U.S. patent application number 10/545115 was filed with the patent office on 2006-10-12 for apparatus for detecting particles.
Invention is credited to Nicholas John Goddard, Kai Freidrich Hoettges, Michael Pycraft Hughes, Martin Bernard McDonnell, Mohammed Zourob.
Application Number | 20060228257 10/545115 |
Document ID | / |
Family ID | 9952953 |
Filed Date | 2006-10-12 |
United States Patent
Application |
20060228257 |
Kind Code |
A1 |
Hughes; Michael Pycraft ; et
al. |
October 12, 2006 |
Apparatus for detecting particles
Abstract
Apparatus for detecting particles in a liquid, comprises an
evanescent optical sensor having an electrically conducting layer,
and means associated therewith for providing an electric field at
or adjacent the sensor surface. Preferably, the optical sensor is a
surface bound sensor based on a metal-clad leaky waveguide
structure. A DC potential applied to the metal layer and a parallel
counter electrode provides a uniform electric field, which drives
deposition of the particles on the sensor surface. Alternatively,
an alternating potential applied to a electrically conducting layer
configured as an electrode and counter electrode induces an
electro-osmotic bulk flow in the liquid which drives deposition of
the particles on the sensor surface.
Inventors: |
Hughes; Michael Pycraft;
(Surrey, GB) ; Hoettges; Kai Freidrich; (Surrey,
GB) ; McDonnell; Martin Bernard; (Wiltshire, GB)
; Goddard; Nicholas John; (Manchester, GB) ;
Zourob; Mohammed; (Manchester, GB) |
Correspondence
Address: |
JOHN S. PRATT, ESQ;KILPATRICK STOCKTON, LLP
1100 PEACHTREE STREET
ATLANTA
GA
30309
US
|
Family ID: |
9952953 |
Appl. No.: |
10/545115 |
Filed: |
February 12, 2004 |
PCT Filed: |
February 12, 2004 |
PCT NO: |
PCT/GB04/00552 |
371 Date: |
April 14, 2006 |
Current U.S.
Class: |
422/82.05 |
Current CPC
Class: |
G01N 21/648 20130101;
G01N 21/553 20130101; G01N 21/05 20130101; G01N 21/6456
20130101 |
Class at
Publication: |
422/082.05 |
International
Class: |
G01N 21/00 20060101
G01N021/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 12, 2003 |
GB |
03033057 |
Claims
1. Apparatus for detecting particles in a liquid, comprising an
evanescent optical sensor having an electrically conducting layer,
and means associated therewith for providing an electric field at
or adjacent the sensor surface.
2. Apparatus according to claim 1, in which the optical sensor
comprises a surface plasmon resonance chip.
3. Apparatus according to claim 1, in which the optical sensor
comprises a metal-clad leaky waveguide chip.
4. Apparatus according to claim 3, in which the means for providing
an electric field comprise a plane surface counter electrode and a
DC potential source.
5. Apparatus according to claim 4, in which the counter electrode
is arranged parallel to the conducting layer.
6. Apparatus according to claim 4, in which the counter electrode
and sensor define a gap there between ranging from 20 to 50
.mu.m.
7. Apparatus according to claim 4, in which the counter electrode
comprises a layer of indium tin oxide provided on a glass
substrate.
8. Apparatus according to claim 3, in which the electrically
conducting layer comprises titanium.
9. Apparatus according to claim 1, in which the optical sensor is a
surface bound sensor.
10. A method for detecting particles in a liquid comprising i)
introducing the liquid to an evanescent optical sensor, including
an electrically conducting layer, having means associated therewith
for providing an electric field at or adjacent the sensor surface
and ii) generating the electric field.
11. A method according to claim 10, in which the optical sensor
comprises a surface plasmon resonance chip.
12. A method according to claim 10, in which the optical sensor
comprises a metal-clad leaky waveguide chip.
13. A method according to claim 12, in which the generated electric
field is uniform.
14. A method according to claim 13, in which a DC potential is
applied to a gap electrode configuration comprised by the sensor
and a parallel planar surface counter electrode at magnitude
ranging from 10 to 100 V for a gap size ranging from 20 to 50 .mu.m
and a flow rate ranging from 50 to 300 .mu.l min.sup.-1.
15. A method according to claim 10, in which the optical sensor is
a surface bound sensor.
16. (canceled)
17. (canceled)
Description
[0001] The present invention is generally concerned with apparatus
for enhanced detection of particles using optical sensors. The
invention is particularly, but not exclusively, directed towards
enhanced deposition of particles, such as bacteria and viruses, on
surface bound optical sensors.
