key: cord-0037746-1x1lebh4
authors: Ugaz, Victor M.
title: PCR in Integrated Microfluidic Systems
date: 2007
journal: Integrated Biochips for DNA Analysis
DOI: 10.1007/978-0-387-76759-8_7
sha: 8c6ca3bcb0d401ac9ccaf49bb1efd0e6e12d2f0e
doc_id: 37746
cord_uid: 1x1lebh4

Miniaturized integrated DNA analysis systems offer the potential to provide unprecedented advances in cost and speed relative to current benchtop-scale instrumentation by allowing rapid bioanalysis assays to be performed in a portable self contained device format that can be inexpensively mass-produced. The polymerase chain reaction (PCR) has been a natural focus of many of these miniaturization efforts, owing to its capability to efficiently replicate target regions of interest from small quantities template DNA. Scale-down of PCR has proven to be particularly challenging, however, due to an unfavorable combination of relatively severe temperature extremes (resulting in the need to repeatedly heat minute aqueous sample volumes to temperatures in the vicinity of 95°C with minimal evaporation) and high surface area to volume conditions imposed by nanoliter reactor geometries (often leading to inhibition of the reaction by nonspecific adsorption of reagents at the reactor walls). Despite these daunting challenges, considerable progress has been made in the development of microfluidic devices capable of performing increasingly sophisticated PCR-based bioassays. This chapter reviews the progress that has been made to date and assesses the outlook for future advances.

Advances in genomic analysis technology continue to be made at a rapid pace and have contributed to the development ofnew instrumentation that is paving the way fur high-throughput low-cost DNA assaysto become commonplace. These technologies have the potential to impact an unprecedented array offieldsincluding medical diagnostics, forensics, biosensing and genome -wide analysis,"! Many ofthese methodologies rely on the ability to replicate selected sub-regions within a larger DNA template. The polymerase chain reaction (PCR) offers a straightforward and highly efficient means to perform this replication, thereby making it one of molecular biology's key enabling technologies.

The PCR process involves repeatedly cycling a reagent mixture containing template DNA, primers, dNTPs, a thermostable polymerase enzyme and other buffering additives, through thermal conditions corresponding to (1) denaturation ofthe double-stranded template (~95'C), (2) annealing ofsingle-stranded oligonucleotide primers at complementary locations flanking the target region (~50-65'C) and (3) enzyme directed synthesis of the complementary strand (~72'C). The number of target DNA copies increases exponentially as this cycling process is repeated, doubling with each cycle under ideal conditions. Although the kinetics associated with each step in the PCRprocess are rapid when considered individually.v" a typical 30-40 cycle replication still requires timescales of order 1-2 hours to complete. These prolonged reaction times are largely a reflection ofthe highly inefficient design ofmany conventional thermocycling instruments, where thermally massive hardware components (e.g., metal thermal blocks) and low-conductivity plastic reaction vessels (e.g., polypropylene tubes) combine to produce an unfavorable coupling between increased thermal energy requirements associated with rapid heating and cooling and the necessity to hold the temperature constant at each step for a sufficiendy long time to ensure that the entire reagent volume attains thermal equilibrium. Progress has been made in the development offaster benchtop-scale thermocyclers, most notably by approaches that involve replacing plastic reaction tubes with thin glassmicro-capillaries and using techniques ranging from forced air circulation to infrared heating for temperature control.I" These configurations allow the reaction mixture to be distributed over a greater surface area for more efficient heat transfer, although overall throughput can still be limited by the capillary loading, sealing and unloading processes.

