Biosensors and Bioelectronics 20 (2004) 246–252
Biological laser printing of genetically modified Escherichia coli
for biosensor applications
J.A. Barron a , R. Rosen b , J. Jones-Meehan a , B.J. Spargo a , S. Belkin b , B.R. Ringeisen a,∗
a
Biological Chemistry Section, Chemistry Division, Chemical Dynamics and Diagnostics Branch, Naval Research Laboratory,
Bldg. 207, 4555 Overlook Avenue SW, Washington, DC 20375, USA
b Environmental Sciences, The Fredy & Nadine Herrmann Graduate School of Applied Science,
Hebrew University of Jerusalem, Jerusalem 91904, Israel
Received 15 October 2003; received in revised form 15 January 2004; accepted 22 January 2004
Available online 3 March 2004
Abstract
One of the primary requirements of cell- or tissue-based sensors is the placement of cells and cellular material at or near the sensing
elements of the device. The ability to achieve precise, reproducible and rapid placement of cells is the focus of this study. We have developed
a technique, biological laser printing or BioLPTM , which satisfies these requirements and has advantages over current technologies. BioLPTM
is capable of rapidly depositing patterns of active biomolecules and living cells onto a variety of material surfaces. Unlike ink jet or manual
spotting techniques, this process delivers small volume (nl to fl) aliquots of biomaterials without the use of an orifice, thus eliminating potential
clogging issues and enabling diverse classes of biomaterials to be deposited. This report describes the use of this laser-based printing method
to transfer genetically-modified bacteria capable of responding to various chemical stressors onto agar-coated slides and into microtiter plates.
The BioLPTM technology enables smaller spot sizes, increased resolution, and improved reproducibility compared to related technologies.
© 2004 Elsevier B.V. All rights reserved.
Keywords: Biological laser printing (BioLPTM ); Whole-cell biosensors; Bacteria-based biosensors
1. Introduction
With the heightened threat of chemical and biological
agents (Atlas, 2002; Iqbal et al., 2000; Hawley and Eitzen,
2001; Niiler, 2002) and increased concern about environmental pollutants (Belkin, 1998; Kohler et al., 2000), there
is a significant need for fast and accurate detection devices
such as cell-based toxicity sensors. Such systems can be
tailored to detect general or specific toxicants, or to monitor chemical and environmental stressors that impact human homeostasis. Due to their large populations, relatively
inexpensive costs, high sensitivity and rapid responses, bacteria are of particular interest for use in cell-based sensors
(Belkin, 2003). Further, with our continuously expanding
understanding of functional genomics and signaling mechanisms of many bacterial systems, it is likely that microbial
∗ Corresponding author. Tel.: +1-202-767-0719;
fax: +1-202-404-8119.
E-mail address: [email protected] (B.R. Ringeisen).
0956-5663/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.bios.2004.01.011
whole-cell biosensors could become an important detection
tool for future environmental, industrial, medical, military
or homeland defense applications (Belkin, 2003; Tao et al.,
1999; Blattner et al., 1997; Niiler, 2002).
For a bacterial cell to function as a sensor, it must be able
to detect an analyte and respond with a detectable signal. In
recent years, it has become easier to genetically manipulate
bacteria and produce strains that emit quantifiable signals
in the presence of an effector analyte. Such strains usually
contain a sensing element (often a gene promoter induced
by the target analyte), fused to a reporter element, a gene
coding for a protein whose activity can be easily quantified
(Daunert et al., 2000; Kohler et al., 2000; Belkin, 2003). The
nature of the detected stress is thus dictated by the choice
of the promoter element, while the character of the emitted
signal is determined by the selected reporter system. It has
been shown that detection levels via bacterial signaling for
a variety of inorganics and organics are comparable to many
of the standard test methods currently used (Daunert et al.,
2000; Kohler et al., 2000).
