Biomed Microdevices (2008) 10:21–33
DOI 10.1007/s10544-007-9106-y
A titer plate-based polymer microfluidic platform for high
throughput nucleic acid purification
D. S.-W. Park & M. L. Hupert & M. A. Witek &
B. H. You & P. Datta & J. Guy & J.-B. Lee & S. A. Soper &
D. E. Nikitopoulos & M. C. Murphy
Published online: 21 July 2007
# Springer Science + Business Media, LLC 2007
Abstract A 96-well solid-phase reversible immobilization
(SPRI) reactor plate was designed to demonstrate functional
titer plate-based microfluidic platforms. Nickel, large area
mold inserts were fabricated using an SU-8 based, UVLIGA technique on 150 mm diameter silicon substrates.
Prior to UV exposure, the prebaked SU-8 resist was flycut
to reduce the total thickness variation to less than 5 μm.
Excellent UV lithography results, with highly vertical
sidewalls, were obtained in the SU-8 by using an UV filter
to remove high absorbance wavelengths below 350 nm.
Overplating of nickel in the SU-8 patterns produced high
quality, high precision, metal mold inserts, which were used
D. S.-W. Park : M. L. Hupert : M. A. Witek : B. H. You : J. Guy :
S. A. Soper : D. E. Nikitopoulos : M. C. Murphy
Center for Bio-Modular Multi-Scale Systems,
Louisiana State University,
Baton Rouge, LA 70803, USA
M. L. Hupert : M. A. Witek : S. A. Soper
Department of Chemistry, Louisiana State University,
Baton Rouge, LA 70803, USA
B. H. You : D. E. Nikitopoulos : M. C. Murphy (*)
Department of Mechanical Engineering,
Louisiana State University,
Baton Rouge, LA 70803, USA
e-mail: [email protected]
P. Datta
Center for Advanced Microstructures and Devices (CAMD),
Louisiana State University,
Baton Rouge, LA 70806, USA
J.-B. Lee
Erik Jonsson School of Engineering and Computer Science,
University of Texas at Dallas,
Dallas, TX 75083, USA
to replicate titer plate-based SPRI reactors using hot
embossing of polycarbonate (PC). Optimized molding
conditions yielded good feature replication fidelity and
feature location integrity over the entire surface area.
Thermal fusion bonding of the molded PC chips at 150°C
resulted in leak-free sealing, which was verified in leakage
tests using a fluorescent dye. The assembled SPRI reactor
was used for simple, fast purification of genomic DNA from
whole cell lysates of several bacterial species, which was
verified by PCR amplification of the purified genomic DNA.
Keywords Titer plate . Multi-well microfluidic platform .
Solid-phase reversible immobilization . UV-LIGA .
Large area mold insert . Micro molding .
Nucleic acid purification
1 Introduction
The standard titer plate, with a 127.76 mm by 85.48 mm
footprint (SBS 2004), is currently used to carry out multiple
reactions in parallel in separate wells for high throughput
applications. Rapid interrogation of many biochemical
samples in the standard titer plate is needed for such
diverse applications as high throughput screening for drug
discovery, biological discovery in genomics and proteomics, medical diagnostics, forensics and homeland defense
(Darvas et al. 2004; Honma et al. 2004). However,
conventional titer plates typically serve only one function,
containment of the sample and appropriate reagents in the
wells with processing implemented by placing the titer
plate in a bench top instrument, such as a thermal cycler,
ultra-centrifuge, or capillary array.
Microfluidic platforms produced via microelectromechanical system (MEMS) fabrication techniques, known
22
as micro total analysis systems (μTAS) or lab-on-a-chip
systems, are expected to be alternatives to the bulky, bench
top commercial instruments currently in use. They offer
many advantages including reduced reagent consumption,
shorter analysis times, the capability for integration of
multiple microfluidic components, potential for automation,
and significantly reduced manufacturing costs via mass
production (Auroux et al. 2002; Erickson et al. 2004;
Huang et al. 2002; Lee and Lee 2004; Reyes et al. 2002).
Microfluidic devices have been widely used in standard
analytical operations and applications such as cell separation, cell lysis, DNA preconcentration and purification,
amplification using the polymerase chain reaction (PCR)
and detection (Auroux et al. 2004; Huang et al. 2002; Liu et al.
2003; Obeid et al. 2003; Paegel et al. 2002; Pal et al. 2005).
Most microfluidic platforms have been manufactured in
Si or glass, because of their excellent mechanical and
chemical properties, with the use of highly precise and
reproducible microfabrication processes including thin film
deposition, lithography, etching, and substrate bonding
(Paegel et al. 2002; Ziaie et al. 2004). Microfluidic devices
can also be realized in polymers from a large selection of
different polymer materials with different combinations of
ease of surface modification, biocompatibility, disposability, and mass producibility. Polymer microfluidic devices
have been successfully demonstrated in materials such as
poly(methyl methacrylate), PMMA (Qi et al. 2002),
polycarbonate, PC (Barrett et al. 2004; Mitchell et al.
2003; Witek et al. 2006), cyclo olefin copolymer, COC
(Kim et al. 2006), and poly(dimethylsiloxane), PDMS (Liu
et al. 2003). These devices can be produced using micromolding techniques, such as injection molding or hot
embossing (Heckele and Schomburg 2004; Kim et al. 2006).
