has also been realized on microchips. To date, three
Nanotechnology, Technical University of Denmark, Bldg 345 east, different types of PCR microchips have been developed,
DK-2800, Kgs. Lyngby, Denmark
featuring either a chamber [15–29], a continuous flow [30–
E-mail: aw@mic.dtu.dk
Fax: 145-45887762
33], or a droplet oscillation design [34]. Until now, only few
real-time PCR microsystems have been reported [35–37].
Abbreviation: CT, cycle threshold value Recently, Gulliksen et al. [36, 37] described a novel real-
time PCR microchip. However, this real-time PCR micro- cal elements. The PCR microchip was tested for real time
chip required an external optical system to detect the flu- PCR detection of the cadF gene of C. jejuni using DNA-
orescent probes. binding dyes.
Corporation, USA) and 25 U/mL of Taq DNA polymerase is kept short (200 mm), as the measured fluorescent signal
(Sigma, USA), 1 mg/mL non-acetylated BSA (Sigma) and intensity is inversely proportional to the detection dis-
DNA template. Two different DNA binding dyes, SYTOX tance squared. Two such pairs of waveguides were inte-
Orange (ex 547 nm/em 570 nm) and TO-PRO-3 grated at different positions inside the reaction chamber
(ex 642 nm/em 661 nm) (Molecular Probes, USA), were to locally monitor the fluorescence intensity. The design
added into the PCR mix (200 nM each) for the real-time with two pairs of integrated waveguides provides the
PCR detections. ability to monitor two different wavelengths in “multiplex”
real-time PCR applications and can easily be modified to
Of the 50 mL prepared PCR master mix, 25 mL were used
detect fluorescent signals at any desired location inside
for the real-time PCR on the PCR microchip while the rest
the PCR reaction chamber.
was used for a conventional real-time PCR using a com-
mercial real-time PCR machine (Chromo4®, MJ Re- All parts of the integrated optical system in the real time
search, USA) as control. The PCR conditions were: 1cycle PCR microchip are made from SU-8, and the selection of
at 957C for 2 min, followed by 40 cycles of 947C for 15 s, suitable DNA-binding dyes according to the optical
507C for 15 s and 727C for 15 s, and ending with a 2-min properties of SU-8 is therefore a crucial step. SYBR
elongation stage at 727C. The PCR chips were used for a Green I is the most common DNA-binding dye used in
single reaction only. real-time PCR [12, 13, 44], and it is excited by a blue light
source (e.g. an Ar-ion laser with a wavelength of 488 nm).
2.4 Optical system However, SYBR Green I is not a suitable dye for the real-
time PCR microchip described here because SU-8 has
For the real-time PCR measurements on chip, a 60-mW very high light absorption and high fluorescence back-
diode pumped solid-state green laser (535 nm) (DPSSL- ground at low wavelengths (less than 500 nm) [45, 46]. To
60, Viasho, P.R. China) and a 5-mW He-Ne laser (633 nm) avoid this problem, two other DNA-binding dyes, SYTOX
(25-LHR-151-230, Melles Griot, USA) were used to pro- Orange (ex 547 nm/em 570 nm) and TO-PRO-3
vide two different excitation wavelengths. Two different (ex 642 nm/em 661 nm) with longer excitation and emis-
types of PMT from Hamamatsu, Japan, a H5784 (for the sion wavelengths were selected for testing the real-time
535 nm excitation light) and a H5784-01 (for the 633 nm PCR microchip. We chose to test two different dyes be-
excitation light), were used to measure the fluorescent cause these dyes had not been tested in real-time PCR
signals at the two different wavelengths. A 5 mm65 mm before. Tests in conventional real-time PCR showed that
62 mm FGL550S long pass filter (cut from a 50 mm the two dyes were thermally stable and showed very low
650 mm filter slide obtained from Thorlabs, USA) was PCR inhibition (unpublished data).
