Abstract Miniaturization of genetic tests has become animportant goal. This review surveys the current progresstowards the miniaturization of tests based on the poly-merase chain reaction (PCR). It examines the different typesof PCR microchip designs, fabrication methods,and the com-ponents of a microchip PCR device. It also discusses theproblems attributable to surface chemistry of microchipcomponents (inhibition of PCR), and the static and dy-namic surface passivation strategies developed for the so-lution of these difficulties

Keywords PCR Microchip · Miniaturization · DNA

Introduction

An important trend in chemical and biological analysisover the past 15 years has been the miniaturization of an-alytical procedures and the development of micro-minia-ture analyzers (microchips) [1, 2, 3, 4]. The ultimate goalof this work is a lab-on-a-chip or a µTAS (micro total an-alytical system) in which all the steps in an analytical pro-cedure are performed in a single chip [5, 6]. The analystwould merely add sample and the chip would automati-cally process the sample, perform the analysis, calculatethe result, and communicate the result to a display or to aninformation system.

Miniaturization of genetic testing has been a particulargoal for many laboratories exploring and developing mi-crochip technology. This type of testing continues to as-sume a greater importance in clinical, forensic, and envi-ronmental studies. Conventional genetic assays are multi-step, manual, and relatively slow. Miniaturization has beenidentified as a viable way to simplify and speed up these

assays and to make them suitable for extra-laboratory ap-plications (e.g., detecting bio-warfare agents, point-of-caregenetic tests).

A key component of most genetic tests is a polymerasechain reaction (PCR) reaction and consequently, consider-able effort has been expended in miniaturizing this reaction[7, 8]. The PCR reaction is a thermal cycling procedurefor amplifying a nucleic acid target. PCR is used to am-plify 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 per-formed at a different temperature.

1. In the first step, double-stranded DNA is denatured ata temperature of approximately 95 °C.

2. Next, each of the two single strands of DNA are hy-bridized (annealed) to pairs of oligonucleotide primersat approximately 55 °C.

3. In the final step, a thermostable magnesium ion-depen-dent polymerase derived from Thermophilus aquaticus(Taq polymerase) synthesizes complementary DNA inthe region flanked by the primers using added de-oxynucleotide triphosphates (dNTP) at approximately72 °C (extension).

This basic cycle is repeated 20–45 times and each cyclegenerates copies of the target sequence. In a RT-PCR re-action the RNA target is first converted into DNA usingreverse transcriptase, then the DNA is amplified using aPCR reaction procedure.

A hand-held battery-powered miniature PCR machinewould have many applications, and there are on-going ef-forts towards this goal. A central component of such a de-vice is a miniaturized PCR chamber (a PCR microchip) oran array of chambers for multiple simultaneous PCR reac-tions. For greatest benefit, the overall cycle time wouldneed to be short and the detection of the PCR ampliconsrapid and sensitive. In addition the PCR microchip shouldbe disposable in order to avoid cross-contamination be-tween specimens, and hence, would need to be relativelyinexpensive in order to make this mode of analysis eco-nomically viable. Other important considerations for any

L. J. Kricka · P. Wilding

Microchip PCR

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

Received: 17 April 2003 / Revised: 16 June 2003 / Accepted: 29 June 2003 / Published online: 19 August 2003

REVIEW

L. J. Kricka (✉) · P. WildingDepartment of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia, PA 19104, USAe-mail: kricka@mail.med.upenn.edu

© Springer-Verlag 2003

PCR-based tests are sample acquisition and the sensitivityrequired in order to detect amplicons generated in a mi-crodevice [2, 7].

This article surveys the development and scope of themicrochip component of a microchip-based PCR analyzer,and explores the progress in the integration of miniatur-ized PCR with other analytical processes in a PCR mi-crochip format. The reader is also directed to related mi-crominiaturization 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 fabricatedusing photolithographic techniques. PCR microchips madefrom PDMS elastomers are fabricated by a molding pro-cess [15], and integrated devices made from polycarbon-ate are produced by computer-controlled machining [16].Microchips for PCR can also be made by low-temperaturefiring of assembled layers of ceramic tape (e.g., DuPontT2000 tapes) produced by mechanical punching or lasercutting [17, 18].