Incorporation of a microfluidic device at each well
location in a standard titer plate format can significantly
enhance the functional capability of the plate for the rapid
analysis of a large number of biochemical samples at
significantly lower costs. Microfabrication of 96-well capillary electrophoresis devices was demonstrated using micromilling and hot embossing (Gerlach et al. 2002a, b; Guber et
al. 2004), but minimum feature dimensions and pattern
densities were limited by the size of the finger mills used.
Feature dimensions and pattern densities can be improved by using either the LIGA or UV-LIGA processes.
Even though LIGA has the potential for use in applications
over large areas, as would be required for a titer-plate
format (10,920 mm2), using a step-and-repeat exposure
would require the implementation of scanning protocols
and the design of the fluidic networks that reduce the
effects of stitching errors at exposure boundaries.
An UV-LIGA technique with SU-8 UV lithography and
nickel electroplating was adopted to realize such titer platebased microfluidic platforms. The SU-8 based UV-LIGA
Biomed Microdevices (2008) 10:21–33
process on 150 mm diameter silicon substrates enabled use
of a single UV-exposure to make polymer electroplating
templates, highly packed with small features (<50 μm)
without any stitching errors. Metal large area mold inserts
(LAMIs) fabricated by a SU-8 UV-LIGA technique were
used to micro mold PC using hot embossing to manufacture
the polymer microfluidic platforms. The quality of the
sealing of the PC chips assembled by thermal fusion
bonding was verified experimentally.
As a demonstration of a functional titer plate-based
microfluidic platform, a 96-well solid-phase reversible
immobilization (SPRI) reactor was designed. SPRI is a
solid phase extraction technique that can be used to purify
nucleic acids of a variety of sizes and formats from
complex biological matrices (DeAngelis et al. 1995; Elkin
et al. 2001; Elkin et al. 2002; Hawkins et al. 1994).
Recently, solid-phase purification of nucleic acids, such as
DNA sequencing fragments and genomic DNA (gDNA)
from whole cell lysates (Witek et al. 2006; Xu et al. 2003),
was demonstrated using UV-exposed PC-based microdevices. These reactors were limited to a single channel format and not appropriate for high throughput applications.
The SPRI reactor concept was extended to a 96-well titer
plate format to enable high throughput sample purification.
2 Methods
2.1 Design and operation of the 96-well SPRI reactor
SPRI technologies are a solid-phase capture approach, so
maximizing the extraction bed surface area is important for
producing a high load of the extracted material. SPRI beds
with arrays of microposts were developed to increase the
available load of purified material without significantly
increasing the footprint of the device. Multi-well SPRI
reactors were designed with two different sizes of microposts; in version 1, the nominal post diameter (d) was
10 μm with a center-to-center spacing of 20 μm; version 2
contained a post diameter and center-to-center spacing of
20 μm and 40 μm, respectively. Figure 1 shows a view of
the 96-well SPRI plate, which is a simple flow-through type
reactor with a standard titer plate footprint. It consisted of
two microfluidic control ports (ports P1 and P2), a microchannel network, and 96 immobilization capture beds and
reservoirs at each well location [Fig. 1(a),(b)]. Using the
standard 96-well SPRI format ensured compatibility with
standard multi-pipettes and/or robotic equipment for loading the plate with samples and reagents.
Upon UV surface modification, the arrays of square
microposts at each of the 96 well locations became
immobilization beds (Witek et al. 2006; Xu et al. 2003)
with sample inlet/outlet reservoirs. Surface modification
Biomed Microdevices (2008) 10:21–33
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Fig. 1 Layout of the first version
of the 96-well SPRI reactor in a
titer plate-based microfluidic
platform: (a) An overall view
(12 columns (1–12) and 8 rows
(A–H), P1 and P2: microfluidic
control ports; (b) Individual immobilization capture bed (R
sample inlet/outlet reservoirs);
and (c) A close-up view of the
entrance/exit section (smallest
square top-view cross section: 10
by 10 μm). Device operating
procedure: (d) introduce nucleic
acid samples at reservoirs (R),
(e) pull them to port P2 using a
vacuum pump, (f) immobilize
nucleic acids, (g) push ethanol
from port P1 to R using a
syringe and pull ethanol to port
P2, (h) dry the capture beds,
(i) push deionized water from
port P1 to R, and (j) collect the
purified nucleic acids
also enhances wettability, resulting in fewer problems with
trapped air bubbles in the microchannels. Each well had a
total surface area of 43.1 mm2 and a volume of 263 nl for
the 10 μm posts and a total surface area of 28.4 mm2 and a
volume of 277 nl for the 20 μm posts. Operation is
straightforward. Once a syringe pump (push-mode) is
connected to port P1 and a vacuum pump (pull-mode) to
port P2, there are two preferential flow paths at each well,
from port P1 to the reservoirs or from the reservoirs to port
P2 [see Fig. 1(c)]. Each well was configured to have the
same pressure drop between ports P1 and P2, so that flow
through the capture beds was controlled simultaneously.
Nucleic acid samples are introduced at each reservoir (R)
by either manual or robotic loading equipment and pulled
to port P2 using a vacuum pump [see Fig. 1(d),(e)]. Nucleic
acids are immobilized on the modified surfaces of the array
of microposts and the majority of the cell debris and
proteins are removed [see Fig. 1(f)]. With immobilization
complete, ethanol is pushed from port P1 to the reservoir,
R, and pulled out through port P2 using a syringe to remove
the remaining cell debris and proteins [see Fig. 1(g)]. After
the capture beds are dried [see Fig. 1(h)], deionized water is
pushed from port P1 to R and the purified DNA collected
for subsequent processing [see Fig 1(i),(j)].
