Note: Descriptions are shown in the official language in which they were submitted.
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METHOD FOR THE PREPARATION OF OPTICAL (BIO)CHEMICAL SENSOR DEVICES
FIELD OF INVENTION
The present invention relates to the preparation of optical (bio)chemical
sensor devices
useful for monitoring a large number of different compounds at the same time.
Other
possible applications are high throughput screening of combinatorial
libraries, food quality
monitoring, process control, gene expression monitoring, and detection of
biological
components, etc. More particularly, the present invention relates to a method
for the
preparation of an optical (bio)chemical sensor device comprising a plurality
of polymeric
(bio)chemical sensor dots.
BACKGROUND OF THE INVENTION
The trend within the field of chemical and biochemical sensors [R. Kellner, M.
Otto, M.
Widmer, Analytical Chemistry: The Approved Text to the FECS Curriculum
Analytical
Chemistry, Wiley-VCH 1998, p 359-360 and 375ff.] is to improve and develop new
ways of
performing classical analytical methods in order to meet the increasing demand
of high
throughput analysis of e.g. environmental and clinical samples as well as
screening of new
compounds for drug development. Especially, miniaturization of chemical and
biochemical
sensing techniques has received a lot of interest, a process which has been
further
supported by the development of new approaches for chemometric data processing
and
neural networks allowing access to information embedded in response patterns
beyond the
sum of individual results.
Chemical and biochemical sensors [i.e. (bio)chemical sensors] have been
defined as
"devices capable of continuously recognizing concentrations of chemical
constituents in
liquids or gases and converting this information in real-time to an electrical
or optical
signal" [R. Kellner, M. Otto, M. Widmer, Analytical Chemistry: The Approved
Text to the
FECS Curriculum Analytical Chemistry, Wiley-VCH 1998]. In this connection, a
chemically
sensitive layer is coupled to a so-called transducer, which converts the
(bio)chemical
information into an optical or electrical signal which is recorded by a data
evaluation unit.
The chemically sensitive layer can either be a surface directly modified with
a recognition
system or a surface covered by a thin film doped with the recognition system;
e.g.
chemically sensitive polymer membranes may be coupled to an electrode to
measure their
potential difference to a sample solution as function of analyte concentration
(or activity);
or they may be coated onto an optical transducer such as an optical fiber to
measure an
optical change (such as absorbance or refraction) as the function of analyte
concentration.
CONFIRMATION COPY
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Bulk optode membranes, as described in EP 0 358 991, are examples of chemical
sensing
layers for optical ion-sensing. Chemical substances that selectively interact
with specific
ions are called ionophores and have traditionally been used in potentiometric
membrane
electrodes to increase or govern their selectivity. Combination of the
ionophore with
further components, in particular, lipophilic ions ("counter ions") and pH-
sensitive dyes
(°chromoionophores"), afford a membrane material that responds to
specific ions, at a
given pH, with a reversible and reproducible color change. Unlike their
electrochemical
cousins, where a reference electrode is prerequisite for measurement, optical
sensors
based on such membranes ("opt(r)odes") function without a reference and are
not
sensitive to electrical interference, which make them much easier to integrate
in
miniaturized systems. Furthermore, optical methods may be combined with
chemometric
techniques, pattern recognition, etc., since more than one parameter can be
deduced from
them: e.g., spectral shape, temporal information or data on both absorbance
and
refraction, could be measured where electrochemical sensors commonly only
yield one
value (such as potential or current).
In order to combine optical sensing using chemically responsive polymers with
the concept
of sensing arrays, individual, small polymer dots need to be arranged on the
substrate in
such a manner that the signal from each individual sensing element can be
distinguished
from one another. One approach is to bundle or array optical fibers as
demonstrated by
Dickinson et al. [Nature 1996, 382, 697-700; cf. also Johnson et al., Anal.
Chem. 1997,
69, 4641-4648]. Representative of the lengths gone to demonstrate the
advantages of
mufti-sensing employing optical changes of polymer probes are the studies from
the group
of Walt (e.g., Ana/. Chem 70 1998 1242-1248). Here, microsphere sensors are
randomly
entrapped in thousands of micrometer-scale wells. These are etched out of the
face of an
optical fiber by hydrofluoric acid, taking advantage of the different etch
rates df fiber cores
and cladding. Alternatively sophisticated site-selective photopolymerisation
have been
employed on such fiber bundle phases by the same group (see review by Steemers
and
Walt, Mikrochim. Acta 131, 99-105 (1999). Such studies strongly confirm the
potential of
miniaturized, polymer array based sensing techniques. However, individual
modification
and subsequent bundling of the fibers clearly makes this approach impractical
for mass-
production of sensor layers or production of sensor layers with many different
sensor
systems. On the other hand, for many practicable devices it will not be
necessary to scale
down the individual sensor elements to the sizes (a few micrometers) achieved
in such
studies. For example, 36 dots of i20 ~m diameter could still easily be
arranged on a 1 cm
x 1 cm sensor area with a 30 wm dot-to-dot distance.
An ideal sensor device should comprise a plurality of different, independently
and spatially
separated polymeric sensor regions (these regions, which in this context are
referred to as
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sensing dots, have a roughly circular shape and microscopic dimensions
(diameter 10-
1000 Nm)). An imaging technique, e.g. camera, or linear or two-dimensional CCD
array,
and suitable software may then be used to distinguish between the responses of
the
different sensing dots. These sensor devices may also show to be more
flexible,
reproducible, and it may be easier to increase the number of sensing regions.
The potential of arrays of optical sensing regions for analysis via imaging
has recently been
demonstrated by Rakow et al. in a colorimetric sensor array for odour
detection [Rakow et
al., Nature 406 (2000) 710-713].
There are a number of different optical sensing schemes that in principle
allow imaging and
thus can be used with arrays of polymer-based sensing dots. In the simplest
case, a
transparent carrier plate could be modified with sensing dots. With a suitable
gasket and
cover plate, a flow channel could be created which allows flowing, e.g., a
ground water
sample over the dots. A light source may be mounted on top of the cover plate
and shining
through both plates and sample, an imaging detector (e.g., camera) below the
lower plate
may then record an image which contains all the information on the color
response of the
individual dots. Of course, it would also be possible to devise similar
systems utilizing
diffuse or total reflection of sensing dots on suitable non-transparent
surfaces.
Fluorescence monitoring would be possible in analogous systems.
More sophisticated optical sensing schemes are those using the phenomenon of
evanescent waves, decaying standing waves that occur at surfaces between two
phases of
different refractive indices upon total reflection of light within the
optically dense medium.
Such waves occur either directly at the totally-reflecting surfaces, at
suitable structures
such as gratings, or via excitation of so-called surface plasmons (collective
electron
oscillations) within a thin film of suitable metals (typically gold or silver)
on related suitable
optical structures. Since such devices allow measurement of optical properties
in the very
proximity of the transducer surface, they can also be combined with arrays of
polymer
sensing dots as described in WO 00/46589 (Vir A/S). Furthermore, evanescent
waves may
be used to excite fluorescence rather than to monitor absorption thereby
allowing
fluorimetric sensing.
Suitable optical transducers are optical waveguides, surface plasmon resonance
films,
reflection grating couplers, optical waveguides, Mach-Zehnder interferometers
or
Hartmann interferometers, allowing detection of changes of optical properties
of the
polymer dots, such as in particular absorption, refractive index or
fluorescent changes
(thus allowing to monitor the chemical response of the polymer dots).
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However, so for there is no automated way of producing arrays of spatially
separated
(bio)chemical sensor dots for optical sensing.
Several techniques for automated dispersion of fluid droplets are available.
