U.S. patent application number 15/423938 was filed with the patent office on 2017-05-25 for particle facilitated testing.
The applicant listed for this patent is UNIVERSITY OF THE WEST OF ENGLAND, BRISTOL. Invention is credited to Janice Helen Kiely, Richard William Luxton, Patrick Wraith.
Application Number | 20170146525 15/423938 |
Document ID | / |
Family ID | 39523355 |
Filed Date | 2017-05-25 |
United States Patent
Application |
20170146525 |
Kind Code |
A1 |
Luxton; Richard William ; et
al. |
May 25, 2017 |
Particle Facilitated Testing
Abstract
Magnetic particles are distributed across a fluid flow by
applied magnetic field to interact with a test substance in fluid.
Alternatively or additionally, particles, which may be magnetic,
are combined with cells and energy, e.g. ultrasonic energy, is
applied to cause the particles to create a lysate. Alternatively or
additionally, the size of a quantity of magnetic particles is
assessed by its impact on the tuning mechanism of a controlled
oscillator that is affected by the particles.
Inventors: |
Luxton; Richard William;
(Bristol, GB) ; Kiely; Janice Helen; (Bristol,
GB) ; Wraith; Patrick; (Bristol, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY OF THE WEST OF ENGLAND, BRISTOL |
Bristol |
|
GB |
|
|
Family ID: |
39523355 |
Appl. No.: |
15/423938 |
Filed: |
February 3, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12532072 |
Dec 22, 2009 |
9610584 |
|
|
PCT/GB2008/000993 |
Mar 25, 2008 |
|
|
|
15423938 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/54326 20130101;
G01N 33/54386 20130101; B01L 99/00 20130101; G01N 33/54306
20130101; C12M 47/06 20130101; G01N 33/54333 20130101 |
International
Class: |
G01N 33/543 20060101
G01N033/543 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 21, 2007 |
GB |
0705428.1 |
Apr 18, 2007 |
GB |
0707480.0 |
Claims
1. Apparatus for lysing a cell, the apparatus comprising a chamber
for holding the cell to be lysed and means for introducing energy
into the chamber, wherein the chamber contains a plurality of
particles which may be excited by the energy to enhance lysing of
the cell.
2. Apparatus according to claim 1 wherein the means for introducing
energy into the chamber comprises means for introducing sound waves
into the chamber.
3. Apparatus according to claim 2 wherein the means for introducing
sound waves into the chamber comprises a sonicator probe.
4. Apparatus according to claim 2 wherein the means for introducing
sound waves into the chamber comprises an ultrasonic
transducer.
5. Apparatus according to claim 1 wherein the means for introducing
energy into the chamber is operable to introduce energy into the
chamber in a pulsed manner.
6. Apparatus according to claim 1 wherein the particles are of a
plastics material.
7. Apparatus according to claim 1 wherein the particles are of
metal.
8. Apparatus according to claim 1 wherein the particles are of a
combination of metal and a plastics material.
9. Apparatus according to claim 1 wherein the plurality of
particles are provided with a binding agent to which components of
a lysed cell may bind.
10. Apparatus according to claim 1 wherein the chamber comprises a
sensor surface provided with a binding agent to which components of
a lysed cell may bind.
11. Apparatus according to claim 9 wherein a label is provided to
identify a complex formed when a component binds to the binding
agent.
12. Apparatus according to claim 11 wherein the label comprises an
enzyme.
13. Apparatus according to claim 1 wherein the plurality of
particles are in the range from approximately 0.1 .mu.m to
approximately 100 .mu.m in diameter.
14. Apparatus according to claim 13 wherein the plurality of
particles are in the range from approximately 1 .mu.m to
approximately 20 .mu.m in diameter.
15. Apparatus according to claim 1 wherein the plurality of
particles are magnetic.
16. Apparatus according to claim 15 wherein the plurality of
particles are of a paramagnetic material.
17. Apparatus according to claim 15 wherein the plurality of
particles are of a ferromagnetic material.
18. Apparatus according to claim 15 wherein the plurality of
particles are of a diamagnetic material.
19. Apparatus according to claim 15 wherein the plurality of
particles are of a super-paramagnetic material.
20. Apparatus according to claim 15 further comprising sensing
means for sensing the magnetic particles.
21. Apparatus according to claim 20 further comprising means for
generating a magnetic field to draw the magnetic particles towards
the sensing surface of the chamber.
22. Apparatus according to claim 21 wherein the means for
generating a magnetic field comprises a permanent magnet.