2.2 Fabrication of the LAMIs
An UV-LIGA process including SU-8 UV lithography and
nickel electroplating was used to fabricate the LAMIs. A
standard 175 mm optical mask, with the design of the 96well SPRI reactor, was purchased from Advanced Reproductions (North Andover, MA, USA).
An epoxy-based, negative photoresist, SU-8 (MicroChem
Corp., Newton, MA, USA), was spin-coated over 150 mm
diameter Si substrates coated with an e-beam evaporated
seed layer of Cr/Au (20 nm/50 nm). Prebaking was done in
two steps, 65°C and 95°C on a hot plate. The SU-8 samples
were exposed using a conventional UV lithography system
(Quintel Ultraline 7000 series mask aligner, Morgan Hill,
CA, USA) with a 1 kW broadband mercury UV lamp. The
exposed samples were also post-baked using two steps,
65°C and 95°C on a hot plate. The samples were developed
in SU-8 developer, propylene glycol methyl ether acetate
(PGMEA, MicroChem Corp.), rinsed with isopropyl alcohol
(IPA, Fisher Scientific, Fairlawn, NJ, USA), and dried in air
to obtain SU-8 templates for electroplating. An oxygen
plasma descum to remove SU-8 residue and trace IPA was
carried out in 100% oxygen at a working pressure of
150 mTorr and an incident power of 100 W for 2–3 min in
a Technics Micro-RIE series 800 reactive ion etcher (RIE)
prior to nickel electroplating to ensure a clean surface at the
seed layer for electrodeposition.
Metal mold inserts were fabricated by overplating nickel
from a nickel sulfamate solution. Table 1 shows the
chemical composition of the electroplating solution and
the electroplating conditions. The E-form (electronic grade
sulfamate nickel concentrate, 180 g/l), Dischem Boric Acid
(minimum purity of 99.8%), and E-liminate Pit (wetting
24
Biomed Microdevices (2008) 10:21–33
Table 1 Chemical composition of the nickel sulfamate electroplating
solution and the electroplating conditions used
Chemicals and parameters
Contents and
conditions
Ni2+ from Ni(SO3·NH2)2 (nickel sulfamate)
H3BO3 (boric acid)
Wetting agent
Total solution volume
Plating temperature
pH
Current density
89 g/l
45 g/l
0.3% volume/l
70 l
50°C
3.8–4.2
7–40 mA/cm2
agent) were purchased from Dischem, Inc. (Ridgway, PA,
USA). A custom-designed electroplating bath, specially
made for the fabrication of the large area 150 mm diameter
mold inserts, consisting of a polypropylene electroplating
tank, a DC power supply (Tequipment.net, Long Branch,
NJ, USA), and a temperature controller (Ross & Pethel,
Baton Rouge, LA, USA) was assembled (Park et al. 2006)
[See Fig. 2(a)]. The electroplating solution was continuously circulated and filtered using a commercially-available
filtration system (Technic Inc., Cranston, RI, USA) with
two different sizes of polypropylene filters (10 μm and
1 μm, Technic Inc.) [see Fig. 2(b)]. Sulfur depolarized
nickel pellets (Inco “S” rounds, Belmont Metal Inc.,
Brooklyn, NY, USA) were used to maintain a supply of
Fig. 2 A custom-designed electroplating setup: (a) Overallview of the setup including the
electroplating bath, a temperature controller, and a DC power
supply; and (b) Schematic topview of the electroplating bath
(F1 and F2 filters, P pump, H
heater, and V1, V2, and V3
valves). Photographs of
(c) an electroplating jig before
assembly and after assembly
(inset); and (d) a top-view of the
electroplating tank showing the
plating jig and the anode basket
immersed and held by titanium
bars in a conductive nickel
sulfamate solution
nickel ions during electroplating. The pellets were encased
in a 250 mm by 213 mm titanium anode basket (Vulcanium
Corp., Northbrook, IL, USA) to ensure an anode-to-cathode
area ratio of 1:1 or greater for maximum plating efficiency.
The anode basket was bagged in a napped polypropylene
bag (Baker Bags, Tamworth, NH, USA). An electroplating
jig with proper insulation was made of polysulfone
(McMaster-Carr, Atlanta, GA, USA) to hold the 150 mm
diameter substrates immersed in the electroplating tank [see
Fig. 2(c)]. The electrical current path for electroplating was
established between the seed layer of the SU-8 mold and
the stainless steel bottom plate of the jig using conductive
copper tape. The plating jig and the anode basket were
supported by titanium bars and immersed in a conductive
nickel sulfamate solution [see Fig. 2(d)].
Upon completion of electrodeposition, the overplated
surface of the mold inserts was milled down to about 3 mm
with a total thickness variation of less than 50 μm over the
entire area. Before removing the Si substrate, the nickel
mold insert was cut to a diameter of 135 mm using a water
jet. The Si substrate was removed in a 25% KOH (Fisher
Scientific, Fairlawn, NJ, USA) solution and the SU-8 was
completely stripped using microwave plasma dry etching
(300 series MW plasma system, PVA Tepla America, Inc.,
Corona, CA, USA). To hold the mold insert, a 135 mm
diameter circular cavity with a depth of 3 mm was
machined in a 6 mm thick stainless steel plate (150 mm
Biomed Microdevices (2008) 10:21–33
25
diameter, Type 316, McMaster-Carr, Atlanta, GA, USA).
