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Microelectronic Engineering 85 (2008) 1484–1489

www.elsevier.com/locate/mee

Patterning a nanowell sensor biochip for specific

and rapid detection of bacteria
Sungkyu Seo a, Maria Dobozi-King b, Ryland F. Young b,
Laszlo B. Kish a, Mosong Cheng a,*
a
Department of Electrical and Computer Engineering, Texas A&M University, College Station, TX 77843-3128, USA
b
Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-3128, USA

Received 3 December 2005; received in revised form 6 October 2007; accepted 9 December 2007
Available online 3 January 2008

Abstract

This paper presents the fabrication process of a SEPTIC (SEnsing of Phage-Triggered Ion Cascades) chip, consisting of two Ti con-
tact pads and a 150 nm wide Ti nanowell device on LiNbO3 substrate. The patterning was fulfilled by combining electron beam lithog-
raphy, contact photolithography and reactive ion etching. When connected to an external preamplifier and spectrum analyzer, the
nanowell can probe nano-scale electric field fluctuations. The use of this chip as nano-scale electric field probe was demonstrated by
detecting the transitory ion efflux from bacteria being infected by phage. Our experiment showed that the electric field noise follows
1/f 2 power spectrum when the bacteria are sensitive to the phage, whereas 1/f noise corresponds to bacteria resistant to the phage.
The use of fluctuation-enhanced sensing can provide sensitivity orders of magnitude higher than conventional sensing that detects
changes only in the mean value of electrical signals.
Ó 2008 Published by Elsevier B.V.

Keywords: Nanofabrication; Biochip; Nanowell; Lithography; Reactive ion etching; Electric field fluctuation; Power spectrum; Bacteria; Bacteriophage;
Ion efflux

1. Introduction inexpensive method for detecting and subtyping bacteria

One of the most imperative needs in clinical and agricul-
tural practice as well as in homeland security applications
is the rapid and sensitive identification of bacteria. For
example, certain strains of the bacterium Escherichia coli
(E. coli) can cause widespread illness if they get into the
food supply. Even though several technologies are avail-
able for the identification of bacteria or viruses in humans,
veterinary and agricultural diagnostic laboratories, such as
culturing and polymerase chain reaction (PCR), these
approaches have difficulty such as time required for cultur-
ing of bacteria, expensive instrumentation or poor selectiv-
ity between living and dead bacteria. A rapid and
*
Corresponding author. Tel.: +1 979 862 4985; fax: +1 979 845 6259.
E-mail address: mcheng@ece.tamu.edu (M. Cheng).
suitable for large-scale surveillance efforts, much less
employed in the field, is not available.
Solid-state biological sensors have the potential to sat-
isfy this need due to their compactness, fast response to tar-
get, potential for integration with digital/analog electronics
and potential advantage in mass production. The majority
of these sensors use a bioaffinity element for sensing. This
involves an antibody, binding protein or receptor protein
that forms a stable complex with a corresponding ligand.
The bioaffinity protein–ligand complex is stable enough
to result in signal transduction. The complex can be
detected with the help of a label such as an enzyme, fluoro-
phore or electroactive substance [1]. Redox enzymes are the
major components in constructing bioaffinity sensors, and
electrochemically active substances are used in signal trans-
duction [1,2]. Examples of bioaffinity sensors include
enzyme field effect transistor (ENFET) [3], ImmunoFET

0167-9317/$ - see front matter Ó 2008 Published by Elsevier B.V.

