Sensors and Actuators B 126 (2007) 508514

On-chip microuidic biosensor for bacterial detection and identication

Douglas A. Boehm
a
, Philip A. Gottlieb
b
, Susan Z. Hua
a,b,
a
Bio-MEMS and Biomaterials Laboratory, Department of Mechanical & Aerospace Engineering,
SUNY-Buffalo, Buffalo, NY 14260, United States
b
Department of Physiology and Biophysics, SUNY-Buffalo, Buffalo, NY 14214, United States
Received 18 January 2007; accepted 29 March 2007
Available online 8 April 2007
Abstract
In this paper, we have developed a simple and rapid method for the detection and identication of bacteria using a microuidic lab-chip. The
microuidic chip utilizes impedance-based measurement to (1) detect cells and (2) identify them when used in conjunction with immobilized
monoclonal antibodies. Bacteria in suspension passing through the microuidic chamber are recognized by antibodies and selectively immobilized
on the functionalized glass surface, thereby increasing the measured impedance within the chamber. Continuous perfusion of bacteria suspension
through the derivatized chamber not only identies specic bacteria but also enhances the chambers detection sensitivity by accumulating bacteria
on the chamber wall over time; this approach would be useful for detecting lowconcentrations of bacteria. To demonstrate this approach, we showed
that the prototype sensor could detect 9 10
5
CFUmL
1
E. coli (BL21(DE3)) in the solution by consecutive perfusions. The chip sensitivity with
immobilized bacteria is governed by height of sensing chamber, and 10
4
CFUmL
1
of E. coli could easily be detected when a shallower chamber
(2 m high) was used. The selectivity of the sensor was tested using a suspension of two bacterial strains, E. coli and M. catarrhalis. The sensor
chip is simple to use, requires minuscule samples, and eliminates extensive cell culture processes. Development of more advanced lab-chips with
multiple chambers containing different antibodies that allow simultaneous detection of different bacteria strains will be a natural extension of this
work.
2007 Elsevier B.V. All rights reserved.
Keywords: Biosensor; Impedance; Bacteria detection; Microuidics
1. Introduction
The rapid detection and identication of bacteria strains has
emerged as a pressing issue in elds ranging from clinical diag-
nostics and monitoring of food-borne pathogens to detection of
biological warfare agents. Conventional methods to detect and
identify bacteria usually require high cell population, which is
achieved by growing an initially small number of bacteria into
high population colonies [1]. Such methods are time consum-
ing, typically requiring more than 24 h. In addition, the lack of
selectivity of sensors in the presence of other species in com-
plex solutions is a common problem that directly affects sensor
sensitivity and the functionality [2]. There is a need for minia-
ture, low-cost sensors capable of rapid detection and accurate
identication of bacteria in a multi-organism suspension. In
this regard, microuidic lab-chip technology provides means

Corresponding author.
E-mail address: zhua@eng.buffalo.edu (S.Z. Hua).
of making portable, fast, and sensitive biosensors with simple
on-chip operation.
Several microuidic on-chip bacteria-sensing modalities
have been developed using electrophoresis [3], labeled or mag-
netic beads [46], cantilevers [7] and membrane lters [8,9].
The signal transduction in these devices is normally achieved via
optical [1013], acoustic [14,15], impedance [16,17], or electro-
chemical measurements [18]. Impedance based sensors offer the
advantages of rapid response, ease of fabrication, and high sen-
sitivity. A number of impedance based on-chip bacteria sensors
have been developed in recent years. The reported modalities
include measuring the impedance change of cell culture media
due to bacteria metabolism [19], detecting the formation of a
conductive polymer layer as a result of antibody-antigen bind-
ing in the presence of bacteria [20], and sensing impedance
change due to direct deposition of bacteria on or across an
electrode array [21]. Here we demonstrate an impedance-based
method to simultaneously identify and detect bacteria in a fully
functionalized microuidic chamber. The lab-chip is capable of
accommodating the antibody/antigen reactions along the ow
0925-4005/$ see front matter 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.snb.2007.03.043
D.A. Boehm et al. / Sensors and Actuators B 126 (2007) 508514 509
pathway, can detect/identify small amounts of bacteria in uid
in a short time. When the sensing chamber surface is function-
alized with antibodies, bacteria in the sample solution can be
selectively captured inside the measuring chamber, thus increas-
ing the chambers resistivity. The sensor chip takes advantage
of ow ltration via surface modication of chamber surface
and simple on-chip electrical measurement, and shows ease
of detection and identication of bacteria in complex sam-
ple solutions. The fabrication steps are straightforward, and
similar to those we have previously developed for impedance-
based microuidic biosensor lab-chips for measuring changes
in cell volume, or electrolytic bubble-based micro-transducers
[22,23].