The ink-jet
technology which is known from e.g. printing technology is characteristic by
that the fluid
is deposited from a capillary. Release of the fluid from the capillary can be
brought about
by different approaches. In the drop-on-demand approach application of a
voltage through
a piezo-actuator, a ceramic collar around the capillary, creates an acoustic
wave in the
capillary and the resulting deformation of the capillary causes release of a
controlled part
of the liquid column as droplets. In the continuous approach, the fluid is
released under
pressure resulting in the generation of a fluid stream and again droplets are
generated and
released from the capillary by the application of a voltage through a piezo-
electric
actuator. The continuous ink-jet technique is widely used for labeling of
products in the
food and pharmaceutical industry. The ink-jet technology has also been used by
Newmann
et al. [Newman et al. Analytical Chemistry 1995, 67, 4594-4599] in the
preparation of
membranes for amperometric biosensors, where, however, the droplets merge to
form a
film. Other related techniques are micro- and nanodispensing instruments, such
as
micropipettes.
None of these techniques are suitable for deposition of fluids with low
surface tension and
high viscosity as application of these solvents offend result in formation of
air bubbles in
the dispensing devices. Heating of the print-head may reduce the viscosity of
the fluid but
it may also cause evaporation of volatile solvent and clogging of the print-
head may be
experienced. It is further known that viscolelasticity causes significant
performance
problems in such printers. Non-Newtonian behavior may occur under the high
shear forces
in the nozzles resulting in unstable drop formation or formation of droplet
satellites.
It is clear from the above brief overview that the ink-jet technology is not a
suitable choice
for deposition of polymer or polymer precursor fluids. The physicochemical
properties of
these fluids will interfere with the deposition process as well as the
aspiration through a
pump into the dispensing mechanism.
Alternative technologies for microarraying of fluids which do not involve
sample aspiration,
pumping and flushing are open deposit units such as pin-printers or arrayers,
e.g. the
quill-printer as described in US 5,807,522. In the pin-printer technology the
fluid to be
deposited is picked up from a small vessel (usually the well of a microtiter
plate). Thus,
the amount of spotting fluid needed to fill the instrument is minimized which
is an
important feature in the preparation of sensing membranes given the high prize
of
(bio)chemical recognition elements. Quill-printers, however, are not suitable
for the
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deposition of polymer and polymer precursor fluids with high viscosity as the
reception of
fluid is based on take-up of fluid into the quill and problems as described
above may arise.
Another disadvantages is the fact that it is very hard to reproduce the size
of the deposited
fluid dots, e.g. some commercially available printers need pre-printing steps
to achieve a
5 constant dot size.
Another example of an open deposit unit is the pin-ring technology as
described in WO
99/36760. The pin-ring technology is developed for the preparation of
reproducible
microarrays of biological samples and is based on surface tension forces as
the basic
mechanism for holding and transferring the fluid.
SUMARY OF THE INVENTION
The present invention relates to a method for the preparation of an optical
(bio)chemical
sensor device as defined in claim 1.
The present invention also relates to such optical (bio)chemical sensor
devices obtainable
by said method, and to methods of using the devices.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1: Photographic image of a plurality of sensor dots obtained by "pin-
ring" deposition
of a spotting fluid comprising PVC/DOS in cyclohexanone (Example 2). The dot
diameter is
approximately 200 Vim.
Figure 2: Fluorescence image of arrays (A-D) of photopolymerized methacrylate
spotting
fluid droplets obtained by deposition of a spotting fluid comprising
methacrylate (Example
4) and subsequent polymerization of the polymer precursors on the suppart
surface.
A second set of arrays was superimposed directly on top of A, C and
photopolymerized
(Example 5.I).
Figure 3: Partially superimposed PVC-DOS dot arrays generated by means of a
"pin-ring"
calibration feature (Example 5.II) (image obtained with fluorescence scanner).
The outer
white box encircles the second array, the inner one the area of dot
superimposition.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method for the preparation of an optical
(bio)chemical
sensor device. The (bio)chemical sensor device includes a plurality of well-
defined spatially
separated (bio)chemical sensor dots arranged on a substrate material. More
specifically,
the optical (bio)chemical sensor device comprises a substrate material having
a planar
surface portion, said planar surface representing a transducer based on an
optical
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phenomenon, said planar surface portion having arranged thereon a plurality of
(bio)chemical sensor dots located at spatially separated predetermined
positions of the
planar surface, said sensor dots comprises (i) a polymer matrix, and (ii) one
or more
(bio)chemical recognition moieties.
The term "plurality" in connection with the expression "plurality of
(bio)chemical sensor
dots" is synonymous with the term "array" which is frequently used in the
technical
literature.
The term "(bio)chemical" is intended to have the same meaning as "biochemical
and
chemical", thereby covering reactions and reagents within the biochemical as
well as the
chemical field.
The term "recognition moieties" is intended to cover chemical groups which
interacts with
an analyte in order to directly, or indirectly, alter the optical properties
of the associated
polymer matrix, as well as chemical groups which, e.g. in a cascade fashion,
are involved
in the alteration of the optical properties. The term "recognition system"
covers a system
comprising one or more recognition moieties which all in all (e.g. by a
cascade reaction) is
responsible for alteration of the optical properties of the associated polymer
matrix.
The sensor device comprises a "substrate material" having a planar surface
portion. The
term "substrate material" is intended to mean a base material optionally
coated with one
or more layers of a surface layer material (see below). It should be
understood that the
planar surface portion of the substrate material should represent a transducer
based on an
optical phenomenon.
The dimension of the planar surface portion of the substrate material is
typically 1-50 mm
wide and 2-100 mm long, such as 2-25 mm wide and 5-50 mm long, e.g. 4-8 mm
wide
and 8-16 mm long.
The substrate material comprises a base material and optionally a surface
layer material
which represent the planar surface portion of the substrate material. In some
embodiments, it is possible to utilize a substrate material wherein the base
material and
the surface layer material is the same material. The substrate may of course
also comprise
multiple layers of the surface layer material.
An important requirement for the method to be applicable for the preparation
of a plurality
of (bio)chemical sensor dots is that the deposited fluids remain localized to
the
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predetermined positions and do not spread to wet the entire substrate
material. The
contact area of a sensor dot defines the size (diameter) of the dot.
The base material constitute an integrated part of the (bio)chemical sensor
device, and is
typically in the form of a smooth planar surface of a material selected from
glass, silica,
dielectric inorganic materials such as SiOZ, PtOX where x = 1 or 2, AI203,
TiOz, Taz05, MgFZ,
or Si3N4, plastics such as acrylics, cycloolefin polymer (TOPAST"~),
polycarbonate,
polyetherimide (ULTEMTM), or silicon with a hydrogen- or deuterium-terminated
surface, in
particular glass and plastics.
In the present context, the term "dielectric" means a material that is a poor
conductor of
electricity and that will sustain the force of an electric field passing
through it.
The base material is often coated with at least one layer of a surface layer
material so as
to govern optical performance of the transducer. Such surface layer materials
are typically
selected from metal (such as gold, silver, copper or platinum), silica and
silicon, preferably
from gold, silver, copper and silicon. .
The surface layer material typically has a thickness of 10-500 nm, such as 20-
80 nm which
is particularly relevant for surface plasmon resonance measurements.
In one embodiment of the invention the substrate material is a mu(tilayered
structure of
one or more metals and a dielectric inorganic material as defined above, the
multilayred
structure may e.g. be a metal-dielectric or a metal-dielectric-metal sandwich
structure.
In one embodiment of the invention the substrate material is transparent
allowing
measurement of the bulk absorption of the (bio)chemical sensor dots.
In another embodiment of the invention the support surface is totally-
reflecting allowing
reflectance-spectroscopic measurement of the optical properties of the
(bio)chemical
sensor dots.