In addition to these benchtop-scale approaches, increasing interest has been focused on performing PCR in miniaturized systems where ultra-small reaction volumes (typically in the nL range) not only gready reduce reagent consumption, but can also be rapidly heated and cooled while simultaneously lowering costs due to gready reduced reagent consumption. Moreover, the use ofphotolithographic microfabrication offers the potential to produce hundreds or thousands ofdevices at once, bringing hardware costs to a level of$l or less.These highly desirable characteristics have stimulated considerable interest in the area ofmicrofabricated thermocycling systems. However the use ofsuch small reaction volumes can pose significant challenges including adverse effects associated with evaporation and nonspecific absorption to the microchannel walls under high surface to volume conditions. Progress continues to be made toward addressing the challenges facing development ofmicrofluidic thermocycling systems and the interested reader is referred to several recent reviews for more details.6. [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] Despite its importance as a key enabling technology, PCR is only one step in a broader sequence of processes that comprise a complete molecular biology assay. Some combination of sample isolation and collection, pre and postreaction purification, subsequent biochemical reactions and product detection and analysis must also be performed. On the rnacroscale, these steps are typically carried out in a conventional laboratory setting, often requiring dedicated instruments and personnel to be employed at each stage along with the need to repeatedly prepare and dispense precise sample and reagent mixtures. These processes can not only be tedious and time consuming, they also introduce multiple opportunities for measurement errors and sample contamination. The ability to adapt a series of these operations to a miniaturized 'lab-on-a-chip' format has the potential to address these limitations, yielding significant reductions in analysis time and cost. Moreover, a fully self-contained portable design offers the potential to minimize tedious manual fluidic manipulations and sample contamination issues.

This enhanced functionality, however, is accompanied by a corresponding increase in the level ofdesign and operational complexity. A myriad ofchallenges must beaddressed, including ensuring biochemical compatibility among successivesteps in the analysisprocess, careful thermal design to ensure that the relatively high temperatures achieved during PCR thermocyclingdo not negatively impact activity on other parts ofthe chip, the ability to accurately dispense and transport nanoliter liquid volumes within a microchannel network and ensuring that reagents can be properly sealed to prevent evaporation during thermocycling. In this chapter, we review some of the remarkable progress that has been made toward developing sophisticated miniaturized devices that incorporate PCR as part ofan integrated molecular biological assaysystem. The review focuses on developments reported in refereed journals, with the understanding that additional studies may be documented in conference proceedings and patent literature.

Considerable progress has been made in the development ofmicrodevices capable ofcombining PCR with postreaction product analysis performed by electrophoretic separation ofthe amplified DNA fragments ( Table 1 ).The Mathies group was the first to demonstrate this approach using a hybrid design consisting ofa 20 ul, microfabricated silicon PCR reactor bonded on top ofa glass electrophoresis microchip." Unfavorable surface interactions at the reactor walls that inhibited PCR amplification were avoided through the useofdisposable polypropylene liners, allowing targets from both plasmid and bacterial genomic DNA templates to be successfully amplified. Following PCR, the products were electrokinetically injected into a separation channel filled with a sieving gel matrix containing a fluorescent intercalating dye and detected using a laser excited confocal imaging system. Total analysis times ranging from 20 to 4S minutes were reported. Subsequent refinements to this basic design involved integrating both the PCR reactor and electrophoresis column into a single continuous glass microchannel network. In one adaptation reagents were loaded and sealed inside a 280 nL reaction chamber using a valve/vent manifold mounted on top of the chip and temperature control was provided by thin film heater and thermocouple elements affixed to the back side." Upon completion ofthe reaction, the manifold was removed and platinum electrode wires were inserted into the accessholes so that the products could be electrokinetically transported to the electrophoresis channel for analysis. This design was used to demonstrate amplification of targets from cloning vector and genomic control templates with sufficient sensitivity to permit single molecule detection." A further improvement to the design involved integration ofmicrofabricated heaters and temperature sensors to actuate thermocycling within a 200 nL reaction chamber.f This device was used to perform multiplex sex determination from human genomic DNA in under 1S minutes. A self-contained portable version was also constructed and used to perform multiplex PCR directly from whole bacterial cells in 30 minutes, with product detection accomplished using fluorescently labeled primers (Fig. lA) .29