doi:10.1016/j.mee.2007.12.046
 S. Seo et al. / Microelectronic Engineering 85 (2008) 1484–1489
[4,5], etc [6–10]. For practical solid-state biosensors, how-
ever, several issues need to be considered, such as the lim-
ited sensitivity due to the poor charge variation caused by
biological processes, non-specific response due to interfer-
ing ions or other substances present in the solution, and
the limited stability of the electrochemically active mem-
brane on the gate of the specific MOSFET [11].
In fact, the detection capability of a solid-state sensor
can be dramatically improved by the aid of fluctuation-
enhanced sensing (FES) [12]. The interaction between a
sensing probe and an analyte is always a dynamic stochas-
tic process. Micro-fluctuations that result from this interac-
tion carry a ‘‘stochastic fingerprint” of the analyte, and
contain much more information than the mean value
alone. Thus fluctuation-enhanced sensing can improve sen-
sitivity, selectivity, and reduce false alarm rates by orders
of magnitude over conventional sensor systems that only
measure the average values [12]. In the simplest version
of FES, the power spectrum density (PSD) of the detected
fluctuations is used as the detected signal [13].
Recently, the authors have developed and demonstrated
a novel sensing technology, named SEPTIC (SEnsing of
Phage-Triggered Ion Cascades), which combines the speci-
ficity and fast response of the bacteriophage with the sensi-
tivity of the nano-scale fluctuation-enhanced sensing [14].
Bacteriophages (‘‘phages”; viruses that specifically detect
and kill bacteria), have become a focus of interest for diag-
nosis, therapy and prophylaxis of bacterial disease in
humans, animals, and plants [15–21]. Typically, when a
phage infects a bacterium, the phage injects its DNA in
the host cell. At the end of the vegetative growth cycle, host
lysis occurs and the progeny virions, typically 100–300 par-
ticles, are released. Many double-stranded DNA bacterio-
phages cause massive, transitory ion leakages from host
cells as a consequence of the phage DNA injection process
[22,23]. This phenomenon is an ideal opportunity for bac-
terial diagnostics, because it not only takes advantage of
the well-known specificity available in bacteriophage but
it can occur, given sufficient phage concentration, within
seconds after admixture of the virions and cells [24]. More-
over, it requires no culturing of the analyte culture but only
that the target cells be physiologically viable (i.e. have ener-
gized membranes or intact membranes capable of energiza-
tion) [23].
To detect the ion efflux due to the DNA injection of a
siphophage or myophage, we constructed a biochip sensor
system, comprising a nanowell for probing electric field
fluctuations, a preamplifier for signal amplification, and a
fast Fourier transform (FFT) spectral analyzer for per-
forming the simplest FES. A nanowell device, consisting
of two metal electrodes with a gap of 5–200 nm in between,
is capable of probing nano-scale electrical fields and has
been used to study electron transport in molecules. [25]
This chip was exposed to a droplet of the analyte solution
containing bacteria and phages. When the bacteria were
resistant to the phages (uninfected bacteria), small voltage
fluctuations were observed in the nanowell displaying a
1485
power spectral density (PSD) of 1/f shape. In the case of
sensitive bacteria (infection occurred), much larger voltage
fluctuations of 1/f 2 shape were observed. No false alarm or
missed detection was observed. The biological experiment
was described in details in Ref. [14]. The detection limit
of SEPTIC based on linear response theory was also dis-
cussed by the authors [26]. This paper is focused on the pat-
terning process of the biochip described in Section 2. The
experimental configuration, procedures and results are
briefly discussed in Section 3. Final conclusions are pre-
sented in Section 4.
2. Nanowell structure and fabrication process
The fabrication of the nanowell device includes two
lithography processes: first, electron beam lithography
(EBL) was used to write a gap, 150 nm wide and 60 lm
long; then contact photolithography was used to pattern
two large contact pads (5 mm  5 mm) and a bridge
(100 lm long, 4 lm wide) connecting the two pads. The
gap interrupts the bridge and forms a nanowell, 150 nm
in width and 4 lm in length. The pads were used for elec-
trical connections with external circuitry and the bridge
connected the pads with the nanowell. The top-down view
of the chip is illustrated in Fig. 1, in which the nanowell is
indicated.
Fig. 2 illustrates the fabrication of the nanowell. First,
100 nm thick Ti layer was deposited on the surface of lith-
ium niobate (LiNbO3) wafer by DC sputtering system with
3200 V/40 lF under the pressure of 5  10 6 Torr. Next,
3% PMMA, 950K (MCB), which had molecule weight
950K and diluted with the solvent monochlorobenzene
made by Brewer Science Inc., was spin coated on the
deposited Ti layer. The spinning took 30 s at 2000 rpm, fol-
lowed by soft baking for 90 s at 185 °C, which resulted in a
200 nm thick PMMA layer. Finally, a 150 nm wide, 60 lm
long trench is written on the PMMA film by EBL and the
PMMA resist is developed in the mixture of MIB-
K:IPA(1:3), for 1 min. The cross-section of the completed
wafer is shown in Fig. 2a.
Nanowell
150nm (150nm*4μm)
Pad 1 Pad 2
(5mm*5 (5mm*5
mm) mm)
4μm
100μm
Ti LiNbO3
Fig. 1. Top-down view of the chip with nanowell, contact pads and
bridges connecting between the pads and the nanowell.
1486 S. Seo et al. / Microelectronic Engineering 85 (2008) 1484–1489

a b R.I.E.