J.A. Barron et al. / Biosensors and Bioelectronics 20 (2004) 246–252
In order for cell-based biosensors to be effective as analytical testing devices, it is necessary that viable cells are
incorporated into the hardware platform that will house
them; the deposition of the cells should be compatible with
high-throughput industrial processes, with high and reproducible accuracy. There are several different methods of
patterning active biomaterials, including soft lithography
and photolithography to produce cell-specific adhesive areas (Spargo et al., 1994; Kane et al., 1999). Other possible
methods of cell transfer include non-lithographic techniques such as ink jet (Wilson and Boland, 2003) or various
laser-based tools (Renn and Odde, 1999; Wu et al., 2003).
Ink jet techniques are preferred by many researchers for use
in high-throughput DNA deposition, mainly due to their
commercial availability and non-contact printing abilities.
Ink jet and other orifice-based techniques, however, suffer
from orifice clogging, irreproducible depositions from variations in solution viscosity, and lowered efficiency due to
adherence of biomaterials to various portions of the fluidic
network. Therefore, it is necessary to develop novel printers for high-throughput applications that are specifically
designed for biologicals, both to maximize efficiency and
minimize irreproducibility and inconsistency.
We have developed a technique, biological laser printing
or BioLPTM (Ringeisen et al., 2004; Barron et al., 2004),
that has many advantages over current deposition technologies. BioLPTM is capable of rapidly depositing patterns of
active biomolecules and living cells onto various surfaces.
Unlike ink jet or manual spotting techniques, our process
delivers small volume (nl to fl) aliquots of biomaterials without using an orifice, thus eliminating potential clogging and
enabling diverse classes of biomaterials to be deposited.
This report describes the results of using this laser-based
printing method to transfer genetically modified bacteria
onto a solid platform. Previously, we have shown the ability to produce mesoscopic patterns of viable Escherichia
coli via a similar laser-based technique, termed matrix assisted pulsed laser evaporation direct write (MAPLE DW;
Ringeisen et al., 2002). Our current laser printing technologies allow for smaller spot sizes, increased resolution, higher
efficiency, and improved reproducibility compared to previously reported results.
2. Methods
2.1. Biological laser printing
BioLPTM is a forward transfer technique that uses focused
laser pulses to transfer material from a carrier support, or
target, onto a receiving substrate (Fig. 1). The target is comprised of three layers: a UV transparent support, a thin light
absorbent coating, and the biological material of interest.
The transparent support allows a laser pulse to be focused at
its interface with the absorbent coating. The laser-material
interaction at this interface produces photoabsorption, which
247
CCD Camera
Energy Meter
Support Layer
Absorption Layer
Laser
Microscope
Objective
Biological
Layer
Material
Ejection
Ribbon
Substrate
Fig. 1. Schematic of the BioLPTM apparatus. The BioLPTM apparatus
uses a microscope objective to focus a laser beam down at the interface
of a quartz substrate and the laser absorptive biopolymer. The focused
beam causes ejection of the biopolymer and cells via photomechanical
and/or photothermal effects.
through photothermal and/or photomechanical effects propels a three-dimensional pixel of the biomaterial towards the
receiving substrate. We are currently performing theoretical
calculations to model the laser-material interaction and the
ensuing biomaterial transfer process in more detail. Preliminary results indicate that there is minimal heat transferred
to the biomaterial layer during the printing process, resulting in less than 5% of the printed biomaterial to be raised
to temperatures above ambient (Barron and Ringeisen, unpublished data). The amount of biomaterial transferred in a
single pixel is a function of several factors including the focused laser spot size, the thickness of the biomaterial layer
on the target, and the laser fluence. The system is completely
automated, allowing for modulation of the laser and scanning of the receiving substrate via CAD/CAM. This allows
for rapid, accurate design of any pattern on a wide variety
of substrates including microtiter plates, microfluidic channels or wells, and microarray slides. Use of a CCD camera
positioned inline with the optical setup allows for real-time
analysis and monitoring of the transfer process.