Another notable family of glass microchips for performing integrated PCR and gel electrophoresis has been developed in the Ramsey group. Here, the basic design consisted ofa 10-20 [-tL reaction reservoir fabricated by drilling a hole in one of the glass substrates. After loading, the PCR reagents were covered with mineral oil or wax to prevent evaporation and thermocycling was performed by placing the chip inside a conventional thcrmocycler.P'" After the reaction was completed, an intercalating dye was added to the sample reservoir and electrophoretic product analysis was performed with laser-induced fluorescence detection. Subsequent improvements included the use of a specially designed thermoelectric fixture with dual thermoelectric heating elements and incorporation ofa porous membrane structure fur injection ofreaction products into the electrophoresis gel." Successful amplification of targets from a variety of templates ranging from lambda DNA to bacterial and mouse genomic DNA have been reported using these devices, with total analysis times of20 minutes or less. Zhou and coworkers also demonstrated a glass microchip design integrating PCR and gel electrophoresis with an external thermoelectric thermocycling apparatus to perform a duplex PCR analysis of24O and 438 base pair (bp) targets associated with the SARS coronavirus." Other examples ofintegration include designs employing hybrid PDMS/glass microchips" and designs interfacing a silicon PCR microchip with integrated temperature control to a glass electrophoresis microchip.l" A particularly novel design reported by Koh et al consisted ofa plastic microfluidic chip with integrated temperature control that incorporated on-chip photopolymerized gel valves that not only sealed reagents inside the PCR reactor but also allowed the products to be electrokinetically extracted through the valve material and directed into an electrophoresis channel for separation and detection. 37 The device was used for successful analysis oftargets from two different bacterial genomic templates with a detection limit on the order ofsix DNA copies.

Recent work by the Landers group has yielded further reductions in analysis time through the use of an innovative noncontact infrared heating technique to actuate thermocycling. This concept was successfully employed in an integrated device capable ofamplifying a 380 bp 13-globin target in a 600 nL reactor followed by electrokinetic injection and electrophoretic separation." In addition, a separate microchip was used to perform solid phase extraction of DNA from whole blood prior to PCR. Subsequent work has resulted in further optimizations to this design by a reduction in reactor volume to 280 nL and through development ofa fluid handling system based on a multilayer hybrid glass/PDMS assembly that provides an addressable array of pneumatic valves capable ofperforming sealing, pumping and injection into the electrophoresis gel matrix (Fig. lB) .39 This design offers an impressive capacity for analysis speed, as demonstrated by the ability to perform integrated PCR and electrophoresis ofbacterial targets with total analysis times ofabout 12 minutes. 

Advances have also been made in the design of microdevices integrating peR with other upstream and downstream sample handling and analysis steps ( Table 2 ). The Wilding group has developed a series ofmicrodevices incorporating micromachined 'weir-type' filtration structures capable ofisolating white blood cells from whole blood samples in the 1-10 ul, range. Filtration occurred as samples were pumped through the chamber where the larger white blood cells were selectively retained by the filter, after which a peR reagent mixture was injected and therrnocycling was performed using a thermoelectric apparatus. 40 ,41 Both hybrid glass/silicon and Plexiglas

IntegratedBiocbipsfOrDNA Analysis substrate materials have been used to construct the devices, and the potential for further integration has been demonstrated by performing product sizing analysis using separate electrophoresis microchips.? An integrated microdevice capable of performing cell lysis followed by PCR was reported by Lee et al. 43 The design consisted ofa hybrid glass/PDMS configuration with PDMS thermal lysisand PCR chambers interconnected by a glassrnicrochannel network in which the lysis products were mixed with PCR reagents and electroosmotically transported to the PCR reactor. The device was used to successfully amplify a 273 bp target from whole bacterial cells. Finally, a portable pathogen detection system integrating DNA purification and real-time PCRdetection has been developed by Cady and coworkers ( Fig. 2A) . 44 Here, samples subjected to off-chip chemical lysiswere pumped through a flow network containing arrays ofetched silicon pillars that allowed purification to be performed via sequential binding and washing steps. The purified samples were then pumped into a PDMS PCR reactor for real-time amplification using a SYBR green fluorescence chemistry. The ability to detect between 10 4 and 10 7 bacterial cells in about 45 minutes was demonstrated in characterization studies involving amplification ofa 544bp target.