PMMA PMMA
Ti (100nm) Ti (100nm)
LiNbO3 LiN bO3

c d R.I.E.

AZ5214
Ti (20nm)
LiNbO3

e f Nanowell
Sensing Area
Pad 1 Nanowell Pad 2

AZ5214
Ti (20nm)
LiNbO 3 LiNbO 3

Fig. 2. Fabrication processes of the nanowell.

With PMMA as hard mask, a 150 nm wide, 60 lm long
trench in the Ti layer was etched anisotropically by reactive
ion etching (RIE) at the pressure of 2  10 5 Torr with
Ar(3 SCCM)/CHF3(30 SCCM)/He(7.5 SCCM) gases.
Since the etching selectivity of Ti to PMMA is only 0.1,
two successive RIE steps were employed. First, all the
200 nm PMMA film and 20 nm of the unprotected Ti layer
were etched, resulting in a thickness of 80 nm in the unpro-
tected Ti region, and 100 nm in the protected region. In the
second step, 80 nm Ti was etched, so finally 20 nm Ti layer
remained in the region originally protected by PMMA. For
the efficient removal of Ti residue, 10 min sonication clean-
ing process was used after each RIE step. The cross-section
of the wafer after the RIE is shown in Fig. 2b.
Next, the whole chip was cleaned by stripper AZ300T at
95 °C for 15 min. Contact pads were defined by conven-
tional contact photolithography with positive resist
AZ5214 on a Karl Suss MJB-6 mask aligner. The pads
were exposed to an illuminating power of 11.0 mW/cm2
for 5.8 s without filter (Fig. 2c). The resist processing
parameters were as follows: spinning 5000 rpm for 30 s,
soft baking 99 °C for 2 min on hot plate. After developing
AZ5214 resist, the Ti layer outside the contact pads was
etched by RIE (Fig. 2d) using the above protocol and the
resist layer was stripped by AZ300T at 95 °C for 15 min
(Fig. 2e). Finally, the whole chip was spin coated with
AZ5214 resist and an 8  6 lm2 window around the nano-
well was opened by photolithography using mask aligner
MJB-6. The resist processing parameters are 5000 rpm
and 30 s spinning, 99 °C and 2 min soft baking in hot plate
and 135 °C and 12 min hard baking in oven (Fig. 2f).
Fig. 3a shows a 200 optical microscope picture of the
fabricated chip with one nanowell device, two contact pads
and two connection bridges. The nanowell was hardly vis-
ible. Fig. 3b shows a scanning electron micrograph (SEM)
of a fabricated nanowell device on silicon substrate, which
is identical to the nanowell on LiNbO3 substrate. However,
the SEM images of the nanowell on LiNbO3 had very poor
contrast, which may be due to the severe charging effect
due to the insulating substrate. When writing the nanowell
with EBL, the charging effect was not an issue because of
the 100 nm thick Ti film covering all of the LiNbO3. But
when taking SEM of the nanowell, which had only 20 nm
thick Ti film covering a small portion of the LiNbO3, the
charging effect became a severe factor limiting the SEM
quality.
It should be noted that the effective device is a Ti-on-
LiNbO3 structure. The LiNbO3 substrate, as an insulator,
prevents direct current flow through the nanowell. In this
sense, a silicon substrate, although less expensive, cannot
be used. An oxidized Si wafer may be usable, but would
complicate the fabrication process. LiNbO3 substrate does
not require a thermal oxidation step, nor any treatment
before Ti deposition. Moreover, LiNbO3 has a stronger
resistance to the employed RIE than SiO2 does, which pre-
vents the possible etching into the substrate.
3. Experiment of probing bacteriophage infection-associated
ion efflux
This biochip was used to perform initial experiments
with bacteriophages kS105 (k Dstf, tfa::CamR cI857
 S. Seo et al. / Microelectronic Engineering 85 (2008) 1484–1489 1487

Fig. 3. (a) Optical microscope pictures of the fabricated nanowell device and contact pads (200); (b) SEM picture of the nanowell (7720). To improve
image contrast, this nanowell was patterned on silicon substrate. The nanowell used in detecting bacteria/phage was patterned on LiNbO3 substrate.

