Reliability of heat transfer

Temperature measurements were performed by installing an adapted measurement LOC chip into the chip-holder of the CytoCycler. Input temperatures, controlled by the software and measured by the chip-holder’s temperature sensor, were compared to output temperatures measured on reaction center B of the measurement LOC chip by a temperature sensor. Thus, the heat transfer from the software to the Peltier element and finally onto the chip surface was validated. Various temperatures in a PCR relevant temperature range from 25°C up to 100°C were tested. Additionally, the performance of an AmpliSpeed slide cycler was tested as a reference PCR system.

The first temperature profile comprised measurements with increasing temperatures, starting from 30°C, and followed by stepwise temperature increments of +5°C up to 100°C, while each temperature was held for about 30 sec. There were slight negative deviations detectable in the upper temperature range from 70-100°C at an average of -0.243°C, but rather randomly distributed than in an increasing linear manner. In the lower temperature range from 30°- 65°C, however, decreasing positive deviations were measured at an average of +0.527°C. Results derived for the CytoCycler were summarized in table 3.

The second temperature profile comprised measurements with temperatures simulating 3-step thermal PCR cycling, starting from 95°C held for 10 min, followed by 3-5 cycles of 94°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec. Two independent runs were performed. Temperature measurement results derived for the CytoCycler at each specific temperature were summarized in table 3. In both runs there were slight but tolerable positive temperature deviations detectable. At 95°C, simulating an initial denaturation step, the averaged positive deviation was about +0.90°C. At 94°C, simulating the denaturation steps at each PCR cycle, positive deviations were measured at an average of +0.98°C. At 60°C, simulating the annealing step at each PCR cycle, the positive deviations were at an average of +0.26°C. And

at 72°C, simulating the extension step of each PCR cycle as well as the final product extension step, averaged positive deviations were about +0.67°C.

Table 3. Temperature deviations between input and output temperature measured at the CytoCycler device. The

particular deviations at each measured temperature are given in brackets and are marked with a delta sign (∆).

Input temperature by software [°C] Averaged output temperature on chip surface [°C] Input temperature by software [°C] Averaged output temperature on chip surface [°C] Averaged output temperature on chip surface [°C] Temperature increment +5°C Simulating PCR cycling

30 31.05 (∆ +1.05) Run 1 Run 2 35 35.92 (∆ +0.92) 95 95.21 (∆ +0.21) 96.60 (∆ +1.60) 40 40.73 (∆ +0.73) 94 94.47 (∆ +0.47) 95.02 (∆ +1.02) 45 45.59 (∆ +0.59) 60 60.21 (∆ +0.21) 60.32 (∆ +0.32) 50 50.43 (∆ +0.43) 72 72.51 (∆ +0.51) 72.86 (∆ +0.86) 55 55.26 (∆ +0.26) 94 94.28 (∆ +0.28) 95.37 (∆ +1.37) 60 60.19 (∆ +0.19) 60 60.37 (∆ +0.37) 60.28 (∆ +0.28) 65 65.05 (∆ +0.05) 72 72.64 (∆ +0.64) 72.68 (∆ +0.68) 70 69.89 (∆ -0.11) 94 95.23 (∆ +1.23) 75 74.69 (∆ -0.31) 60 60.22 (∆ +0.22) 80 79.73 (∆ -0.27) 72 72.67 (∆ +0.67) 85 84.84 (∆ -0.16) 94 95.26 (∆ +1.26) 90 89.87 (∆ -0.13) 60 60.22 (∆ +0.22) 95 94.78 (∆ -0.22) 72 72.70 (∆ +0.70) 100 99.50 (∆ -0.50) 94 95.23 (∆ +1.23) 60 60.18 (∆ +0.18) 72 72.63 (∆ +0.63)

Values of input temperatures were plotted against output temperatures and illustrated in a graphical image (figure 14 A). The distinct deviation values were related to measured temperatures and were shown graphically in an additional plot (figure 14 B).

Figure 14. Temperature measurements of the LOC CytoCycler. A) Graphical display of measured input

temperature (via software) versus output temperature (via hardware on the chip surface). Slight deviations could be detected at each measuring point. The drift of both graphs clearly showed positive deviations at lower temperatures and negative deviations at higher temperatures. B) Graphical illustration of the deviations of each particular temperature. Input temperatures were plotted against measured temperature deviations.

The PCR-simulated temperature profile of 94°C, 60°C and 72°C was applied to the AmpliSpeed slide cycler as well. Slight deviations of constantly about ±0.5°C were recorded for the slide cycler, featuring this cycler as an ideal reference system for the LOC system, especially for performing negative and positive control reactions during PCR performances. According to an optimal setup based on contacting surfaces of the Peltier element and the chip underside, the CytoCycler provided a stable heat transfer onto the chip surface. Besides an efficient and optimal temperature transfer, a stable temperature support could be provided by this setup. Repeated temperature measurements were performed in two independent runs. Temperature output was quite constant at simulated PCR cycling any time tested, accounting for reproducible temperature measurements. In several independent measuring approaches, constantly slightly positive temperature deviations close to the desired input temperature were detected. These positive temperature deviations represented an optimal temperature transfer effectivity from the Peltier element to the LOC chip surface. This optimal temperature output on the chip surface validated the CytoCycler for reliable PCR performances on the LOC system. This setup of direct contact between the heating element and the microchip was comparable to the used multi LV-PCR microdevices, which were applied on conventional thermocycler devices using an appropriate in situ adapter, like e.g. reported in Schmidt U et al. (2006), Proff C et al. (2006), Lutz-Bonengel S et al. (2007) and Schmidt U et al. (2008). This contacting setup was also applied in the special thermocycler designed for slide-based PCR (AmpliSpeed slide cycler, Advalytix AG/Beckman Coulter Biomedical GmbH, Munich, Germany). This commercially available AmpliSpeed slide cycler device provided a comparable contact heating method for performing low-volume PCR amplification reactions using a multi LV-PCR microdevice. Thus, it was taken as reference system for all conducted measurements. As temperature measurement results were quite comparable between both devices, the AmpliSpeed slide cycler was taken as a permanent reference PCR system for validating the results obtained from the LOC CytoCycler PCR performances.