Microchip Pcr

Anal Bioanal Chem (2003) 377 : 820–825 DOI 10.1007/s00216-003-2144-2

REVIEW

L. J. Kricka · P. Wilding

Microchip PCR

Received: 17 April 2003 / Revised: 16 June 2003 / Accepted: 29 June 2003 / Published online: 19 August 2003 © Springer-Verlag 2003

Abstract Miniaturization of genetic tests has become an important goal. This review surveys the current progress towards the miniaturization of tests based on the polymerase chain reaction (PCR). It examines the different types of PCR microchip designs, fabrication methods,and the components of a microchip PCR device. It also discusses the problems attributable to surface chemistry of microchip components (inhibition of PCR), and the static and dynamic surface passivation strategies developed for the solution of these difficulties Keywords PCR Microchip · Miniaturization · DNA

Introduction An important trend in chemical and biological analysis over the past 15 years has been the miniaturization of analytical procedures and the development of micro-miniature analyzers (microchips) [1, 2, 3, 4]. The ultimate goal of this work is a lab-on-a-chip or a µTAS (micro total analytical system) in which all the steps in an analytical procedure are performed in a single chip [5, 6]. The analyst would merely add sample and the chip would automatically process the sample, perform the analysis, calculate the result, and communicate the result to a display or to an information system. Miniaturization of genetic testing has been a particular goal for many laboratories exploring and developing microchip technology. This type of testing continues to assume a greater importance in clinical, forensic, and environmental studies. Conventional genetic assays are multistep, manual, and relatively slow. Miniaturization has been identified as a viable way to simplify and speed up these

L. J. Kricka (✉) · P. Wilding Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia, PA 19104, USA e-mail: [email protected]

assays and to make them suitable for extra-laboratory applications (e.g., detecting bio-warfare agents, point-of-care genetic tests). A key component of most genetic tests is a polymerase chain reaction (PCR) reaction and consequently, considerable effort has been expended in miniaturizing this reaction [7, 8]. The PCR reaction is a thermal cycling procedure for amplifying a nucleic acid target. PCR is used to amplify DNA targets and a reverse transcriptase-PCR (RT-PCR) is used for RNA targets. PCR is a three step process in which each step is performed at a different temperature. 1. In the first step, double-stranded DNA is denatured at a temperature of approximately 95 °C. 2. Next, each of the two single strands of DNA are hybridized (annealed) to pairs of oligonucleotide primers at approximately 55 °C. 3. In the final step, a thermostable magnesium ion-dependent polymerase derived from Thermophilus aquaticus (Taq polymerase) synthesizes complementary DNA in the region flanked by the primers using added deoxynucleotide triphosphates (dNTP) at approximately 72 °C (extension). This basic cycle is repeated 20–45 times and each cycle generates copies of the target sequence. In a RT-PCR reaction the RNA target is first converted into DNA using reverse transcriptase, then the DNA is amplified using a PCR reaction procedure. A hand-held battery-powered miniature PCR machine would have many applications, and there are on-going efforts towards this goal. A central component of such a device is a miniaturized PCR chamber (a PCR microchip) or an array of chambers for multiple simultaneous PCR reactions. For greatest benefit, the overall cycle time would need to be short and the detection of the PCR amplicons rapid and sensitive. In addition the PCR microchip should be disposable in order to avoid cross-contamination between specimens, and hence, would need to be relatively inexpensive in order to make this mode of analysis economically viable. Other important considerations for any

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PCR-based tests are sample acquisition and the sensitivity required in order to detect amplicons generated in a microdevice [2, 7]. This article surveys the development and scope of the microchip component of a microchip-based PCR analyzer, and explores the progress in the integration of miniaturized PCR with other analytical processes in a PCR microchip format. The reader is also directed to related microminiaturization of PCR reactions in capillaries [9, 10, 11] and on the surface of microarrays [12, 13, 14].