Additional through-holes were drilled in the cavity area to
enable mounting of the nickel mold inserts in the circular
cavity of the stainless steel plate using laser welding.
sealing while minimizing microstructure deformation as
observed by visual inspection under a microscope.
2.3 Micro molding and assembly of the PC chips
Two PMMA connectors, which were used to mount
capillary tubes and syringes to the device, were bonded to
the top and bottom microfluidic control ports for leakage
testing. To investigate the effectiveness of the sealing of the
assembled PC chips, the microfluidic channels were filled
with a 5 μM solution of fluorescein (Sigma-Aldrich,
Milwaukee, WI, USA) in 1X TBE (Tris–borate–EDTA)
buffer (BioRAD, Hercules, CA, USA) using a syringe. The
fluorescent intensity was imaged using a Nikon Optiphot-2
microscope equipped with a mercury lamp. Images were
recorded with a color Nikon DXM-1200 CCD camera
using Nikon ACT-1 software.
PC substrates were micro molded using hot embossing
(Jenoptik Mikrotechnik HEX 02, Jena, Germany). PC sheets
(Lexan 9034 sheet, GE Structured Products, Pittsfield, MA,
USA) with a thickness of 5 mm were used for hot
embossing. The PC stock sheets were cut into 200 mm wide
octagons and dried in a convection oven at 80°C for 12 h.
Prior to embossing, a mold release agent (Mold Wiz, F57NC, Axel Plastics Research Laboratory Inc., Woodside,
NY, USA) was applied to the nickel mold insert. A molding
pressure of about 200 psi was applied for 2 min at a mold
temperature of 190°C followed by demolding at a temperature of 140°C with a cycle time of approximately 15 min/
part. After hot embossing, the PC chips were cut into the size
of the standard 96-well titer plate and two 1 mm diameter
holes were drilled for the microfluidic control ports. The hot
embossed PC chips and 500 μm thick PC cover plates were
thoroughly cleaned in a dilute 1% solution of Liqui-Nox
(Jersey City, NJ, USA), a critical-cleaning liquid detergent,
in deionized (DI) water followed by a DI water rinse,
isopropyl alcohol rinse, and a final DI water rinse. The PC
chips and covers were dried in a convection oven at 75°C for
12 h and then inspected under a microscope to confirm the
absence of debris prior to sealing.
The surfaces of the PC SPRI chips were modified by UV
radiation using an UV exposure system (Model 60 DUV
Exposure System, AB-M, Inc., San Jose, CA, USA) at a
wavelength of 254 nm and a light intensity of 15 mW/cm2
for 30 min. The top PC cover and bottom molded PC chips
were sandwiched between two 5 mm thick glass plates
(Borosilicate glass, McMaster-Carr, USA), clamped using
spring clips and placed in a convection oven for 30 min. A
parametric study of fusion bonding temperatures was
carried out for temperatures from 146°C to 160°C in order
to determine the temperatures that yielded acceptable
2.4 Leakage testing
2.5 PCR and gel electrophoresis
All bacterial cell lines used (see Table 2) were purchased
from Sigma-Aldrich (Milwaukee, WI). Polyethylene
glycol (PEG, MW=8,000), NaCl, and ethanol were all
purchased from Sigma. All reagents were used as
received unless stated otherwise. The oligonucleotide
primers used for the polymerase chain reactions (PCRs)
were obtained from Integrated DNA Technologies, Inc.
(Coralville, IA).
PCR was performed on the isolated gDNA samples
using 1 μl of the purified material from the whole cell
lysate. PCR was performed using a GeneAmp® PCR
reagent kit with AmpliTaq® DNA polymerase (Applied
Biosystems, Foster City, CA). PCR cocktail mixtures
consisted of 1 μl of each of the primers (final concentration
1 μM), 2 μl dNTP’s (final concentration of each 0.2 mM),
10 μl of PCR buffer, 1 μl of SPRI-purified gDNA, 1 μl of
Taq DNA Polymerase and 78 μl of DI water. The PCR
mixture was amplified using a commercial thermal cycling
machine (Techne, Burlington, NJ) with cycles consisting of
an initial denaturation at 94°C for 5 min followed by 30
cycles of the following: 94°C for 30 s; 69°C for 40 s; 72°C
Table 2 Sequences of the primers used in the studies
Species
Abbrev. used
Primers 5′→3′
Length (bases)
Amplicon size (bp)
Bacillus subtilis
B. subtilis
S. aureus
Escherichia coli type B
E. coli
19
20
19
20
22
22
159
Staphylococcus aureus wood 46
F: CTC ATC GAT TGG TTG CTG C
R: CTT CCT CCT CCA GTG GGT TC
F: GGC TCG GAC TTT TAT GGC G
R: CGT TAA CAT GGG TTC ACC TC
F: ATG GCA AAC CCG GAA CAA CTG G
R: CGC TGC TAT CTG GAA ACG ACC G
F Forward primer, R reverse primer
204
600
26
for 60 s. A final extension at 72°C for 7 min was followed
by cooling to 4°C.
PCR products were electrophoresed on an ethidium
bromide stained 3% agarose gel (Bio-Rad Laboratories,
Hercules, CA). Amplicons were indexed against a DNA
sizing ladder (50–1,000 bp, Molecular Probes, Eugene,
OR). Separation was performed at 4.8 V/cm in 1X TBE
buffer (Bio-Rad Laboratories). Images of the gels were
collected using a Logic Gel imaging system (Eastman
Kodak Company, Rochester, NY).