23. Apparatus according to claim 21 wherein the means for
generating a magnetic field comprises an electromagnet.
24. A method of lysing a cell, the method comprising introducing
the cell into a chamber containing a plurality of particles and
introducing energy into the chamber to excite the plurality of
particles.
25. A method according to claim 24 wherein the energy introduced
into the chamber comprises sound waves.
26. A method according to claim 25 wherein the sound waves are
introduced into the chamber using a sonicator probe.
27. A method according to claim 25 wherein the sound waves are
introduced into the chamber using an ultrasonic transducer.
28. A method according to claim 25 wherein the energy is introduced
into the chamber in a pulsed manner.
29. A method according to claim 24 wherein the particles are of a
plastics material.
30. A method according to claim 24 wherein the particles are of
metal.
31. A method according to claim 24 wherein the particles are of a
combination of metal and a plastics material.
32. A method according to claim 24 wherein the plurality of
particles are provided with a binding agent to which components of
a lysed cell may bind.
33. A method according to claim 24 wherein the chamber comprises a
sensor surface provided with a binding agent to which components of
a lysed cell may bind.
34. A method according to claim 33 wherein a label is provided to
identify a complex formed when a component binds to the binding
agent.
35. A method according to claim 34 wherein the label comprises an
enzyme.
36. A method according to claim 24 wherein the plurality of
particles are in the range from approximately 0.1 .mu.m to
approximately 100 .mu.m in diameter.
37. A method according to claim 36 wherein the plurality of
particles are in the range from approximately 1 .mu.m to
approximately 20 .mu.m in diameter.
38. A method according to claim 24 wherein the plurality of
particles are magnetic.
39. A method according to claim 38 wherein the plurality of
particles are of a paramagnetic material.
40. A method according to claim 38 wherein the plurality of
particles are of a ferromagnetic material.
41. A method according to claim 38 wherein the plurality of
particles are of a diamagnetic material.
42. A method according to claim 38 wherein the plurality of
particles are of a super-paramagnetic material.
43. A method according to claim 38 wherein sensing means are used
to sense the magnetic particles.
44. A method according to according to claim 43 wherein a magnetic
field is generated to draw the magnetic particles towards the
sensor surface of the chamber.
45. A method according to claim 44 wherein the magnetic field is
generated using means comprising a permanent magnet.
46. A method according to claim 44 wherein the magnetic field is
generated using means comprising an electromagnet.
Description
FIELD
[0001] The invention relates to apparatus for, and methods of,
testing fluid, and to apparatus for and methods of lysing cells
using particles.
BACKGROUND
[0002] It is known to utilise magnetic particles to capture an
analyte in a solution under test. Conventionally, the magnetic
particles are coated with a substance to which the analyte will
attach. A sensor surface in contact with the solution is provided
with a similar coating and a magnetic field is applied to urge the
magnetic particles onto the surface. Analyte bound to the magnetic
particles then becomes attached to the sensor surface also. Thus,
magnetic particles that have picked up analyte become immobilised
on the sensor surface. An inductor located near to the sensor
surface is used to quantify the number of magnetic particles that
are so immobilised. The inductor forms part of a resonant
electrical circuit. The resonant frequency of this circuit is
determined in part by the inductance of this inductor and the
inductance of the inductor is determined in part by the quantity of
immobilised magnetic particles.
[0003] In the investigation of cell organelles and measurement of
intracellular proteins, cells need to be disrupted or lysed,
releasing the intracellular components for study. Freeze-thaw
methods are commonly used to lyse both bacterial and mammalian
cells. These methods involve freezing a cell suspension using a dry
ice/ethanol bath or freezer and then thawing the material at room
temperature or 37.degree. C. This method of lysis causes cells to
swell and ultimately break as ice crystals form during the freezing
process and then contract during thawing. Multiple cycles are
necessary for effective lysis, and the process can be time
consuming. However, the freeze/thaw methods have been shown to
release proteins located in the cytoplasm of bacteria effectively,
and are recommended for the lysis of mammalian cells in some
protocols.
[0004] Another approach commonly used to disrupt cells is to
solubilise the cell membrane using a detergent. This has the added
advantage of releasing membrane bound proteins but may dissociate
protein complexes. Classically, physical methods have been used to
disrupt cells, such as grinding tissue in a pestle and mortar or
using a blade either as a scalpel or a liquidiser. There are some
inherent disadvantages to mechanical lysis methods such as
localized heating within a sample leading to protein denaturation
and aggregation.