A KrF excimer laser (MPB Technologies, PSX-100,
248 nm, 2.5 ns FWHM) was used for all BioLPTM transfers. A fixed laser spot of 5000 ␮m2 at the material–target
interface was used, and the incident laser fluence was varied from 100 to 200 mJ/cm2 . The laser was directed onto
the target surface at an incident angle of 90◦ , with the receiving substrate positioned 0.25–10 mm below the target.
Movement of the substrate was achieved via a high-speed
translation stage (Aerotech, ATS 15010), with X, Y, and Z
travel dimensions.
2.2. Target and receiving substrate preparation
The target consists of an optically transparent quartz disk
that has been coated with a metal or metal oxide via standard ion assisted electron beam processing techniques (Thin
Films Research, Boston, MA). Current studies have used ei-
248
J.A. Barron et al. / Biosensors and Bioelectronics 20 (2004) 246–252
ther Au (thickness 350 Å) or Ti (thickness 750 Å) metals as
absorption layers. A solution of bacteria is then spread on
top of the absorption layer prior to printing. Bacterial culture coating solution volume was controlled by deposition
via a micropipette into 40 ␮m deep wells on the disk. The
wells were made by applying a SU-7 photoresist to the disk
and exposing the well area through a mask to produce wells
varying in size from 6 to 1 mm.
Genetically modified E. coli cells were suspended in a solution of 80% standard Luria–Bertani (LB; Difco Laboratories, Sparks, MD) broth with either kanamycin or ampicillin
and 20% glycerol (v/v). The quartz disks were rinsed with
sterile water, sterilized with 70% (v/v) ethanol and dried
by N2 gas prior to use. The receiving substrate, either an
agar-coated glass slide, a solid LB Petri dish, or a microtiter
plate, was sealed with parafilm after transfer.
2.3. Microbial cultures, media and assay conditions
Two E. coli strains were used in this study, both harboring a plasmid-borne fusion of the recA gene promoter
to a fluorescent protein gene, in a RFM443 host (Menzel,
1989): (a) strain RR2R, harboring the pRR2 plasmid containing a recA::DsRed fusion (red fluorescent protein) and
(b) strain RR1R, containing a recA::EGFP fusion. Details
of construction of both reporter strains were reported elsewhere (Sagi et al., 2003). The recA gene promoter, a part
of the E. coli SOS DNA repair system, was shown to be a
sensitive indicator of the presence of genotoxicants (mutagens, etc.) (Vollmer et al., 1997; Davidov et al., 2000). Cultures were grown overnight at 30 ◦ C with shaking at 150 rpm
in LB broth, supplemented with either kanamycin or ampicillin (50 ␮g/ml) to ensure plasmid maintenance. Glycerol
was then added (to 20% v/v) and the suspensions were used
to coat the quartz “target” disks as described above. The receiving substrates for the laser transfer were either LB agar
plates containing the appropriate antibiotic or sterile glass
slides with a thin film (approximately 1 mm) of LB agar plus
the appropriate antibiotic. The agar-coated glass slides were
stored in sterile glass Petri dishes. After the laser transfer,
agar plates and slides were incubated overnight at 30 ◦ C,
and then shipped to Jerusalem, Israel where they were maintained at 4 ◦ C until tested (1–2 weeks). Temperature during
shipment was not controlled.
Expression of enhanced green fluorescent protein (EGFP)
and DsRed in the laser-transferred bacterial cells was
confirmed using a microtiter plate fluorimeter (Victor2 ,
Wallac, Finland) with excitation/emission wavelengths of
550/590 nm for DsRed and 485/510 nm for EGFP. Cells
were tested either “as transferred” (collected from the deposition surface, suspended in fresh LB, and directly tested)
or after re-growth for a few generations (“refreshed”) in LB
medium. The kinetics of fluorescence development in both
“as transferred” and “refreshed” cells were studied following induction by the known SOS-inducer nalidixic acid.