[0002] As used herein the term "surface bound sensor" describes an
optical sensor including a sensing medium comprising a biological
material capable of binding the particles to be detected--such as
an antibody or lectin.
[0003] Optical sensors often comprise waveguide structures in which
an evanescent wave, associated with an optical mode existing in the
structure, extends into the sensing layer. A change in the
refractive index of the sample by interaction with a particle leads
to a change in an optical property of the mode, which can be
readily detected.
[0004] One such optical sensor uses the phenomenon of surface
plasmon resonance (SPR). Light incident a dielectric prism having
an upper surface coated with a thin metal layer of gold or silver
and a sensing layer comprising the biological sample, is coupled at
a certain "resonant" angle or angles to oscillations of the
electron cloud in the metal layer. A surface optical mode is
propagated at the interface of the prism and metal layer and a drop
in the amount of reflected light is recorded at a detector. The
surface optical mode generates an evanescent field that extends
into the sensing medium. A particle binding to the sample leads to
a change in the refractive index of the medium affecting the
surface mode and the angle at which resonance is excited.
[0005] The basic structure of "leaky" waveguide sensors, described
in International Patent Application WO 99/44042, is similar but
offers improved sensitivity in that an optical mode is supported in
the bulk of the sensing layer.
[0006] International patent application WO 01/42768A describes flow
cell apparatus in which detection of surface bound particles on an
SPR sensor is improved by monitoring light scattered or emitted
there from.
[0007] Improved detection is also obtained using metal-clad or
dye-clad leaky waveguide sensors described in our co-pending
international patent application PCT/GB02/045045 incorporated by
reference herein. Here, an evanescent wave extends above the
sensing surface and can be "tailored", by choice of refractive
indices in the stucture, to optimise overlap with the particles to
be detected.
[0008] However, the sensitivity of flow cell apparatus
incorporating these optical sensors is inherently limited by slow
diffusion of the particles in the liquid to the sensor surface,
even with stirring or agitation. Consequently, there is still a
need to improve the detection of particles at surface bound
sensors.
[0009] The present invention generally aims to improve detection at
optical sensors, and in particular, surface bound optical sensors
by providing an electric field acting on the particles so as to
direct them to the sensor surface.
[0010] Under suitable fluid flow conditions, the electric field may
act on charged particles or on an induced dipole on the particles
to enhance the deposition of the particles to the sensor
surface.
[0011] Accordingly, in one aspect, the present invention provides
apparatus for detecting particles in a liquid, comprising an
evanescent optical sensor, including an electrically conducting
layer, and means associated therewith for providing an electric
field at or adjacent the sensor surface.
[0012] It will be apparent, that the electrically conducting layer
can in itself comprise an electrode. In a preferred embodiment of
the present invention, therefore, the means providing an electric
field comprise a plane surface counter electrode arranged in
parallel with the conducting layer, a DC potential source and means
for applying the potential across the electrode and the conducting
layer. In this embodiment, the conducting layer and the plane
surface electrode together define a gap electrode configuration in
which the sensing layer partially extends.
[0013] It will be understood that the DC potential provides a
uniform electric field capable of charging the particles and
directing them to the sensor surface (electrophoresis). The
magnitude of the DC potential, the extent of the gap between the
plane surface electrode and the conducting layer, the flow rate of
the liquid containing the particles across the sensor surface will
all be determined having regard to each other and to the particle
size. It will be appreciated that the force acting on the particles
should be sufficient to overcome flow and drag effects.
[0014] In a preferred embodiment of the present invention, the
optical sensor comprises a surface bound sensor such as the MCLW
sensor mentioned above and described in our co-pending
international patent application PCT/GB02/045045. Suitable
electrically conducting layers comprise metals, in particular,
aluminium, tantalum, zirconium, titanium or chromium, or
crystalline dye materials.
[0015] Suitable applied potentials for the detection of bacillus
subtilis var. Niger (bacillus globbiggi, BG) spores according to
this embodiment range from 10 to 100 V, typically about 30 V for a
gap size ranging from 20-50 .mu.m and flow rates ranging from 50 to
300 .mu.l min.sup.-1.
[0016] The plane surface counter electrode may comprise a
conducting, metal or metal oxide layer arranged on an inert
substrate. Preferably, the counter electrode comprises a layer of
indium tin oxide (ITO) arranged on a glass substrate since it is
clear and known to resist optical degradation on prolonged
polarisation by application of an alternating potential.