[0005] Ultrasound has also been used as a method of physical cell
disruption which is based on the generation of high frequency
pulses of pressure. Sonication (i.e. the process of disrupting the
cell using sound waves) generates heat which may denature proteins,
so the process should be performed in an ice bath. Some studies
have shown that lysis using detergents to solubilise the cell
membranes is more efficient at releasing intracellular protein than
ultrasound.
BRIEF SUMMARY
[0006] According to an aspect of the invention, there is provided
apparatus for lysing a cell, the apparatus comprising a chamber for
holding the cell to be lysed and means for introducing energy into
the chamber, wherein the chamber contains a plurality of particles
which may be excited by the energy to enhance lysing of the
cell.
[0007] The means for introducing energy into the chamber may
comprise means for introducing sound waves into the chamber.
[0008] The means for introducing sound waves into the chamber may
comprises a sonicator probe.
[0009] Additionally or alternatively, the means for introducing
sound waves into the chamber may comprise an ultrasonic
transducer.
[0010] The means for introducing energy into the chamber may be
operable to introduce energy into the chamber in a pulsed
manner.
[0011] The particles may be of a plastics material.
[0012] Alternatively, the particles may be of metal.
[0013] Alternatively, the particles may be of a combination of
metal and a plastics material.
[0014] The plurality of particles may be provided with a binding
agent to which components of a lysed cell may bind.
[0015] The chamber may comprise a sensor surface provided with a
binding agent to which components of a lysed cell may bind.
[0016] A label may be provided to identify a complex formed when a
component binds to the binding agent.
[0017] The label may comprise an enzyme.
[0018] The plurality of particles are preferably in the range from
approximately 0.1 .mu.m to approximately 100 .mu.m in diameter.
[0019] The plurality of particles are more preferably in the range
from approximately 1 .mu.m to approximately 20 .mu.m in
diameter.
[0020] In certain embodiments, the plurality of particles may be
magnetic.
[0021] For example, the plurality of particles may be of a
paramagnetic, ferromagnetic, diamagnetic or super-paramagnetic
material.
[0022] The apparatus may further comprise sensing means for sensing
the magnetic particles.
[0023] The apparatus may further comprise means for generating a
magnetic field to draw the magnetic particles towards the sensing
surface of the chamber.
[0024] The means for generating a magnetic field may comprise a
permanent magnet.
[0025] Additionally or alternatively, the means for generating a
magnetic field may comprise an electromagnet.
[0026] According to a further aspect of the invention, there is
provided a method of lysing a cell, the method comprising
introducing the cell into a chamber containing a plurality of
particles and introducing energy into the chamber to excite the
plurality of particles.
[0027] The energy introduced into the chamber may comprise sound
waves.
[0028] The sound waves may be introduced into the chamber using a
sonicator probe.
[0029] Additionally or alternatively the sound waves may be
introduced into the chamber using an ultrasonic transducer.
[0030] The energy may be introduced into the chamber in a pulsed
manner.
[0031] The particles may be of a plastics material.
[0032] Alternatively the particles may be of metal.
[0033] Alternatively, the particles may be of a combination of
metal and a plastics material.
[0034] The plurality of particles may be provided with a binding
agent to which components of a lysed cell may bind.
[0035] The chamber may comprise a sensor surface provided with a
binding agent to which components of a lysed cell may bind.
[0036] A label may be provided to identify a complex formed when a
component binds to the binding agent.
[0037] The label may comprise an enzyme.
[0038] The plurality of particles are preferably in the range from
approximately 0.1 .mu.m to approximately 100 .mu.m in diameter.
[0039] The plurality of particles are more preferably in the range
from approximately 1 .mu.m to approximately 20 .mu.m in
diameter.
[0040] In certain embodiments, the plurality of particles may be
magnetic.
[0041] For example, the plurality of particles may be of a
paramagnetic, ferromagnetic, diamagnetic or super-paramagnetic
material. Sensing means may be used to sense the magnetic
particles.
[0042] A magnetic field may be generated to draw the magnetic
particles towards the sensor surface of the chamber.
[0043] The magnetic field may be generated using means comprising a
permanent magnet.
