Microfabricated reaction and separation systems.
ABSTRACT Over the past year there have been a number of recent advances in the fields of miniaturized reaction and separation systems, including the construction of fully integrated 'lab-on-a-chip' systems. Microreactors, which initially targeted DNA-based reactions such as the polymerase chain reaction, are now used in several other chemical and biochemical assays. Miniaturized separation columns are currently employed for analyzing a wide variety of samples including DNA, RNA, proteins and cells. Although significant advances have been made at the component level, the realization of an integrated analysis system still remains at the early stages of development.
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ABSTRACT: A micromachined capillary electrophoresis chip is described for simultaneous measurements of glucose, ascorbic acid, acetaminophen, and uric acid. Fluid control is used to mix the sample and enzyme glucose oxidase (GOx). The enzymatic reaction, a catalyzed aerobic oxidation of glucose to gluconic acid and hydrogen peroxide, occurs along the separation channel. The enzymatically liberated neutral peroxide species is separated electrophoretically from the anionic uric and ascorbic acids in the separation/reaction channel. The three oxidizable species are detected at the downstream gold-coated thick-film amperometric detector at different migration times. Glucose can be detected within less than 100 s, and detection of all electroactive constituents is carried out within 4 min. Measurements of glucose in the presence of acetaminophen, a neutral compound, are accomplished by comparing the responses in the presence and absence of GOx in the running buffer. The reproducibility of the on-chip glucose measurements is improved greatly by using uric acid as an internal standard. Factors influencing the performance, including the GOx concentration, field strength, and detection potential, are optimized. Such coupling of enzymatic assays with electrophoretic separations on a microchip platform holds great promise for rapid testing of metabolites (such as glucose or lactate), as well as for the introduction of high-speed clinical microanalyzers based on multichannel chips.Analytical Chemistry 07/2000; 72(11):2514-8. · 5.70 Impact Factor
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ABSTRACT: A homogeneous binding assay for the detection of biotin in picoliter vials was developed using the photoprotein aequorin as the label. The binding assay was based on the competition of free biotin with biotinylated aequorin (AEQ-biotin) for avidin. A sequential protocol was used, and modification of the assay to reduce the number of steps was examined. Results showed that detection limits on the order of 10(-14) mol of biotin were possible. Reducing the number of steps provided similar detection limits but only if the amount of avidin used was decreased. These binding assays based on picoliter volumes have potential applications in a variety of fields, including microanalysis and single-cell analysis, where the amount of sample is limited. In addition, these assays are suitable for the high-throughput screening of biopharmaceuticals.Analytical Chemistry 07/2000; 72(11):2590-4. · 5.70 Impact Factor
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ABSTRACT: Liquid crystalline solutions of Pluronic copolymers are versatile alternatives to solutions of entangled, random coil polymers as replaceable media for capillary gel electrophoresis (CGE). Pluronic copolymers are tri-block polymers of poly(ethylene oxide) [(EO)x] and poly(propylene oxide) [(PO)y] with the general formula (EO)x(PO)y(EO)x. Large micelles form in aqueous solutions in which central, hydrophobic cores of (PO)y segments are surrounded by "brushes" of hydrated (EO)x tails. Solutions of Pluronic F127 (BASF Performance Chemicals) in a concentration range of about 18-30% are liquids at refrigerator temperatures (< or = 5 degrees C) and are easily introduced into capillaries. A self-supporting, gel-like liquid crystalline phase is formed as the temperature is raised to > or = 20 degrees C. This liquid crystalline phase consists of spherical micelles with diameters of 17-18 nm which pack with local cubic symmetry. CGE in Pluronic F127 liquid crystals separates species within several chemical classes as varied as nucleoside monophosphates and organic dyes, oligonucleotides of 4-60 nucleotides, DNA fragments of 50-3000 base pairs (bp), and supercoiled plasmid DNAs of 2000-10,000 bp. Mechanisms of molecular sieving in polymer liquid crystals must differ in fundamental ways from separations in random polymer gels because molecules move around uncrosslinked obstacles that are larger than the smallest dimensions of typical analytes. Molecular sieving in Pluronic liquid crystals is envisioned to occur as molecules squeeze between hydrated (EO)x strands of micelle brushes, or through brushtips and interstitial spaces between micelles. Small molecules such as nucleotides appear to separate by a different mechanism involving partitioning between hydrophilic and hydrophobic environments. This process is termed "hydrophobic interaction electrophoresis". The unique structures of Pluronic copolymers and their liquid crystalline phases provide new challenges and opportunities in separations science.Journal of Chromatography 08/1998; 817(1-2):287-95. · 4.61 Impact Factor
Over the past year there have been a number of recent
advances in the fields of miniaturized reaction and separation
systems, including the construction of fully integrated ‘lab-on-a-
chip’ systems. Microreactors, which initially targeted DNA-
based reactions such as the polymerase chain reaction, are
now used in several other chemical and biochemical assays.
