The Preparation and Hybridization Analysis of DNA/RNA from E. Coli on Microfabricated Bioelectronic Chips
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Cheng et al.
The preparation and hybridization analysis of DNA/RNA from E. coli on microfabricated bioelectronic chips
Jing Cheng*, Edward L. Sheldon, Lei Wu, Adam Uribe, Louis O. Gerrue,
John Carrino, Michael J. Heller and James P. O′Connell
Nanogen Inc., 10398 Pacific Center Court, San Diego, California, USA.
*Author for Correspondence:
Jing Cheng, Ph.D.
Nanogen Inc.
10398 Pacific Center Court
San Diego, CA 92121, USA
Tel: (619) 546-7700
Fax: (619) 546-7718
email: [email protected]
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Abstract
Dielectrophoretic separation of E. coli bacteria from a mixture containing
blood cells followed by electronic lysis was performed on a
microfabricated bioelectronic chip by applying a series of high voltage
pulses. Bacteria were isolated from human blood cells using
dielectrophoresis to direct the prokaryotic cells to 25 microlocations
directly above individually-addressable platinum microelectrodes on a
chip. The surface of the chip, including the platinum electrodes and the
rest of the non-metallized area, was coated with an agarose permeation
layer to prevent the direct contact of cells with the electrode and also to
minimize the non-specific adhesion of cells and lysate to the chip
surface. The platinum electrodes were 80 µm in diameter and had a
center-to-center spacing of 200 µm. Analysis of the electronically-lysed
bacterial mixture showed that it contained a spectrum of nucleic acids
including RNA, plasmid DNA and genomic DNA. The lysate was then
further analysed by electronically-enhanced nucleic acid hybridization on
different bioelectronic chips made for specific DNA and RNA
hybridization analyses. Dielectrophoretic cell separation followed by
electronic lysis has significant potential as a sample preparation process
for chip-based electronic-hybridization assays in an integrated DNA/RNA
analysis system.
Keywords: bioelectronic chip, dielectrophoresis, electronic lysis, RNA
hybridization, system integration
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Introduction
Use of microfabricated chips for biological assays, especially in DNA
hybridization, holds significant potential for an integrated system which
includes both sample preparation and detection1. The front end of such
a system, sample preparation, has proven to be the most difficult part to
integrate and therefore has attracted much attention recently.
In the ongoing efforts directed toward system integration, several
methods for separating cells have been developed using microfabricated
devices. Microfabricated silicon-glass filter chips have been used to
isolate human white blood cells from whole blood2. Lysates released
from the isolated white blood cells by heating have been directly used in
the same chip for PCR amplification3. Cell analysis has also been
achieved by electrophoresis using a microfabricated, mechanical sieving
bed which consists of many micro-sized posts etched on the silicon
substrate4. A recent report has described cell transportation by
electroosmotic and electrophoretic pumping and chemical lysis of cells in
microfabricated structures5. The separation of cells by dielectrophoresis
is one of the most broadly applied methods among all chip-based cell
separation technologies6. It is well known that dielectrically polarizable
particles, including those with no net charge, are subject to a
“dielectrophoretic” force in a non-uniform electrical field as long as the
effective polarizability of the particles is different from that of the
surrounding medium. The direction of migration of the different cell
types is determined by: (1) electrical double layers associated with
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surface charges, (2) the conductivity and permittivity of the membranes
and any cell walls, and (3) morphologies and structural architectures6.
Dielectrophoresis has been employed for the selective separation of such
populations as viable and nonviable cells7, Gram-positive and Gram-
negative bacteria8, viruses 9, and cancer cells10.
We report here the development of a sample preparation process and also
the electronically enhanced hybridization of RNA and DNA on
microfabricated chip-based devices. The sample preparation process
begins with the separation of E. coli from blood cells by dielectrophoresis
on a silicon chip covered with an array of 25 individually addressable
microelectrodes. The separated bacteria are retained above the
electrodes after the blood cells have been washed off. The procaryotic
cells isolated this way are then lysed electronically by applying a series of
high voltage pulses to release a full spectrum of nucleic acids. The
released RNA and DNA are then analyzed by bioelectronic chips11.
