Download A ZEPTO MOLE DNA MICRO SENSOR *

A ZEPTO MOLE DNA MICRO SENSOR*
Tza-Huei Wang, Sylvain Masset, and Chih-Ming Ho
Mechanical & Aerospace Engineering, University of California
Los Angeles, CA 90095-8357, USA
With the introduction of molecular beacon, DNA detection
with high specificity can be performed directly in a microchannel
(inchannel detection) that reduces the necessary sample volume by
several orders of magnitude compared with most of the other DNA
sensors [3,4] and also simplifies the processes of integrating a
biosensor into a micro total analysis system(µ-TAS). In this paper,
microchannels with different crosssection geometries were
fabricated and their sensitivity of DNA detection were compared
to determine the optimum design. Metals with high reflectance
like Al and Au were deposited and patterned to form mirror-like
sidewalls on the detection region of the channels to enhance the
surface reflectance and increase the fluorescence signal level.
ABSTRACT
Using molecular beacons (MB) as highly sensitive and
selective nucleic acid probes, we eliminate two of the major but
cumbersome steps, probe immobilization and washing, of genebased biosensors. By integrating sidewall mirrors with
microchannels, we can increase the signal-to-noise ratio of the
optical detection. The detection specificity is achieved using a MB
based DNA hybridization technique. Molecular beacons become
fluorescent only upon hybridization with target DNA/RNA
molecules as the quencher is separated from the fluorophore. We
can hence eliminate washing and immo bilization steps and
establish the inchannel detection technique. This technique
reduces the detection volume to 36 pL. Microchannels coated with
metal films with high reflectance are used to increase the signal
level in the Laser Induced Fluorescence(LIF) system. By using the
side-mirror channel with inchannel sensing technique, the
detection limit is 0.07 zmol which is at least 3 orders of magnitude
lower than many other DNA detection schemes.
DESIGN OF MOLECULAR BEACON PROBES
Molecular beacons are single stranded DNA molecules
with a stem-and-loop structure.(Fig.1) The loop portion of the
beacon can form a double stranded structure in the presence of its
complementary nucleic acid strand. The two ends of the stems of a
MB are labeled with a fluorophore and a quencher.
INTRODUCTION
(a)
DNA/RNA analysis plays an extremely important and
fundamental role in the rapid development of molecular
diagnostics, genetics, and drug discovery. One of the fastest
growing areas in DNA/RNA analysis is the development of DNAbased biosensors. A variety of biosensors, both optical and
electrochemic al, have been developed for gene sequence analysis
and biological pathogen detection[1,2] based on the DNA
hybridization technique. In DNA hybridization, the target gene
sequence is identified by a DNA probe that can form a double stranded hybrid with its complementary nucleic acid with high
efficiency and extremely specificity.
For a typical DNA hybridization based biosensor, it requires
the steps of immobilization of DNA probes on the sensor surface
and washing away the non-specific molecule binding t o ensure
specificity. The non-perfect surface modification from
immobilization and incomplete washing are the main sources of
noise and hence determine the ultimate sensitivity[3,4]. To
immobilize probe molecules on the sensor surface and to achieve
efficient washing require more fluidic devices to deal with
excessive solutions if automation is desired. These are also time
and power consuming steps. In addition, the immobilized
monolayer could be destroyed by a high temperature condition that
limits the post fabrication (chip bonding) choices if a closed
sensor is to be developed. All of these issues, which comes from
these two cumbersome steps, add complexities to the lab-on-chip
design. To remove the immobilization and washing steps, a
molecular beacon (MB) based RNA -DNA hybridization technique
[5,6,7] was implemented for DNA detection. The molecular
beacons are oligonucleotide probes that become fluorescent only
upon hybridization with target DNA/RNA molecules. By
implementing this technique, the biosensor can still provide high
specificity even without washing away the excess non-hybridized
probes which are not fluorescent (if they exist in the solution).
(b)
Hybrid
Molecular Fluorophore
Beacon
Quencher
Fig. 1 (a) Before hybridization, the Molecular Beacon
remains non-fluorescent because the fluorophore is
quenched by the quencher, (b) Molecular Beacon
becomes fluorescent after hybridization with targets
The sequences of the two stems are five to eight bases
long and are complementary to each other. Due to the
hybridization of the two stems, the fluorophore and quencher are in
close proximity to each other, causing the fluorescence to be
quenched by the quencher (Fig. 1(a)). The sequence of the loop,
which is typically twenty to thirty bases long, is designed to be
complementary to sequence of the target DNA molecules. In the
presence of the target DNA molecules, the stronger binding force
between the longer loop structure and target DNA will unbind the
shorter/weaker stem structures and separate the quencher from the
fluorophore(Fig. 1(b)).