Miniaturized separation columns are currently employed for
analyzing a wide variety of samples including DNA, RNA,
proteins and cells. Although significant advances have been
made at the component level, the realization of an integrated
analysis system still remains at the early stages of development.
Department of Chemical Engineering and Department of Biomedical
Engineering, 2300 Hayward, 3022 HH Dow, University of Michigan,
Ann Arbor, MI 48109-2136, USA
Current Opinion in Biotechnology 2001, 12:92–98
0958-1669/01/$ — see front matter
© 2001 Elsevier Science Ltd. All rights reserved.
Photolithographic microfabrication is a mature technology
developed and optimized by the microprocessor industry.
Semiconductor microfabrication serves as an excellent plat-
form for developing miniature integrated analysis systems
with short analysis times, reduced sample-volume require-
ments and cost efficiency. These microanalysis devices can
be classified into two broad categories based on the com-
plexity of the fluidics involved: microarray-based (DNA or
protein immobilized on the chip) and microfluidic-based
(DNA or proteins being transported, reacted and separated
on the chip) microdevices . In this review, we focus on
recent developments in microfluidic systems for the analy-
sis of DNA, proteins and other biomolecules.
There has been a burst of activity in the analysis of biomol-
ecules other than DNA and proteins using microdevices.
Biosensors have been developed for the detection and analy-
sis of physiologically relevant molecules, such as glucose,
lactic acid and ascorbic acid [2•], and detection of environ-
mental agents and herbicides, such as 2,4-diphenoxyacetic
acid . In the area of DNA analysis, reaction volumes range
from a few microliters [4•] to a few hundred nanoliters ,
and although reaction volumes can easily be reduced further,
speculation persists on whether this is indeed a viable ven-
ture when developing diagnostic devices for the detection of
infectious agents. This caution is because of the fact that
sample volume statistically limits the number of available
targets for the detection in a given assay .
In this review, we discuss recent advances in various com-
ponents of the microfluidic analysis system and also report
attempts at constructing integrated microanalysis systems.
Microfabricated reaction systems
The general trend in the area of microfabricated DNA
analysis is toward devices with multiple functions that per-
form multiple reactions in series and/or parallel. The major
operations now performed in DNA analysis devices
include cell lysis, sample concentration and enzymatic
reactions such as reverse transcription, PCR, DNase diges-
tion and terminal transferase labeling [4•,7] (Figure 1).
Several groups also report combined PCR and elec-
trophoretic analysis of reaction products [5,7,8]. Sanger
DNA sequencing was performed in a solid-phase nanore-
actor directly coupled to capillary gel electrophoresis .
With the current interest in performing PCR in a
microchip, the choice of substrate material has become an
important issue. The trade-off in materials reduces the
need for low-thermal conductivity for thermal isolation in
a multiple reaction device versus the need for higher con-
ductivity for effective heat removal for rapid cycling in
PCR. Polycarbonate and glass devices have been reported
for on-chip PCR in conjunction with other reactions [4•]
and an electrophoresis module [5,7]. In addition to thermal
isolation, a significant challenge in performing PCR in
microfabricated devices is associated with controlling the
evaporation of the reaction. This problem has been
addressed through the use of diaphragm valves [4•].
An important area in DNA analysis is the analysis of gene
expression. Gene expression analysis on the microscale has
traditionally been performed by hybridization in microarrays.
A recent development on the conventional method of pas-
sive, non-active hybridization is dynamic hybridization using
DNA probes pumped through target-bearing paramagnetic
beads . Traditional hybridization systems are not elec-
tronically active; Radtkey et al. [11•] describe a method for
the discrimination of short tandem repeat (STR) alleles
based on active microarray hybridization. A rapid analysis of
STR alleles using passive hybridization techniques (required
in non-active microarrays) is currently very difficult.