[0017] In another embodiment of the present invention, the
electrically conducting layer of the optical sensor is itself
configured, as described in our co-pending GB patent application
No. 0303305.7--incorporated by reference herein, so as to provide a
planar electrode and planar counter electrode.
[0018] In this embodiment, the means providing an electric field at
or adjacent the sensor surface comprise an alternating potential.
The alternating potential when applied at a predetermined frequency
and magnitude to the layer induces a non-uniform electric field
that can induce an electro-osmotic flow in the bulk of the liquid
so as to focus particles onto the sensor surface (abnormal
dielectrophoresis).
[0019] It will be understood that, in some embodiments of the
present invention, the fluid layer in contact with the upper
surface of the sensor comprises a sensing layer. The fluid layer,
which is semi-infinite, will contain the particles to be detected.
However, a surface bound sensor is preferred, particularly where it
comprises a sensing layer of an antibody or lectin.
[0020] In a second aspect, the present invention provides a method
for detecting particles in a liquid comprising i) introducing the
liquid to an evanescent optical sensor, including an electrically
conducting layer, having means associated therewith for providing
an electric field at or adjacent the sensor surface and ii)
generating the electric field.
[0021] In a preferred embodiment, the means providing an electric
field comprise a plane surface counter electrode arranged in
parallel with the conducting layer, a DC potential source and means
for applying the potential across the electrode and the conducting
layer. In this embodiment, the conducting layer and the plane
surface electrode together define a gap electrode configuration in
which the sensing layer partially extends.
[0022] The magnitude of the DC potential, the extent of the gap and
the flow rate of the liquid containing the particles across the
sensor surface will all be determined having regard to each other
and to the force necessary for the particles to overcome flow and
drag effects.
[0023] In a particularly preferred embodiment, the optical sensor
comprises a surface bound optical sensor such as the MCLW sensor
described in our co-pending international patent application
PCT/GB02/045045.
[0024] The DC voltage may be applied to one or both of the
electrodes. Preferably, however, the counter electrode is earthed.
Suitable applied potentials for the detection of bacillus subtilis
var. Niger (bacillus globbiggi BG) spores according to this
embodiment from 10 to 100 V, typically about 30 V for a gap size
ranging from 20-50 .mu.m and flow rates ranging from 50 to 300
.mu.l min.sup.-1.
[0025] Other embodiments in this aspect of the present invention
will be apparent from the foregoing description. In addition, it
will be apparent that a combination of uniform and non-uniform
fields (electrophoresis and dielectrophoresis) maybe used where it
is desired to manipulate different particles.
[0026] The present invention will now be described by reference to
several embodiments and the following examples and drawings in
which
[0027] FIG. 1 is a schematic illustration of one embodiment of the
present invention;
[0028] FIGS. 2a) to c) are photographs showing electrophoretic
enhancement for the collection of bacillus globiggi BG spores on
the embodiment of FIG. 1;
[0029] FIG. 3 is a graph showing the enhancement at various
concentrations of spores;
[0030] FIG. 4 is a graph showing the capture of spores on a
preferred embodiment of the present invention; and
[0031] FIGS. 5a) and b) show the scattering image and fluorescence
image of the spores captured on the preferred embodiment.
[0032] Having regard now to FIG. 1, a basic MCLW chip 11 comprises
an upper surface of a 300 nm silica sol layer 12 (n=1.43) provided
on a thin layer 13 (8.5 nm) of titanium coating a 1 mm glass
substrate layer 14 (n=1.5). The thickness and refractive index of
the silica sol layer 12 is chosen to support a single sharp-guided
optical mode at a wavelength of incident light of 685 nm or 488 nm
and to optimise the extent of the evanescent field above the
surface to about 1.5 to 2.0 .mu.m.
[0033] As mentioned previously, a final sensing layer (not shown)
may comprise a liquid layer containing the particles to be
analysed. Alternatively or additionally the sensing layer can
comprise an antibody layer deposited on the silica sol layer.
[0034] A counter electrode comprises a glass substrate 15 coated
with a conducting layer 16 of ITO. The counter electrode is joined
to the chip 11 by a 30 .mu.m (gap size) double-sided adhesive tape
17 through which a 12 mm by 2 mm section, defining a flow cell 18,
has been cut. Two 1.5 mm holes drilled in the counter electrode
provide inlet and outlet means for the flow of liquid through a
delivery and collection tube 19 to the cell 18.
[0035] The titanium layer 13 of the sensor chip is provided with a
silver loaded epoxy contact (not shown) at one end of the chip 11.