Several groups have also explored integration of microchip PCR with DNA hybridization analysis (Table 3 ). Anderson and coworkers demonstrated this concept using a device containing a credit card sized polycarbonate fluidic cartridge interfaced with an Affymetrix GeneChip microarray," The integrated system was capable of performing sequential reverse transcription, PCR, enzymatic reactions and hybridization. Further progress in microarray integration was reported by Liu et al who presented a fully self-contained biochip device that incorporated the ability to perform sample preparation (cell capture, concentration, purification and lysis), PCR and hybridization using an integrated Motorola eSensor microarray (Fig. 2B) . 46 This device was used to perform pathogenic bacteria detection from whole rabbit blood and single-nucleotide polymorphism analysis from diluted human blood samples. Another example ofintegration with hybridization involved employing an array ofhybrid glass/silicon microreactors with integrated on-chip heaters and temperature sensors." The bottom surface ofeach microreactor was patterned with hybridization oligonucleotides allowing reaction products to be detected with a confocal fluorescence scanner. Finally, Liu et al demonstrated a polycarbonate microdevice where PCR and hybridization are performed in separate fluidically interconnected reactors." Here, reagents were sealed in the PCR chamber during thermocycling by employing phase change valves based on Pluronic F127, a block copolymer that liquefies at low temperature (~5·C) but becomes a solidified gel at higher temperatures. Operation of the device was demonstrated by using it to perform a bacterial detection assay.

Increases in device complexity that accompanies simultaneous miniaturization and integration ofmultiple sample processing and analysis steps can pose daunting challenges, however progress is steadily being made toward addressing many of these issues resulting in the development of increasingly sophisticated designs (Table 4 ). An early compelling example illustrating the power of miniaturized genomic analysis systems was the hybrid glass-silicon design developed in the Burns group," capable of performing a series of liquid metering, thermal reaction and analysis operations. This pioneering device was used to amplify a 106 bp DNA target from a bacterial genomic template via an isothermal strand displacement amplification (SDA) process followed by gel electrophoresis with integrated photodetection ofthe fluorescently labeled reaction products. A greatly improved version of this design was recently reported that is capable of performing a complete genotyping assayinvolving two sequential reactions followed by an electrophoretic separation in an ultra-compact 1.5x 1.6cm hybrid glass/silicon microfluidic chip (Fig. 3A) .50 Sample DNA and PCR reagents were loaded into the chip and pneumatically propelled into a reaction zone where they were sealed using paraffin phase change valves.Thermocyclingwas actuated using an array ofintegrated resistive heaters and temperature sensors, with the reaction zone thermally isolated from the rest of the chip. Upon completion ofthe PCR reaction, the sealing valveswere opened and the sample was propelled forward, mixed with additional reagents and incubated to perform a restriction enzyme digestion reaction. Upon completion of the second reaction, the products were directed into a gel electrophoresis column for analysis. This system was used to perform a PCR-RFLP assayfor influenza A virus detection, illustrating tremendous potential as a generic platform suitable for use in a wide range of genotyping applications. Another notable recent advancement has been reported by the Mathies group, where a multilayer glass/PDMS device was used to perform integrated Sanger cycle sequencing of DNA-a reaction requiring similar thermal cycling parameters as PCR (Fig. 3B )Y Sequencing reactions were performed in 250 nL reactors, after which the products were electrokinetically transported into a novel capture gel for purification. Here, oligonucleotides complementary to the region immediately adjacent to one of the sequencing primers were immobilized in a sparsely crosslinked polyacrylamide gel plug, such that the sequencing products could be retained inside the gel while unincorporated primers and other reagents passed through. Upon heating the gel to a temperature above the primer melting point, the hybridized products were released and electrokinetlcally injected into the separation channel. Read lengths of 556 bases were achieved with a 99% base call accuracy, demonstrating tremendous potential for significant reductions in cost and time scales associated with genome sequencing.

