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 poly-
merase chain reaction (PCR). It examines the different types
of PCR microchip designs, fabrication methods,and the com-
ponents of a microchip PCR device. It also discusses the
problems attributable to surface chemistry of microchip
components (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 analysis
over 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 goal
of 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 analyst
would merely add sample and the chip would automati-
cally 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 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 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: kricka@mail.med.upenn.edu
assays and to make them suitable for extra-laboratory ap-
plications (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, consider-
able 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 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 at
a temperature of approximately 95 °C.
2. Next, each of the two single strands of DNA are hy-
bridized (annealed) to pairs of oligonucleotide primers
at approximately 55 °C.
3. In the final step, a thermostable magnesium ion-depen-
dent polymerase derived from Thermophilus aquaticus
(Taq polymerase) synthesizes complementary DNA in
the region flanked by the primers using added de-
oxynucleotide 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 re-
action 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 ef-
forts towards this goal. A central component of such a de-
vice is a miniaturized PCR chamber (a PCR microchip) or
an array of chambers for multiple simultaneous PCR reac-
tions. 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 be-
tween specimens, and hence, would need to be relatively
inexpensive in order to make this mode of analysis eco-
nomically viable. Other important considerations for any
 821

PCR-based tests are sample acquisition and the sensitivity

required in order to detect amplicons generated in a mi-
crodevice [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 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 fabricated

using photolithographic techniques. PCR microchips made Fig. 1 A pille of silicon-glass PCR microchips (17 mm × 14 mm)
from 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-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 as-
sembled into a leak-proof final device. Silicon-glass mi-
crochips are usually assembled using a high temperature
anodic bonding process. An alternative method involves Fig. 2 Schematic of a flow-through-type of PCR microchip. The
gluing the two components with a UV glue [19, 20]. An- serpentine reaction microchannel crosses each of three zones (T1,
other approach is to place the glass cover on top of the sil- T2, T3) each of which is set at a different temperature
icon chip, seal the edges with varnish and maintain the seal
by placing a weight on top of the glass cover [21]. Silicon Table 1 Microchip PCR vessel designs
chips can be bonded with a low temperature curing poly- Vessel Material Reference
imide [22]. In this process, glass covers are sealed onto architecture
glass microchips by first hydrolyzing the glass surfaces and
then thermally bonding the assembled microchip [23]. PDMS- Reservoir
glass microchips are assembled by sealing the PDMS com- glass [23, 25, 26, 27]
ponent onto the glass (e.g., a cover slip) at an elevated tem- Channel
perature (e.g., 80 °C) [15]. The two parts of polycarbonate Linear polytetrafluoroethylene [28]
integrated microchip devices can be bonded by ultrasonic (PTFE)
welding or using adhesives, or held together with a clamp- Serpentine glass [29]
ing device [16]. Finally, the contents of a microchip can silicon glass [30]
be protected against evaporation and the external environ- polycarbonate [16]
ment by simply covering the microchamber with oil [24]. co-fired ceramics [17, 18]
Designs for PCR microchips range from wide cham- Chamber
bers of varying sizes and depths (Fig. 1) to narrow chan- Single glass [31, 32]
nels (linear or serpentine) (Fig. 2), and can have a single silicon [22]
reaction chamber or arrays of chambers for multiple si- silicon-glass [33, 34, 35, 36, 37, 38]
multaneous reactions (Table 1) [25, 26, 27, 28, 29, 30, 31, polydimethylsiloxane [39]
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45]. Vol- (PDMS)-glass
umes inside the microchips vary over the nL to µL range, ceramic tape [18]
and devices with volumes as low as 12 nL have been pro- polyimide [40]
duced [46]. The physical dimensions of microchips also Multiple silicon [24, 41]
vary widely but most are approximately 1 cm×1 cm as a silicon-glass [19, 21, 43, 44, 45]
matter of convenience for handling the chips. The prospect
of even smaller device is inherent in the fabrication pro-
cesses available, but ultra-miniature devices would be more The ways of performing PCR on a microchip have
difficult to handle and manipulate in a research and devel- been classified into a time domain and a space domain ap-
opment process. proach [46].
822
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 temperature
zones. The advantage of this strategy is that the device
does not have to be heated and cooled and this facili-
tates faster cycling. In one embodiment a rotary device
continually cycles reaction mixture contained in a loop
over different heaters [46]. A serpentine channel de-
sign 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 vari-
ant of this general design incorporates outlets at differ-
ent 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 cool-
ing 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 fea-
tures, such as grooves and air spaces [17, 24] designed to
isolate 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 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 feed-
back to the temperature controller. Another way of assess-
ing 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 crys-
tals suspended in a liquid sample have been used to deter-
mine the temperature uniformity of a 3×6 array of PCR
microchambers (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 operating
temperature 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 hue
of 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 density
of 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. This
did, however, facilitate assessment of temperature varia-
tions 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 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 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 cy-
cling 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 mi-
crochip 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-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 in
a microchip is significant. Secondly, the surface area/vol-
ume 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 mix-
ture (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 poly-
imide 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 dif-
ferent types: Type I – Static passivation and Type II – Dy-
namic passivation (Table 2).