Often a PCR microchip is a composite of two or morecomponents made of different materials that must be as-sembled into a leak-proof final device. Silicon-glass mi-crochips are usually assembled using a high temperatureanodic bonding process. An alternative method involvesgluing the two components with a UV glue [19, 20]. An-other approach is to place the glass cover on top of the sil-icon chip, seal the edges with varnish and maintain the sealby placing a weight on top of the glass cover [21]. Siliconchips can be bonded with a low temperature curing poly-imide [22]. In this process, glass covers are sealed ontoglass microchips by first hydrolyzing the glass surfaces andthen thermally bonding the assembled microchip [23]. PDMS-glass microchips are assembled by sealing the PDMS com-ponent onto the glass (e.g., a cover slip) at an elevated tem-perature (e.g., 80 °C) [15]. The two parts of polycarbonateintegrated microchip devices can be bonded by ultrasonicwelding or using adhesives, or held together with a clamp-ing device [16]. Finally, the contents of a microchip canbe protected against evaporation and the external environ-ment by simply covering the microchamber with oil [24].

Designs for PCR microchips range from wide cham-bers of varying sizes and depths (Fig. 1) to narrow chan-nels (linear or serpentine) (Fig. 2), and can have a singlereaction chamber or arrays of chambers for multiple si-multaneous reactions (Table 1) [25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45]. Vol-umes inside the microchips vary over the nL to µL range,and devices with volumes as low as 12 nL have been pro-duced [46]. The physical dimensions of microchips alsovary widely but most are approximately 1 cm×1 cm as amatter of convenience for handling the chips. The prospectof even smaller device is inherent in the fabrication pro-cesses available, but ultra-miniature devices would be moredifficult to handle and manipulate in a research and devel-opment process.

The ways of performing PCR on a microchip havebeen classified into a time domain and a space domain ap-proach [46].

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

Fig. 2 Schematic of a flow-through-type of PCR microchip. Theserpentine 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 Material Referencearchitecture

Reservoirglass [23, 25, 26, 27]

ChannelLinear polytetrafluoroethylene [28]

(PTFE)

Serpentine glass [29]silicon glass [30]polycarbonate [16]co-fired ceramics [17, 18]

ChamberSingle glass [31, 32]

silicon [22]silicon-glass [33, 34, 35, 36, 37, 38]polydimethylsiloxane [39](PDMS)-glassceramic tape [18]polyimide [40]

Multiple silicon [24, 41]silicon-glass [19, 21, 43, 44, 45]

1. Time domain PCR – In this format the reaction mix-ture is kept stationary and the temperature of the sur-rounding reaction chamber is cycled between the dif-ferent temperatures.

2. Space domain PCR – In this format the reaction mix-ture is moved between different fixed temperaturezones. The advantage of this strategy is that the devicedoes not have to be heated and cooled and this facili-tates faster cycling. In one embodiment a rotary devicecontinually cycles reaction mixture contained in a loopover different heaters [46]. A serpentine channel de-sign is used for continuous-flow microchip PCR. Thechannel runs back and forth across three heaters (60 °C,77 °C, 95 °C) to provide the required cycling [29]. A vari-ant of this general design incorporates outlets at differ-ent distances along the serpentine channel that allowproduct collection after 20, 25, 30, 35, and 40 cycles.Successive samples can be analyzed simultaneously byisolating 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 requiresrapid cycles of heating and cooling. The source of heat fora microchip PCR reaction can be an external heating block,or non-contact heating by infrared radiation [40], or heatersfabricated directly onto the surface of the microchip (e.g.,tungsten or platinum film) [17, 20, 48], Cooling can beachieved via forced air using a fan [49], or by means of aPeltier heater-cooler device [34]. It is highly desirable tohave the highest possible ramp rates for heating and cool-ing in order to minimize cycle times, and values as high as80 °C s–1 have been obtained for heating and 40 °C s–1 forcooling [44]. Some microchips incorporate specific fea-tures, such as grooves and air spaces [17, 24] designed toisolate the PCR chamber and minimize lateral heat trans-fer from the chamber to the bulk of the microchip.

Monitoring the temperature of a PCR microchip is im-portant because of the critical dependence of this reactionon accurate temperature control during the different cycles.A thermal sensor is often fabricated onto a chip along withthe heaters in order to monitor temperature and provide feed-back to the temperature controller. Another way of assess-ing temperature in a chip during thermocycling is using aninfrared camera. This remote monitoring method has theadvantage of not compromising the thermal properties ofthe microchip. Encapsulated thermochromic liquid crys-tals suspended in a liquid sample have been used to deter-mine the temperature uniformity of a 3×6 array of PCRmicrochambers (2-µL volume) and as a tool for optimiz-ing the thermal design of the device [43]. In one study,two formulations were employed, one with an operatingtemperature range of approximately 1 °C centered at 55 °C,and the other with an operating temperature range of ap-proximately 2 °C centered at 95 °C. The change in the hueof the liquid crystals with temperature was recorded with

a video camera. One minor disadvantage is that the den-sity of the encapsulated crystals did not match the densityof the fluid in the microchambers. The lighter high tem-perature crystals floated, and the more dense lower tem-perature crystals sank to the bottom of the chamber. Thisdid, however, facilitate assessment of temperature varia-tions at the floor and ceiling of the vessels. Modeling andsimulation have also been used to investigate aspects ofPCR microchip design [50].