[0044] Additionally or alternatively, the magnetic field may be
generated using means comprising an electromagnet.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] By way of example only, certain embodiments of the invention
will now be described with reference to the accompanying drawings,
in which:
[0046] FIG. 1 provides an overview of a fluid analysis system;
[0047] FIG. 2 shows in more detail the test unit of the system
shown in FIG. 1;
[0048] FIG. 3 illustrates the distribution of magnetic particles
within the measurement chamber of the test unit of FIG. 2 under
certain magnetic field conditions;
[0049] FIG. 4 illustrates schematically the underside of the plate
that is disposed at the bottom of the measurement chamber that is
shown in FIG. 2;
[0050] FIG. 5 illustrates schematically the main elements of the
measurement unit of the test unit that is shown in FIG. 2;
[0051] FIG. 6 illustrates the type of response that can be obtained
from the measurement unit of FIG. 5 during an assay;
[0052] FIG. 7 illustrates a plot, for various test substance
concentrations, of a metric that can be derived from an assay of
the type shown in FIG. 6;
[0053] FIG. 8 illustrates a modified form of the test unit shown in
FIG. 2;
[0054] FIG. 9 illustrates a modified form of fluid analysis system
containing several test stations;
[0055] FIG. 10 illustrates a modified form of the test unit of FIG.
8 in which the measurement chamber is formed as a removable
unit;
[0056] FIG. 11 illustrates schematically an alternative embodiment
of a test unit;
[0057] FIG. 12 illustrates schematically the main elements of an
alternative measurement unit which is used in the test unit that is
shown in FIG. 11
[0058] FIG. 13 illustrates an apparatus for cell lysis;
[0059] FIG. 14 illustrates a modified apparatus for cell lysis;
[0060] FIG. 15 shows the apparatus of FIG. 14 after lysis has
occurred;
[0061] FIG. 16 shows the apparatus of FIGS. 14 and 15 with an
external magnetic field applied in a first direction;
[0062] FIG. 17 shows the apparatus of FIG. 14 when the direction of
the magnetic field is subsequently reversed;
[0063] FIG. 18 shows experimental results for the amount of total
protein released after lysis of Jurkat cells in the apparatus of
FIGS. 12-15 when no particles are present, when particles of 2.8
.mu.m diameter particle are present and when particles of 1 .mu.m
are present;
[0064] FIG. 19 shows a plot of the dose response of a
magneto-immunoassay to prostatic specific antigen (PSA) released
from LNCAP cells by particle enhanced sonication using the
apparatus of FIGS. 13-16;
[0065] FIG. 20 shows a Scanning Electron Microscope (SEM) image of
Jurkat cells sonicated with no paramagnetic particles present,
using the apparatus shown in FIG. 11;
[0066] FIG. 21 shows a SEM photo of Jurkat cells sonicated in the
presence of 2.8 .mu.m particles; and
[0067] FIG. 22 shows a SEM photo of Jurkat cells sonicated in the
presence of 1 .mu.m particles.
DETAILED DESCRIPTION
Analyte Detection
[0068] FIG. 1 shows an overview of a fluid analysis system 10. A
pump 12 is provided with an inlet 14 for acquiring a sample of
liquid that is to be examined by a test unit 16. After a sample of
liquid has been acquired through inlet 14, the pump then operates
to repeatedly circulate the liquid sample through the test unit 16
via tubes 18 and 20. The pump 12 pumps the liquid to the test unit
16 through tube 18 and the liquid returns from the test unit 16 to
the pump 12 through tube 20. The test unit 16 is configured to
detect the presence of a particular antigen in the liquid that is
being pumped through the test unit 16. Henceforth, this antigen
shall be referred to as the target antigen.
[0069] The test unit 16 is shown in more detail in FIG. 2. The main
structure of the test unit 16 is provided by a block 22 of plastics
material. A spherical measurement chamber 24 is formed in the
centre of the block 22. Two bores 26 and 28 are formed in the block
22 to connect the measurement chamber 24 with the exterior of the
block. The mouth that bore 28 presents to the exterior of the block
22 is connected to tube 18 and the mouth that bore 26 presents to
the exterior of the block 22 is connected to tube 20. In this way,
the pump 12 can pass the test liquid through the measurement
chamber 24. The measurement chamber 24 is populated with particles
of paramagnetic material, which are denoted in FIG. 2 by the black
dots lying within the measurement chamber 24. The paramagnetic
particles are treated with a coating of a particular antibody to
which the target antigen will bind.
[0070] The measurement chamber 24 is preferably shaped so as to
reduce the speed of the test liquid as it flows through the
measurement chamber 24, to reduce disturbance to the paramagnetic
particles, which will usually be manipulated to form specific
configurations, as will be described below.
[0071] A square plate 31 is mounted in the bottom of the
measurement chamber 24. The plate 31 has upper and lower major
surfaces facing towards and away from the centre of the measurement
chamber, respectively. The upper major surface of the plate 31 is
covered with a coating 30 of the same antibody that has been
applied to the paramagnetic particles. The lower major surface of
the plate 31 is provided with an electrical coil which is connected
to a measurement unit 32 by means of electrical connection 34.