Table 2 Passivation agents and procedures
Method
Static passivation
Bovine serum albumin (BSA)
Silicon oxide
Silanization
Silanization + acrylamide polymer
Parylene-C
Polydimethylsiloxane (PDMS)
Epoxy poly(dimethylacrylamide)(EPDMA)
Polypropylene plastic liner
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
Type I – Static passivation
823
Adverse surface properties of an otherwise PCR friendly
Reference
material can arise during microchip manufacture. Resid-
ual chromium from chrome masks used to manufacture glass
microchips on the surface of the glass is inhibitory to a PCR
[15, 31] reaction and must be removed by strenuous washing pro-
[34, 36, 37, 51] cedures [56].
[30, 34, 36, 52, 53]
[32]
[16] Targets and detection of amplicons
[44]
[53] A wide range of targets have been amplified in a micro-
[54] chip by PCR and its variants, RT-PCR and DOP-PCR [57].
Amplicons are detected by removing the reaction mixture
[31, 32, 42, 55] from the microchip, and then analyzing it by gel electro-
[40, 53] phoresis, capillary electrophoresis [10, 23, 25, 31], or
MALDI-TOF [58].
[53] The transparent nature of a glass cover on a microchip
facilitates optical readout of the progress of a PCR reac-
tion in the microchip in real time using a TaqMan assay
[31, 45] [41, 45, 52, 59] or by monitoring the increase in fluores-
[30] cence due to intercalation of ethidium bromide into the
[38] double-stranded amplicons [52]. Amplicon yields in mi-
crochip PCR that are superior and inferior to conventional
thermocycling yields have been reported [22, 35, 36].

In this type of passivation, the surface of a microchip is pre- Reusability

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 of
the oxide method is that it can be performed at the wafer
stage during microchip fabrication and it does not inter-
fere with subsequent capping of the microchips with Pyrex
glass using an anodic bonding process [51]. Other exam-
ples 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 pary-
lene-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 proce-
dure 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.
Most microchips are designed with clinical testing in
mind and hence should be disposable. Our experience has
been that microchips are difficult and sometimes impossi-
ble 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 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 integrated
onto the same microchip device [60]. These include pre-
PCR separation and isolation of white blood cells using
filters 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 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, multi-
plex PCR and capillary electrophoresis analysis were per-
formed sequentially and shown to be effective for the analy-
824
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 been
developed 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 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 devel-
opment of microchip-based PCR analyzers are nearing
completion, few have been commercialized. One example
of a PCR microchip-based device is the miniature analyt-
ical 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 agri-
cultural 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 mi-
crochip-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 in-
tellectual property aspects of this branch of miniaturiza-
tion. 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 mi-
crochip-based PCR analyzers are now available. The cen-
tral role of PCR in genetic tests, and the emerging need to
perform 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 integrated
analyzers that incorporate PCR microchips. However, cur-
rently the macroscale PCR devices dominate and the wide
scale 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 step
prior to PCR.
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