Buoyancy driven natural convection phenomena have also been investigated as a means of accelerating the thermocycling process. By designing reaction chambers that harness an imposed static temperature gradient to generate a circulatory convective flow field, PCR reagents can be automatically transported through temperature zones associated with denaturing, annealing and extension conditions. Convective flow thermocyclers may be broadly classified as cavity-based or loop-based systems, depending on the nature of the flow field generated. Cavity-based designs typically consist of reactor geometries in which the PCR reagents are enclosed between upper and lower surfaces maintained at annealing and denaturing temperatures, respectively.52'54 When the aqueous PCR reagent mixture is heated from below, an unstable "top heavy" arrangement is created which can provide sufficient driving force to establish a continuous circulatory flow in much the same fashion as in an ordinary lava lamp. Here, thermocycling parameters (e.g., cycling time, residence time within each temperature zone) are controlled by an interplay between reactor geometry (height to diameter aspect ratio) and the magnitude ofthe imposed temperature gradient. Krishnan et al successfully demonstrated this concept by amplifying a 295 bp~-actin target from a human genomic DNA template in a 35 ul, cylindrical reactor (Fig. 4A) . 52 In subsequent work, this simple design has been adapted to perform amplification of a 191 bp target associated with membrane channel proteins M 1 and M2 ofthe influenza-A virus with cycling times ranging from 15 to 40 minutes in a multiwell cartridge format that offers potential for use in high throughput settings. 53.55 Convectively driven PCR of a 96 bp target from a bacterial genomic template has also been demonstrated by Braun et al in low aspect ratio cylindrical cavities using either focused infrared heating or a micro immersion heater inserted at the center ofa low aspect ratio cavity to drive the flow (Fig. 4B) . [56] [57] [58] Successful PCR amplification has also been demonstrated in closed-loop convective flow systems. These designs are attractive because oftheir ability to generate unidirectional flows along a closed path enablingcycling parameters to be precisely controlled. Wheeler and coworkers designed a novel disposable polypropylene reactor in which opposite sides ofa racetrack-shaped flow loop were maintained at 94 and 55°C respectively to amplify targets ranging from 58 to 160 bp from a bacterial genomic template (Fig. 4C) ,59 while Chen et al employed a triangular arrangement with three independently controlled temperature zones maintained at 94, 55 and 72°C to amplify 305 and 700 bp targets from a bacterial genomic template (Fig. 4D) . 60 Krishnan ec al employed both triangular and racetrack shaped designs to amplify 191 and 297 bp targets from control and human genomic DNA targets respectively." Most recently, Agrawal et al have demonstrated a triangular design in which flow loops are constructed using disposable plastic tubing (Fig. 4£) . 61 Here, two opposing sides of the loop are maintained at denaturing and extension temperatures using independently controlled thermoelectric heaters while the third side passivelyattains annealing conditions. This system is capable ofperforming single and multiplex PCR for targets ranging from 191 bp to 1.3 kb within 10 to 50 minutes using 10 to 25 ul, reaction volumes, highlighting 

Impressive progress continues to be made in the development ofintegrated microfluidic systems capable of performing PeR-based bioanalysis assays. These advances are largely driven by the compelling benefits offered by miniaturization, including lower hardware costs arising from mass production using microfabrication technology, extremely rapid analysis times and greatly reduced reagent requirements. A number ofchallenges remain, however, including development ofimproved miniaturized fluid handling technology capable ofprecisely metering, transporting and sealing nanoliter liquid volumes ro prevent evaporation without relying on bulky off-chip mechanical hardware. Potential inhibitory effects that arise under ultra-high surface to volume geometric conditions are also likely to remain a serious consideration as reactor volumes continue to shrink. Finally, improvements are still needed in the area ofproduct detection, where the necessity to employ benchtop-scale fluorescence imaging systems often limits portability. Developments in these areas are ongoing and ultimately have the potential to greatly expand the use ofgenomic analysis technology by making the capability to perform an increasingly sophisticated array of assaysaccessible for use in a wide range of settings by those who need it most .

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