[0072] Two cavities 36 and 38 are provided in the upper and lower
surfaces of the block 22. A permanent magnet 40 is slidably mounted
within cavity 36. A shaft 42 connects magnet 40 to a drive unit 44.
The drive unit 44 is configured to act on the shaft 42 to vary the
position of magnet 40 within cavity 36. That is to say, the drive
unit can raise and lower the magnet 40 in the cavity 36 so as to
vary the distance of the magnet 40 from the measurement chamber 24.
Analogously, a permanent magnet 46 is slidably mounted in cavity 38
and can be moved by drive unit 48 by means of rod 50. The positions
of the magnets 40 and 46 within the cavities 36 and 38 are governed
by a control unit 52 that applies control signals to the drive
units 44 and 48 through connections 54 and 56. Surface 58
constitutes the north pole of magnet 40 and surface 60 constitutes
the south pole of magnet 46. The magnets 40 and 46 are closely
fitted to their corresponding cavities 36 and 38 so that the pole
faces 58 and 60 and the major surfaces of the plate 31 remain
parallel with one another as the magnets are moved.
[0073] The positions of the magnets 40 and 46 relative to the
centre of the measurement chamber 24 dictate the magnetic field
that is experienced by the paramagnetic particles that are located
within the measurement chamber. In order to promote the capture of
any target antigen that is present within the test liquid that is
flowing through the measurement chamber 24, the magnets 40 and 46
are positioned so as to generate within the measurement chamber 24
a magnetic field that causes the paramagnetic particles to
distribute themselves across the measurement chamber in the manner
of a sieve acting on the test liquid that is flowing through the
measurement chamber 24. In this configuration, the paramagnetic
particles form strands that extend across the flow within the
measurement chamber 24 and generally attempt to extend between the
pole faces 58 and 60, following the lines of magnetic force
extending between the pole faces 58 and 60. These strands are
illustrated schematically in FIG. 3 which shows the central portion
of the block 22, focussing on the measurement chamber 24.
[0074] In FIG. 3, the strands are denoted by irregular vertical
lines within the measurement chamber 24. Some strands, e.g. 62, may
form extending from the upper surface of the measurement chamber.
Other strands, e.g. 64, form extending from the base of the
measurement chamber, which is effectively provided by the plate 31.
Yet other strands, e.g. 66, may extend entirely between the base
and upper surface of the measurement chamber 24. In this condition,
the paramagnetic particles are distributed across the flow of the
test liquid through the measurement chamber 24 which facilitates
the paramagnetic particles' capture, via their antibody coating, of
target antigen in the test liquid. Accordingly, the state of the
magnetic field required to place the paramagnetic particles in this
condition shall be referred to as the "capture state". The precise
positions of the magnets 40 and 46 that are required to transform
the magnetic field in the measurement chamber 24 into the capture
state will depend upon various parameters of the precise design of
the equipment and can be determined through experimentation.
Examples of such parameters include the material and size of the
paramagnetic particles, the material and size of the magnets 40 and
46 and the diameter of the measurement chamber 24.
[0075] The magnetic field within the measurement chamber 24 can
also be adjusted to a so-called "collection state" in which the
paramagnetic particles are drawn down to collect over the upper
major surface of the plate 31. The collection state of the magnetic
field can be achieved by moving the magnets 40 and 46 to their
maximum and minimum distances, respectively, from the centre of the
measurement chamber 24. When the magnetic field in the measurement
chamber 24 is in the collection state, the paramagnetic particles
are urged onto the antibody coating 30 on the plate 31. Some of the
paramagnetic particles in contact with the coating 30 will have
target antigen bound onto them. These particles can then become
linked to the coating 30 by the target antigen that they carry and
therefore become immobilised on the plate 31.
[0076] In order to examine the test liquid for the presence of the
target antigen, the magnets 40 and 46 are moved to cycle the
magnetic field in the measurement chamber 24 between the capture
and collection states. When desired, the quantity of paramagnetic
particles that have become attached to the antibody coating 30 on
the plate 31 can be assessed electronically, as will now be
explained.
Sensing Arrangement
[0077] FIG. 4 shows the lower major surface of the plate 31. An
electrical coil 68 is provided on the lower major surface of the
plate 31. A pair of supply conductors 70 extend from the coil and
provide the connection 34 to the measurement unit 32. The coil 68
forms part of a voltage controlled oscillator (VCO), the remainder
of which is housed within the measurement unit 32.