PCR microchip fabrication and designs Most glass or silicon microchips for PCR are fabricated using photolithographic techniques. PCR microchips made from PDMS elastomers are fabricated by a molding process [15], and integrated devices made from polycarbonate are produced by computer-controlled machining [16]. Microchips for PCR can also be made by low-temperature firing of assembled layers of ceramic tape (e.g., DuPont T2000 tapes) produced by mechanical punching or laser cutting [17, 18]. Often a PCR microchip is a composite of two or more components made of different materials that must be assembled into a leak-proof final device. Silicon-glass microchips are usually assembled using a high temperature anodic bonding process. An alternative method involves gluing the two components with a UV glue [19, 20]. Another approach is to place the glass cover on top of the silicon chip, seal the edges with varnish and maintain the seal by placing a weight on top of the glass cover [21]. Silicon chips can be bonded with a low temperature curing polyimide [22]. In this process, glass covers are sealed onto glass microchips by first hydrolyzing the glass surfaces and then thermally bonding the assembled microchip [23]. PDMSglass microchips are assembled by sealing the PDMS component onto the glass (e.g., a cover slip) at an elevated temperature (e.g., 80 °C) [15]. The two parts of polycarbonate integrated microchip devices can be bonded by ultrasonic welding or using adhesives, or held together with a clamping device [16]. Finally, the contents of a microchip can be protected against evaporation and the external environment by simply covering the microchamber with oil [24]. Designs for PCR microchips range from wide chambers of varying sizes and depths (Fig. 1) to narrow channels (linear or serpentine) (Fig. 2), and can have a single reaction chamber or arrays of chambers for multiple simultaneous reactions (Table 1) [25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45]. Volumes inside the microchips vary over the nL to µL range, and devices with volumes as low as 12 nL have been produced [46]. The physical dimensions of microchips also vary widely but most are approximately 1 cm×1 cm as a matter of convenience for handling the chips. The prospect of even smaller device is inherent in the fabrication processes available, but ultra-miniature devices would be more difficult to handle and manipulate in a research and development process.

Fig. 1 A pille of silicon-glass PCR microchips (17 mm × 14 mm)

Fig. 2 Schematic of a flow-through-type of PCR microchip. The serpentine reaction microchannel crosses each of three zones (T1, T2, T3) each of which is set at a different temperature Table 1 Microchip PCR vessel designs Vessel architecture

Material

Reference

glass

[23, 25, 26, 27]

polytetrafluoroethylene (PTFE)

[28]

glass silicon glass polycarbonate co-fired ceramics

[29] [30] [16] [17, 18]

glass silicon silicon-glass polydimethylsiloxane (PDMS)-glass ceramic tape polyimide

[31, 32] [22] [33, 34, 35, 36, 37, 38] [39]

silicon silicon-glass

[24, 41] [19, 21, 43, 44, 45]

Reservoir Channel Linear Serpentine

Chamber Single

Multiple

[18] [40]

The ways of performing PCR on a microchip have been classified into a time domain and a space domain approach [46].

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1. Time domain PCR – In this format the reaction mixture is kept stationary and the temperature of the surrounding reaction chamber is cycled between the different temperatures. 2. Space domain PCR – In this format the reaction mixture is moved between different fixed temperature zones. The advantage of this strategy is that the device does not have to be heated and cooled and this facilitates faster cycling. In one embodiment a rotary device continually cycles reaction mixture contained in a loop over different heaters [46]. A serpentine channel design is used for continuous-flow microchip PCR. The channel runs back and forth across three heaters (60 °C, 77 °C, 95 °C) to provide the required cycling [29]. A variant of this general design incorporates outlets at different distances along the serpentine channel that allow product collection after 20, 25, 30, 35, and 40 cycles. Successive samples can be analyzed simultaneously by isolating the individual samples by segments of diluent [47].

Microchip thermocycling The excellent thermal conductivity of silicon (≈150 W °C–1) makes it ideal in an application such as PCR that requires rapid cycles of heating and cooling. The source of heat for a microchip PCR reaction can be an external heating block, or non-contact heating by infrared radiation [40], or heaters fabricated directly onto the surface of the microchip (e.g., tungsten or platinum film) [17, 20, 48], Cooling can be achieved via forced air using a fan [49], or by means of a Peltier heater-cooler device [34]. It is highly desirable to have the highest possible ramp rates for heating and cooling in order to minimize cycle times, and values as high as 80 °C s–1 have been obtained for heating and 40 °C s–1 for cooling [44]. Some microchips incorporate specific features, such as grooves and air spaces [17, 24] designed to isolate the PCR chamber and minimize lateral heat transfer from the chamber to the bulk of the microchip. Monitoring the temperature of a PCR microchip is important because of the critical dependence of this reaction on accurate temperature control during the different cycles. A thermal sensor is often fabricated onto a chip along with the heaters in order to monitor temperature and provide feedback to the temperature controller. Another way of assessing temperature in a chip during thermocycling is using an infrared camera. This remote monitoring method has the advantage of not compromising the thermal properties of the microchip. Encapsulated thermochromic liquid crystals suspended in a liquid sample have been used to determine the temperature uniformity of a 3×6 array of PCR microchambers (2-µL volume) and as a tool for optimizing the thermal design of the device [43]. In one study, two formulations were employed, one with an operating temperature range of approximately 1 °C centered at 55 °C, and the other with an operating temperature range of approximately 2 °C centered at 95 °C. The change in the hue of the liquid crystals with temperature was recorded with