In yet another embodiment of the invention the support surface is diffusely-
reflecting to
allow diffuse-reflectance spectroscopic measurement of the optical properties
of the
(bio)chemical sensor dots.
The size, shape and adherence of the (bio)chemical sensor dots may be further
controlled
by modification of the surface of the substrate material thereby reducing or
increasing the
capacity of the spotting fluid to wet the surface or become chemically bonded
thereto.
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In one embodiment of the invention the planar surface of the substrate
material is
chemically modified by treatment with a bifunctional reagent:
X-Z-Y
wherein X is selected from -OR', asymmetric or symmetric disulfides (-SSR'Y', -
SSRY),
sulfides (-SR'Y', -SRY), diselenide (-SeSeR'Y', -SeSeRY), selenide (-SeR'Y', -
SeR'Y'), thiol
(-SH), selenol (-SeH), -N--__C, -NO2, trivalent phosphorous groups, -NCS, -
OC(S)SH,
thiocarbamate, phosphine, thio acid (-COSH), dithio acid (-CSSH), -
Si(OR/R/H)3, and
halogen;
each of the substituents R and R' independently are selected from optionally
substituted
Cl_3o-alkyl, optionally substituted Cz_3o-alkenyl, optionally substituted
CZ_3o-alkynyl, and
optionally substituted aryl;
Y and Y' are selected from hydroxyl, carboxyl, amino, formyl, hydrazine,
carbonyl, epoxy,
vinyl, allyl, acryl, epoxy, and methacryl,
Z is a linker (biradical) between the two functional groups and typically
designates
optionally substituted Cl_12-alkylene, optionally substituted CZ_iz-
alkenylene, and optionally
substituted C~_lz-alkynylene which may be interrupted by heteroatoms such as
N, S, O and
Si.
In the present context, the term "Cl_3o-alkyl" means a linear, cyclic or
branched
hydrocarbon group having 1 to 30 carbon atoms, such as methyl, ethyl, propyl,
iso-propyl,
cyclopropyl, butyl, tent-butyl, iso-butyl, cyclobutyl, pentyl, cyclopentyl,
hexyt, cyclohexyl,
hexadecyl, heptadecyl, octadecyl, nonadecyi, likewise the term "Cl_6-alkyl"
means a liners,
cyclic or branched hydrocarbon group having 1 to 6 carbon atoms, such as
methyl, ethyl,
propyl, iso-propyl, butyl, tert-butyl, iso-butyl, pentyl, cyclopentyl, hexyl,
cyclohexyl, in
particular methyl, ethyl, propyl, iso-propyl, tert-butyl, iso-butyl and
cyclohexyl.
Similarly, the terms "CZ_3o-alkenyl" is intended to mean a linear, cyclic or
branched
hydrocarbon group having 2 to 30 carbon atoms and one or more unsaturated
bonds,
likewise the terms "Cz_6-alkenyl" is intended to mean a linear, cyclic or
branched
hydrocarbon group having 2 to 6 carbon atoms and one or more unsaturated
bonds.
Examples of alkenyl groups are vinyl, allyl, butenyl, pentenyl, hexenyl,
heptenyl, octenyl,
heptadecaenyl. Examples of aikadienyl groups are butadienyl, pentadienyt,
hexadienyl,
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heptadienyl, heptadecadienyl. Examples of alkatrienyl groups are hexatrienyl,
heptatrienyl,
octatrienyl, and heptadecatrienyl.
Similarly, the term "Cz_3o-alkynyl" is intended to mean a linear or branched
hydrocarbon
group having 2 to 30 carbon atoms and comprising a triple bond. Examples
hereof are
ethynyl, propynyl, butynyl, octynyl, and dodecaynyl.
In connection with the terms "alkyl", "alkenyl", and "alkynyl", the term
"optionally
substituted" means that the group in question may be substituted one or
several times,
preferably 1-3 times, with groups) selected from hydroxyl, Cl_6-alkoxy,
carboxyl, Cl_s-
alkoxycarbonyl, Cl_6-alkylcarbonyl, formyl, aryl, aryloxycarbonyl,
arylcarbonyl, heteroaryl,
amino, mono- and di(Cl_6-alkyl)amino, carbamoyl, mono- and di(Cl_6-
alkyl)aminocarbonyl,
amino-Cl_6-alkyl-aminocarbonyl, mono- and di(Cl_6-alkyl)amino-Cl_6-alkyl-
aminocarbonyl,
Cl_6-alkylcarbonylamino, cyano, carbamido, halogen, where aryl and heteroaryl
may be
substituted 1-5 times, preferably 1-3 times, with Cl_4-alkyl, Cl_4-alkoxy,
nitro, cyano,
amino or halogen. Especially preferred examples are hydroxyl, Cl_6-alkoxy,
carboxyl, aryl,
heteroaryl, amino, mono-. and di(Cl_6-alkyl)amino, and halogen, where aryl and
heteroaryl
may be substituted 1-3 times with Cl_4-alkyl, Cl_4-alkoxy, nitro, cyano, amino
or halogen
(such as fluoro, chloro, bromo, and iodo).
In the present context the term "aryl" means a fully or partially aromatic
carbocyclic ring
or ring system, such as phenyl, naphthyl, 1,2,3,4-tetrahydronaphthyl,
anthracyl,
phenanthracyl, pyrenyl, benzopyrenyl, fluorenyl and xanthenyl, among which
phenyl is a
preferred example.
In connection with the term "aryl", the term optionally substituted" means
that the group
in question may be substituted 1-5 times, preferably 1-3 times, with Cl_4-
alkyl, Cl_4-alkoxy,
nitro, cyano, amino or halogen.
The term "heteroaryl" means a fully or partially aromatic carbocyclic ring or
ring system
where one or more of the carbon atoms have been replaced with heteroatoms,
e.g.
nitrogen (=N- or -NH), sulphur, and/or oxygen atoms. Examples of such
heteroaryl groups
are oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrrolyl, imidazolyl,
pyrazolyl, pyridinyl,
pyrazinyl, pyridazinyl, piperidinyl, coumaryl, furyl, quinolyl,
benzothiazolyl, benzotriazolyl,
benzodiazolyl, benzooxozolyl, phthalazinyl, phthalanyl, triazolyl, tetrazolyl,
isoquinolyl,
acridinyl, carbazolyl, dibenzazepinyl, indolyl, benzopyrazolyl, phenoxazonyl.
The functional group Y is often chosen to interact with a polymer or polymer
precursors. In
some embodiments, Y and Y' are the same.
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Chemical modification of the surface changes the capability of a spotting
fluid to wet the
substrate material and this material may therefore be tailored to a specific
composition of
a spotting fluid affording well-defined sensor dots.
5
In one embodiment of the invention Y represent an amino-groups which may react
with
polyvinylchloride affording polyvinylchloride dots covalent bound to the
substrate material.
In another embodiment of the invention treatment of a gold or silver coated
surface with
10 allyl- or methacroyl thiol affords a surface which may react with polymer
precursors such
as methacrylate or acrylate during polymerization to afford a plurality of
methacrylate- or
acrylate sensor dots covalently bound to the substrate material.
In yet another embodiment of the invention treatment of a glass or silicon
oxide surface
with an allyl- or methacroyl silane affords a surface which may react with
polymer
precursors such as methacrylate or acrylate during polymerization to afford a
plurality of
methacrylate- or acrylate sensor dots covalently bound to the substrate
material.
In yet another embodiment of the invention treatment of a gold or silver
coated surface
with a hydroxyl-terminated aliphatic thiol such as 11-mercaptoundecanol
affords a surface
which allows deposition of stable droplets of a spotting fluid containing
dodecyl
methacrylate and 1,6-hexanediol dimethacrylate.
In an alternative embodiment of the invention, wetting of the substrate
material is
controlled by microstructures such as small wells on the surface of the
substrate material.