2. Materials and methods
2.1. Sensor design and construction
The impedance-based bacteria sensor is based on the mea-
surement of resistance of a microuidic testing chamber. The
microfabricated chamber has a xed volume and is attached to
an input and output for uid perfusion, as shown schematically
in Fig. 1. Since the membrane of bacterial cells act as insula-
tor at low signal frequency, the presence of bacteria cells in the
solution can be detected via resistance change in the chamber
as they displace an equivalent volume of conducting solution
in the chamber. Fig. 1 shows schematically the testing chamber
with suspended cells (Fig. 1a) and immobilized cells (Fig. 1b).
As shown in Fig. 1, the measured resistivity for the suspended
cells depends on its volume concentration in the testing solution,
while for immobilized cells it depends on both the concentration
of cells and the height of the chamber - shallower the chamber,
higher the sensitivity.
The prototype sensor chip has a testing chamber 15 m
deep and 1.5 mm wide. The distance between the two sens-
ing electrodes is 2.4 mm giving an active chamber volume
of 70 nL. A microuidic channel with an inlet and an out-
let port is used for sample loading and solution exchange.
Thin lm platinum/platinum-black electrodes in the cham-
ber form a four-point probe for measuring the chamber
resistance. The chip was fabricated using standard silicon tech-
Fig. 1. Schematic drawing of impedance based bacteria sensor for suspended
(top) and attached cells (bottom).
nology. Detailed microfabrication steps are described elsewhere
[22].
Glass slides with derivatized surfaces were used to cap the
microuidic chamber by pressing it against the chip using a
mechanical clamp that applies a uniform pressure of 12 Psi.
For the electrical measurements, an active current source pro-
vides 1 A, 50 Hz sinusoid to the two outer electrodes in Fig. 1.
We chose low frequency stimulation to minimize the dielec-
tric loss through silicon and to reduce the demands on the
common mode rejection of the voltage amplier. The voltage
between the two inner electrodes was measured using a home-
made instrumentation amplier with input currents <1 pA to
reduce polarization. A lock-in amplier provided rectication
and ltering. The image of bacteria on the cover glass was
obtained using a Zeiss Axiovert 200 inverted microscope and
recorded with a digital camera.
2.2. Materials and reagents
All general chemicals and buffer reagents were purchased
from Sigma Company, St. Louis, MO. Green uorescent
nucleic acid stain SYTO-16 and uorescence spheres were
acquired from Molecular Probes Company, Eugene, OR. Cross
linking reagents methyl N-succinimidyl adipate (MSA) and 1-
ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride
(EDC), 2-(N-morpholino)ethanesulfonic acid (MES), amino-
propyltriethoxysilane (ATEPS), N-hydroxysuccinimide (NHS),
and phosphate buffered saline (PBS) were acquired from Pierce
biotechnology Inc., Rockford, IL. Rabbit polyclonal IgG Anti-
body (ab13626) was purchased from Abcam, Cambridge, MA;
another antibody, mAb 3F5-5E5, was a gift of Dr. A. Campag-
nari, SUNY at Buffalo.