Fluorescence intensity is reported in the fluorimeter’s arbitrary relative fluorescence units (RFU) and the response ratio is defined as the ratio of the fluorescence in the presence
of a given inducer concentration to that in its absence. Fluorescence readings were taken for 0.1 s at 10 min intervals.
This degree of exposure had no adverse effects on the cells
or on their activity. Each experiment was repeated at least
three times, in duplicate. Data provided are an average of
duplicates in single representative experiments. Deviations
between duplicates and between individual experiments did
not exceed 5 and 15%, respectively.
3. Results
The ability to rapidly deposit bacteria onto the active area
of a biosensor and to deposit a large number of different bacterial types on an array may be vital for the next generation of
cell-based biosensors. Fig. 2 shows an array of bacterial suspension drops deposited onto a non-coated microscope slide
via the BioLPTM technique. The array shows excellent reproducibility and consistent spot sizes, whose average diameter
is 70 ␮m (σ = ±6 ␮m, N = 27). The volume of these spots
is approximately 5 pl, based on profilometry measurements
and previous calculations of the spot size and volume of material transferred (Barron and Ringeisen, unpublished data).
Transfer of the bacterial suspension onto either an
agar-coated slide or plate with subsequent incubation at
30 ◦ C allowed for observation of cell viability and growth
via light and fluorescent microscopy (Fig. 3A and B). The
bacterial deposition was carried out using both single and
multiple laser shots per transfer spot. In all cases, cells
were found to be viable in the transfer areas as indicated by
colony formation within 24 h. Fig. 3A shows such a colony
of an E. coli containing EGFP, 17 h after deposition on an
agar-coated slide. The fluorescence (EGFP: λabs
max = 485 nm,
λem
=
508
nm)
was
observed
via
excitation
at 485 nm.
max
The transferred bacteria spot is approximately 200 ␮m in
diameter after 17 h of incubation. Fluorescence was observed only in the area where the transferred material was
deposited. In Fig. 3B similarly displays DsRed containing
em
bacteria (DsRed: λabs
max = 558, λmax = 583 nm) at 400×
magnification, with individual cells clearly observed.
Following shipment to Israel, both the recA::DsRed2- and
recA::EGFP-containing E. coli strains were tested for inducible activity. Cells were exposed to SOS-inducing agent
nalidixic acid, and the results are depicted in Fig. 4. The kinetic dose-dependent response (Fig. 4A) is similar to what
has been previously been reported for this strain (Sagi et al.,
2003). When the cells were allowed to re-grow for a few
generations in fresh medium before exposure to the inducer,
the duration of the lag period preceding measurable activity
was significantly reduced, but the magnitude of the response
was similar (Fig. 4B). The induction ratios (fluorescence in
the presence of nalidixic acid over that in its absence) were
very similar in all cases (Fig. 4C).
J.A. Barron et al. / Biosensors and Bioelectronics 20 (2004) 246–252
249
Fig. 2. Array of 70 ␮m diameter droplets of E. coli in LB media:glycerol (50:50 v/v) transferred by BioLPTM to a glass slide.
Fig. 3. (A) A 17 h post transfer photograph of E. coli containing a green fluorescent protein (EGFP) under (a) white light and (b) epi-fluorescence at 100×
magnification. (B) A 17 h post transfer photograph of E. coli containing a red fluorescent protein (DsRed2) under (a) white light and (b) epi-fluorescence
at 400× magnification.