The spotting fluid is deposited into the wells wherein it is allowed to
spread. The diameter
and the depth of the wells define the size and the height of the resulting
sensor dots. In
one preferred embodiment of the invention the diameter of the wells is between
50 and
1000 Nm, and the depth of the wells is between 1 and 50 pm.
The substrate material has a planar surface portion. It should be understood
that not all of
the substrate material needs to represent a planar surface, or planar surfaces
within the
device. The substrate material may have other portions with gratings, rims
which expand
above the planar surtace, holes for mounting, etc. What is important in
connection with the
present invention is that the substrate material has at least one planar
surface portion on
which the plurality (bio)chemical sensor dots are established.
This planar surface portion represents a "transducer based on an optical
phenomenon".
The term "optical phenomenon" is intended to cover refraction, reflection,
diffuse
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reflectance, attenuated reflectance, transmission, spectral changes, color
changes,
absorption, critical angle of reflection, evanescent wave phenomena such as
surface
plasmon resonance, fluorescence, and fluorescence quenching, preferably
transmission,
fluorescence, and surface plasmon resonance, in particular surface plasmon
resonance.
These phenomena form the basis for a number of transducer technologies such as
spectroscopy, spectrophotometry, photometry, SPR technology, Total Internal
Reflection
Fluorescence (TIRE) sensing, Grating Coupler Sensing (GCS), Resonant Mirror
sensing,
Reflectometric Interference Spectroscopy (RIFS), Tntegrated Optical Devices
(Waveguides), Integrated-Optical Interferometers, critical angle
refractometry, etc.
The term "change in optical properties" and similar terms are intended to
encompass
changes of the optical phenomena mentioned above, allowing detection of
changes of
optical properties of the (bio)chemical sensor dots, such as in particular
absorption,
refractive index or fluorescent changes (thus allowing to monitor the chemical
response of
the polymer dots).
The individual (bio)chemical sensor dots may have the same composition, but
typically the
sensor dots are not all identical. Thus, the device prepared according to the
invention
typically comprises at least 5, such as at least 15, different sensor dots.
This being said,
the polymer matrix of the different sensor dots is typically identical,
whereas the one or
more (bio)chemical recognition moieties are different, thereby rendering it
possible to
identify a plurality of analytes on the same device. In a preferred embodiment
each of the
spatially separated (bio)chemical sensor dots comprise different (bio)chemical
recognition
moieties.
Alternatively, the composition of the some of the sensor dots may be identical
so as to
image the distribution of an analyte in an inhomogeneous sample.
The (bio)chemical sensor devices formed by the method according to the
invention
comprise a plurality of (bio)chemical sensor dots in spatially separated
predetermined
positions in the x-y plane of the planar surface portion of the substrate
material. For
practical purposes, it is often desirable to deposit the sensor dots with a
uniform distance
between the sensor dots in the x-direction and a uniform distance between the
sensor dots
in the y-direction, where the distance between the dots in the x- and y-
direction may be
the same or different. In a preferred embodiment of the invention the
distances between
the centers of the (bio)chemical sensor dots in the x- and y-direction
independently are in
the range of 1.1-10 times the diameter of the (bio)chemical sensor dots.
In order to establish a plurality of (bio)chemical sensor dots, the method
comprising
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(a) providing a substrate material having a planar surface portion;
(b) providing one or more spotting fluids) each comprising at least one of
(i) a polymer and/or polymer precursor; and
(ii) a component representing one or more (bio)chemical recognition moieties;
(c) depositing either simultaneously or sequentially the one or more spotting
fluids) at the
spatially separated predetermined positions of the planar surface portion of
the substrate
material by means of a "pin-ring" deposition mechanism and allowing the
spotting fluid (s)
to consolidate.
The term "consolidation" is intended to include polymerisation,
polycondesation,
crosslinking, sol-gel processing, evaporation of solvent(s), e.g. upon
exposure to heat,
irradiation with ultraviolet light, irradiation with visible light, or by
means of electron
induced excitation.
The method comprises spotting a fluid comprising one or more polymers or
polymer
precursors, hereinafter termed "spotting fluid", onto the planar surface
portion of a
substrate material, by means of a "pin-ring" depositing technique. Subsequent
consolidation of the spotting fluid droplets on the support surface, either by
means of
evaporation of a solvent, polymerization of polymer precursors or a
combination thereof,
affords a plurality of spatially separated sensor dots.
The "pin-ring" depositing technique applied in the present invention was
originally
introduced by Genetic MicroSystemsT"" (WO 99/36760) as a method for
preparation of in
particular microarrays of biological materials where the biological materials
were either
adherently or covalently bound to a two-dimensional surface. The present
inventors have
now found that the "pin-ring" depositing system can advantageously be used for
the
preparation of a plurality of (bio)chemical sensor polymer dots where the
(bio)chemical
recognition moieties are comprised in the three-dimensional matrix of or on
the surface of
spatially separated sensor dots.
The "pin-ring" depositing technique relies on surface tension forces as the
basic
mechanism for holding and transferring fluids. The key mechanical component
consists of
a circular open "ring" which is oriented parallel to the substrate, and which
is held in place
by a vertical rod running perpendicular to the ring. A vertical pin is centred
on the ring.
Both the ring rod and the pin are attached to control devices so that each
part can be
moved separately in the z-axis, while both are kept in constant relation to
one another in
the x-y plane. When the ring is dipped into a spotting fluid and lifted, it
withdraws an
aliquot of sample, which is held in the centre of the ring by surface tension.
The pin-ring
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13
mechanism is then moved to any desired location in the x-y plane. When one
desires to
make a dot on the substrate material, the pin is driven down through the ring.
When the
pin passes through the ring, a portion of the spotting fluid is transferred
from the interior
ring meniscus to the bottom of the pin, forming a new pendant drop on the
lower surface
of the pin. The pin continues to move downward until the fluid on the pin
makes contact
with the substrate material. The pin is then lifted, and the combined forces
of gravity and
surface tension causes the spotting fluid to be deposited on the substrate
material as a
dot.
Neither impact nor mechanical contact between the pin and substrate are
required for fluid
transfer.
Movement of the pin through the internal meniscus of the ring does not destroy
the
meniscus until enough aliquots of fluid have been removed such that some
minimal
volume threshold has been passed. Given the volumes presented in the ring and
on the
pin, the pin driving process can be repeated many times, so that a very large
number of
similar dots can be created from a single moving pin-ring assembly.
The volume of the deposit fluid is dependent on pin dimensions and is roughly
equal to the
volume of a hemisphere with a radius equal to the radius of the pin which,
with today's
available hardware, is in the range of 50 to 500 wm. This is typically
desirable for the
embodiments described herein.
Characteristic for the present invention is that at least one of the spotting
fluids)
comprises a polymer and/or polymer precursor, and that at least one of the
spotting fluids
comprises a component representing one or more (bio)chemical recognition
moieties.
In one embodiment, only one spotting fluid comprising the polymer and/or
polymer
precursor as well as the components representing one or more (bio)chemical
recognition
moieties is utilized.
In a preferred embodiment, at least two spotting fluids are utilized; a first
spotting fluid
comprising a polymer and/or polymer precursor, and a second spotting fluid
comprising a
component representing one or more (bio)chemical recognition moieties. In a
preferred
embodiment, the first spotting fluid is deposited before the second spotting
fluid.