2.3. Surface derivatization
The surface of cover glass slides was derivatized with a layer
of monoclonal antibodies that target specic bacteria. The sur-
face modication protocol is as follows. The glass was rst
cleaned in a 50% ethanol in DI water for 10 min ultrasonically,
followed by a mixture of 1:1 methanol and HCl for 20 min. The
cleansed glass slides were then treated with 4% ATEPS solu-
tion in acidic-methanol (0.3% glacial acetic acid) for 20 min to
create an amine reactive surface. After this step, the glass was
rinsed with methanol followed by DI water, and dried with N
2
,
after which it was ready for attachment of a linker. A reagent
called MSAwas introduced to treat the surface for 30 min to 1 h,
and this specically reacts with amino groups leaving a carboxy
ester. The carboxylic acid group was then exposed by phos-
phate buffer with pH 9.5 for 1 h followed by washing with DI
water and drying. The compound, EDC, was used to covalently
link the desired antibody to the glass surface. A buffer solu-
tion containing 0.1 M MES, 0.5 M NaCl, 0.4 mg/mL EDC and
0.6 mg/mL NHS was applied to the surface of the glass slides
for 15 min. The antibody was then applied to the glass slides and
allowed to sit at room temperature for 3 h. A rabbit polyclonal
IgG Anti-E. coli (0.1 mg/mL in PBS) was used for immobiliz-
ing E. coli (BL21(DE3)) cells, and Mab 3F5-5E5 (0.1 mg/mL
510 D.A. Boehm et al. / Sensors and Actuators B 126 (2007) 508514
in PBS) antibody was used for M. catarrhalis cells. The glass
slides were thenstoredat 4

C, 70%humidityina coldroomuntil
needed.
2.4. Bacterial culture condition
Bacteria were grown in standard culture media LB broth in
the presence of appropriate antibiotics. The cells were incubated
at 37

C to full conuence and then diluted in LB broth for the

measurements.
3. Results and discussion
3.1. Sensor calibration using uorescence beads
The sensitivity of the impedance based microuidic sen-
sor is governed by two factors, (1) the detection limit for the
smallest volume change in the testing chamber and (2) the
capability of increasing the volumetric ratio of bacteria to test-
ing chamber. The former reects the intrinsic measurement
limit of electronics and latter results from the chamber design
Fig. 2. (a) (left panel) Fluorescence images of sensor region containing 2 m uorescence beads of various concentrations, 0.05%, 0.12% and 0.22%, respectively.
(b) (right panel) Corresponding sensor output. (c) Sensor output via volume percent of beads in the testing chamber for different bead sizes.
D.A. Boehm et al. / Sensors and Actuators B 126 (2007) 508514 511
(chamber height) and surface functionality for bacteria immo-
bilization.
We characterized the resolution of the sensor to detect change
in conducting volume by perfusing uorescence beads with var-
ious sizes and different concentrations in the chamber. We chose
uorescence beads with diameters of 0.5 m, 1 m, 2 m, and
4 mto closely match the size of the bacteria. In this experiment,
the beads with known size and various volume concentrations
were perfused into the chamber through the microuidic inlet
channel, and the chamber resistance (as an output voltage) was
monitored. We then stopped the ow to count the number of
beads in the measuring chamber and calculated the fractional
volume occupied by the beads. Fig. 2a (left panel) shows the
optical images of the test region with 2 m beads of various
volume concentrations, 0.05%, 0.12% and 0.22%, respectively.
Fig. 2b (right panel) shows the corresponding sensor output.
The calibration results using the beads with different sizes are
summarized in Fig. 2c. The results show that a 0.05% change
in volume in the testing chamber can be distinguished via resis-
tance measurement. Aconsistent sensor response with respect to
volume was observed when the beads size is larger than 1 m. In
the case of smaller beads (smaller than 1 m) some aggregation
can make the count less accurate.
3.2. Sensitivity characterization
To test the chip performance for bacteria detection, we used
the functionalized glass slide to cover the chip in order to immo-
bilize the targeted bacteria in the chamber. The cover glass
slides were derivatized with rabbit polyclonal IgG antibody for
immobilizing E. coli cells. Testing solution was the LB broth
containingvarious amounts of E. coli cells dilutedfromsaturated
cell culture.
In this test, we rst perfused the chamber with plain LBbroth
without bacteria to establish the base line resistance, and then
switched to a broth media with cultured bacteria at a given con-
centration. We then stopped the ow for 10 min to allow the
bacteria to interact with the functionalized surface. After the
immobilization period, the ow of plain LB broth was rein-
troduced for resistance measurements. Fig. 3ac shows sensor
Fig. 3. Left panel: Sensor output resulting from the change in conducting volume when perfused with a solution with 9 10
6
CFUmL
1
(a), 2 10
7
CFUmL
1
(b), and 1 10
8
CFUmL
1
(c) of E. coli. The chamber was modied by immobilizing antibodies. (d) As a control, a solution containing 1 10
8
CFUmL
1
of E.
coli. was perfused through the chamber that was not modied with antibodies. Right panel: corresponding optical image of E. coli attachment on the cover glasses.
The scale bars represent 20 m.