The sequence of the loop structure is designed according
to a portion of the sequence of 16 s rRNA in E.coli (MC41000)
and is 22 bases long. The 5' end is labeled with Fluorescein and the
3' end is labeled with Dabycl quencher. The complete sequence is
5' Fluorescein -GCTCG TATTA ACTTT ACTCC CTTCC
TCCGA GC - 3’Dabycl.
*Patent pending
0-7803-5998-4/01/$10.00 @2001 IEEE
Target
431
Metal (2200A)
(a)
(a)
(b)
(b)
Cr/Au 2200Å
Pyrex Glass
Glass
Si
SiO2 5000Å
(c)
SiO2 (5000Å )
(d)
Fig. 4 Anodic bonding of Pyrex and SiO2 with a metal
layer partially in between
(a)Before bonding, a space of 2200Å between glass and
SiO 2 (b)After bonding, the metal is squeezed in between
Fig. 2 Cross sections of microchannels bonded
with glass (with silicon dioxide and metal layers
in between). Sidewalls are coated with a metal layer
to make reflection mirrors
(a)rhombus channel, (b) v-groove channel
(c)trapezoid channel,(d) rectangular channel
(a)
(b)
Au
SiO2
Air
Water
(a)
Air
(b)
Fig. 5 Completely bonded channels
(a)Channel is empty (b)Channel partially filled with water
and no leak is observed in the Au/SiO2 interface and top
Au edges of the channel
100 µm
100 µ m
between the channel chip and glass is strong enough to squeeze the
metal layer tightly between those two substrates and minimize the
clearance in the interface between the non-bonded metal and
bonded SiO 2 areas. A complete bonded chip is then injected with
water, and no leak is observed along the Au and SiO 2 interface as
well as in the squeezed top metallic edges of channel (Fig. 5).
(d)
(c)
100 µ m
100 µm
PMT
Fig. 3 Microscopic pictures of channel crosssections
(a)rhombus channel, (b) v-groove channel,
(c)trapezoid channel, (d) rectangular channel
Focusing Lens
Band pass
filter
Laser
Input
FABRICATION
Microchannels with different geometry, and metal
coatings on the sidewalls are fabricated to maximize signal-to–
noise ratio. Channels with v -groove, trapezoid, and rectangular
cross sections were fabricated by KOH and DRIE etching, and
channels with rhombus cross sections were made by DRIE pre etching followed by KOH etching (Fig. 2 and 3).
The channel width varies from 10µm to 150µm and its
depth changes from 20 µm to 100µm. After 5000Å of thermal
silicon oxide is grown for electrical isolation, a 2200 Å thin Al or
Cr/Au layer is deposited by sputtering or e -beam evaporation. To
pattern electrodes and sidewall mirrors in t he deep channels, 10
µm AZP4620 PR is coated, over-exposed and developed to make
etching masks or perform lift -off as the case requires.
The channel chip is then bonded to a pre -drilled Pyrex
glass plate using anodic bonding technique to form a closed
channel. In spite of a spacing 2200 Å between the channel chip
and the glass plate due to the metallic layer as shown in Fig. 4 (a),
the electric field is large enough to pull the two plates together and
form a completely bonded channel (Fig. 4(b)). Also even if the Al
or Au metal layer does not bond with glass, the bonding strength
Beam Expander
Band pass filter
Beam Splitter
Mirror
Achromatic Objective
DNA sensor chip
Fig. 6 Setup of a Laser Induced Fluorescence System
INSTRUMENTATION
A Laser Induced Fluorescence (LIF) system (Fig. 6) is
used for biosensor characterization. An excitation beam (2mW)
from an air-cooled Ar ion laser (ILT,100mW) passes into a beam
expander (Melles Griot, 09LBZ010) and a band pass filter
(Omega, XF1073). It then reflects from a dichroic beam spliter
432
(Omega, XF2037) to a 20 x 0.50 N.A. objective (Rolyn Optics
Company, 80.3080), which focuses the beam to a 30 µm spot
within the channel. Fluorescence is collected by an objective,
passes through the dichroic beam splitter, filtered by a bandpass
filter (Omega, XF3003), focused by a focusing lens (Newport,
PAC052), and finally collected by a PMT (Hamamatsu, HC12001). The signal from the PMT is transmitted to a data acquisition
card and analyzed by a Lab View program.