S105). As kS and kR strains low motility Escherichia coli
(E. coli) W3110 DfhuF and W3110 DfhuF DlamB were used,
respectively. The experimental details and results are pre-
sented in Ref. [14]. The basic experimental protocol was
to mix the purified phage stock (about 2  1010 pfu/ml)
with the host cells (mid-log phase cells, washed and resus-
pended in 5 mM MgSO4), incubate at 37 °C for various
times and measure the voltage fluctuations in the nanowell.
The isogenic host mutant strain (E. coli W3110 DfhuF
DlamB) was used as negative control, for which we did
not anticipate injection leakage of ions.
During the experiment, a 5 ll droplet of the analyte
containing bacteria and bacteriophage was placed in and
around the nanowell, as indicated by the dotted circle in
Fig. 3a. Two probes, providing input to an external voltage
amplifier, were firmly pressed on the contact pads. In order
to prevent short circuit between the two pads and reduce
unwanted noise caused by background ions in the solution,
the surface of the chip, except for the nanowell sensing
area, was covered with resist AZ5214. A 6 lm  8 lm rect-
angular window was then opened by photolithography.
Finally, the resist was hard baked. This way only the
opened sensing window outlined in Fig. 3a was exposed
to liquid during experiment. Fig. 4 shows the schematic
representation of the experimental apparatus. The voltage
fluctuations induced on the nanowell device by the electri-
cal field was amplified by a low-noise preamplifier SR560
with high input impedance (100 MX) and fed into signal
1488 S. Seo et al. / Microelectronic Engineering 85 (2008) 1484–1489

SIGNAL ACQUISITION UNIT *

PREAMPLIFIER **

8 um
150 nm
Probe 1 Probe 2

Pad 1 Pad 2

AZ5214 LiNbO3 * ML750 Powerlab / 4SP

** SR560
Ti Analyte Droplet

Fig. 4. The schematic representation of the experimental apparatus.

Fig. 5. Response of sensitive and resistant bacteria with phages. In the

acquisition unit ML750 PowerLab/4SP as illustrated in case of kR bacteria (E. coli W3110 DfhuA DlamB, negative response, blue
Fig. 4. The power density spectrum of the fluctuations line), phage infection did not occur, so the spectrum of voltage
was calculated by a Dynamic Signal Analyzer SR785. The fluctuations in the nanowell follows approximately 1/f shape. In the case
of positive response of kS bacteria (E. coli W3110 DfhuA, red), phage
nanowell was placed in a double screening box (Amuneal
infection occurred, so the fluctuations are enhanced, resulting in a steeper
Manufacturing Corp.) to prevent electromagnetic distur- spectrum with a 1/f 2 shape. Comprehensive data are given by Ref. [14].
bance. The double screening box and the preamplifier were (For interpretation of the references to colour in this figure legend, the
placed on an anti-vibration platform 100BM-2 (Nano-K) to reader is referred to the web version of this article.)
avoid potential artifacts caused by vibrations. The time
window of the determination of the power density spec-
The detection sensitivity can be significantly improved if
trum Su(f ) was 2 min.
the external preamplifier is integrated into the same chip so
An important rule is that the analyte droplet should not
that the noise due to external cable and input stage is elim-
touch the two probes connected to the external preampli-
inated. An estimation of the minimum number of bacteria
fier, otherwise large unwanted noise would result.
that can be detected is shown in Fig. 6, based on linear
When exposed to air or to a solution without any organ-
response theory [26]. When a JFET preamplifier with ther-
ism, the voltage fluctuations in the nanowell were thermal
mal noise reduction technique is integrated into the chip,
noises, which appeared as white noises. In the mixture of
the SEPTIC technology is projected to detect the presence
non-adsorbing phages and bacteria (the phage does not
of two bacteria. For certain phages, the sensitivity is even
infect the bacteria), the voltage fluctuations in the nanowell
100 times higher, i.e. SEPTIC can detect a single bacterium
were small, displaying PSD of approximately 1/f shape. On
even if the number of ions leaking from the bacterium were
the other hand, in the solution where the phages invade the
bacteria (i.e. the bacteria is sensitive to the phage), we
observed large and slow stochastic waves with various time
10 6
with Ur - λ (Number of bacteria)