Examples of suitable polymers are plastic resins which comprise polyacrylates
such as
poiy(methyl propenoate) or poly(2-methyl propenoate), polyanilines,
poly(butadiene),
polyethylene, polyethylene-co-vinyl acetate), polymethacrylates such as
poly(methyl
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14
methacrylate), poly(octyl methacrylate), poly(decyl methacrylate) or
poly(isodecyl
methacrylate), polystyrenes such as polystyrene, poly(4-tert-butyl styrene) or
poly(4-
methoxy styrene), polypyrroles, polythiophenes, polyurethanes such as Tekoflex
p EG 80
A, polyvinyl acetate), polyvinyl alcohol), polyvinyl chloride), epoxy novolac
resins such
as SU 8 from Shell, and co- or terpolymers of the above mentioned polymers
such as
polyethylene-co-vinyl acetate). Particular examples are poly(decyl
methacrylate),
poly(isodecyl methacrylate), Tekoflex~ EG 80 A, and polyvinyl chloride).
In the present context the term "polymer precursor" designates monomers,
dimers,
oligomers, prepolymers, as well as crosslinkers which upon polymerization,
polycondensation, and crosslinking to form a macromolecular, polymeric
structure.
Examples of plastic monomers are monomeric acrylates such as acrylic acid, n-
butyl
acrylate, isodecyl acrylate, acrylamide, hexanediol diacrylate,
cyclohexanediol diacrylate,
N, N' methylene bisacrylamide or tripropylene glycol diacrylate, monomeric
methacrylates
such as methacrylic acid, methyl methacrylate, ethyl methacrylate, n-butyl
methacrylate,
isobutyl methacrylate, hexyl methacrylate, nonyl methacrylate, decyl
methacrylate,
dodecyl methacrylate, hydroxyethyl methacrylate, glycidyl methacrylate,
trifluoroethyl
methacrylate, ethylene glycol dimethacrylate, triethylene glycol
dimethacrylate or 1,6-
hexanediol dimethacrylate. Particular examples of plastic monomers are n-butyl
acrylate,
isodecyl acrylate, decyl methacrylate, and 1,6-hexanediol dimethacrylate.
Another class of monomeric units comprised by the invention is metal or
semimetal
compounds such as organosilanes, e.g. tetramethoxysilane, 3-
aminopropyltrimethoxy
silanes, and tetramethylorthosilicate, which upon hydrolysis of the alkyl-O-Si
bonds afford
silanols (SiOH-groups) followed by polycondensation to form sol-gels (-Si-O-Si-
). In this
instance a sol-gel ("polymer" precursor) is utilised in combination with an
alcohol a as
solvent, water and an acid.
Examples of oiigomers are aliphatic urethane diacrylate oligomers such as
Ebecryl 230
(MW 5000) and Ebecryl 270 (MW 1500) (from UCB chemicals), and proteins such as
bovine serum albumine (BSA) which in combination with a crosslinker, e.g.
glutardialdehyde, form a water-insoluble macromolecular, polymeric structure
which may
physically entrap biochemical recognition elements.
As may be apparent, the spotting fluid may further comprise one or more
solvents. The
solvent or solvent mixture should be selected so that the polymers and/or
polymer
precursors stay dissolved or suspended therein during the depositing process
and so that
consolidation of the spotting fluid does not occur until the spotting fluid
has been deposited
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as droplets onto the substrate material. Preferably, the solvent or solvent
mixture
evaporates spontaneously after deposition of the spotting fluid onto the
substrate material.
However, for some non-volatile solvents or solvents mixtures it may be
necessary to apply
heat or reduced pressure in order to ensure proper and rapid evaporation of
the solvent or
5 solvent mixture and following consolidation of the sensor dots.
Suitable solvents are ketones such as acetone, butanone, 4-methyl-2-pentanone,
cyclopentanone or cyclohexanone, hydrocarbons such as n-hexane, n-pentane,
benzene,
toluene or xylene, esters such as ethyl acetate, propyl acetate, butyl acetate
or diethyl
10 sebacate, alcohols such as methanol, ethanol, glycerol, ethanolamine or
phenol, acides
such as formic acid, or acetic acid, amides such as N,N-dimethyl formamide,
N,N-dimethyl
acetamide or N-methyl pyrrolidon, halogenated hydrocarbons such as
dichloromethane,
chloroform, tetrachlorethane or chlorobenzene, nitromethane, nitrobenzene,
water and
mixtures thereof.
The spotting fluid may further comprise one or more plasticizer. Examples of
suitable
plasticizers are esters such as bis(1-butylpentyl) adipate, bis(1-
butylpentyl)decane-i,10-
diyl diglutarate, bis(2-ethylhexyl) adipate, bis(2-ethylhexyl) phtalate, bis(2-
ethylhexyl)
sebacate, dibutyl phtalate, dibutyl sebacate, 10-hydroxydecyl butyrate,
tetraundecyl
benzhydrol-3,3',4,4'-tetracarboxylate, tetraundecyl benzophenone-3,3',4,4'-
tetracarboxylate, tris(2-ethylhexyl) trimellitate, dibutyltin dilaureate,
dioctyl
phenylphosphonate, isodecyl diphenyl phosphate, tributyl phosphate or tris(2-
ethylhexyl)
phosphate, ethers such as dibenzyl ether, benzyl 2-nitrophenyl ether, 2-
cyanophenyl octyl
ether, dodecyl 2-nitrophenyl ether, dodecyl [2-(trifluoromethyl)phenyl] ether,
[12-(4-
ethylphenyl)dodecyl] 2-nitrophenyl ether, 2-fluorophenyl 2-nitrophenyl ether,
2-
nitrophenyl phenyl ether, 2-nitrophenyl octyl ether, 2-nitrophenyl pentyl
ether or octyl [2-
trifluoromethyl)phenyl] ether, alcohols such as 1-decanol, 1-dodecanol, 1-
hexadecanol, 1-
octadecanol, 5-phenyl-1-pentanol or 1-tetradecanol, halogenated hydrocarbons
such as 1-
chloronaphtalene or chloroparaffin, phosphin oxides such as trioctylphosphine
oxide, and
mixtures thereof. Particular examples are bis(2-ethylhexyl) sebacate, dodecyl
[2-
(trifluoromethyl)phenyl] ether, and 2-nitrophenyl octyl ether.
In one embodiment of the invention the plasticizer constitutes the solvent.
A particular example of a spotting fluid according to the invention comprises
polyvinyl
chloride) (PVC) and bis(2-ethylhexyl) sebacate (DOS) in the ratio of from 1:1
to 1:4 such
as around 1:2, dissolved in cyclohexanone. Cyclohexanone evaporates at a
suitably slow
rate allowing deposition of about 100-400 fluid droplets onto the substrate
material
without clogging of the depositing mechanism.
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When the spotting fluid comprises polymer precursors, polymerization (one type
of
consolidation) of the polymer precursors is required in order to obtain
polymer dots.
Preferably, the polymerization/consolidation process does not takes place
until after
deposition of the spotting fluid droplets onto the substrate material and
occurs either
spontaneously or initiated by exposure of spotting fluid droplets to heat,
irradiation with
ultraviolet or visible light, or by means of electron induced excitation.
However, for some
polymer precursors the polymerization process will not be initiated or will be
undesirable
slow unless a polymerization initiator is present. Thus, the spotting fluid
may further
comprise a polymerization initiator. An example of a polymerisation initiator
is the radical
initiator, a,a-dimethoxy-a-phenylacetophenon, which may further be combined
with a
photosensitizer such as benzophenone or benzoyl peroxide.
Likewise, polycondensation of polymer precursors, as in the formation of a sol-
gel, may
I5 require the presence of water and/or acids which may be comprised by the
spotting fluid.
In some instance polycondensation may be initiated by exposure of the
deposited soi-gel
precursor spotting fluid to water and/or acid vapor.
The function of the polymer matrix is to provide a carrier for the
(bio)chemical recognition
system. In the present invention, (bio)chemical recognition system relates to
a complex
that may be comprised of one or more components, which upon exposure to a
particular
analyte induces a change in the physical property, e.g. the optical property,
of the polymer
matrix. The planar surface portion of the substrate material forms a suitable
transducer
which thereby facilitates detection of the change in the physical (optical)
property of the
polymer matrix, thereby, allowing the detection and quantification of a
particular analyte.
