Development And Optimization Of A Microchip PCR System Using Fluorescence Detection

Abstract

Microfabricated thermal cyclers for nucleic acid amplification by using polymerase chain reaction (PCR) have been demonstrated by several groups over the last decade, with improved cycling speed and smaller volumes when compared to conventional bench-top cyclers. However, high fabrication costs coupled with difficulties in temperature sensing and control remain impediments to commercialization. In this study we have used a silicon-glass device that takes advantage of the high thermal conductivity of silicon but at the same time utilizes minimum number of fabrication steps to make it suitable for disposable applications. The thermal cycler is based on noncontact induction heating developed in this group. The microchip reaction kinetics is studied for the first time in-situ during PCR, using a real-time fluorescence block that is capable of data acquisition every 0.7 s from the microchip. The fluorescence information from SYBR green I dye is used to optimize microchip amplification reactions and confirm the product by melting curve analysis. We have also developed a novel non-contact temperature sensing technique using SYBR green fluorescence that can be used for miniaturized PCR devices. The thesis is organized into the following chapters. In chapter 1 we introduce the basic biology ideas that are required to understand DNA amplification. DNA based analysis requires amplification of low initial concentrations to above detectable limits using a technique known as polymerase chain reaction (PCR). In this process, the sample is cycled through three thermal steps for 3040 times to produce multiple copies of DNA. In microchip PCR, conventional polypropylene tubes using 2050 µL volume are replaced by miniaturized devices using ~1 µL sample volumes. The device response improves in terms of ramp rate and total analysis time due to the small volume and smart design of the materials. In this chapter we summarize some of the issues important for miniaturized PCR devices and compare them with commercial tube PCR systems. In chapter 2 we describe the induction heating technique that was developed by our group for miniaturized devices. Induction heating is a noncontact heating technique unlike resistive heating which has been commonly used for microchip PCR. Though resistive heating is very efficient in terms of heat transfer efficiency, it is not suitable for disposable devices and requires multi-step microfabrication. Other non-contact heating techniques such as hot air and IR heating require larger size arrangements that are not suitable for miniaturized devices. The heating was verified by using a thermocouple soldered at the back of the secondary plate that was also used for feedback to the comparator circuit for control. The simple on-off circuit was able to control within ±0.1 ◦C with heating and cooling ramp rates of 25 ◦C/s and 2.5 ◦C/s respectively. In this chapter, we also describe the design and fabrication of the silicon-glass microchip fabricated in our lab. We have used silicon-glass hybrid device for PCR in which glass with a 2 mm drilled hole is anodically bonded to an oxidized silicon surface. The hole formed the static reservoir for 3 µL volume of amplification solution. During PCR, the solution needs to be cycled to high temperature of ~95 ◦C. Hence it was necessary to seal the tiny droplet of liquid against evaporation at this temperature. The devices after being filled by sample were covered by 4 µL of mineral oil to serve as an evaporation barrier. It was easy to recover the whole sample after amplification for further testing. Chapter 3 describes the development of a fluorescent block for SYBR green I dye (SG) used for real-time monitoring of the amplification. The block contains a blue LED for excitation, a dichroic beamsplitter, and silicon photodiode along with filters and focusing optics. Signal levels being weak, we incorporated lock-in detection technique. A TTL at 190 Hz was used to pulse the excitation source and detect the emission at the same frequency using a commercial lock-in amplifier. The block was first characterized using a commercial thermal cycler and polypropylene tubes with different dilution of initial template copy number, and the results crosschecked with agarose gel electrophoresis. Performing continuous monitoring every 0.7s within cycles, we discovered interesting features during extension which have not been studied previously. During the constant temperature extension step, the fluorescence shows a rise and then saturates until the temperature is cycled to the next set point. We have confirmed the same behavior in single cycle extension control experiments and established its connection with polymerase extension activity. We were thus able to extract the activity rate for two different kinds of polymerase in-situ during PCR. By monitoring PCR reactions with different fixed extension times, we were able to determine the optimum conditions for tube PCR. Chapter 4 implements the ideas of fluorescence monitoring from tube that was explained in the previous chapter for the silicon-glass microchip. Since the microchip uses parameters such as sample volume, ramp rates, stay time etc. which are different from tube PCR, we performed several initial test experiments to establish key capabilities such as low volume detection, 3 µL amplification, surface passivation of silicon-glass etc. The same fluorescence block was used to obtain DNA melting point information by continuously monitoring ds-DNA with SG while the temperature is ramped slowly (melting curve analysis). Depending on ds-DNA present, the fluorescence gives a melting temperature (TM ), which was used to calibrate the mix temperature with respect to the thermocouple sensor. After successfully calibrating the microchip, we confirmed complete chip PCR in silicon-glass devices using induction heater. The continuous monitoring of chip PCR gave similar curves as obtained previously for tubes except that the signal level was lower in silicon devices. Extension fluorescence information was used to find an optimum temperature for microchip that shows a maximum activity rate. Similarly the reaction time was optimized in-situ during PCR by using continuous fluorescence data in a feedback experiment. The commercial lock-in amplifier was also replaced by a homemade circuit to successfully pickup fluorescence signal from the microchip during melting curve analysis. In chapter 5, we describe a novel technique to sense the temperature from the microchip without touching the sample volume. Usually the temperature is monitored by a sensor spatially separated from the mix and it has always been challenging to measure the exact temperature accurately. Most of the sensors are not biocompatible and too large in size to be placed inside the small volume of liquid. We have developed a protocol that involves SG fluorescence with addition of excess sensor DNA to the amplification solution. The sensor DNA added into the mix is non specific to the primer used for amplification of the template. It therefore does not participate in the amplification and its number remains unchanged throughout the 3040 cycles of PCR. If the amount of sensor DNA is titrated accurately, it will saturate the fluorescence envelope which then shows very reproducible thermal response with cycling. We have used this thermal response of the fluorescence for feedback as a temperature sensor. The fluorescence feedback was shown to produce identical amount of product in comparison to thermocouple feedback. The product can also be verified by melting curve analysis if the sensor DNA is chosen carefully depending on the product. In this chapter we also discuss some preliminary experiments with smart devices that will use dye based temperature sensor and control along with fluorescence based amplification monitoring. Chapter 6 summarizes the thesis and discusses some of the future areas which can be explored in the field of microchip PCR devices.