and amplitude scales. These fluctuations had PSD of

approximately 1/f 2 shape in the frequency range of 1–
Projected detection limits

10 5
10 Hz, which is conjectured to be due to the ion efflux from
the bacteria and possibly the Brownian motion of the 10 4
charged bacteria. Further investigation is ongoing to study
10 3
the nature of the observed 1/f 2 noise. Fig. 5 shows an
example of PSD plots corresponding to sensitive and resis- 10 2
tant bacteria that were incubated for 3 min and then mixed
with kS105 phages. 10 1
Note that other sources of electric field noise may also
exist, including temperature fluctuations, background ions, 10 0
Bipolar JFET JFETpreamp
etc. However, our experiment showed that these effects preamp preamp with thermal
were much weaker than the transitory ion leakage due to noise reduction
phage invasion. Therefore, our fluctuation-enhanced nano- Fig. 6. Projected sensitivities, based on the assumption of linear response
scale electric field detection system was successfully applied with the bacteriophage kUR. Figure is based on data extracted from [26].
in all our experiments. With some types of phages, the sensitivity limits are about 100 times lower.
 S. Seo et al. / Microelectronic Engineering 85 (2008) 1484–1489
100 times less [26]. The theoretical exploration on the
detection sensitivity was detailed in Ref. [26].
4. Conclusion
During a phage infection process, the myophage or
siphophage injects its DNA into the host cell, resulting in
a transitory, massive ion efflux. This phenomenon provides
a perfect opportunity for the rapid and specific detection of
bacteria, due to the fact that a certain type of phage can
only invade a specific type of bacteria, resulting in an ion
efflux within seconds of phage DNA injection. To this
end, we have successfully fabricated a biochip whose core
element is a nanowell device on LiNbO3 substrate, which
comprises two 4 lm wide Ti electrodes with a 150 nm gap
between them. The fabrication was performed by combin-
ing electron beam lithography, contact photolithography
and RIE without using lift-off process. Detection experi-
ments were conducted by mixing phage with sensitive and
resistant bacteria, respectively, on the nanowell region
(no voltage is applied to the nanowell) and analyzing the
voltage fluctuations across the surface. In mixtures where
the bacteria were sensitive to the phage, large 1/f 2 noise
(1–10 Hz) was observed, while with mixtures where bacte-
ria were resistant to the phage, only small 1/f noise due
to preamplifier was observed. The large 1/f 2 noise is
believed to be due to temporary charging fluctuations of
the bacteria due to phage-induced ion leakage. Our preli-
minary experiment showed 100% success rate in identifica-
tion of bacteria on the scale of minutes, whereas other
known technologies require hours to days of bacteria cul-
turing and are often not specific. The sensitivity is expected
to be at the single bacterium level. Ultimately, this technol-
ogy could prove invaluable in clinical, veterinary and agri-
culture practice, as well as in applications to
microbiological threat detection and reduction in biode-
fense applications.
Acknowledgements
Valuable discussions with Dr. Sergey Bezrukov at NIH,
Prof. Bob Biard and Prof. Henry Taylor at TAMU EE are
appreciated. The authors are also grateful to Prof. Win-
fried Teizer and Arlene Ford at TAMU Physics, Robert
Atkins of the TAMU ISEE for valuable technical support.
This work was supported in part by the TAMU Informa-
tion Technology Task Force (LBK). MDK was supported
1489
in part by the US Army Medical Research and Material
Command Disaster Relief and Emergency Medical Ser-
vices program and by a Program of Excellence award (Pro-
gram of Membrane Structure and Function) from the
TAMU Life Sciences Task Force to RY. The nano-well
developments have been supported by funds from the Col-
lege of Engineering to MC.
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