It should be understood that not all the (bio)chemical recognition moieties
have to interact
directly with the analyte, but that their combination (e.g. in a cascade
fashion) bring about
a change in the physical property of the polymer matrix.
The components representing the (bio)chemical recognition moieties are
retained near the
interface or in the matrix of the sensor dot either by physical entrapment
within the
polymeric network, by covalent linkage to the polymer backbone, by ionic
interaction with
charged groups on the polymer, or by physical dissolution in the polymeric
phase.
In an alternative embodiment of the invention one or more components of the
(bio)chemical recognition system are directly immobilized on the surface of a
sensor dot.
This is particularly interesting for biochemical recognition moieties
comprising enzymes,
antibodies, catalytic antibodies, proteins, nucleic acids and derivatives
thereof such as PNA
(protein nucleic acid), or LNA (locked nucleic acid), aptamers, receptors, or
cell- and tissue
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segments. However, these biochemical recognition moieties may also be retained
near the
interface or in the matrix of the sensor dot as described above. It should
however be
understood that such recognition moieties attached to the surface of the
polymer matrix
are only considered a part of the recognition system if there is a direct
chemical link to the
remaining (embedded) components of the recognition system.
In a preferred embodiment of the invention the sensor device prepared by the
method
comprises a plurality of optode membranes. An optode membrane is considered as
a
single, thermodynamic homogeneous phase, which responds reversibly to the
activity of
an analyte. An optode membrane consists of a polymer matrix which serves as a
carrier for
the chemical recognition moieties. The chemical recognition system may
comprise a ligand
(ion carrier, ionophor, indicator, complexing agent) which is either
chemically bound or
physically entrapped in the polymer matrix. An optical signal is generated
upon interaction
of the ligand with the analyte, whereupon the ligand itself or an additional
compound
(chromoionophore, fluoroionophore, indicator dye) changes its optical
properties upon
complexation with another ion.
In one embodiment of the invention the sensor device prepared by the method is
a
plurality of ion-selective optode membranes, where the chemical recognition
system
comprises, e.g., an ion-selective, electrically neutral ionophore and an H+-
selective
electrically neutral chromoionophore as well as lipophilic anionic sites. The
membrane
changes its color upon exchanging a hydrogen ion against the anaiyte cation.
This change
of the spectral properties is used for optical detection. To ensure constant
amount of ions
present within the polymer matrix, lipophilic anionic sites are added.
Examples of ionophores are those selected from the group consisting of ion
specific
ionophores such as the lithium specific ionophores N, N'-diheptyl-N,N',5,5-
tetramethyl-3,7-
dioxanonanediamide, or N,N,N',N'-tetraisobutyl-cis-cyclohexane-1,2-
dicarboxamide,
the sodium specific ionophores N,N',N"-trimethyl-4,4',4"-propylidyne tris
(3-oxabutyramide), 4-octadecanoyloxymethyl-N,N,N',N'-tetracyclohexyl-1,2-
phenylenedioxydiacetamide, or 4-tart-butylcalix[4]arena-tetraacetic acid
tetraethyl ester,
the potassium specific ionophores Valinomycin, 2-dodecyl-2-methyl-1,3-
propanediyl bis[N-
[5'-nitro(benzo-15-crown-5)-4'-yl]carbamate], or 4-tart-butyl-2,2.14,14-
tetrahomo-
2a,14a-dioxacalix[4]arene-tetraacetic acid tetra-tart-butyl ester,
the ammonium specific ionophore 4-[N-(1-adamantyl)carbamoylacetyl]-13-[N-
(n-octadecyl)carbamoylacetyl]-1,7,10,16-tetraoxa-4,13-diazacyclooctadecane,
the
cesium specific ionophore calix[6]arena-hexaacetic acid hexaethyl ester,
the magnesium specific ionophores N,N"-octamethylene-bis(N'-heptyl-N'-methyl-
methylmalonamide), N,N"-octamethyfene-bis(N'-heptyl-N'-methyl-malonamide),
N,N',N"-
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tris[3-(heptylmethylamino)-3-oxopropionyl]-8,8'-iminodioctylamine, 7-[(1-
adamantylcarbamoyf)acetyl]-16-[(octadecylcarbamoyl)acetyl]-1,4,10,13-tetraoxa-
7,16-
diazacyclooctadecane, the calcium specific ionophores (-)-(R,R)-N,N'-bis[11-
(ethoxy-
carbonyl)undecyl]-N,N'-4,5-tetra methyl-3,6-dioxaoctane-diamide, calcimycin,
10,19-bis-
[(octadecylcarbamoyl)methoxyacetyl]-1,4,7,13,16-pentaoxa-10,19-
diazacycloheneicosane,
the barium specific ionophore N,N,N',N'-tetracyclohexyl-oxybis(o-
phenyleneoxy)diacetamide, the heavy metals specific ionophores o-
xylylenebis(N,N-
diisobutyldithiocarbamate) (particularly copper), S,S'-methylenebis(N,N-
diisobutyldithiocarbamate) (particularly silver), O,O"-bis[2-
(methylthio)ethyl]-tert-
butylcalix[4]arene(particularly silver), methylene bis(2-thiobenzothiazole)
(particularly
silver), 5-tetradecyl-1,4-dioxa-8,11-dithia cyclotetradecane (particularly
silver), 7-
tetradecyl-6,9-dioxa-2,13-dithia tetradecane (particularly silver),
tetrabutylthiuram
disulfide (particularly zinc), N-phenyl-iminodiacetic acid N'-N'-dicyclohexyl-
bis-amide
(particularly zinc), N,N,N'N'-tetrabutyl-3,6-dioxaoctanedi(thioamide), [1,1'-
bicyclohexyl]-
1,1'-2,2'-tetrol (particularly cadmium), N,N-dioctadecyl-N',N'-dipropyl-3,6-
dioxaoctanediamide (particularly lead), N,N,N',N'-tetradodecyl-3,6-
dioxaoctanedithioamide
(particularly lead), tert-butylcalix[4]arene-tetrakis(N,N-
dimethylthioacetamide)
(particularly lead), tert-butylcalix[6]arene ethyleneoxydiphenylphosphine
(particularly
lead), N,N,N',N'-tetradodecyl-3,6-dioxaoctane-1-thio-8-oxadiamide
(particularly lead),
5,7,12,14-tetramethyldibenzotetraazaannulene (particularly lead), 1,10-
dibenzyl-1,10-
diaza-18-crown-6 (particularly lead), O-methyldihexylphosphine oxide O'-hexyl-
2-
ethylphosphoric acid (particularly uranyl ions), anion specific ionophores
such as
tridodecylmethylammonium chloride, or the fluoride and chloride specific
ionophores chloro
(2,3,7,8,12,13,17,18-octaethylporhyrinato) gallium(III), chloro (5,10,15,20-
tetraphenylporphyrinato)gallium(III), hydroxo (5,10,15,20-tetrakis(o-
pivalamidophenyl)porphyrinato)gallium(III), chloro (2,3,7,8,12,13,17,18-
octaethylporhyrinato) indium(III), chloro (5,10,15,20-
tetraphenylporphyrinato)indium(III),
hydroxo (5,10,15,20-tetrakis(o-pivalamidophenyl)porphyrinato)indium(III),
chloro
(2,3,7,8,12,13,17,18-octaethylporhyrinato) thallium(III), chloro (5,10,15,20-
tetraphenylporphyrinato)thallium(III), [N,N-[4,5-bis(dodecyloxy)-1,2-
phenylenebis[nitrilomethylidyne (2-hydroxy-1,3-phenylene)]acetamide]-N,N'O,O']
dioxouranium, 4,5-dimethyl-3,6-dioctyloxy-1,2-phenylene bis(mercury
trifluoroacetate),
3,6-didodecyloxy-4,5-dimethyl-1,2-phenylene bis(mercury chloride),
[9]mercuracarborand-3, ruthenium(II) (2,3,7,8,12,13,17,18-octaethylporhyrin)
carbonyl,
trioctyltin chloride, tricyclohexyltin chloride, other ionophores are the
triiodide specific
ionophore 2,4,6,8-tetraphenyl-2,4,6,8-tetraazabicyclo[3.3.0]octane, nitrite
specific
ionophores cyano aqua cobyrinic acid heptakis(2-phenylethyl ester), dicyano
cobyrinic acid
heptapropyl ester, aquo-cyano-cobinamide, the carbonate and sulfide specific
ionophors
3,12-bis(trifluoroacetobenzoyl) cholic acid, trifluoroacetyl-p-butylbenzene,
octadecyl 4-
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formylbenzoate, and the sulfate specific ionophores dibecain sulfate, and a,a'-
bis(n'-
phenylthioureylene)-m-xylene. Particular examples are 4-tert-
butylcalix[4]arene-
tetraacetic acid tetraethyl ester, 2-dodecyl-2-methyl-1,3-propanediyi bis[N-
[5'-
nitro(benzo-15-crown-5)-4'-yl]carbamate], 4-[N-(1-adamantyl)carbamoylacetyl]-
13-[N-
(n-octadecyl)carbamoylacetyl]-1,7,10,16-tetraoxa-4,13-diazacyclooctadecane, (-
)-(R,R)-
N,N'-bis[11-(ethoxy-carbonyl)undecyl]-N,N'-4,5-tetramethyl-3,6-dioxaoctane-
diamide,
tridodecylmethylammonium chloride, hydroxo (5,10,15,20-tetrakis(o-
pivalamidophenyl)porphyrinato)indium(III), and cyano aqua cobyrinic acid
heptakis(2-
phenylethyl ester).
