Surrounding instrumentation improvements

Temperature on the chip

As previously described, heaters below the chip control the chip temperature in different zones. A programmable temperature controller regulates the temperature on each heater. The temperature in the PCR and RT-PCR chip plays a key role for

successful amplification, especially in the case of the lower temperature heater (50– 60◦C) compared to the high temperature one (95◦C). Stable annealing and reverse transcription (in the case of RT-PCR) temperatures are essential for effective PCR performance. However, the denaturing temperature allows higher temperature variations (approximately 90–97 ◦C) without altering the amplification process.

Verifying the temperature in the area of interest is a difficult task in microfluidic systems without altering the process in a substantial manner. To determine the actual temperature inside the chip for a given heater temperature, a thermal mea- surement chip was built for a previous chip generation which had the same shape as chip generations 1 and 2. The chip is shown in Figure 4.20, it has equidistant measurement channels located in a similar depth as the PCR microfluidic chan- nel. A thermocouple is embedded in high temperature silicone in every channel. The channels are sealed using a regular cover slip. Six thermocouples measure the temperature in the middle area of the chip while the other six provide temperature values of the outer area of the chip. Thermocouples were read out using a digital thermometer. For different given temperatures, the temperatures of the thermo- couples inside the measurement chip were recorded. Approximately, for the 60◦C heater temperature, the temperature inside the chip was 2 ◦C below; while for a heater temperature of 95 ◦C, the temperatures inside the chip were in the range 90–92 ◦C. Given these results, for on-chip experiments, the heaters were set 2 ◦C above the target temperature. In the case of target temperature of 95 ◦C inside the chip, heaters were set to 97–98 ◦C to prevent boiling effects.

Figure 4.20: Thermal measurement chip with 12 thermocouples. A) Schematic drawing. Numbers 1–6 show the inner thermocuples and 7–12 the outer ther- mocuples. B) Picture.

Optical system

As previously stated, optical methods are the most common methods to detect am- plification products in real-time. Among them, the fluorescence-based technique is the most popular one due to its high sensitivity, high selectivity, and commer- cial availability of fluorophores.18 _{Commercial real-time PCR machines monitor}

fluorescence in approximately a total volume of 20 µl; however, for on-chip-PCRs, only a small section of each PCR cycle was monitored. As previously described, for the chip generations with 50 µm channel depth (PCR chip generations 2, 3, 4 and both RT-PCR chip generations) the approximate monitored volume within the optical field of view was a scant 50 nl. In the case of the 1st PCR chip, the monitored volume was 350 nl due to its higher channel depth (350 µm).

Fraunhofer CMI developed an optical detection system previous to the start of this PhD thesis. The system was composed of a light-emitting diode (LED) source which excited fluorescence through a standard microscope FITC fluores- cence excitation and emission filter set. The excitation light was directed to the sample and the fluorescence generated by the sample was collected and directed to a charge-coupled device (CCD) camera placed into the sample image plane. Photo images were taken with the camera, which were latter analyzed using Image J pro- gram. Unfortunately, this system was not sensitive enough for monitoring on-chip amplification, and post sample processing through gel electrophoresis was required to determine the success of the amplification. A schematic drawing of the optical system and a CAD drawing are shown in Figure 4.21 A and B respectively.

In order to increase the sensitivity of the system, a photodetector was incor- porated. Collected light was split into two detection paths using a broadband beam splitter with 30/70 intensity split ratio. In the first detection path, a CCD camera was placed into the sample image plane. In the second detection path, a photodetector surface to maximize intensity response was located. The changes in the photodetector current were measured using an optical power meter. The camera was used to pre-align the target channel onto the opening in the second detection path. A schematic drawing of the optical system and a CAD drawing are shown in Figure 4.22 A and B respectively.

Figure 4.21: First version of the optical system. A) Schematic drawing. B) CAD drawing. For both A and B figures numbers are as follows. 1) LED light. 2) Excitation filter. 3) Sample. 4) Dichroic filter. 5) Emission filter. 6) Camera.

Figure 4.22: Second version of the optical system after the addition of a pho- todetector. A) Schematic drawing. B) CAD drawing. For both A and B figures numbers are as follows. 1) LED light. 2) Excitation filter. 3) Sample. 4) Dichroic filter. 5) Emission filter. 6) Beam splitter. 7) Camera. 8) Photodetector.

The use of the photodetector surface increased the sensitivity of the system and allowed real-time on-chip measurements. Using this optical set up, several additional improvements were made to enhance the sensitivity, improve data collec- tion, and data accuracy. Initially, values were directly read out from the power meter; however, the rapidly fluctuating values led to inaccurate data collection. By connecting the output of the power meter to a computer this problem was solved and fluorescence intensity was read-out through the computer. Moreover, data collection was improved with the incorporation of a computer program. This program allowed for a more consistent data acquisition by collecting a desired number of data points in an established time interval. During the optical system improvement process, an attenuating effect in the fluorescence values due to the platform on top of the chip was discovered. The platform was reshaped to avoid interferences in the fluorescence data collection. Additionally, the sensitivity of the system was improved by placing in the photodetector path a rectangular-shaped opening into the sample image plane limiting throughput of the fluorescence to a single channel.

In Figure 4.23, the on-chip PCR graphs associated with different optical sys- tem iteration are shown. It can be observed that the different iterations have very similar profiles and the CT value is very similar for the second and third iterations.

The last iteration allows for consistent data collection and provides high quality and sensitive fluorescence curves. It should be noted that since all the system im- provement experiments –regarding the chip, system and reagents– were performed at the same time as the improvements in the optical system, the on-chip PCR data shown during the improvement of the different sections varies depending on the available optical system.

The next steps to further improve the optical system should be focused in reducing the overall costs. For example, the microscope stage could be removed if the chip could be positioned in the same identical plane. In the case of the CCD camera, it could also be eliminated if an integrated system that automatically moves through the chip channels was available.

Figure 4.23: Iterations to improve the optical system. Experiments conducted with E. coli O157:H7 and initial template concentration 104 _{DNA copies/µl. IDT}

primers used. Iteration 1: optical data were directly read out from the power meter. No error bars were obtained. Iteration 2: optical data read out from the power meter connected to a computer. Attenuating effect in the fluorescence values due to interference with the chip platform can be observed. Iteration 3: optical data collected through a computer program consistently.