3 Results and discussion
3.1 Fabrication of the LAMIs
The negative photoresist, SU-8, was chosen for the 150 mm
UV-LIGA process. Since first introduced by IBM in 1989
(Gelorme et al. 1989), SU-8 has been widely used for
general MEMS applications (Lorenz et al. 1998), including
the fabrication of high aspect ratio microstructures
(HARMs) due to its excellent sensitivity and high resolution. A peak aspect ratio of nearly 15:1 was demonstrated
with a conventional UV lithography system by Lorenz et al.
(1998) using SU-8. In addition, the choice of SU-8 was
verified by the observation that this resist has been shown
to be a good candidate material for the fabrication of
Fig. 3 Scanning electron microscope (SEM) images for SU-8 UV
lithography of the first version of
the SPRI: (a) View of an individual capture bed and a close-up
view of the entrance/exit section
(inset) and (b) cross-sectional
side-view showing the vertical
sidewalls of the posts. Layout of
a 96-well SPRI device: (c) before
placing dummy rectangular patterns, and (d) after placing dummy rectangular patterns
Biomed Microdevices (2008) 10:21–33
electroplating molds due to its thermal and chemical
stability (Kim 2004).
The lithographic performance of SU-8 over a 150 mm
diameter area is greatly influenced by the total thickness
variation (TTV) of the SU-8 film, which can introduce a
significant nonuniform gap between an optical mask and
the SU-8 film. Flycutting was used to reduce TTV of the
SU-8 film over the large area Si substrates. After a 150–
200 μm thick layer of SU-8 was initially spin-coated onto
the Si substrate and prebaked, the TTV was 25–50 μm. The
SU-8 films were then machined down to a thickness of
50 μm with a TTV of less than 5 μm over the 150 mm
diameter Si substrates by flycutting (Optimum 120,
Precitech Inc., Keene, NH, USA).
The 1 kW mercury UV lamp installed in the Quintel
7000 series mask aligner used emits over a broad range of
UV wavelengths. It was necessary to filter out high
absorbance UV wavelengths below 350 nm to avoid the
formation of excessively cross-linked thin layers of SU-8 at
the top surface area, which is called the T-topping effect
(Kim 2004; Lu et al. 2007), because SU-8 is optimized for
near UV (350–400 nm) exposure. The flycut SU-8 samples
were exposed through an UV filter (Kopp 9345, Kopp
Glass Inc., Pittsburgh, PA, USA) to remove the UV
wavelengths below 350 nm during exposure. The use of
flycutting and an UV filter during SU-8 lithography
greatly enhanced the lithographic performance over the
Biomed Microdevices (2008) 10:21–33
27
Table 3 Summary of metrology results for the width of the microposts and microchannels on the optical mask, SU-8 plating mold, and
LAMI from the first version of the 96-well SPRI reactor
Design value
(μm)
Optical mask
(μm)
SU-8 mold
(μm)
LAMI
(μm)
Microposts: 10
Channels: 100
10±0.2
100±0.2
10.5±0.2
102.2±0.5
10.2±0.2
102.2±0.5
entire 150 mm diameter surface with excellent verticality
of the 50 μm thick SU-8 micropost structures as shown
by the scanning electron microscopy (SEM) images in
Fig. 3(a),(b).
Nickel was used as the material for the metal mold
inserts due to its ease of processing and excellent corrosion
Fig. 4 Nickel LAMI for the
first version of the 96-well
SPRI reactor: SEM images of
(a) LAMI before SU-8 removal
and (b) after SU-8 removal and
a close-up view of micro
recesses (inset) and photographs
of the LAMI (c) before mounting and (d) after mounting in the
hot embossing fixture. SEM
images of the hot embossing
results: overall views of an
individual capture bed and a
close-up view of the entrance/
exit section (inset) hot embossed
from (e) the first version of the
96-well SPRI reactor, and (f) the
second version of the 96-well
SPRI reactor
resistance. Nickel sulfamate was selected as the electrolyte
because of its very small grain size and low internal stress,
which yielded very flat metal mold inserts. Nickel oxide
formed at the interface between the electroplated nickel and
the seed layer on the substrate is known to be a source of
weak adhesion for nickel structures directly electroplated
onto a substrate (Mair et al. 2006). Although the use of a
Wood’s strike bath can improve the adhesion of nickel
structures, there is still a relatively weak bond at the
interface between the nickel structures and the substrate.
Consequently, overplating of nickel at the cost of longer
plating times is preferred to make mold inserts. For
overplating of nickel, once the cavities in the polymer
plating pattern are filled, it is often necessary to coat a seed
layer on the top surface of the SU-8 to enable contiguous
28
electrodeposition to produce the base of the overplated
mold insert. This introduces the possiblility of the formation of nickel oxide between processing steps. To eliminate
this problem, dummy rectangular patterns with a spacing of
0.5–1 mm were placed over the entire 150 mm diameter
substrate area in the layout of the 96-well SPRI device to
ensure continuous electroplating [see Fig. 3(c),(d)]. The
current density for electroplating in the cavities was initially
set at 7–10 mA/cm2 until they were filled, and increased to
20–40 mA/cm2 to complete the overplating of the base of
the large area mold inserts. The dummy rectangular patterns
expedited the overplating, resulting in about 3.5 mm thick
mold inserts at a plating rate of ∼40 μm/h.