SNR Enhancement (%)
1000.0
865.3
Al Coating
800.0
Au Coating
579.9
566.2
600.0
421.2
400.0
200.0
79.5
34.7
EXPERIMENTAL PROCEDURES
20.6
0.0
The eight hundred bases long nucleic acid targets used
for detection were synthesized by polymerase chain reaction
(PCR). The sense (5' –CAGAT GGGAT TAGCT AGTAG GTG3') and antisense (5' –GTCTC ACGGT TCCCG AAGGC AC -3')
primers derived from the most conserved region of 16s rRNA of
2E.coli.(MC41000) were identical to those used in the previously
reported study[7].
Initially, 50 µl DNA solution with
concentration of 0.2 µM and 50 µl MB solution of the same
concentration were mixed for biosensor characterization. The
signal to noise ratio (SNR) and detection limit of channels with
various geometries and surface coatings are determined by testing
the different channels with the serially diluted solution. Because
of the introduction of molecular beacons, two of the major but
cumbersome steps, immobilization and washing, for typical DNA
hybridization technique were eliminated. This greatly simplified
the preparation steps for DNA detection. The mixed solution was
pipetted into the inlet of the drilled glass hole on the sensor
chip(Fig. 7), and the surface tension force pulls the solution to the
coated detection region. A 488 nm light beam form the Ar ion laser
was focused onto the coated region, and the existence of target
DNA can be identified by checking the intensity of the emitted
fluorescence light (λ=512nm) which comes from stretched
Fluorescein labeled MB probes.
20
2
0.2
15.0
0.02
Concentration (nM)
Fig. 8 SNR enhancement due to Al & Au coating
for different sample concentration
SNR
25.00
20.00
15.00
10.00
5.00
0.00
Retangular
Rhombus
Trapzoid
V-groove
Fig. 9 Comparison of SNR for different channels.
The concentration of MB-DNA solution used for
testing is 2 nM. Sidewalls in the detection regions
of the four channels were coated with an Al layer.
The channel width is 80 µm.
in the experiment which was 512 nm in air (385 nm in aqueous
solution), the average reflectance (normal incidence) for Al thin
flim is 0.92, for Au thin film is about 0.40, and for SiO2 thin film
is less 0.20 [8]. The magnitude order of refle ctance agrees with the
experimental SNR enhancement data.
Comparing the SNR of rectangular, V-groove, trapezoid,
and rhombus channels, it is found that SNR of the trapezoid
channel is 3.0 times higher than that of retangular channel, and is
2.4 times higher than that of rhombus channel (Fig. 9). The
experiments are performed by injecting 2 nM sample solution
(DNA -MB hybridization products) into the various channels with
the same width of 80 µm.
It is obvious that channels fabricated by KOH etching
have mu ch smoother sidewalls than those made by DRIE etching
(Fig.10(a), (b)), and have higher overall reflectivity for the same
surface coating condition. For the KOH etched channels, due to the
deposition and patterning difficulties in creating a uniform metal
coating on the four sidewalls of the rhombus channels(Fig.11), the
SNR of the rhombus channels is lower than those of trapezoid and
V-groove channels.
According the above results, the trapezoid channel is
determined to be the optimum design for detection based on the
currently employed fabrication techniques. Thus, trapezoid
channels coated with Al, Au, and thermal oxide are tested to
compare the detection limits. As shown in Fig. 12, the detection
limit for a channel with thermal oxide coating is 200 p M while for
a channel with Au coating is 20 pM. And for an Al coated channel,
Detection channel
Reservoir
Detection
Mirror
Electrode
Fig.7 A sensor chip with 8
detection channels.
RESULTS
For the same geometry of microchannels, those with Al
coating in the detection region was found to have the highest signal
to noise ratio over those coated with Au or SiO 2 in the same
detection condition. In addition, micrchannels with Au coating has
higher SNR than those without any metallic coating which is SiO 2
surface. The SNR enhancement for the Al and Au coated channels
over SiO2 coated channels ranges from 80% to 860% and from
14% to 420% respectively for different concentrations of sample
solution as shown in Fig. 8. It is believed that the enhancement is
because of the improvement of reflectance which was due to the
metallic coatings. In the wavelength range of emitted fluorescence
433
(a)
(b)
technique, the concentration detection limit is 0.07 zmol with
SNR=3 as a threshold. This is approximately three orders of
magnitude lower than that for many other DNA detections. It is
possible to visualize a single light-emitting molecule but not for
DNA detection with high specificity[9,10], thus the 0.07 zmol
(about 50 molecules) detection limit is a significant advancement.