The ITO layer 16 of the counter electrode is provided with similar
contacts (not shown) at positions along its length so as to reduce
voltage gradient due to the resistance of the metal and ITO layers.
The contacts connect the sensor chip and counter electrode to a DC
potential source.
[0036] The assembly is used in conjunction with an equilateral,
coupling prism (not shown 30 mm) of BK 7 glass and refractive index
1.510. The interrogation of particle deposition at the upper
surface of the chip may be conducted using the basic arrangement
including an optical source and detection means described in our
co-pending international patent application PCT/GB02/045045.
EXAMPLE 1
[0037] The sensor surface was blocked by exposure to 0.1% w/v BSA
in PBS/Tween.RTM. 20 and stored overnight at 4.degree. C. BG spores
(4.7.times.10.sup.7 spores/ml) in 50 mM histidine buffer were
introduced to the flow cell using a MINIPULS-3, MP4 peristaltic
pump (Gilson, Canada) at 50 .mu.l min.sup.-1. A positive potential
of 30 V was applied to the metal sensor layer with respect to the
counter electrode for a total of 2 min. After a further period, a
negative potential of 1.5 V was applied to the metal sensor layer
with respect to the counter electrode.
[0038] FIG. 2 shows the distribution of spores before (a) and on
(b) applying the positive potential. As may be seen, the number of
cells incident the sensor surface is greatly increased during the
application of the potential. The application of the negative
potential (c) highlights negligible non-specific adsorption of the
spores to the chip surface.
[0039] These effects were repeated for BG spore concentrations
ranging from 10.sup.3 to 10.sup.6 spores/ml. An increase in the
electrical conductivity of the buffer (to 150 mM NaCl), however,
greatly decreased the number of cells incident the sensor
surface--presumably through ionic screening and restriction of the
electric field to the double charge layer adjacent the
electrodes.
EXAMPLE 2
[0040] A direct immunoassay utilised two DC potential application
steps. A biotinylated-labelled anti BG capture antibody was
deposited on the chip by repeated introduction of 50 .mu.g/ml
suspension in water to the flow cell (200 .mu.l/min) and applying a
positive potential of 20 V. Unbound antibody was removed by washing
with 50 mM histidine buffer. A BG spore solution (4.times.10.sup.7
spores in 50 mM histidine buffer) was introduced to the flow cell
at a flow rate of 50 .mu.l/min. A positive potential of 30 V was
applied to the metal sensor layer with respect to the counter
electrode during 2 min. After a short period, a negative potential
(1.5 V) was applied to the metal sensor layer with respect to the
counter electrode. The assay was visualised in real time by
detection of scattered light from captured BG spores using a CCD
camera. These effects were repeated for BG spore concentrations
ranging from 10.sup.3 to 10.sup.6 spores/ml (FIG. 3).
[0041] FIG. 4 shows the number of cells captured by subtraction of
the number of spores removed from the sensor surface on application
of the negative potential from the number of spores. The values are
in good agreement with the number of captured spores remaining on
the surface. Comparison of the number of captured spores with the
number of spores captured using the MCLW chip alone revealed a 30
fold enhancement equal to an increase in local concentration from
10.sup.3 to 10.sup.8 spores.
EXAMPLE 3
[0042] A sandwich format immunoassay utilised three DC potential
application steps. A biotinylated-labelled anti BG capture antibody
was deposited on the chip and BG spores were captured on the
antibody layer as previously described. Finally a suspension of
CY5-labelled BG antibody (10 .mu.g ml.sup.-1 in 50 mM histidine
buffer) was introduced to the cell at a flow rate of 50 .mu.l
min.sup.-1 over 2 min. A positive potential of 30 V was applied to
the metal sensor layer with respect to the counter electrode during
2 min. Unbound CY5-labelled antibody was washed from the flow cell
by purging with 50 nM histidine buffer for 3 min at 200 .mu.l
min.sup.-1.
[0043] FIG. 5 shows the scattering image of captured BG spores and
the fluorescence image following exposure to CY5 labelled antibody.
Although not shown it is found that the fluorescence emitted from
the labelled antibody is greater when attached to the captured
spores than when the labelled antibody alone is deposited at the
sensor surface.
[0044] Repetition of the experiment with varying concentration of
labelled antibody (3, 10, 20 .mu.g ml.sup.-1) showed that the
optimum for detection of captured spores by fluorescence using this
method was 10 .mu.g ml.sup.-1.
* * * * *