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Results
The experimental set-up for dielectrophoresis and electronic lysis is
depicted in Fig. 1. The dielectrophoretic separation of E. coli from the
blood cells, the electronic lysis of the isolated E. coli, and the digestion of
proteins using proteinase K were all performed on one microfabricated
bioelectronic chip contained in a flow chamber. The illustrations of
‘checkerboard’ and ‘square-wall’ patterns of addressing of the five-by-five
arrays of electrodes and the corresponding computer models of AC field
distribution are shown in Fig. 2 (a)-(d). In the square wall pattern of
addressing, the electrodes on the same square frame have the opposite
bias from electrodes on the nearest neighboring square frame. In the
checkerboard pattern of addressing, each electrode has the opposite bias
from its nearest neighbor. The field distribution model indicates that a
uniform distribution of the electric field can be obtained using the
checkerboard addressing format with the field maxima at the electrode
and the field minima in the areas between the electrodes.
Prior to the dielectrophoretic separation of E. coli cells from blood, the
chip was washed by the separation buffer to wet the permeation layer10
coating and remove any air bubbles. In the first experiment, square-wall
addressing was used. Fig. 2(e) shows the picture of the coated chip after
the pre-washing step. After the cell mixture was introduced into the flow
cell, the separation was finished in approximately 4 min. The separation
results shown in Fig. 2(g) match very well with the field distribution
model [see Fig. 2(c)]. In this case it is shown that the E. coli cells
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occupied the entire area of the electrodes with the blood cells
accumulated between the electrodes. Next, a separation buffer was used
to wash away the blood cells loosely held at the field minima between the
electrodes. During the washing process the separated E. coli cells were
retained on the electrodes at the field maxima because the A.C. power
remained on. When the washing process was completed, an image was
taken showing the isolated E. coli cells on all 25 electrodes [Fig. 2(i)]. In a
second experiment, checkerboard addressing was used. Fig. 2(f) shows
the picture of the coated chip after the pre-washing step. After the cell
mixture was introduced into the flow cell, the separation was finished in
approximately 4 min. The separation results shown in Fig. 2(h) matches
very well with the field distribution model [see Fig. 2(d)]. Blood cells
accumulating at the field minima were removed using the wash step.
When the washing process was completed, an image was taken showing
the isolated E. coli cells on 25 electrodes [Fig. 2(j)].
An analysis of nucleic acids released from the isolated cells by electronic
lysis is shown in Fig. 3. The results of agarose gel electrophoresis (see
Fig. 3) reveal that both RNA and DNA (plasmid DNA and genomic DNA)
have been released from the cells without causing any obvious damage to
the nucleic acids. RNA bands are identified by comparing lanes 4 (RNase
treated) and 5 (untreated). Also, the plasmid DNA released remained as
the supercoiled form (unnicked).
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The hybridization result in Fig. 4(a) shows the control where an
oligonucleotide derived from an HLA coding region11 end-labeled with the
fluorophore Bodipy Texas Red was captured by the immobilized probe,
indicating that the immobilization chemistry for the oligonucleotide probe
worked as expected. The signal-to-noise ratio obtained is above 4. Here
the hybridization signal measured at the microlocations attached with
specific probes were subtracted from the background signal measured at
the locations without any probes and recorded as true signal. The signal
measured at the locations attached with non-specific probes were
subtracted from the background signal and recorded as noise signal.
The results in Fig. 4(b) show that thermally denatured plasmid DNA
obtained from electronic lysis could be used directly for hybridization-
based assays with high reproducibility (pads 1-1, 1-3, 2-1, 2-3, 4-1, 4-3,
5-1, 5-3). The intensity of the hybridization signal obtained from the
three-fold diluted lysate was comparable to that of the specific
oligonucleotide target.