After electrodeposition, it was essential to remove the
SU-8 to reveal the metal microstructures. A wet removal
using 1-methyl-2-pyrrolidone (Alfa Aesar, Ward Hill, MA,
USA) with strong agitation at 95°C can be used to strip
SU-8, but this wet stripping process can leave unwanted
SU-8 residue between small structures. In order to avoid this
problem, an isotropic plasma dry etch using a microwave
plasma asher was used for SU-8 removal. The optimum
conditions for microwave plasma ashing were found to be
25% CF4 (75% O2) at 700 mTorr with an incident power of
500 W resulting in an etch rate of 50–60 μm/h.
Variation of the feature dimensions from the original
design to the final nickel mold insert was investigated by
comparing dimensions of the features on the optical mask,
the SU-8 templates, and the nickel mold inserts. A Nikon
MM-22U Measuroscope was used for these measurements.
The critical features were the widths of the microposts and
microchannels. Table 3 shows the summary of these
measurements, confirming the high integrity of the feature
dimensions from design to the LAMI. Further inspection of
the nickel mold inserts was carried out using a SEM,
revealing 50 μm tall nickel structures on the high quality
mold insert [see Fig. 4(a),(b)]. An array of square micro
Fig. 5 Comparison of the hot
embossing results at each well:
(a) an array of 10 μm posts; and
(b) an array of 20 μm posts
Biomed Microdevices (2008) 10:21–33
cavities with feature dimensions as small as 10 μm was successfully fabricated after complete removal of the SU-8
[see Fig. 4(b)]. Figure 4(c),(d) show photographs of the
nickel mold insert in a stainless steel hot embossing fixture
before and after mounting by laser welding.
3.2 Micro molding, assembly, and fluidic performance
of the PC chips
Hot embossing is a very efficient molding technique for
forming microstructures over large surface areas due to the
relatively short polymer flow paths, which leads to lower
stress and less shrinkage than injection molding (Heckele
and Schomburg 2004; Mekaru et al. 2004). With careful
control of the molding temperature and pressure, acceptable
replication fidelity can be obtained over the entire mold
insert area.
Figure 4(e),(f) shows SEMs of representative hot embossed
SPRI reactors from each of the two versions of the 96-well
SPRI reactor, clearly showing well-formed square microposts
down to the smallest 10 μm posts. In order to evaluate the
polymer filling effectiveness during the hot embossing experiments, the heights of the 10 and 20 μm square microposts
were inspected. The heights of the microposts were measured
by focusing sequentially on the bottom surface of the PC and
the top surface of the chip under an optical microscope with a
vertical measurement resolution of 5 μm or less (less than
10% of the height of the posts). While the 10 μm microposts
in the center of the plate were completely formed [see
Fig. 5(a)], those at the edges of the chip were only 65–70%
filled due to insufficient polymer flow in the smallest recesses.
Microposts of 20 μm were well-formed regardless of the
location of the well [see Fig. 5(b)]. The 20 μm square
microposts hot embossed in PC showed excellent micro
molding with nearly 100% replication quality over the entire
150 mm diameter mold insert area.
Biomed Microdevices (2008) 10:21–33
A nominal well-to-well spacing of 9 mm is required in
the micro molded PC titer plate to take advantage of
existing multi-channel pipettes or robotic equipment for
sample and reagent handling used in conventional 96-well
titer plate platforms. The well-to-well spacing of the large
area, nickel mold insert was designed at 9.054 mm to accommodate an estimated shrinkage of ∼6 μm/mm because
PC undergoes an average shrinkage of 5–7 μm/mm during
hot embossing (Edwards et al. 2000; Murakoshi et al.
2003). The actual well-to-well spacing for the hot embossed
PC, measured with a Nikon MM-22U Measuroscope, was
found to be 8.982±0.004 mm, which corresponded to an
average shrinkage of 8 μm/mm for the micro molding
conditions used. The measured shrinkage was used to
correct the spacing in a second mold insert and realize the
required well-to-well spacing of 9 mm. After accounting for
the shrinkage, well-to-well spacing of the hot embossed PC
chips was measured as 8.999±0.004 mm.
Post-processing for access holes was carried out on the
backside of the 5 mm thick hot embossed fluidic network
[see Fig. 6(a)]. A micro-milling machine (KERN MMP
2522, KERN Micro- und Feinwerktechnik GmbH & Co.
KG, Germany) (Hupert et al. 2006) was used to precisely
drill 96 reservoirs with a depth of 4 mm. Laser-drilling
(Resonetics Rapid X® 1000 Series excimer laser system,
Nashua, NH, USA) was then used to break through the 96
micromilled holes to the fluidic network without formation
of polymer burrs [see Fig. 6(b)]. Alternatively, double-sided
hot embossing with two master molds (Datta et al. 2006)
can be used to define the additional access holes and
reservoirs simultaneously during micro molding to reduce
the post-processing overhead and accommodate mass
production of the desired parts.
Hot embossed PC chips and cover plates were exposed
to UV radiation with a wavelength of 254 nm at an UV
intensity of 15 mW/cm2 for 30 min. For PC, UV surface
modification reduces the glass transition temperature of the
surface and enables thermal fusion bonding at a lower
bonding temperature (Witek et al. 2004). Surface modification also results in the formation of carboxylate groups on
the surface of the microposts and channel walls (Witek et
al. 2006; Xu et al. 2003), which can be used as an effective
immobilization bed for the solid-phase purification of
nucleic acids when placed in a solution containing PEG
and NaCl.