In the area of protein analysis in microchips, a device has
been demonstrated that performs enzymatic reactions,
Microfabricated reaction and separation systems
Madhavi Krishnan*, Vijay Namasivayam†, Rongsheng Lin‡, Rohit Pal§and
Mark A Burns#
Microfabricated reaction and separation systems Krishnan et al. 93
electrophoretic separation of the reactants from the prod-
ucts, and post-separation labeling of proteins and peptides
prior to detection . In this work, the authors performed
tryptic digestion of the insulin B chain and reduction of the
disulfide bridges of insulin on a microchip . Another
area that appears to have benefited significantly from
increasing interest in the development of microanalysis
devices for a wide variety of biomolecules has been the
area of immunoassays. Immunoassays typically require
very high specificity and are time-consuming and expen-
sive. Reports on chip-based immunoassays usually focus
on separation of the free form of the antigen from the anti-
gen–antibody complex by capillary electrophoresis (CE);
there have been few reports on the antigen–antibody reac-
tion performed on-chip . In a recent report on a
microdevice-based immunosorbent assay, detection of
secretory human immunoglobulin A was performed on
polystyrene beads in a microchip . The time required
for the assay was reduced by two orders of magnitude com-
pared to the traditional assay in microtiter plates.
In clinical diagnostics, although they return chemical infor-
mation rapidly, microanalysis devices have limits in
versatility and scope because of minimal sample-handling
capability (associated with their limited surface chemistry).
Notwithstanding, there have been reports in the literature
on bioassays for clinically relevant molecules. One such
report is a reaction/electrophoresis chip for simultaneous
bioassays of glucose, uric acid, ascorbic acid and aceta-
minophen [2•]. A binding assay for biotin has been
reported in a microfabricated picoliter vial . Results
indicate that detection limits of the order of 10–14 mol of
biotin are possible. These binding assays based on pico-
liter volumes have potential applications in a variety of
fields including microanalysis and single-cell analysis
(where the amount of sample is limited) as well as in high-
throughput screening of biopharmaceuticals. Along these
lines, from the perspective of environmental microdevices,
the sensing of biological substances based on the bending
of microfabricated cantilevers has been demonstrated in
the detection of the herbicide 2,4-dichlorophenoxyacetic
(a) An integrated device that automatically
performs a multi-step HIV genotyping assay.
(b) The steps include RNA extraction, RT-
PCR, nested PCR, DNase fragmentation and
dephosphorylation, terminal transferase
labeling, dilution and hybridization, washing,
phycoerithrin staining and washing. S1–S6
are the reagent storage chambers, R1–R6 are
the reaction chambers and M1–M4 are the
intermediate product storage chambers.
Reproduced from [4•] with permission.
Intermediate product samples
RT-PCRPCR Fragm ent Label
R3R4 R2R1 R5
Target preparation zonesHybridization zone
acid (2,4-D) . The cantilevers were coated with 2,4-D
and the deflection was measured while continuously rins-
ing with a solution containing monoclonal antibody.
On another front, there has been significant activity in the
recent past in the area of culturing cells and tissues in micro-
fabricated devices. An important and interesting advance in
cell culture in microdevices is the neurochip, a silicon micro-
machined multielectrode device upon which cultured
mammalian neurons can be continuously and individually
monitored and stimulated. The neurochip significantly
improves on earlier methods by associating individual elec-
trodes with the cell bodies of each of the neurons in a small
network [16••]. The neurochip is based on a 4 ×4 array of
metal electrodes, each of which has a caged well structure
designed to hold a single mature cell body while permitting
normal outgrowth of neural processes. The device is capable
of maintaining cell survival, and the electrodes can both record
and stimulate electrical activity with no crosstalk between
channels. In addition, surface microfabrication techniques
have been widely used for the spatial control of cells in culture.
Many strategies have employed variations in surface charge,
hydrophilicity and topology to regulate cell functions such as
attachment. Ito has published a good review in this area .