a video camera. One minor disadvantage is that the density of the encapsulated crystals did not match the density of the fluid in the microchambers. The lighter high temperature crystals floated, and the more dense lower temperature crystals sank to the bottom of the chamber. This did, however, facilitate assessment of temperature variations at the floor and ceiling of the vessels. Modeling and simulation have also been used to investigate aspects of PCR microchip design [50].

Fluidic connections Operation of a microchip requires convenient fluidic connections so that µL or sub-µL volumes of fluid can be introduced into the microchip and, if required, removed after completion of the PCR reaction. In some protocols the microchip is filled by capillary action by simply pipetting the reaction mixture onto one of the entry ports. Other protocols attach a pump to the inlet port and the reaction mixture is pumped into the microchip. Effective sealing of the microchip during thermal cycling is important in order to avoid leakage of the contents during the period of thermal cycling. Another issue is bubble formation due to leaks or evaporation of the microchip contents. Bubbles may be relatively benign but can cause significant differences in temperature (4–5 °C) and prevent effective PCR [48].

Materials and surface chemistry Two factors complicate the design and construction of PCR microchips. First, the PCR reaction is a multi-component reaction that includes reagents with diverse properties – metal ions, buffers, oligonucleotide primers, dNTPs, enzymes. Hence the possibility of at least one of these components binding to some degree with an internal surface in a microchip is significant. Secondly, the surface area/volume ratio is high in microdevices and this further increases the possibility of adverse interactions between the inner surface of a microchip and components of the reagent mixture (e.g., denaturation of Taq polymerase) or the sample (e.g., irreversible binding of the target). For example the surface area/volume ratio of a PCR microchip can be 20-fold or greater than the surface area/volume ratio of a conventional Microamp tube. PCR microchips have been mostly fabricated from glass or glass and silicon, although other materials such as polyimide and PDMS have also proved suitable construction materials (Table 2) [15, 16, 30, 31, 32, 34, 36, 37, 38, 39, 40 ,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53]. Early work with silicon-glass microchips revealed the problems of adverse surface interactions and led to the development of passivation procedures to render the internal surfaces of microchips “PCR friendly”[34, 36, 36, 51]. Passivation procedures can be classified into two different types: Type I – Static passivation and Type II – Dynamic passivation (Table 2).

823 Table 2 Passivation agents and procedures Method

Reference

Static passivation Bovine serum albumin (BSA) Silicon oxide Silanization Silanization + acrylamide polymer Parylene-C Polydimethylsiloxane (PDMS) Epoxy poly(dimethylacrylamide)(EPDMA) Polypropylene plastic liner

[15, 31] [34, 36, 37, 51] [30, 34, 36, 52, 53] [32] [16] [44] [53] [54]

Dynamic passivation BSA Polyethyleneglycol (PEG) (molecular weight ≈8,000) Polyvinylpyrrolidone (PVP) (molecular weight ≈1 million) Combined static and dynamic passivation BSA + BSA BSA + silanization Silicon oxide layer + BSA

[31, 32, 42, 55] [40, 53] [53]

[31, 45] [30] [38]

Adverse surface properties of an otherwise PCR friendly material can arise during microchip manufacture. Residual chromium from chrome masks used to manufacture glass microchips on the surface of the glass is inhibitory to a PCR reaction and must be removed by strenuous washing procedures [56].

Targets and detection of amplicons A wide range of targets have been amplified in a microchip by PCR and its variants, RT-PCR and DOP-PCR [57]. Amplicons are detected by removing the reaction mixture from the microchip, and then analyzing it by gel electrophoresis, capillary electrophoresis [10, 23, 25, 31], or MALDI-TOF [58]. The transparent nature of a glass cover on a microchip facilitates optical readout of the progress of a PCR reaction in the microchip in real time using a TaqMan assay [41, 45, 52, 59] or by monitoring the increase in fluorescence due to intercalation of ethidium bromide into the double-stranded amplicons [52]. Amplicon yields in microchip PCR that are superior and inferior to conventional thermocycling yields have been reported [22, 35, 36].