[0078] In FIG. 5, the circuitry that supplements coil 68 to form
the VCO is indicated 72. From another perspective, coil 68 is
simply an inductor that forms part of a VCO design and which has
been located remote from the other components of the design. It
should be noted, however, that the coil 68 and the measurement unit
are, preferably, physically close to the measurement chamber 24.
VCO designs that are suitable for adaptation in this manner are
known to the skilled person. The VCO incorporates a variable
capacitor 90, which is, for example, a variable capacitance diode.
Together, the coil 68 and the capacitor 90 determine the frequency
of the VCO's output signal.
[0079] The measurement unit 32 also includes a crystal oscillator
74. The crystal oscillator 74 produces a 70 MHz output signal on
line 76. The voltage controlled oscillator produces a signal on
line 78 whose frequency the measurement unit 32 endeavours to
maintain locked to a frequency of 70 KHz away from the output
signal of the crystal oscillator 74. To achieve this end, output
signals of the VCO and the crystal oscillator 74 are mixed together
in a mixer 80 and resulting signal is provided on line 82 as one
input to a phase detector 84. The other input to the phase detector
84 is provided over line 79 and is the output signal of a variable
oscillator 81. The variable oscillator 81 and the phase detector 84
are integrated into a single package 85, which may also contain the
other elements of the system of FIG. 5, with the exception of the
crystal oscillator 74. The variable oscillator 81 is tuned so that
the output signal that it provides on line 79 has a frequency of 70
KHz. The phase detector 84 produces a DC voltage on line 86 that is
proportional to the phase difference between its two input signals.
This DC voltage is sensed on line 88 (and is referred to henceforth
as a detection signal) and is also applied via line 92 to the
variable capacitor 90 within the VCO. This voltage controls the
capacitance of the variable capacitor 90, thereby tuning the
frequency of the VCO's output. It will be apparent to the skilled
person that the elements shown in FIG. 5 are formed into a phase
locked loop (PLL), or more accurately a frequency locked loop (FLL)
for the purpose of locking the frequency of the output signal of
the mixer 80 to 70 KHz, i.e. to the frequency of the output of the
variable oscillator 81. This means that the PLL acts to maintain
the output signal of the VCO at 69.93 MHz.
[0080] As mentioned earlier, coil 68 forms part of the voltage
controlled oscillator that is the object of the PLL. The frequency
of the output of the VCO that is supplied over line 78 is governed
in part by the inductance of coil 68. In turn, the inductance of
coil 68 is governed by the distribution of the paramagnetic
particles within the measurement chamber 24 and in particular by
the immobilisation of target antigen carrying paramagnetic
particles on the coating 30. Accordingly, the voltage of the output
of the phase detector 84 that is sensed on line 88 contains
information about the behaviour of the paramagnetic particles and,
in turn, about test antigen in the measurement chamber 24. In order
to make deductions about test antigen in the measurement chamber
24, the voltage of the output of the phase detector 84 is recorded
over time as the magnetic field within the measurement chamber 24
is varied. A typical assay will now be described.
Results
[0081] FIG. 6 shows a plot of a sandwich assay performed using
apparatus according to the present embodiment of the invention.
FIG. 6 plots the detection signal versus time. At the beginning of
the measurement process, the measurement chamber 24 contains just a
buffer solution and the detection signal value is A. Then, the
magnets 40 and 46 are positioned so as to bring the magnetic field
in the measurement chamber 24 into the collection state, which
causes the detection signal value to change to B. Next, a quantity
of liquid containing antibody coated paramagnetic particles mixed
with target antigen is added to the buffer solution in the
measurement chamber 24. This causes a marked drop in the detection
signal value to C. At this point, the paramagnetic particles are
clumped on the upper surface of the plate 31. The magnets 40 and 46
are then repositioned to change the magnetic field within the
measurement chamber 24 to the capture state. This reduces the
detection signal value to D. After 30 seconds, the magnets 40 and
46 are repositioned to change the magnetic field to the collection
state, whereupon the detection signal value changes to E. After 30
seconds, the magnets 40 and 46 are repositioned to change the
magnetic field back to the capture state such that the detection
signal value changes to F. After 30 seconds, the magnets 40 and 46
are repositioned to change the magnetic field to the collection
state, whereupon the detection signal value changes to G. After a
further 30 seconds, the magnets 40 and 46 are drawn back as far as
possible from the measurement chamber 24 and the detection signal
value changes to H and the paramagnetic particles are allowed to
relax on the upper surface of the plate 31 for 30 seconds. Then,
magnet 40 is driven to its point of closest approach to the chamber
24 whilst magnetic 46 is kept remote from the chamber 24. This
causes the detection signal value to change to I. During this time,
the proximity of magnet 40 causes any paramagnetic particles that
are not bound to the coating on plate 31 to move away from the
plate. Then, after 30 seconds, magnet 40 is retracted as far as
possible from the sample chamber 24 such that the detection signal
value changes to J.