The hybridization result (pad 1-5) in Fig. 5(a) shows the control where
RCA5, end-labeled with Bodipy Texas Red, was captured by the
immobilized probe ATA5 indicating that the immobilization chemistry for
the oligonucleotide probe worked as expected. The signal-to-background
ratio [positive control (pad 1-5) vs. negative control (pad 1-4)] was above
4. The results in Fig. 5(b) showed that thermally-treated ribosomal RNAs
(3× dilution on pad 1-1 and 1-3, and 5× dilution on pad 2-1, 2-3, 3-1 and
3-3) obtained from electronic lysis could be used directly for
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hybridization-based assays with high reproducibility (2-1, 3-1) where
pads 1-1, 2-1 and 3-1 show the hybridization results after electronic
stringency wash and pads 3-1, 3-2, and 3-3 show the hybridization
results before electronic stringency wash. The intensity of the
hybridization signal obtained from the three-fold diluted lysate was
comparable to that of the specific oligonucleotide target.
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Discussion
The separation of E. coli from normal human blood cells was
accomplished using the bioelectronic chip originally designed for
electronically enhanced nucleic acid hybridization assays. Both the
theoretical modeling and the experimental results suggest that
dielectrophoretic separation of cells using checkerboard addressing
generates more uniform electric field distribution than using the square-
wall addressing. The consequence of the uniform distribution of the
applied field is that it made the washing off of the undesired cells much
easier. Although the chip used in this study had only 25 electrodes,
preliminary results indicated that E. coli cells separated by this method
can provide enough plasmid DNA and RNA for some hybridization
assays. Further, we believe that by using arrays with an increased
microelectrode density, much higher recovery yields of desired cells could
be achieved in less time.
The chip surface was coated with an agarose permeation layer, which
has three advantages. First, the coating reduces cell adhesion where the
field is at a minimum and therefore facilitates the washing away of the
unwanted cells. Second, the isolated cells are kept away from the metal
electrode and are therefore less likely to be damaged by the
electrochemistry occuring at the surface of the electrode. Third, for the
capture of a particular type of cell the agarose layer can be functionlized
to provide a attachment chemistry. For example, streptavidin can be
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covalently bound to the agarose and used to immobilize a biotinylated
cell-specific monoclonal antibody.
The non-specific background fluorescence was greater with RNA target
than with DNA target. The electronic stringency washing was very
effective in removing background signal, even with low electric field
conditions, when the Tris-phosphate buffer was used. The level of the
hybridization signal obtained with DNA from the lysate diluted three-fold
is comparable to that of the oligonucleotide target. This result suggests
that in cases where it is necessary to screen multicopy plasmid DNA,
direct signal detection could be used, without the necessity of involving
an enzymatic amplification process.
The results obtained in the present study indicate that the separation
and isolation of E. coli cells from blood cells, the electronic lysis of the
isolated E. coli and the hybridization of the electronically released DNA or
RNA are feasible using microfabricated bioelectronic chips coated with a
permeation layer. The lysate contains a full spectrum of nucleic acids,
including RNA, supercoiled plasmid DNA and genomic DNA all without
apparent degradation. In addition, specific hybridization signals were
obtained using the released plasmid and RNA. Gene expression studies
using microarrayed chips have recently received considerable attention12-
16. The hybridization of RNA using microarrayed chips has significant
potential applications in gene expression monitoring. The high
concentration of RNA observed also suggests that the bacterial cells were
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viable after dielectrophoresis. The method used here may prove to be
useful in gene expression studies where a small number of cells of a
specific type are to be separated from a large number of other cells for
studying the RNA of a specific subpopulation.
The chips used for cell separation were originally designed for
hybridization-based DNA analysis. Chips with greater numbers of
electrodes should facilitate recovery of a greater number of the targeted
cells and, therefore, result in a higher yield of nucleic acids. Cell
separation using dielectrophoresis followed by electronic lysis has
significant potential as the front-end sample preparation process for
chip-based electronic hybridization assays in a future integrated system.
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Experimental protocol
The fabrication and coating of the bioelectronic chip, the manufacture of
the chip cartridge assembly, and the computer modeling of the field
distribution were same as previously described10.