placed in front of the aperture of the PMT SMA adapter
(E5776-51, Hamamatsu) for filtering of 535 nm excitation In initial experiments, the melting curves were determined
light, while a 5 mm65 mm63 mm LP645 long pass filter by mixing the DNA binding dyes with the DNA fragment
(cut from a 50 mm650 mm filter slide obtained from from a PCR reaction. At the melting point of the DNA
Melles Griot) was placed in front of the aperture of another fragment, where the dsDNA melts to ssDNA, the fluores-
photomulitplier tube (PMT) SMA adapter for filtering of cent signals decrease significantly because the DNA-
633 nm excitation light. To avoid photo bleaching of the binding dyes are only fluorescent when bound to dsDNA.
fluorescence dyes, a custom-modified chopping blade Using the real-time PCR microchip with a temperature
with a 3% duty cycle was placed in front of the lasers. A gradient (from 357C to 957C with 27C/min), the melting
SR540 chopper controller (Stanford Research Systems, curve of the DNA fragment could be determined. The
USA) was used to manipulate the chopper. result of such an experiment with SYTOX Orange-labeled
DNA is shown in Fig. 3. The melting point was determined
by differentiating the registered melting curve, and the
3 Results and discussion melting point (837C) measured on the real-time PCR chip
was the same as measured on the conventional
In this report, a real-time PCR microchip with an inte- Chromo4® real-time PCR thermal cycler. The presented
grated thermal system and a polymer-based optical approach shows good sensitivity for the fluorescence
detection system is presented. The fluorescence from the measurements during the thermal process.
real-time PCR is measured using a pair of waveguides.
The waveguides, one for introducing excitation light, and DNA-binding dye binds to the dsDNA of any PCR prod-
the other for receiving the fluorescent signals, are placed uct. Therefore, specific fluorescence probes (labeled
perpendicular to each other to avoid collection of too ssDNA oligonucleotides) are normally required to distin-
much excitation light. Together they define a detection guish the different products in a multiplex real-time PCR.
point (Fig. 2). The distance between two waveguide ends The costs of the fluorescent DNA-binding dyes are,
During the real-time PCR experiments, two different Figure 4. Typical on-chip real-time PCR data profile
colored fluorescent DNA-binding dyes (SYTOX Orange using TOPRO-3. Due to the thermal dependence of the
and TO-PRO-3) were added into the PCR mixture to fluorescent intensity of the dyes, the three stages of the
monitor the reactions on chip. A typical data profile of the PCR cycle can be clearly distinguished by fluorescence
real-time PCR labeled with TO-PRO-3 is shown in Fig. 4. measurements using the integrated waveguides. The flu-
The three stages of a PCR cycle (15 s denaturation at orescence signal traces correspond perfectly to the
947C, 15 s annealing at 507C and 15 seconds elongation measured temperature profile in the chip.
at 727C) can clearly be distinguished.
Possible surface-induced inhibition is always a critical
In this approach, the temperature deviation during the
issue in microfabricated PCR devices [42, 48–51]. To
temperature switching is less than 0.57C. Such tiny devia-
avoid any inhibition from the SU-8 surface, 1 mg/mL non-
tions are mainly caused by overcompensation through the
acetylated BSA was added to the PCR mix. The BSA
PID control algorithm. A more accurate feedback control
suppresses the interaction of the PCR reagents with the
algorithm may eliminate it. The PCR thermocycling has
SU-8 surface and thus prevents inhibition. The non-
been optimized previously in order to decrease the cycling
acetylated BSA did not add to the background fluores-
time [47]. The achievement of fast cooling (20 6 27C/s) and
cence. Furthermore, the applied dye concentrations in
heating (11 6 17C/s) rates reduced the whole PCR pro-
the PCR mixture are optimized and limited to 200 nM to
cess on-chip to only 30–40 min in comparison to 1.5 h on
avoid any PCR inhibition effects.
a conventional PCR thermocycler (Chromo4®, MJ Re-
search, USA). Therefore, this prototype can be developed By taking the mean fluorescent signal value during the
towards a portable lab-on-a-chip system for rapid elongation period of each thermal cycle, the relative PCR
screening of pathogens in the field. product concentration for each PCR cycle can be deter-
CT = mLog(Conc0) 1 b (2)
e = 1021/m 2 1 (3).