[0082] In an alternative method, the paramagnetic particles are
added to the buffer solution in the measurement chamber 24 before
the magnets 40 and 46 are positioned so as to bring the magnetic
field in the measurement chamber 24. In this method, the magnets 40
and 46 are positioned so as to cause the magnetic field to be in
the capture state, causing the detection signal value to change to
D immediately.
[0083] Various metrics can be derived from the time varying
detection signal value shown in FIG. 6. For example, the following
metrics could be used: [0084] J-H [0085] J-A [0086] (J-H)/A [0087]
(J-H)/B [0088] (J-H)/(B-C)
[0089] The time varying detection signal value is typically
normalised and smoothed prior to calculating the metrics.
[0090] FIG. 7 plots various values of the metric J-H for different
known concentrations of target antigen. A curve 96, which has been
fitted to the results, is shown. Such a curve can thereafter be
employed to estimate the target antigen concentration in live test
situations.
Further Analyte Detection Arrangements
[0091] Another embodiment of the invention is shown in FIG. 8. In
this embodiment, the permanent magnets 40 and 46 have been replaced
with electromagnets 98 and 100, the energisation of which is
controlled by the control unit 52 in order to vary the magnetic
field within the measurement chamber 24, e.g. to change the
magnetic field from the collection state to the capture state.
[0092] FIG. 9 shows a further embodiment of the invention in which
several sample chambers are used. Elements 104 to 110 each denote a
test unit similar to test unit 16 of FIG. 1. However, in the
embodiment of FIG. 9, the control and measurement functions
associated with the plurality of sampling locations are collected
into a single unit 112. This means, for example, that a single
crystal oscillator can be used to provide a reference frequency for
the VCOs associated with each of the sample chambers. Pump 102
takes in a volume of test liquid and circulates it through elements
104 to 110. It will be appreciated that elements 104 and 106 are
placed in parallel whilst 108 and 110 are placed in series. The
elements 104 to 110 need not all test for the same antigen. It is
possible to utilise a temperature control system to keep multiple
measurement chambers at the same temperature, should this be
necessary given the types of test performed in those chambers (e.g.
the temperature could be held the same within a group of chambers
testing for the same antigen using the same antibody). A
temperature control system can also be used to stabilize the
temperature of those electronic components whose electrical
properties or performance are temperature dependent (for example,
components such as coil 68).
[0093] In the foregoing embodiments, a charge of the fluid under
test is recirculated through the measurement chamber (or, as the
case may be, chambers), and this is useful when attempting to
detect a very low concentration of the target antigen. In other
embodiments, however, it is possible to arrange that a given charge
of test fluid is passed through a given measurement chamber just
once. Additionally or alternatively, it is possible to hold a
charge of test fluid with a given measurement chamber for a
protracted period before perhaps processing another charge.
[0094] In the foregoing embodiments, antibody coated particles with
attached antigen adhere to an antibody coating on a plate. Over
time, it is possible that all of the magnetic particles will become
adhered, or that no more particles can become adhered, resulting in
the exhaustion of the measurement chamber. The measurement system
can be configured to detect this condition (by monitoring the
behaviour of the VCO that incorporates the coil that is associated
with plate in question) and issue an appropriate indication to a
user, who can take action to replenish the system. An embodiment in
which replenishment is facilitated shall now be described.
[0095] FIG. 10 illustrates a variant 116 of the test unit of FIG. 8
in which the measurement chamber is formed as a removable unit 114.
In FIG. 10, elements carried over from FIG. 8 retain the same
reference numeral and shall not be described in detail again. Of
course, the concept of rendering the measurement chamber
replaceable is not limited to the particular type of test unit
shown in FIG. 8 and could be applied to any type of test unit,
within reason.
[0096] In test unit 116, there is a removable cell 114 in block 22.