In the case of a more diluted solution, the metallic mirror can be
also used as an electrode to apply positive potential for
concentrating negatively charged DNA in order to further improve
the performance of the sensor.
SiO 2
SiO 2
Al
Al
Fig. 10 SEM pictures of channels with Al coating
(a) KOH etched trapezoid channel, sidewall and bottom are smooth
(b) DRIE etched rectangular channel, sidewall and bottom are very
rough
ACKNOWLEDGMENTS
This work is supported by DARPA MTO under a Contract
N66001-96-C-83632 managed by SPAWAR. Advice offered by
Dr. Jeffery Miller and Mr. Minghsun Liu’s on MB probes and PCR
primers are appreciated.
Fig. 11 SEM picture of KOH
etched rhombus channel with
Al coating. The coating is not
uniform on the sidewall which
affects the SNR of detection.
REFERENCES
1.
M. Yang, M.E. McGovern, and M. Thompson, "Review
Genosensor technology and the detection of interfa cial
nucleic and chemistry", Analytica Chimica Acta, 346(1997),
259-275
2. D. Ivnitski, I. Abdel-Hamid, P. Atanasov, and E. Wilkins,"
Review Biosensors for detection of pathogenic bacteria",
Biosensors & Bioelectronics 14 (1999), 599-624
3. Y.F. Chen, J.M. Yang, J.J. Gau, C.M. Ho, and Y.C.
Tai.”Microfluidic System for Biological Agent Detection,”
The 3rd International conference on the interaction of Art and
Fluid Mechanics, Zurich, Switzerland, 2000.
4. J. Gau, E. Lan, et al., Proceedings of the Fourth International
Symposium on m-TAS (2000), 509-512
5. X. Liu, W. Farmerie, et al., "Molecular Beacons for DNA
Biosensors with Micrometer to Submicrometer Dimensions",
Anal. Biochem. 283(2000), 56-63
6. S. K. Poddar, "Detection of adenovirus using PCR and
molecular beacon", J. Virological Methods 82 (1999), 19-26
7. T.H Wang, Y. F. Chen, S. Masset, C. M. Ho, Y.C. Tai,
"Molecular Beacon Based Micro Biological Detection
System," proceedings of METMBS'00, pp295-300
8.
M. Bass, “Handbook of Optics”, 2 nd Edition, V.II, Mc-Graw
Hill
9. W .P. Ambrose, P.M. Goodwin.,et al. “Single -molecule
detection with total internal reflection excitation: comparing
signal-to-background and total signals in different
geometries.”Cytometry 36(1999), 224-231
10. C. Zander, K.H. Drexhage,” Single -molecule counting and
identification in a microcapillary”, Chemical Physics Letters
286(1998), 457-465
Relative Signal Level
30
Al Coating
25
Au Coating
SiO2 Coating
20
Back Ground
15
10
5
0
1 -19
7x10
20,000
2 -20
3 -21
7x10
7x10
2,000 200
4 -22
7x10
20
5 -23
7x10
mole
2
pM
Fig12 Sensitivity check of channels with different
coatings. KOH etched trapezoid channels are used.
Channel width and depth are 80 µm and 50µm.
The detection limit for Al coated channel is 7x10-23
mole which is about 50 DNA molecules.
the detection limit is as low as 2pM which is about only 0.07 zmol
(0.07 x 10-21 mole, about 50 DNA molecules) in the 36 pL probe
volume (based on a 30 µm dia. focusing spot and 50 µm channel
depth). This is approximately three orders of magnitude lower than
that for many other DNA detections[1,2].
CONCLUSIONS
Inchannel specific DNA detection was achieved by using
molecular beacon based DNA hybridization. This process greatly
reduces the sample volume required for detection and could
facilitate the integration of a biosensor to a µ-TAS. Microchannels
coated with metal films with high reflectance are fabricated that
increase the signal level in a Laser Induced Fluorescence(LIF)
system. By using the side-mirror channel with inchannel sensing
434