Concproduct ¼ Conc0 ð1 þ eÞCEnd ¼ Conc0 k (4) Figure 5. (A) Results from real-time PCR on chip to
amplify the 398-bp amplicon on the Campylobacter jejuni
where Concproduct is the concentration of the PCR product cadF gene using SYTOX Orange. 0, negative control on
after CEnd thermo cycles and k = (1 1 e)Cend. Equation (4) is chip; 1–5, real-time PCR on chip for detection of the
strictly only valid for cases where the reaction is stopped Campylobacter jejuni DNA template series (2, 10, 20, 100,
in the exponential phase, as in our experiments. For most and 200 ng/mL). (B) Results for gel electrophoresis on an
Agilent Bioanalyzer DNA500 chip. L: DNA marker ladder
PCR, however, the reaction proceeds from exponential to
(15–600 bp); –, negative control in tube; 1, positive con-
linear and finally a plateau phase before the reaction is trol in tube; numbers correspond to the samples men-
terminated and in such cases, the equation is not valid. tioned above.
The raw data has a relatively high noise level due to the
fluorescent background level and the light losses asso-
ciated with SU-8, even with the new dyes. To find the CT and the DNA template concentration (Fig. 6A), and the
value, the raw data (Fig. 5A) were smoothened using an expected linear correlation between the PCR product
eight-point adjacent average function (OriginPro 7.5, data concentration and the DNA template concentration
not shown) and for each graph, a baseline and a line for (Fig. 6B) for both dyes. Linear regression of the CT vs.
the linear amplification range were drawn. The CT value Log(Conc0) plot according to Eq. (2) yields m = 23.95
was then determined as the intercept of these lines. The and b = 30.7 for SYTOX Orange, and m = 24.41 and
results obtained from the real-time PCR microchips b = 34.7 for TO-PRO-3. By using Eq. (3), the reaction
showed the expected logarithmic correlation between CT efficiency can be calculated to be 79 and 69% for SYTOX
4 Concluding remarks
To our knowledge, this is the first time monitoring of real-
time PCR using integrated optical elements in a lab-on-a-
chip system has been demonstrated. All the integrated
polymer optical systems were defined in the same SU-8
layer as the PCR reaction chamber, without any extra
mask step. By using two fluorescent DNA-binding dyes
with suitable excitation and emission spectra (SYTOX
Orange and TO-PRO-3), the progression of the PCR
amplification could be followed efficiently. The measured
CT value on the chip provided more accurate quantitative
information about the initial DNA template concentrations
than the conventional post-PCR analysis by CE. The
integrated optical system described in this study allows
real-time monitoring of the reaction dynamics at any
Figure 6. Two DNA binding dyes (SYTOX Orange and location inside the micro reaction system and can be
TO-PRO-3) with different excitation wavelengths were
integrated into various types of microchips to facilitate
used for detection of the Campylobacter jejuni cadF gene
on the real-time PCR chip. (A) The internal assay (CT) for real-time monitoring for a number of different purposes.
both dyes shows good linear relationships with the loga- Integration of thermal control and polymer waveguides for
rithm of the DNA template concentration series. (B) The real-time PCR is thus an important step towards a port-
external end-point assay results (obtained via DNA gel able microchip system for pathogen detection.
electrophoresis) can only indicate the trend of the DNA
template concentrations. We would like to thank Dr. Klaus B. Mogensen for useful
suggestions for the integrated optical system design. This
research was supported by the Danish Technical Re-
Orange and TO-PRO-3, respectively. Control PCR reac- search Council (STVF) (Grant No. 26-02-0307) and EU
tions in tubes on a conventional PCR thermocycler were STREP project OptoLabCard.
performed in parallel. The results of these controls were:
m = 23.56 and b = 28.1 for SYTOX Orange, and m = 23.59
and b = 32.4 for TO-PRO-3, corresponding to a reaction 5 References
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