512 D.A. Boehm et al. / Sensors and Actuators B 126 (2007) 508514
output measured from solutions containing 9 10
6
, 2 10
7
and 1 10
8
CFUmL
1
E. coli, respectively. In Fig. 3ac, the
culture media containing bacteria was perfused into the cham-
ber during the time periods indicated by the two dashed lines.
Since the microbial metabolism usually results in an increase
in both conductance and capacitance, causing a decrease in
impedance, we have only used measurements when the system
is perfused with LB broth. The amount of attachments on the
glass cover-slide was conrmed by an optical microscope. The
right panel of Fig. 3 shows corresponding optical image from
each run. Using this direct measurement we were able to iden-
tify the presence of bacteria in the range of 10
8
10
6
CFUmL
1
.
This demonstrates the ability and functionality of impedance-
based sensing approach. The detection limit can be reduced
to 10
5
CFUmL
1
and lower simply by reducing the chamber
height to 2 m. In order to establish a control and to differen-
tiate specic bonding from unspecic bonding, we conducted
the same tests using an unfunctionalized glass slide. Fig. 3d
shows the measured sensor output and the corresponding optical
image. As seen from Fig. 3d the signal is at zero-level after per-
fusing the chamber with bacteria, and the optical image shows
very few bacteria due to nonspecic bonding. Factors affect-
ing the amount of bacteria that become attached to the surface
include the treatment of the surface and the amount of bacteria
that interact with the surface. Several independent experiments
have been performed to conrm the consistency. A minimal
variation (10%) was seen between runs using different glass
slides.
3.3. Surface immobilization and bacterial accumulation
The uidic nature of the sensor chip combined with a func-
tionalized chamber surface allows us to advance the detection
limit by accumulating bacteria on the chamber wall through
multiple perfusions. The ultimate detection limit, therefore,
depends on the binding afnity of the antibody for antigen
and the concentration of conjugate, in addition to the nature
of the transducer. The optimal sensing speed relies on reaction
kinetics.
For the given dimensions of the testing chamber, the diffusion
time is largely reduced; capture of targets is simply limited by
reaction kinetics, governed by Langmuir equation [24]. How-
ever, for a small dimension of testing chamber and a stop-ow
condition during the reaction, the depletion of the target analytes
need to be considered. We have estimated the time required to
deplete the bacteria in a closed chamber for various concentra-
tions in sample solution, and found that the required time can be
5 min for concentrated solution (10
8
CFUmL
1
) to 15 min
for diluted solutions (10
5
CFUmL
1
). For detecting very low
concentrations of bacteria, multiple perfusing into the chamber
was conducted. Fig. 4 shows the test results from multiple per-
fusions with a solution containing 9 10
5
CFUmL
1
E. coli.
After perfusing the chamber with plain broth media (time period
A in Fig. 4), the sample solution was perfused into the chamber
with ve stopped ow intervals, (seen as spikes in time period
B in Fig. 4) followed by plain broth perfusion during which
measurements were made (time period C in Fig. 4). A 15 mV
Fig. 4. Resistivitychanges after multiple perfusions tothe chamber withsolution
containing 9 10
5
CFUmL
1
E. coli. Each cycle of attachment contained ve
stopped ow intervals (B, D), seen as spikes. After each set of ve cycles the
change in baseline was determined using a broth solution (C, E), and compared
to the original baseline (A).
signal was observed at this stage. The same process was repeated
to further increase the sensing output, and a higher signal of
21 mVwas observed (time period E in Fig. 4). This increase in
signal was attributed to an increase in both bacterial attachment
and their subsequence growth.
3.4. Specicity tests
Identication of bacteria was accomplished through selective
immobilization of bacteria to functionalized antibody. Nonspe-
cic binding of other objects during measurements can cause
false signals and an inaccurate result. We enhanced the surface
specicity by introducing monoclonal antibodies on the surface
of the measuring chamber, as described in the previous section.
To evaluate the surface selectivity, two types of slides were
prepared, one set was derivatized with rabbit polyclonal IgG
Anti-E. coli for immobilizing E. coli, and the other with Mab
3F5-5E5 antibody for immobilizing M. catarrhalis. We deter-
mined the specicity of the derivatized surfaces by allowing
the bacteria to react with either surfaces and assessing specic
and non-specic binding. Fig. 5a and b shows the attachment
of E. coli on glass slides derivatized with IgG Anti-E. coli and
Mab 3F5-5E5 antibodies, respectively. Fig. 5c and d shows the
attachment of M. catarrhalis on IgG Anti-E. coli and Mab 3F5-
5E5 antibody, respectively. Results show a much higher levels
of attachment on slides treated with the corresponding specic
bacteria.
