Farhan Ahmad

Ph.D. Candidate

2006-contd.

CV

ahmadfa3@egr.msu.edu

Flow-through PCR: A Tool for Rapid and Parallel Microbial Detection

Nucleic acid (DNA & RNA) analysis techniques have a major impact in diverse areas, such as molecular biology, environmental monitoring, medical diagnostics, forensic applications, pathogen detection etc. One of the ways of nucleic acid analysis is by performing polymerase chain reaction (PCR), which exponentially amplifies the specific DNA sequence under repetitive temperature cycling. Although conventional PCR is a reliable technique but high through-put and rapid nucleic analysis is not possible, which is highly desirable in bio-defense and infectious diseases related research. Miniaturization of the nucleic acid analysis platforms also referred as µTotal Analysis System or Lab-on-a-Chip can provide the following benefits as shown in Figure 1. Among such systems are Handheld Advanced Nucleic Acid Analyzer (HANNA) and Autonomous Pathogen Detection System (APDS).1-2

                        

Figure1. The above schematic shows the key benefits of the miniaturization of the nucleic acid analysis platform.

PCR in the micro-environment is commonly performed in two configurations as shown in Figure 2.

Figure2. (a) Static chamber PCR (where the sample is kept stationary in the chamber and the temperature is recycled) (b) Flow-through PCR (where is sample moves through three constant temperature zones).

Extensive literature review for both the systems shows that PCR can be performed in minutes in micro-environment together with the positive features of miniaturization. However the problems related to evaporation, sample cross talk, time taken in temperature ramping (high thermal mass of system), and multiplexing also exists. In the case of static chamber PCR the major time is utilized in temperature cycling, which can be controlled by reducing the thermal mass of the system but can not be rooted out completely. Therefore, even the handheld systems (HANNA and APDS) based on the temperature cycling takes around half an hour for the complete analysis. In contrast, the flow-through system, where the major time is utilized in the flow of sample, which can be decreased by increasing the sample flow rate or by decreasing the channel dimensions. Figure 2 shows that the amplification time can be decreased by decreasing the channel cross section area per unit flow rate. Kopp et. al. was able to achieve the PCR in approximately 1.5 minutes by choosing an optimum channel cross-sectional area and the flow rate.3 However multiplexing was not addressed in their work. Obeid et. al. inserted a gas bubble between different samples to address the problem of multiplexing and performing PCR in 5 minutes.4 However, both the cases were just the proof-of-concept and would not be applicable for real time nucleic acid analysis.

The above analysis shows that the nucleic acid analysis by the flow-through system can be performed in approximately one minute by optimizing the flow rate and channel dimensions. Moreover, the specific system design can provide the parallel nucleic acid amplification. It is surprising that no effort has ever been taken to develop a handheld system based on the flow-through configuration.

The objective of this project is to develop a handheld system based on the flow-through configuration for the rapid (~1min), real time detection, and quantification of 10-20 waterborne pathogens. Moreover, the weight of the system is expected to be around one pound after integrating the different components like pump, heater, camera etc. This project is divided into following steps:

a: Designing of a flow-through chip with 10-20 inlet channels and open area for real time
    detection.

b: Designing of integrated heaters (maintained at 950C, 720C, and 550C)

c: Performing simulations to optimize the design.

c: Sorting out the right pumping mechanism (displacement pump or electro-kinetic flow)

d: Integrating the components to achieve the handheld system with a weight of 1lbs.

Figure3.  The schematic shows the flow-through chip with multiple (10) inlet channel integrated with fluid pump system, heaters, and detection system. (Note: dimensions are not up to scale and 20-25 loops are required to achieve sufficient nucleic acid amplification).


Acknowledgement: The project is supported in part by grants from the Michigan Economic Development Corporation (GR-476 PO 085P3000517) and Environmental Protection Agency (RD83162801-0 & RD83301001).


References:

1: Belgrader et. al. ‘Rapid pathogen detection using a microchip PCR array instrument’, 
    Clinical Chemistry 44 (1998) 2191-2194.

2: Higgins et. al. ‘A handheld real time thermal cycler for bacterial pathogen detection’,
    Biosensors and Bioelectronics 18 (2003) 1115-1123.

3. Kopp et. al. ‘ Chemical amplification: Continuous-flow PCR on a chip’ 280 (1998)
    1046-1048.

4. Obeid et. al. ‘Microfabricated device for DNA and RNA amplification by continuous-
    flow through polymerase chain reaction and reverse transcription-polymerase chain
    reaction with cycle number selection’ 75 (2003) 288-295.