250
J.A. Barron et al. / Biosensors and Bioelectronics 20 (2004) 246–252
50,000
Nalidixic acid (mg/l)
1.25
0.6
80,000
0.3
0
40,000
0
0
100
(A)
200
300
400
500
10
Laser transferred, refreshed
40,000
Control, direct
Control, refreshed
30,000
20,000
10,000
0
600
0
50
8
Refreshed
Control
6
4
2
0
100 150 200 250 300 350 400
(B)
TIME (min)
Direct
Laser transferred, direct
RESPONSE RATIO
120,000
FLUORESCENCE (RFU)
FLUORESCENCE (RFU)
160,000
0
1
(C)
TIME (min)
2
3
4
5
6
NALIDIXIC ACID (mg/l)
Fig. 4. Fluorescent response to nalidixic acid of E. coli strain RR1R cells harboring a plasmid containing a recA::EGFP fusion. (A) Kinetics of fluorescence
development following exposure to various nalidixic acid concentrations of cells directly off the laser-transfer plate. (B) Comparison of cells directly off
the laser-transfer plate (direct) and following re-growth in fresh medium before exposure (refreshed). Control—cells that have not been laser-transferred,
and were tested either directly off a solid LB agar plate (direct) or following several generations in liquid LB (refreshed). Nalidixic acid concentration
was 0.6 mg/l. (C) Response ratio of direct, refreshed and control cells in response to increasing nalidixic acid concentrations (600 min after exposure).
10
45,000
Plate-transferred
Glass-slide transferred
Control (fresh cells)
30,000
15,000
0
(A)
12
RESPONSE RATIO
FLUORESCENCE (RFU)
60,000
0
100
200
300
400
500
6
4
2
0
600
TIME (min)
8
(B)
0
1
2
3
4
5
NALIDIXIC ACID (mg/l)
Fig. 5. Fluorescent response to nalidixic acid of E. coli strain RR2R cells harboring a plasmid containing a recA::DsRed2 fusion. (A) Fluorescence
development in response to 4 mg/l nalidixic acid of cells laser-transferred onto a glass or agar surface. (B) Dose dependency of the response on nalidixic
acid concentrations (600 min after exposure).
Data for the strain containing the red fluorescent protein
as the reporter are presented in Fig. 5, which also serves
to compare between cells that were laser-transferred either
onto an agar plate or to a glass surface. Fluorescence was
practically identical in cells deposited onto the two surfaces
tested and, as for the EGFP strain, its intensity was dependent upon inducer concentration. It may be observed that
with this reporter strain, a longer lag period was needed before the accumulation of detectable fluorescent protein concentrations; for non-refreshed cells, this lag period was even
longer (data not shown). Quite clearly, the nature of the deposition surface did not affect retention of activity. Furthermore, the laser-transfer process appears not to have exerted
a detectable effect on fluorescence output.
4. Discussion
Detection of chemical and biological stressors can be done
via physical, chemical or biological means. While physical and chemical tests are able to provide the most accurate
analytical data, they are unable to show the biological impact that a stressor has on living systems. Biosensors fill
this void by providing biological tests that cannot only detect the stressor, but also provide an account of its bioavailablity and biological effects. This has been studied using the
basic active areas of a cell, such as proteins, DNA, RNA,
or enzymes. While it is possible to obtain excellent detection levels with these components, only through measurable
changes in cell physiology it is possible to see the potential effect of a chemical or biological stressor on a living
system (Belkin, 2003). The ideal cell type for these studies must possess a sensitivity and response spectrum that is
relevant to human exposure risk. Such cells should also be
inexpensive, easy to reproduce, and amenable to biochemical and molecular manipulation. In many cases, bacteria can
successfully fulfill these roles.
The laser-based aspect of the BioLPTM technique allows
for great flexibility in terms of materials and volumes transferred. As an orifice-free technique, it is able to rapidly
switch cell types without concern about cleaning cycles or
potential contamination, allowing for patterning different
J.A. Barron et al. / Biosensors and Bioelectronics 20 (2004) 246–252
cell types adjacently with little increase in design time. The
orifice-free aspect may also be useful in the design of an
off-the-shelf bacterial biosensor that can be stored for many
years without loss of activity. Orifice-free allows for transfer
of lyophilized, or freeze-dried, bacteria that pose difficulties
for most pin or orifice-based techniques. By changing the
laser focus and energy, it is possible to change the volume
of material transferred and the transfer spot size. BioLPTM
allows for patterning picoliter (pl) volumes of biological solution onto areas of substrate smaller than 50 ␮m × 50 ␮m.