The quality of the bond resulting from thermal fusion
bonding is determined primarily by the bonding temperature (Mair et al. 2006). A parametric study of fusion
bonding of the UV-modified PC was carried out by varying
the bonding temperature from 146°C to 160°C. During
bonding, the pressure was maintained by the application of
a constant number of clips and the bonding time was fixed
at 30 min. Fusion bonding temperatures below 150°C did
29
not provide complete sealing between the cover plate and
fluidic substrate. Successful bonding of the cover plate
and PC substrate components occurred at temperatures of
150°C or above. However, bonding temperatures >150°C
resulted in some visible deformation of the microposts, so
a bonding temperature of 150°C was selected for thermal
fusion bonding of the 96-well SPRI chips.
In order to assess whether there were any leaks between
the cover plate and the hot embossed PC chip in the
microfluidic channel network, fluorescein was pushed
through the microfluidic channels and wells [see Fig. 7(a)].
A close-up view of one of the 96-wells at 20× magnification shows that no fluorescent signal was observed in the
areas outside of the microfluidic channels indicating proper
sealing between the hot embossed PC chip and the cover
plate [see Fig. 7(b)].
The distribution channels were designed to be of the
same length and have the same cross sectional dimensions
in order to maximize the probability that the fluid will be
Fig. 6 (a) Post-processing sequence of a molded PC chip (R reservoir);
and (b) SEM image of a laser-drilled hole
30
evenly distributed to each branch in terms of flow rate and
have the same travel time. In reality, because of manufacturing tolerances including LAMI fabrication, micro molding, and thermal fusion bonding, this was not true. The flow
in the entire chip is laminar, so the relationship between the
pressure drop and flow rate is linear, with the proportionality factor representing the hydraulic resistance. The
hydraulic resistance is very sensitive to the variability of
the channel cross sectional area, and above all the
narrowest dimension of the cross section. The manufacturing variability for the height (50 μm) of the distribution
channels used was estimated, based on measurements, to be
±5%. For the width, which is larger, the percentage
variation was smaller. A simple calculation of the hydraulic
resistance using well-known laminar solutions (White
1974) and propagation of the dimensional variation leads
to a corresponding variation in the hydraulic resistance of
each channel branch is on the order of ±15%. The hydraulic
resistance of the capture beds was estimated by treating
each one of them as a porous medium and using a simple
model based again on the laminar flow channel solution
(Bear 1988). Using such a model, variability of the
hydraulic resistance of the capture bed was most sensitive
to the height of the flow path (the net flow width is much
larger—on the order of 500 μm considering the 1 mm total
width of the capture bed and assuming 50% average
blockage). The expected variation of the hydraulic resisFig. 7 Photographs of the leakage test: (a) An overall view of
an assembled PC chip with
fluorescein-filled microchannels
pushed through PMMA connectors; and (b) a close-up view at
20× magnification showing no
leakage. Photographs of (c) a
PPC SPRI chip; and
(d) gDNA purification setup
(P1 and P2: microfluidic control
ports)
Biomed Microdevices (2008) 10:21–33
tance of the capture bed was of the same order as for the
straight channels.
Given the estimated variation of the hydraulic resistance
of each element, and the number of channel splits in the
distribution system network necessary to deliver fluid to the
96 different capture beds, an estimate of the variation of
the flow rate (pulling or pushing) through a typical capture
bed would be on the order of 37%; the hydraulic resistance
variability multiplied by the square root of the number of
splits. Through similar considerations, the variation of the
linear velocity of the fluid in each capture bed was
approximated as ±24%. Variability in the filling and
emptying time should also be of that order.
For a total flow rate of 2 ml/min, the estimated pressure
drop through the distribution system up to a typical capture
bed and under ideal conditions was ∼169 kPa, while
through the well itself it was ∼10 kPa, for a total pressure
drop of ∼179 kPa. These estimates were made using the
laminar flow solutions mentioned above (White 1974) and
include estimates of losses because of hydraulic elements
such as tees and bends (White 1986).
The chip is initially full of air, so when it is filled for the
first time, the effect of the variation in the capillary
resistance on the flow rates (and velocities) of the fluid in
each branch should be considered. This is much more
difficult to estimate, because it is sensitive to the variation
of the smallest dimension of the cross section as well as the
Biomed Microdevices (2008) 10:21–33
wall-surface properties (e.g. roughness, wettability). The
latter are very difficult to quantify. However, an estimate of
the contributing capillary pressure inside the smaller
distribution channels was ∼1.5–2.0 kPa, while in the
capture bed between posts it was ∼8 kPa. These values,
obtained by using the Young–Laplace equation, were quite
small compared to the total pressure drop, which builds up
as the vias fill, and should not affect the filling rate of the
capture bed significantly.
Experimental observations confirmed that the 96 capture
beds had different filling times even though the nominal
distances from port to reservoirs are designed to be all the
same. For example, the measured fluid volume for
deionized water pushed into the 96 beds was 15.4±5.2 μl,
34% RSD. The 96 capture beds also exhibited different
pulling speeds (by vacuum pump) at different capture beds.
The observed variation of the flow during initial aspiration
of the nucleic acid samples through the immobilization
beds did not influence the amount of nucleic acids captured.