Examples of chromoionophores are those selected form the group consisting of 9-
(diethylamino)-5-(octadecanoylimino)-5H-benzo[a]phenoxazine, 9-dimethylamino-5-
[4-
(16-butyl-2,14-dioxo-3,15-dioxaeicosyl)phenylimino] benzo[a]phenoxazine, 9-
(diethylamino)-5-[(2-octadecyl)imino] benzo[a]phenoxazine, 5-octadecanoyloxy-2-
(4-
nitrophenylazo)phenol, 9-(diethylamino)-5-(naphthoylimino)-5H-
benzo[a]phenoxazine,
4',5'-dibromofluorescein octadecyl ester, fluorescein octadecyl ester, 4-
(octadecylamino)azobenzene, and N-2,4-dinitro-6-(octadecyloxy)phenyl-2',4'-
dinitro-
(trifluoromethyl)phenylamine. Particular examples are 9-(diethylamino)-5- '
(octadecanoylimino)-5H-benzo[a]phenoxazine, and 9-dimethylamino-5-[4-(16-butyl-
2,14-
dioxo-3,15-dioxaeicosyl)phenylimino] benzo[a]phenoxazine.
Examples of the complex lipophilic inorganic ions are those selected form the
group
consisting of tetrakis[3,5-bis(trifluoromethyl)phenyl]borate, tetrakis(4-
chlorophenyl)borate, tetrakis(4-fluorophenyl)borate, and tetradodecylammonium.
Particular examples are tetrakis[3,5-bis(trifluoromethyl)phenyl]borate, and
tetradodecylammonium.
In one embodiment of the invention the chemical recognition system comprises
one
ionophore, one chromoionophore, and one complex lipophilic inorganic ion.
In another embodiment of the invention, the biochemical recognition system
comprises an
enzyme or enzymes and a color reagent, e.g. an enzyme (e.g. glucose oxidise)
which in
the presence of oxygen oxidizes an analyte (e.g. glucose) when bound to the
enzyme. The
recognition system may contain further components that may interact with
either the
reaction product of this oxidation, gluconic acid or hydrogen peroxide (e.g.,
the former
could protonate a pH indicator or the latter could oxidize a dye) in order to
facilitate the
optical detection.
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In one embodiment of the invention, the optical phenomenon is surface plasmon
resonance, and the substrate material is prepared from a plastic base material
and a metal
surface layer material, the sensor dots being prepared from a
polyvinylchloride or cross-
linked acrylate comprising a plasticizer. In particular, the metal is gold and
the base
5 material is polyetherimide.
In an alternative embodiment of the invention chemical recognition is brought
about by the
polymer structure itself. Polymeric materials, which responds reversibly to
the activity of
an analyte and which are characterized by having a polymeric structure
comprising cavities
10 are referred to as molecular imprinted polymers. Molecular imprinted
polymers are
prepared by polymerization of a polymer precursor, e.g. acrylates, in the
presence of a
template molecule, often the analyte itself. Subsequent, extraction of the
template
molecule from the polymer matrix affords cavities in the polymer matrix which
constitute
analyte specific binding sites. Binding of an analyte in the cavity lead to
changes in the
15 optical/physical properties of the polymer matrix. In this particular
embodiment of the
invention the template molecule (here "recognition moiety" although a
"negative") is
comprised in the spotting fluid and the chemical recognition site/system is
subsequently
formed upon washing of the consolidated sensor dots.
20 As indicated above introduction of a (bio)chemical recognition system into
the polymer
matrix of a sensor dot can be accomplished in different ways comprising one or
more
subsequent steps. These steps may comprise one or more washing steps, one or
more
"pin-ring" depositing steps, as well as one or more consolidation steps, e.g.
by exposure to
heat, vacuum, or irradiation with different sources.
In one embodiment of the invention the components of the (bio)chemical
recognition
system are contained in the same spotting fluid as the polymer and/or polymer
precursor.
In one embodiment two or more spotting fluids are sequentially deposited at
each
predetermined position of the planar surface, and wherein the spotting fluids
are allowed
to consolidate after the last deposition of a spotting fluid.
In another embodiment two or more spotting fluids are sequentially deposited
at each
predetermined position of the planar surface, and wherein the spotting fluids
are allowed
to consolidate after deposition of each of the spotting fluids.
In another embodiment of the invention the chemical or biological recognition
system may
be introduced into (or onto) the polymer matrix of a sensor dot by
superimposition of one
or more fluids comprising one or more components of the (bio)chemical
recognition
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21
system, by means of the "pin-ring" depositing technique, onto the pre-formed
sensor dot.
In this embodiment the fluids comprising the (bio)chemical recognition
elements may
further comprise a solvent and/or a plasticizer.
This approach may not only be advantageous for the introduction of recognition
elements
that may be damaged when exposed to polymerization conditions. It may also
show to be
an economical way of producing (bio)chemical sensor~devices with different
patterns.
In one embodiment of the invention the chemical recognition system is
introduced into the
polymer matrix of a sensor dot in the following way: A first spotting fluid
comprising a
polymer and/or polymer precursor and a plasticizer is deposited onto a
substrate material.
The spotting fluid is allowed to consolidate. The plasticizer is re-extracted
by washing of
the consolidated sensor dot with a suitable solvent. A second spotting fluid
comprising the
(bio)chemical recognition system in the form of components representing one or
more
(bio)chemical recognition moieties and a plasticizer is deposited on top of
the consolidated
polymer matrix by re-plasticizing of the polymer matrix. The combination is
then allowed
to consolidate
In one embodiment of the invention the (bio)chemical sensor dots may be
composed on
the support surface by successive superimposition of one or more fluids
comprising one or
more components selected from the group consisting of solvent, plasticizer,
polymerization
initiator, and (bio)chemical recognition components, onto droplets of
consolidated as well
as non-consolidated sensor dots. Each of the successive superimposition steps
may be
followed by consolidation such as exposure of the sensor dots to heat, vacuum,
or
irradiation by different sources, by or washing.