Cell culture. A 296 bp fragment was amplified from the SpaO region of
Salmonella enteritidis, then cloned into plasmid pCR 2.1 (3890 bp) using
the Invitrogen T/A Cloning Kit (Invitrogen, San Diego, CA). Ligation was
performed according to kit instructions and the ligation product used to
transform INVαF′ competent E. coli. Transformants were grown up on LB
plates supplemented with ampicillin (100 µg/ml), 80 µl of x-gal (20
mg/ml), and 4 µl of isopropylthio-B-D-galactoside (40 mM) for blue/
white screening. Positive colonies were screened by PCR using SpaO
specific primers and the amplicons were examined by gel electrophoresis.
One positive clone from the PCR screen was grown up at 37°C overnight
in LB and ampicillin (100 µg/ml) liquid culture with shaking at 225 rpm.
Preparation of cell mixture. The cell separation buffer consists of
0.05× TBE (4.5 µM Tris, 4.5 µM boric acid, 0.1 µM EDTA, pH 8.2), 250
mM sucrose, pH 8.2. The conductivity of the buffer was 114 µS/cm
measured by an Accumet pH Meter 50 (Fisher Scientific, Pittsburgh, PA).
The conductivity of the cell separation was chosen carefully to ensure
that the E. coli cells would be subjected to positive dielectrophoresis and
all normal human blood cells would be subjected to negative
dielectrophoresis. The cultured E. coli cell suspension (1 ml) was
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centrifuged at 325× g for 4 min and the supernatant removed. The cell
pellet was washed in the cell separation buffer (1 ml) and pelleted using
the same conditions described above. The cells were then resuspended
in the cell separation buffer (1 ml). Fresh EDTA-anticoagulated human
blood (20 µl) was added to the E. coli cell suspension.
Dielectrophoresis system. The dielectrophoresis system used in the
current study is shown in Fig. 1. The cartridge was placed on the stage
of the Nikon phase contrast microscope. Illumination was through a ring
light (Precision MicroOptics, Irvine, CA). Image signals were collected by
a CCD/RGB color video camera (Sony DXC-151, Mikron Instruments,
San Diego, CA) through a 10× objective. Images were recorded using a
Sony VCR and monitored by a Sony TV monitor. The pumping of fluids
was accomplished by using a peristaltic pump (Model RP-1, Rainin
Instruments, Woburn, MA). The signals on electrodes were generated
from a function/arbitrary waveform generator (Model HP33120A,
Hewlett-Packard, Santa Clara, CA) and monitored by an oscilloscope
(Model HP54600, Hewlett-Packard).
Dielectric characterization. The frequency at which the E. coli cells
were subjected to the positive dielectrophoretic force and blood cells the
negative dielectrophoretic force was empirically determined by subjecting
the cell mixture to different conditions. The investigation was conducted
by gradually increasing the frequency of the sinusoidal signal (10 volts
peak-to-peak) starting from 5 KHz. When the frequency reached 10 KHz,
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evident separation of E. coli cells and the rest of the human blood cells
was observed. These electrical parameters were later used for the
isolation of the E. coli cells.
Dielectrophoretic separation of cells. To perform the dielectrophoretic
separation of E. coli cells from blood, an unused cartridge was employed.
The chip was first washed by pumping the separation buffer through the
flow cell. Next, the cell mixture was pumped into the flow cell and then
the pump was switched off. The entire 5×5 array of electrodes was
addressed in a checkerboard bias format by applying a sinusoidal signal
of 10 volts peak-to-peak at 10 KHz. The separation of E. coli cells from
the human blood cells was complete in approximately 4 min. Afterwards,
the pump was switched on to start the washing process. When the
sample mixture was nearly drained from the sample/buffer reservoir, the
separation buffer was added to wash the rest of the sample through the
flow cell while the A.C. signal was still on. After the wash, the separation
buffer (300 µl) containing proteinase K (490 µg, Boehringer Mannheim,
Indianapolis, IN) was pumped into the flow chamber.