Fluidic connections

Operation of a microchip requires convenient fluidic con-nections so that µL or sub-µL volumes of fluid can be in-troduced into the microchip and, if required, removed af-ter completion of the PCR reaction. In some protocols themicrochip is filled by capillary action by simply pipettingthe reaction mixture onto one of the entry ports. Otherprotocols attach a pump to the inlet port and the reactionmixture is pumped into the microchip.

Effective sealing of the microchip during thermal cy-cling is important in order to avoid leakage of the contentsduring the period of thermal cycling. Another issue isbubble formation due to leaks or evaporation of the mi-crochip contents. Bubbles may be relatively benign butcan cause significant differences in temperature (4–5 °C)and prevent effective PCR [48].

Materials and surface chemistry

Two factors complicate the design and construction ofPCR microchips. First, the PCR reaction is a multi-com-ponent reaction that includes reagents with diverse prop-erties – metal ions, buffers, oligonucleotide primers, dNTPs,enzymes. Hence the possibility of at least one of these com-ponents binding to some degree with an internal surface ina microchip is significant. Secondly, the surface area/vol-ume ratio is high in microdevices and this further increasesthe possibility of adverse interactions between the innersurface of a microchip and components of the reagent mix-ture (e.g., denaturation of Taq polymerase) or the sample(e.g., irreversible binding of the target). For example thesurface area/volume ratio of a PCR microchip can be20-fold or greater than the surface area/volume ratio of aconventional Microamp tube.

PCR microchips have been mostly fabricated from glassor glass and silicon, although other materials such as poly-imide and PDMS have also proved suitable constructionmaterials (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]. Earlywork with silicon-glass microchips revealed the problemsof adverse surface interactions and led to the developmentof passivation procedures to render the internal surfacesof microchips “PCR friendly”[34, 36, 36, 51].

Passivation procedures can be classified into two dif-ferent types: Type I – Static passivation and Type II – Dy-namic passivation (Table 2).

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Type I – Static passivation

In this type of passivation, the surface of a microchip is pre-coated with a substance, usually during microchip fabri-cation. For silicon-glass microchips deposition of an ox-ide coating and silanization are effective. An advantage ofthe oxide method is that it can be performed at the waferstage during microchip fabrication and it does not inter-fere with subsequent capping of the microchips with Pyrexglass using an anodic bonding process [51]. Other exam-ples of this type of passivation are silanization of internalsurfaces. This is accomplished by filling a chip with asilanizing agent and incubating the filled microchip for aperiod of time, then emptying and washing the microchip[36, 51]. A further variant is to insert an inner sleeve of aninert material into a microchip and so eliminate contactbetween the chip surface and the reaction mixture [54].Uncoated injection molded polycarbonates surfaces arereported to be inert in PCR although a coating of pary-lene-C was found to improve reproducibility [16]. Attemptsto passivate silicon surfaces with silicon nitride have beenunsuccessful [36, 51].

Type II – Dynamic passivation

This is a type of passivation that occurs during the filling andoperation of a microchip. It is accomplished by includingthe passivation reagent in the reaction mixture. Examplesof substances effective in this type of passivation proce-dure include polymers (e.g., PEG, PVP [29]), and proteinssuch as BSA (see Table 2). Presumably, these substancesbind preferentially to the inner surface of the microchip andprevent binding by components of the sample or reagentmixture.

Adverse surface properties of an otherwise PCR friendlymaterial can arise during microchip manufacture. Resid-ual chromium from chrome masks used to manufacture glassmicrochips on the surface of the glass is inhibitory to a PCRreaction and must be removed by strenuous washing pro-cedures [56].

Targets and detection of amplicons

A wide range of targets have been amplified in a micro-chip by PCR and its variants, RT-PCR and DOP-PCR [57].Amplicons are detected by removing the reaction mixturefrom the microchip, and then analyzing it by gel electro-phoresis, capillary electrophoresis [10, 23, 25, 31], orMALDI-TOF [58].