The specicity of the sensor chip was further tested using
a solution with mixed population of bacteria. In this experi-
ment, the E. coli was stained with SYTO-16 dye while the
M. catarrhalis does not stain. A mixed population of bacte-
ria was introduced into testing chambers functionalized with
two different antibodies, the total attachment (specic and non-
specic) was observed by uorescence microscopy. A high
degree of E. coli attachment to the chamber walls for E.
coli specic antibodies was observed, as shown in Fig. 5e,
while binding of E. coli on the glass derivatized with M.
D.A. Boehm et al. / Sensors and Actuators B 126 (2007) 508514 513
Fig. 5. (ad) Optical image of bacteria attachment on functionalized glass surfaces with different antibodies. (a) E. coli reacted with IgG Anti-E. coli antibodies; (b)
E. coli reacted with Mab 3F5-5E5 antibodies; (c) M. catarrhalis reacted with IgG Anti-E. coli antibodies; (d) M. catarrhalis reacted with Mab 3F5-5E5 antibodies.
(e and f) Fluorescent images of E. coli immobilized on the cover glasses functionalized with IgG Anti-E. coli antibodies (e) and Mab 3F5-5E5 antibodies (f). The E.
coli cells were stained with SYTO-16 dye while the M. catarrhalis was not stained in (e) and (f). The dashed lines in (e) and (f) indicate the boundary of the sensor
chamber. The scale bars represent 20 m.
catarrhalis specic antibodies (pointed by arrows in Fig. 5f) was
negligible.
4. Conclusions
We have described a simple method to identify and detect
bacteria in a microuidic measuring chamber. By coupling an
impedance based detection system with surface derivatization
of the sensing chamber using monoclonal antibody, bacterial
samples can be identied within minutes. Even very dilute sus-
pensions can be used because the derivatized surface serves to
concentrate the specic bacteria strain in the sensing chamber.
The biosensor currently can detect 9 10
5
CFUmL
1
E. coli
using multiple perfusion of cellular suspension. High selectivity
of the sensor is demonstrated by testing the solution with mixed
stains of bacteria, E. coli and M. catarrhalis. By immobilizing
different antibodies to parallel chambers, this approach can be
easily extended to a multiple chambers Lab-on-a-Chip to detect
and identify various bacteria in a complex solution.
Acknowledgements
This work was supported by National Science Foundation
Grant No. CMS-0201293 and CMS-0509723. We thank Pro-
fessors Frederick Sachs and Harsh Deep Chopra for useful
discussions. This work was performed, in part, at the Cornell
NanofabricationFacility, whichis supportedbythe National Sci-
ence Foundation Grant No. ECS-9731293, Cornell University
and industrial afliates.
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Biographies
Douglas A. Boehm received his BS in mechanical engineering from the Uni-
versity at Buffalo in 2004. He continued studying at UB to earn his MS in
mechanical engineering (2006) under the supervision of Dr. Susan Hua, where
his research focused on microuidic detection of bacteria. He currently works
as a Product Test Engineer for Bureau Veritas, a multinational company, where
he evaluates toys and other consumer products for compliance with government
safety standards.
Philip A. Gottlieb earned his BS in physics from Northeastern University in
1976. He then attended the Weizmann Institute of Science where he received his
MS and PhD in bioorganic chemistry under the supervision of Dr. Mati Fridkin.
Following this, he studied nucleic acid chemistry and protein-DNA interactions
with Dr. Marvin Caruthers at the University of Colorado. He started as an assis-
tant professor at the University of Delaware and is currently a research professor
in the Department of Physiology and Biophysics at the State University of New
York at Buffalo where his main interest is mechanically active ion channels.
Susan Z. Hua is an assistant professor in the Department of Mechanical
and Aerospace Engineering, and Department of Physiology and Biophysics at
SUNY-Buffalo. She received her PhD in 1993 from University of Maryland,
College Park. Her research interests include microuidics, lab-chips for appli-
cations in biology such as cell volume regulation, cell based drug screens, and
single molecule detection.