Type I – Static passivation In this type of passivation, the surface of a microchip is precoated with a substance, usually during microchip fabrication. For silicon-glass microchips deposition of an oxide coating and silanization are effective. An advantage of the oxide method is that it can be performed at the wafer stage during microchip fabrication and it does not interfere with subsequent capping of the microchips with Pyrex glass using an anodic bonding process [51]. Other examples of this type of passivation are silanization of internal surfaces. This is accomplished by filling a chip with a silanizing agent and incubating the filled microchip for a period of time, then emptying and washing the microchip [36, 51]. A further variant is to insert an inner sleeve of an inert material into a microchip and so eliminate contact between the chip surface and the reaction mixture [54]. Uncoated injection molded polycarbonates surfaces are reported to be inert in PCR although a coating of parylene-C was found to improve reproducibility [16]. Attempts to passivate silicon surfaces with silicon nitride have been unsuccessful [36, 51]. Type II – Dynamic passivation This is a type of passivation that occurs during the filling and operation of a microchip. It is accomplished by including the passivation reagent in the reaction mixture. Examples of substances effective in this type of passivation procedure include polymers (e.g., PEG, PVP [29]), and proteins such as BSA (see Table 2). Presumably, these substances bind preferentially to the inner surface of the microchip and prevent binding by components of the sample or reagent mixture.

Reusability Most microchips are designed with clinical testing in mind and hence should be disposable. Our experience has been that microchips are difficult and sometimes impossible to clean for resuse, and validation of the effectiveness of cleaning poses considerable difficulties. A continuous flow borosilicate glass microchip has been successfully reused (2 weeks of continuous usage). Blockages that clogged the 55-µm deep channels were encountered but the microchip could be cleaned by heating at 300 °C for 1 h. This process burns organic material, such as denatured protein from inside the channels [47].

Integration and pre-PCR and post-PCR Various analytical steps that precede a PCR or are performed after completion of a PCR have been integrated onto the same microchip device [60]. These include prePCR separation and isolation of white blood cells using filters within a PCR chamber [38], and post-PCR capillary electrophoresis or capillary gel electrophoresis analysis of the PCR reaction mixture [23, 25, 31, 54]. Microarrays have also been incorporated in PCR microchips for hybridization-based analysis of the PCR reaction products [55]. The arrays of oligonucleotides were printed onto the bottom of each of four 3-µL volume microchambers prior to sealing the glue coated-silicon microchip with a glass cover. In one microchip device, the steps of cell lysis, multiplex PCR and capillary electrophoresis analysis were performed sequentially and shown to be effective for the analy-

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sis of 154-, 264-, 346-, 410-, and 550-bp DNA target sequences in whole Escherichia coli cells [25, 26]. A microdevice that incorporates a PCR chamber has also been developed for integrated multi-step genetic analysis [16]. The device integrates the serial steps of extracting and concentrating nucleic acids from a sample, PCR or RT-PCR, enzymatic fragmentation of amplicons, and a nucleic acid hybridization assay. The effectiveness of this device has been demonstrated in an assay for the 1.6-kb region of the HIV genome.

Products Several prototype microchip-based PCR analyzers have been described (e.g., advanced nucleic acid analyzer (ANAA)) [41]. However, despite strong indications that the development of microchip-based PCR analyzers are nearing completion, few have been commercialized. One example of a PCR microchip-based device is the miniature analytical thermal cycling instrument (MATCHI) system (Smart Cycler see www.cepheid.com). This is a battery-powered portable, real-time, integrated analytical system based on PCR performed in an array of silicon microchambers [52, 59, 61]. The entire system fits inside a briefcase for ease of transport. Applications identified for this nucleic acid analysis device include forensic, environmental and agricultural analyses and detecting biowarfare agents [62, 63, 64].

Patents There are a series of issued patents directed at the general area of miniaturized reactors and more specifically at microchip-based amplification reactions, such as PCR [65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75]. The patents cited are intended as a starting point for information on the intellectual property aspects of this branch of miniaturization. The interested reader is directed to the appropriate web sites for further and more comprehensive information on patents and patent applications (see www.uspto.gov and www.delphion.com).

Conclusions Microchip PCR has developed rapidly and now represents an important application for miniaturization, and microchip-based PCR analyzers are now available. The central role of PCR in genetic tests, and the emerging need to perform PCR in the field as part of efforts to detect infectious agents will provide continued impetus for the development of miniature PCR analyzers and totally integrated analyzers that incorporate PCR microchips. However, currently the macroscale PCR devices dominate and the wide scale implementation of microchip PCR depends on an industry commitment to the development and commercialization of these devices, especially devices that integrate

other aspects of testing such as the sample preparation step prior to PCR.

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