The transparent nature of a glass cover on a microchipfacilitates optical readout of the progress of a PCR reac-tion in the microchip in real time using a TaqMan assay[41, 45, 52, 59] or by monitoring the increase in fluores-cence due to intercalation of ethidium bromide into thedouble-stranded amplicons [52]. Amplicon yields in mi-crochip PCR that are superior and inferior to conventionalthermocycling yields have been reported [22, 35, 36].

Reusability

Most microchips are designed with clinical testing inmind and hence should be disposable. Our experience hasbeen that microchips are difficult and sometimes impossi-ble to clean for resuse, and validation of the effectivenessof cleaning poses considerable difficulties. A continuousflow borosilicate glass microchip has been successfullyreused (2 weeks of continuous usage). Blockages thatclogged the 55-µm deep channels were encountered butthe microchip could be cleaned by heating at 300 °C for 1 h. This process burns organic material, such as dena-tured protein from inside the channels [47].

Integration and pre-PCR and post-PCR

Various analytical steps that precede a PCR or are per-formed after completion of a PCR have been integratedonto the same microchip device [60]. These include pre-PCR separation and isolation of white blood cells usingfilters within a PCR chamber [38], and post-PCR capil-lary electrophoresis or capillary gel electrophoresis analy-sis of the PCR reaction mixture [23, 25, 31, 54]. Microar-rays have also been incorporated in PCR microchips forhybridization-based analysis of the PCR reaction products[55]. The arrays of oligonucleotides were printed onto thebottom of each of four 3-µL volume microchambers priorto sealing the glue coated-silicon microchip with a glasscover.

In one microchip device, the steps of cell lysis, multi-plex PCR and capillary electrophoresis analysis were per-formed sequentially and shown to be effective for the analy-

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Table 2 Passivation agents and procedures

Method Reference

Static passivationBovine serum albumin (BSA) [15, 31]Silicon oxide [34, 36, 37, 51]Silanization [30, 34, 36, 52, 53]Silanization + acrylamide polymer [32]Parylene-C [16]Polydimethylsiloxane (PDMS) [44]Epoxy poly(dimethylacrylamide)(EPDMA) [53]Polypropylene plastic liner [54]

Dynamic passivationBSA [31, 32, 42, 55]Polyethyleneglycol (PEG) [40, 53](molecular weight ≈8,000)Polyvinylpyrrolidone (PVP) [53](molecular weight ≈1 million)

Combined static and dynamic passivationBSA + BSA [31, 45]BSA + silanization [30]Silicon oxide layer + BSA [38]

sis of 154-, 264-, 346-, 410-, and 550-bp DNA target se-quences in whole Escherichia coli cells [25, 26]. A mi-crodevice that incorporates a PCR chamber has also beendeveloped for integrated multi-step genetic analysis [16].The device integrates the serial steps of extracting and con-centrating nucleic acids from a sample, PCR or RT-PCR,enzymatic fragmentation of amplicons, and a nucleic acidhybridization assay. The effectiveness of this device hasbeen demonstrated in an assay for the 1.6-kb region of theHIV genome.

Products

Several prototype microchip-based PCR analyzers have beendescribed (e.g., advanced nucleic acid analyzer (ANAA))[41]. However, despite strong indications that the devel-opment of microchip-based PCR analyzers are nearingcompletion, few have been commercialized. One exampleof a PCR microchip-based device is the miniature analyt-ical thermal cycling instrument (MATCHI) system (SmartCycler see www.cepheid.com). This is a battery-poweredportable, real-time, integrated analytical system based onPCR performed in an array of silicon microchambers [52,59, 61]. The entire system fits inside a briefcase for easeof transport. Applications identified for this nucleic acidanalysis device include forensic, environmental and agri-cultural analyses and detecting biowarfare agents [62, 63,64].

Patents

There are a series of issued patents directed at the generalarea of miniaturized reactors and more specifically at mi-crochip-based amplification reactions, such as PCR [65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75]. The patents citedare intended as a starting point for information on the in-tellectual property aspects of this branch of miniaturiza-tion. The interested reader is directed to the appropriateweb sites for further and more comprehensive informationon patents and patent applications (see www.uspto.govand www.delphion.com).

Conclusions

Microchip PCR has developed rapidly and now representsan important application for miniaturization, and mi-crochip-based PCR analyzers are now available. The cen-tral role of PCR in genetic tests, and the emerging need toperform PCR in the field as part of efforts to detect infec-tious agents will provide continued impetus for the devel-opment of miniature PCR analyzers and totally integratedanalyzers that incorporate PCR microchips. However, cur-rently the macroscale PCR devices dominate and the widescale implementation of microchip PCR depends on an in-dustry commitment to the development and commercial-ization of these devices, especially devices that integrate

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

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