Electronic lysis. To lyse the cells, a series of pulses (500 V, 50 µs pulse
width) was applied between the 4 counterelectrodes and the 25 smaller
cell-collecting electrodes. The pulses were applied in such a way that the
polarity were alternated every 20 pulses between the two groups of
electrodes. A total of 400 pulses were applied for each lysis process. The
lysate mixture was incubated at 50°C for 20 min to allow the DNA-
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contaminating proteins to be digested. The lysate was collected by
pumping it out of the flow chamber.
The combination of dielectrophoretic separation of cells and electronic
lysis was repeated six times and all the lysates were pooled together.
The pooled lysate was centrifuged at 16,000× g for 5 min. Next, the
supernatant was recovered and two volumes of chilled ethanol (-20°C)
were added to the above solution, mixed, and spun at 16,000× g for 10
min. The supernatant was removed and the pellet was dried in air.
Then the pellet was redissolved in 0.05× TBE buffer (300 µl). A fraction
of the redissolved solution (30 µl) was combined with a solution
containing RNase (3 µl, 10 mg/ml, Boehringer Mannheim). The
combined solution was incubated at 37°C for 30 min.
Gel electrophoresis. A 1.2% agarose gel was prepared by melting 600
mg of agarose in 50 ml of 1× TBE buffer. Ethidium bromide (2.5 µg) was
added to the agarose solution before it became solidified. Proteinase K
treated lysate samples with or without RNase treatment were loaded onto
the gel along with the marker DNAs.
DNA hybridization assay. An oligonucleotide capture probe (39-mer)
specific for the spaO region of the plasmid DNA was synthesized with
biotin in its 5′ end. The probe was diluted in 50 mM L-histidine buffer to
make a final concentration of 500 nM. The capture probe was
immobilized in agarose covering the electrodes containing streptavidin
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with the underlying electrodes positively biased. The electric biasing was
performed by maintaining the current on each selected electrode at 200
nA for 1 min. Afterwards, the remaining probe solution was removed
and the chip washed with 50 mM L-histidine buffer. The immobilization
of the probes for the biotinylated control oligonucleotides ATA5 and GAS
probe was accomplished using the same protocol described above. ACA5
is complementary to RCA510 and the GAS probe is derived from the speB
gene of S. pyogenes. The sequence of the GAS probe is as follows: 5′-
biotin-GGTAGAGTATCCTAGAATTTCTGGAGAACGTTTATCTCCTGAAACG-
3′.
To facilitate the hybridization of DNA, a fraction of the proteinase K
treated lysate was diluted three and five times respectively in the L-
histidine buffer. The tubes containing the diluted lysate samples were
placed in a 100°C water bath for 10 min to fragment the DNA (the final
size of DNA ranged between 300 bp to 1 Kb) and also to make the DNA
single stranded. To test the control, the synthesized RCA5
oligonucleotide target10 specific for capture probe ATA5 has been
electronically hybridized to both the immobilized ATA5 probe and the
adjacent non-specific GAS probe in parallel by keeping the positive
current at each electrode at 450 nA for 3 min. When the hybridization
processes were finished, fresh L-histidine buffer was loaded to perform
the electronic stringency wash. The washing was performed by applying
150 D.C. pulses to a row negatively biased at a time, 1 µA per pad with
0.1 sec on and 0.2 sec off. To conduct the sandwich assay the reporter
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probe hybridization was performed as follows. The chip was overlayed
with 1× STE buffer (10 µl) containing sonicated, denatured calf thymus
DNA (100 µg/ml, Sigma, St. Louis, MO) at room temperature for 5 min.
Afterwards, the buffer was removed and 10 µl of mixed solution (500 nM
reporter probe in 1× STE buffer containing calf thymus DNA) was put on
the chip and kept at room temperature for 5 min. The chip was then
washed five times with 10 µ1of 1% SDS buffer made with 0.2× STE then
soaked at room temperature in 5 ml of the same buffer for 10 min. The
chip was finally washed five times with 0.2× STE.