In an even more preferred embodiment one or more of the sensor dots represent
a
reference sensor dot containing a reference polymer matrix which is responsive
to the
unspecific changes due to effects from temperature, aging, analyte, bulk
solution refractive
index, swelling of the polymer matrix, ionic strength, or to fluctuations in
the light source
employed by the sensor transducer. The reference sensor dot may comprise all
the
components of the sensor dots to which it is a reference except from one or
more of the
(bio)chemical recognition elements.
The diameter of the (bio)chemical sensor dots is typically 1-1000 Nm, more
preferably
150-250 Nm, and the height of the dots is 0.1-1000 Nm, preferably i-5 pm. The
number of
fluid superimposition steps normally controls the diameter and the height of a
sensor dot.
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A (bio)chemical sensor device prepared according to the method of the present
invention
may be used for parallel detection and quantification of two or more analytes
comprised in
the same sample. The skilled person in the art will recognize the broad scope
of potential
application of the invention.
EXAMPLES
Example 1
Modifications of a commercially available "Pin-Rin "-arrayer
A commercially available "Pin-Ring"-arrayer (Affymetrix 417, formerly from
Genetic
Microsystem as GMS 417) is adapted for the deposition of spotting fluids
comprising
polymer or polymer precursors rather than biological or biochemical fluids,
i.e. adapted for
continuous use with organic solvents like ethanol for washing of the pins.
Tubings - commonly silicone - are exchanged for more solvent-resistant FEP
(fluorethylen-
propylene) tubing. In a similar manner, the pumps (AS Thomas) that transport
the
washing liquid into the wash stations are removed and replaced by the same
model in the
"chemically resistant" version. The protective lock of the door is deactivated
to allow
access to the pins for manual washing with tetrahydrofuran using a wash
bottle. Flow
restrictors rather than clamps (or in addition to clamps) are mounted onto the
washing
solvent tubing to allow increased control of the solvent spurting out of the
nozzle in the
wash station. Protective foil can be used to cover the inside of the
transparent front door
of the instrument to prevent it from damage in case of minor wash solvent
splashing.
The outlet of the vacuum pump which removes the washing fluid from the bath by
aspiration is connected to a laboratory air ventilation system (hood) to
prevent significant
introduction of solvent vapor into the work environment. The samples are
introduced in the
arrayer in the wells of a microtiter plate. Common polystyrene plates are not
resistant to
many organic solvents, which is why polypropylene plates are chosen.
Example 2
Preparation of a plurality of miniaturized PVC dots on glass materials
33 mg of polyvinyl chloride) (PVC) (high molecular weight) and 66 mg
plasticizer bis(2-
ethylhexyl) sebacate (DOS) are dissolved in 800 NL cyclohexanone. 35 NL of the
resulting
spotting fluid are filled in well A1 of a 256-well polypropylene microtiter-
plate. Using the
GMS 417 arrayer with 125 Nm-pins, demonstration arrays of PVC dots can easily
be
deposited on substrates such as commercially available glass or gold-coated
glass
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23
microscope slides (Figure 1). Other support surfaces may be placed in the
instrument by
employing custom-made metal adapter plates. The pin is washed with
tetrahydrofuran in
order to remove PVC-DOS residues. This may be done manually, or suitable
solvents may
be used in the washing lines and bath in a correspondingly adapted instrument.
Example 3
Preparation of a plurality of miniaturized sodium-selective (bio)chemical
sensor dots
2.9 mg of 9-(diethylamino)-5-octadecanoylimino)-5H-benzo[a]phenoxazine, 4.6 mg
sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate, 10.0 mg 4-tert.-
butylcalix[4]arene-
tetraacetic acid tetraethyl ester, 139.2 mg bis(2-ethylhexyl) sebacate, and
69.1 mg
polyvinyl chloride) (high molecular weight) are dissolved in 2.0 ml
cyclohexanone. 35 pL
of the resulting spotting fluid is filled in well A1 of a 256-well
polypropylene microtiter-
plate. Using the GMS 417 arrayer with 125um-pins, plasticized PVC based sodium-
selective
(bio)chemical sensor dots was prepared on gold-coated microscope glass slides.
Functionality of the sensing dots. i.e. response to target ion sodium in
buffered solution,
can be verified by means of fiber optical absorbance spectroscopy or surface
plasmon
resonance spectroscopy, respectively. The latter detects the refractive index
changes in
the membrane, that are related to spectral /absorbance changes by the Kramers-
Kronig
relation.
Example 4
Preparation of a pluralitLr of sensor dots using a spotting-fluid comprising
methacr~rlate
uL of spotting fluid made from 160 mg 1,6-hexanediol dimethacrylate, 100 mg
dodecyl
methacrylate, 200 mg bis(2-ethylhexyl) sebacate and a radical initiator, e.g.,
1 mg of a,a-
dimethoxy-a-phenylacetophenon, or 2.5 mg of benzoyl peroxide with 5 mg of
30 benzophenone as photosensitizer, is filled in well A1 of a 2S6-well
polypropylene
microtiter-plate. Using the GMS 417 arrayer with 125Nm-pins, demonstration
arrays of
photopolymerized methacrylate sensor dots was made on commercially available
microscope slides. After deposition of the spotting fluid, the pin was washed
with a suitable
solvent such as ethanol. After deposition, the spotting fluid droplets were
35 photopolymerized by exposure to UV-light in an inert-gas atmosphere,
typically for 10 - 20
minutes. Such an experiment demonstrates convincingly that methacrylate
cocktail dots
can be produced in high number with high accuracy and photopolymerized
immedialety
afterwards. It is obvious to the person skilled in the art that addition of
(bio)chemical
recognition components (e.g., an ionophore, a chromoionophore, complex
lipophilic
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inorganic ions) to the spotting fluid in concentrations of few % (w/w) will
result in arrays of
sensing dots without affecting the deposition process. However, since many of
these
components are photobleachable, replasticizing can be chosen as an alternate
route to
introduce the sensing components. Towards this end, plasticized dots without
any
recognition elements are deposited and subsequently treated with
tetrahydrofuran to
extract the plasticizer from the material. Afterwards, droplets of a fluid
comprising
(bio)chemical recognition components (e.g., an ionophore, a chromoionophore,
complex
lipophilic inorganic ions) in bis(2-ethylhexyl) sebacate may be deposited
directly on top of
the polymer dots. Given sufficient time, the plasticizer and with it the
sensing components
are taken up by the polymer matrix, resulting in arrays of functional ion-
selective
(bio)chemical sensor dots.
Example 5
Superimposition of Polymer Dots
5.I
A spotting fluid analogous to that in example 4 was deposited on a glass
microscope slide
in such a manner that four arrays of three times three dots A, B, C, D were
obtained. The
dots were then photopolymerized by UV irradiation in an inert gas atmosphere.
Subsequently, two arrays were superimposed on top of arrays A and C and
polymerized.
An image of the sensor dots was taken with the Affymetrix 418 fluorescence
scanner
(Figure 2) and confirms the successful superimposition (note the
superimposition that
failed due to an error of the experimentation in array B, where the two
individual arrays
are shifted).
5.II
Furthermore, a spotting fluid analogous to that in example 2 was deposited in
a 20 by 20
array, using a feature in the calibration software of the arrayer intended for
an alignment
test. The depositing was repeated with shifted dot location. The scanner image
in Figure 3
shows the two arrays and the area in which they overlap. The slight difference
in array
appearance is most probably caused by use of a different surface of the
microscope slides.