Separation and detection systems
Microfabricated separation systems have become integral
components for chemical analysis after the critical evalua-
tion of the benefits of miniaturization was presented by
Manz et al. in 1993 . A large number of articles published
in recent years offer insights into fabrication procedures,
choice of materials, design considerations and mode of oper-
ation of microchips for separations applied to a wide variety
of analytes [19–22,23•]. DNA continues to be the main mol-
ecule of interest; other molecules being studied include
RNA, proteins and peptides. A noticeable trend in
microseparations has been the dominance of electrophoret-
ic techniques over chromatographic techniques. This trend
is predominantly because pumping liquids in chromato-
graphic separation systems requires more engineering effort
than electrophoretic systems; electrokinetic systems can be
operated by simply changing the applied voltages. Another
significant trend is the constant push towards improving
separation power (higher resolution, faster analysis times)
while keeping the separation lengths short.
One principal area of interest is improving the quality of
separation by altering the type, composition and the quality
of sieving media. A wide variety of existing polymers and
novel materials are being investigated to achieve enhanced
resolution and quality of separation [24–29]. For DNA sep-
arations, both crosslinked and non-crosslinked gels are
being investigated. Crosslinked gels offer the advantage of
shorter separation lengths and lower applied voltages but
suffer from nonreusability [28,29]. Non-crosslinked gels
operate under relatively high voltages but offer the desirable
feature that the sieving matrices can be exchanged after
each run [24–27]. Single base pair resolution up to 800 bases
using denaturing linear polyacrylamide solutions run under
optimized conditions has been reported [30•]. Capillary
array electrophoresis for massive parallelization of elec-
trophoresis has also been demonstrated [31,32••] (Figure 2).
Separation resolution also depends on the sample injection
and applied voltage. Several types of injection geometries
including simple T, cross and double T configurations have
been reported for sequencing DNA . Microfabricated
porous membrane structures have been constructed to con-
centrate DNA samples before injection [34•]. High voltages
required for running non-crosslinked CE systems can easi-
ly be attained by microfabricating metal electrodes within
the microchannel. Many researchers are investigating the
most optimal conditions to obtain high speed/resolution
sequencing; several papers provide fundamental insights
on band dispersion and suggest ways to reduce band
Schematic of a 96-channel radial capillary electrophoresis microplate.
The device has separation channels (100 µm wide and 50 µm deep)
with 200 µm twin-T injectors. Detection is performed using a laser-
excited galvoscanner. In this system, 96 different DNA samples can be
injected, separated and detected in less than 8 min. Reproduced from
[32••] with permission.
Microfabricated reaction and separation systems Krishnan et al.95
Advances in microfabrication and nanofabrication techniques
have opened doors for novel ways of separating biomolecules.
Cell sorting has been demonstrated in microfabricated arrays
. Long DNA molecules have been separated in microfab-
ricated asymmetric obstacle courses [39••] and entropic trap
arrays [40••]. Single mol-ecule sizing and sorting devices
made out of silicone elastomer have been developed .
Sodium dodecyl sulfate (SDS) capillary gel electrophoresis of
proteins has been demonstrated in planar microchannels
resulting in faster separations while retaining macroscale res-
olution . A microfluidic diffusion-based separation system
has been reported for extracting small molecules from
blood [43••]. In the future we will probably witness DNA
sequencing and oligonucleotide separations performed in
novel, custom-made media constructed from materials such
as carbon nanotubes. Also, microseparation devices made out
of plastic substrates by simple injection molding and/or hot
embossing techniques will become more widespread and will
reduce fabrication costs considerably .
The power of a miniaturized chemical analysis system is
ultimately limited by the ability to detect low concentra-
tions of the analyte. Microseparation systems currently rely
on one of the three major detection modes: fluorescence,
electrochemical (EC) detection or chemiluminescence (CL)
detection . Fluorescence detection is an accurate, time-
tested technique, and the dyes currently used are extremely
sensitive to the analytes, permitting low concentration
detection in femtoliter samples. The primary method of this
detection system for DNA detection is laser-induced fluo-
rescence, which can detect single molecules of DNA on CE
chips [46•]. Improvements have been made by introducing
optical fiber liquid core waveguides [47–49]; however,
fluorescence detection requires a large and expensive sup-
porting optical system that minimizes the advantage of cost
and portability. Development of on-chip fluorescence
detectors has been reported and will aid in the realization of
integrated ‘lab-on-a-chip’ systems .