RNA hybridization The oligonucleotide capture probe 5′-biotin-
GAGTTGCAGACTCCAATCCGGACTACGACGCACTTTA-3′ specific for 16S
RNA was incorporated with biotin at its 3′ end. The probe was diluted in
50 mM L-histidine buffer to make a final concentration of 500 nM. The
capture probe was immobilized on the test microlocations containing
streptavidin with the underlying electrodes positively biased. The
positive electric biasing was performed by maintaining the current on
each electrode at 200 nA for 1 min. Afterwards, the probe solution left
was removed and the chip washed with 50 mM L-histidine buffer. The
immobilization of the probes for the controls (ATA5 and GAS probe) was
accomplished using the same protocol described above.
To test the control, the synthesized RCA5 oligonucleotide target specific
for capture probe was electronically hybridized to both the immobilized
probe and the adjacent non-specific probe in parallel by keeping the
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current at each electrode at 450 nA for 3 min. The capture of the
synthesized oligonucleotide target 5′-AATGGCGCATACAAAGAGAAGCG
ACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTG
GAGTATGCAACTCG-3′ was performed as above. To facilitate the
hybridization of 16S RNA, a fraction of the proteinase K treated lysate
was diluted three and five times respectively in the L-histidine buffer.
The diluted lysate samples used above for plasmid DNA hybridization
were also used here. When the hybridization processes were finished,
fresh Tris-phosphate buffer (20 mM Tris-base, 20 mM di-basic sodium
phosphate, pH 9.0) was loaded to replace the old L-histidine buffer to
perform the electronic stringency wash. The washing was performed by
applying 70 D.C. pulses to a row negatively biased at a time, 750 nA per
pad with 0.1 sec on and 0.2 sec off. To conduct the sandwich assay the
reporter probe hybridization was performed as follows. The chip was
overlayed with 1× STE buffer (10 µl) containing sonicated, denatured calf
thymus DNA at room temperature for 5 min. Afterwards, the buffer was
drained and 15 µl of mixed solution (500 mM reporter probe in 1× STE
buffer containing above mentioned calf thymus DNA) was put on the chip
and kept at room temperature for 5 min. The chip was then washed five
times with 15 µ1 of 1% SDS buffer made with 0.2× STE then soaked at
room temperature in 5 ml of the same buffer for 10 min. The chip was
finally washed five times with 0.2× STE.
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ACKNOWLEDGEMENT
This work was supported by grant number 95-08-009 under the
Advanced Technology Program from the Department of Commerce. The
authors would like to thank Drs. R. G. Sosnowski and J. Diver for
reading the manuscript and comments, and Drs. E. Mather and P.
Swanson for technical assistance. Excellent reproduction of the
photographs was made by Mr. K. Roy.
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Figure Legends
Figure 1. The illustration of the set-up for dielectrophoresis and
electronic lysis experiments.
Figure 2. (a)-(d) the illustration of square-wall and checkerboard
addressing of the five by five electrodes and the corresponding computer
models of the A.C. field distribution. The field distribution indicates that
a uniform distribution of the electric field can be obtained using the
checkerboard addressing format. (e) and (f) show the picture of the
coated chip after the pre-wetting step. (g) and (h) illustrate the
separation results under different addressing formats. (i) and (j) show the
results when the washing process was completed.
Figure 3. The analysis of nucleic acids released from the E. coli cells by
electronic lysis. Lanes 1 and 6 contained the λDNA Hind III digest
marker and ΦX 174 Hae III digest marker, respectively. Lanes 2 and 3
contained the control samples of supercoiled plasmid DNA and linear
plasmid DNA. Lanes 4 and 5 contained the electronic lysate with and
without RNase treatment.
Figure 4. The results of hybridization of plasmid DNA released from E.
coli by electronic lysis. (a) The hybridization control for electronically-
enhanced target capturing. (b) The sandwich assay result from the
plasmid DNA hybridization.
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Figure 5. The hybridization results of RNA released from E. coli by
electronic lysis. (a) The control for electronically-enhanced target capture
hybridization. (b) The sandwich assay result from ribosomal 16S RNA
hybridization.
24