For a given sample and immobilization buffer, nucleic acid
capture efficiency depends primarily on the capture bed
characteristics, including the active surface area and the
interpost distance. The uniformity of the flow during the
final elution of nucleic acids influences, however, the final
concentration of the nucleic acids (ng/ml) in the eluted
solution, which can be adjusted if necessary for downFig. 8 Agarose gel electrophoresis images of the PCR products generated from 96
amplification reactions with purified (a) B. subtilis, (b) S.
aureus, and (c) E. coli gDNA
using the second version of the
96-well SPRI microfluidic platform. Lane m represents DNA
sizing ladder (50, 150, 300, 500,
750, 1,000 bp)
31
stream applications. The observed nonuniformity of the
flow for different capture beds does not affect the function
of the device for high throughput purification of nucleic
acids.
The fluidic control ports P1 and P2 can be complementary to each other, so the syringe and vacuum pumps can be
interchanged without altering the overall fluidic performance. A series of fluidic performance tests were carried
out by interchanging the fluidic control ports. The variability in the filling and emptying times for each capture bed
showed similar trends in either configuration.
3.3 Purification of genomic DNA from whole cell lysates
The performance of the 96-well SPRI reactor using photoactivated polycarbonate (PPC) chips [see Fig. 7(c),(d)] was
tested for the purification of genomic DNA (gDNA) from
different bacterial species (Bacillus subtilis, Staphylococcus
aureus, and Escherichia coli). A simple and fast purification protocol for the chip was developed and the quality of
the purification verified with PCR and electrophoresis
(Witek et al. 2006; Xu et al. 2003; Park et al. 2007).
Whole cell lysates of several bacterial species (B.
subtilis, S. aureus, and E. coli) in an immobilization buffer
(3% PEG, 0.4 M NaCl) were introduced into each of the 96
sample reservoirs and drawn through the capture beds to
32
port P2 by using vacuum. Upon completion of sample
immobilization, ethanol was pushed through the extraction
beds from port P1 to the sample reservoirs and pulled out
through port P2 to remove cell debris and proteins.
Captured DNA was released from the PPC surface by
pushing DI water from port P1 to the sample reservoirs,
where it was collected in 96 micro-tubes. This unique
operating procedure allowed effective gDNA purification in
a high throughput, automated format with a closed
architecture that could eliminate potential contamination.
PCR was performed on the isolated gDNA samples
using 1 μl (∼10 ng) of the SPRI/PPC purified material. The
PCR products were electrophoresed on an ethidium
bromide stained 3% agarose gel. Figure 8 shows the
fluorescence image of the PCR amplicons generated during
the amplification reaction with a DNA sizing ladder in lane
m. Purified gDNA was clearly evident from the 159, 204,
and 600 bp PCR amplicons in lanes a–c for the B. subtilis,
S. aureus, and E. coli, respectively. Successful capture and
purification of gDNA was obtained at all 96 capture beds in
the 96-well PPC SPRI chip, demonstrating its high
throughput purification capability.
4 Conclusions
A titer plate-based polymer microfluidic platform was
demonstrated by incorporating functional microfluidic
devices at each well location to realize high throughput
purification of nucleic acids. To demonstrate the potential
of this format, a 96-well solid-phase reversible immobilization (SPRI) reactor was designed to fit the footprint of a
titer plate; the prototype allowed simultaneous fluidic
control of samples at 96 separate immobilization beds
using only two control ports. By using the standard titer
plate format, the system was compatible with multichannel
pipettes or robotic equipment for loading samples and
reagents. The device was replicated from a 150 mm
diameter LAMI fabricated using a SU-8 based UV-LIGA
technique. Excellent lithography results for the SU-8 electroplating templates, particularly the vertical sidewalls,
were obtained over the entire 150 mm diameter surface
area by using flycutting to reduce the SU-8 film total
thickness variation to less than 5 μm and exposure through
an UV filter to remove absorbance of UV wavelengths
below 350 nm. A custom-designed electroplating apparatus
was then used to effectively control overplating of nickel
and to fabricate high quality, high precision, metal mold
inserts. Hot embossing of PC was demonstrated resulting in
good replication fidelity over the large surface area. The
micro molded PC chips had high integrity in all feature
locations, particularly for the required well-to-well spacing
of 9 mm that would allow the platform to take advantage of
Biomed Microdevices (2008) 10:21–33
existing sample and reagent liquid handling technologies.
Thermal fusion bonding of the hot embossed PC chips at
150°C yielded good sealing, which was verified by leakage
testing using fluorescence microscopy. All 96 capture beds
in the SPRI reactor successfully purified gDNA from whole
cell lysates of bacterial species in a highly parallelized
fashion.
Efforts are currently underway to optimize the molding
process parameters in order to achieve uniform filling of the
10 μm, or smaller, microcavity arrays over the complete
surface area of the LAMI using extensive simulation and
molding tool design (Worgull and Heckele 2004; Worgull
et al. 2005). Alternative polymer materials with better flow
characteristics are also being explored to address this issue.
The 96-well photo-activated PC SPRI reactor in the titer
plate-based polymer microfluidic platform will open up
new avenues for low cost, disposable DNA/RNA sample
purification and many other options for high throughput
analysis once other micro-analytical systems are implemented in the multi-well format.
Acknowledgements This work was supported by the National
Science Foundation and the State of Louisiana Board of Regents
Support Fund under grant number EPS-0346411, and the State of
Louisiana Board of Regents Support Fund, Industrial Ties Program
through grant number LEQSF(2005-08)-RD-B-04. The authors thank
the staff of the Center for Advanced Microstructures and Devices
(CAMD) at Louisiana State University for the microfabrication
support. J. Guy was funded by a Louisiana Governor’s Biotechnology
Initiative grant.
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