This, along with changes in the concentration, allows for
control of cell density on the sensor platform. It is theoretically possible to transfer single E. coli cells with each laser
shot providing the ultimate in homogeneous population.
As previously shown (Sagi et al., 2003), while the turn-on
time for fluorescent genetic activation monitoring is slow
compared to other possible detection methods, such as bioluminescence, the fluorescent protein is highly stable and
its cumulative effect and allowable detectable signals to be
observed with inducer/activator concentrations lower than
1 ppm. Fluorescent sensors may thus be more suitable for
long-term exposure, while bioluminescent sensors may be
preferable for rapid on-line responses. Both types of cells
could be deposited using the BioLPTM technique, which appeared to have little to no effect on the sensing ability of the
fluorescent sensors used here as a model. Cells transferred
by laser and then refreshed in LB broth, had similar backgrounds, turn-on times, and relative fluorescence intensities
for the inducer concentration used for activation/induction.
The data presented in Figs. 4 and 5 clearly indicate:
(a) Following laser deposition, a 48 h shipping process and
an up to 2 week 4 ◦ C storage, activity of the reporter
cells was maintained. This was demonstrated for strains
harboring either the green (Fig. 4) or the red (Fig. 5)
fluorescent reporter proteins.
(b) The activity measured was similar in cells deposited onto
glass or agar surfaces; in the former case, the lag period
preceding significant signal detection was longer.
(c) To obtain optimal response of the laser deposited cells,
they should be allowed to regain exponential growth by
a short exposure to a non-toxic medium before exposure
to the tested sample. Nevertheless, even without such
treatment detection sensitivity was not affected.
Taken together, these observations clearly indicate the
biocompatibility of the BioLPTM technique, and demonstrate its potential for high-throughput deposition of live
cells onto active biosensor surfaces. In the future, this potential may be manifested not only in single strain deposition, as demonstrated here, but also in the patterning of
whole cell arrays composed of diverse libraries of live reporters (Belkin, 2003). Additional future studies should also
include the deposition of cells in a dormant state. The use
of such cells, in which long-term stability has been acquired
either by lyophilization or by encapsulation in a solid matrix
(Premkumar et al., 2001), may significantly increase biosen-
251
sor lifetime. The orifice-free BioLPTM system, which allows
the transfer of materials directly from a dried state, may thus
prove to be highly suited for the design and development of
future off-the-shelf whole-cell biosensors.
4.1. Conclusions
For future production of whole-cell biosensor systems,
a large number of biomaterials will need to be accurately
placed onto high-throughput platforms such as microtiter
plates or microchips in small volumes. In order to achieve
quantitative assessment of the fluorescent signal, it is also
necessary to deposit a reproducible number of cells on each
active surface of the sensor. BioLPTM is an excellent technique for this type of work, as it is able to transfer very small
volumes of material in a precise and accurate manner. The
transfer of genetically modified E. coli in a LB broth/glycerol
mixture demonstrate the power of the BioLPTM technique.
Both the green and red fluorescent reporter strains were reproducibly transferred in pl aliquots onto agar-coated substrates. In addition, both sensor strains retained functionality
as tested via the addition of various concentrations of the
model SOS-inducer nalidixic acid.
Acknowledgements
This work was funded by DARPA through the Metabolic
Engineering and Tissue-Based Biosensor programs. J.A.B.
would like to thank the National Research Council for support through a NRC Postdoctoral Fellowship. Work in the
Belkin laboratory was supported by Defense Advanced Research Projects Agency (DARPA) of the US Department of
Defense (Grant N00173-01-1-G009).
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