EC detection offers considerable promise for detection in
micromachined chips because of the remarkable sensitivi-
ty, tunable selectivity and low volume required. CE chips
with integrated EC detectors were made in 1998 .
Subsequently, Wang et al.  reported a different
microchip CE-EC system with thick-film EC detectors
that provided lower detection limits than those previously
reported. Detection of single-stranded DNA by using
polymer-modified electrodes has also been demonstrated
. Recently, Martin et al.  reported a CE-EC
microchip, fabricated in polydimethylsiloxane that
employs dual electrodes for detection.
CL detection has been widely used for the analysis of metal
ions and immunoassays, because no light source is required in
CL measurements and the instruments for CL are much
simpler than those for optical methods. This method has the
potential to be integrated onto a chip for analyte detection
. Hostis et al.  reported an electrochemiluminescence
(ECL) detector and a microenzymatic reactor combining
Schematic and photograph of an integrated
device with a nanoliter liquid injector, a
sample mixing and positioning system, a
temperature-controlled reaction chamber, an
electrophoretic separation system and
fluorescence detectors. The device is capable
of measuring aqueous reagents and DNA-
containing solutions, mixing the solutions
together, amplifying or digesting the DNA to
form discrete products and separating and
detecting those products. Reproduced from
 with permission.
silicon and polymer technologies. The use of CL is not wide-
spread; more research is needed to produce robust and/or
universal probes to increase the technique’s applicability.
The extraction of useful analytical information, such as
DNA sequence, from a sample involves a series of chemi-
cal manipulations. These manipulations include metering
and mixing of reagents, thermal cycling, labeling and frag-
ment analysis. An integrated miniaturized chemical
analysis device is a system capable of performing all of the
above operations in microscale and nanoscale volumes.
Microfluidic components such as channels, valves, and
pumps form the core of integrated devices. A wide variety
of micromachined valves and pumps have been reported
[56,57]; however, to date, none of them have been integrat-
ed onto complex analysis systems involving reaction and
separation components. The key solution to integration is
to have robust valves and pumps that involve simple fabri-
cation procedures and (preferably) do not involve moving
parts . Electro-osmotic pumps have found wide accep-
tance in separation devices mainly for this reason .
Though the power of integration is widely appreciated ,
very little progress towards constructing integrated devices has
been reported [61,62]; however, several functional integrated
devices have been reported that employ macroscale assembly
of components as opposed to fabricating all the components
on the same platform [7,8,12]. Also, there have been very few
published reports on sample preparation, a potentially impor-
tant area for integrated microdevices (Figure 3).
In the future we shall witness advances at the component
level: higher resolution separation systems, smaller volume
reaction chambers, and ultrasensitive detectors. Novel
materials will be used for fabrication: plastic microchannels,
nanostructured sieving matrices, and polymer-based light
emitting diodes (LEDs) and detectors. Trends in commer-
cialization show the push towards plastic substrates that can
be processed by cheaper fabrication techniques like injec-
tion moulding, hot embossing, and casting [63••,64–66];
however, conventional photolithographic machining of sili-
con and glass will still have a place in several applications,
particularly where active on-chip electronics is essential.
To actually accomplish genetic or other biochemical analy-
sis, individual micromachined components are more
powerful when linked together to function as an integrat-
ed device; this will continue to be the focus of many
research groups. Component development without some
integration will not find widespread commercial applica-
tions of microchips. However, not all components need to
be integrated for all applications.
The greatest impact of these integrated microchips will
most likely be in the area of personalized healthcare involv-
ing the analysis of infectious diseases, the identification of
genetic predisposition to diseases, and treatment of the
same using custom-designed drugs. Personal identification
based on DNA sequence will eventually be accomplished
using microchip scanners. Portable chemical analysis
devices will find applications in forensics, agriculture, and
the exploration of space. In the not too distant future,
microfabricated chemical reaction and separation systems
will be as prevalent as microprocessors are today.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
••of outstanding interest
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Wang J, Chatrathi MP, Tian B, Polsky R: Microfabricated
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electrophoretic separations, and amperometric detection is demonstrated.
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A method is described for the discrimination of short tandem repeat alleles
based on active microarray hybridization. An essential factor in this method
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