This cell contains the measurement chamber 24, and the paramagnetic
particles and plate 31 within it, and also parts of connection 34,
bore 26 and bore 28. The cell 114 and the block 22 are provided
with appropriate electrical connectors at the interface between the
cell and the block in order to complete connection 34 when the cell
is installed in the block Likewise, fluid-tight connectors are
provided at that interface to complete bores 26 and 28 when the
cell 112 is installed in the block 22. Thus, an incumbent cell 114
can be replaced at will, e.g. with a fresh cell of the same type
(when it is desired to refresh an exhausted measurement chamber) or
with a cell of a different type in which the paramagnetic particles
and the plate 31 are coated differently (in order to switch to
testing for a different antigen). During fabrication of such a
cell, the paramagnetic particles and the plate are given coatings
appropriate for the antigen that the cell is to detect. The
paramagnetic particles can be dried into the measurement chamber of
the cell with suitable stabilising agents to allow rapid dispersal
of individual particles when they are rehydrated by test fluid
entering the chamber. Examples of suitable stabilising agents
include sucrose, trehalose, and other poly-ionic compounds.
[0097] FIG. 11 shows an alternative embodiment of a test unit of
the system shown in FIG. 1. In this embodiment, the measurement
chamber 120 is generally cylindrical, which helps to cause a
controlled flow of fluid through the measurement chamber 120 and to
reduce turbulence. The measurement chamber 120 is formed in the
centre of a block 122 of a plastics material. Two bores 124, 126
are formed in the block 122 to connect the interior of the
measurement chamber 120 with the exterior of the block 122, and the
bores 124, 126 are tapered to assist in causing a controlled flow
of fluid through the measurement chamber 126 and to reduce
turbulence in the fluid. The mouth that bore 124 presents to the
exterior of the block 122 is connected to tube 18 and the mouth
that bore 126 presents to the exterior of the block is connected to
tube 20. In this way, pump 12 can pass the test liquid through the
measurement chamber 120. The measurement chamber 120 is populated
with particles of paramagnetic material, which are denoted in FIG.
11 by the small circles lying within the measurement chamber 120.
The paramagnetic particles are treated with a coating of a
particular antibody to which the target antigen will bind.
[0098] A plate 128 is mounted in the bottom of the measurement
chamber 120. The plate 128 has upper and lower major surfaces
facing towards and away from the centre of the measurement chamber
120 respectively. The upper major surface of the plate 128 is
covered with a coating 130 of the same antibody that has been
applied to the paramagnetic particles. Disposed beneath the plate
128, externally of the measurement chamber 120, is an electrical
coil 131 which is connected to a measurement unit 132 by means of
an electrical connection 134.
[0099] Two cavities 136, 138 are provided in the upper and lower
surfaces of the block 122. A permanent magnet 140 is slidably
mounted within cavity 136. A shaft 138 connects permanent magnet
140 to a servo 144. The servo 144 is configured to act on the shaft
142 to vary the position of the magnet 140 within the cavity 136.
That is to say, the servo 144 can raise and lower the magnet 140 in
the cavity 136 so as to vary the distance of the magnet 140 from
the measurement chamber 120. Analogously, a permanent magnet 146 is
slidably mounted in cavity 138 and can be moved by a servo 148 by
means of a shaft 150. The positions of the magnets 140, 146 within
the cavities 136, 138 are governed by a control unit 152 that
applies control signals to the servos 144, 148 through connections
154, 156. Surface 158 constitutes the north pole of the magnet 140
and surface 160 constitutes the south pole of the magnet 146. The
magnets 140, 146 are closely fitted to their corresponding cavities
136, 138 so that the pole faces 158 and 160 and the major surfaces
of the plate 128 remain parallel with one another as the magnets
140, 146 are moved.
[0100] As is the case for the test unit shown in FIG. 1, in the
embodiment shown in FIG. 11 the positions of the magnets 140, 146
relative to the centre of the measurement chamber 120 dictate the
magnetic field that is experienced by the paramagnetic particles
that are located within the measurement chamber 120. The magnets
140, 146 may be positioned so as to cause the paramagnetic
particles to adopt a sieve-like configuration by forming into
strands that extend across the flow within the measurement chamber
120.
[0101] The electrical coil 131 in this embodiment is positioned
outside of the measurement chamber 120, but performs the same role
as the electrical coil of the embodiment of FIG. 2 in detecting the
number of paramagnetic particles that are bound to antibody coating
130 of the plate 128.
[0102] The system of FIG. 11 uses a detection unit, which is shown
schematically in FIG. 12. The detection unit 160 is similar to the
detection unit shown in FIG. 5, and thus like elements have the
same reference numerals in FIG. 12. However, in the detection unit
160, the variable oscillator 81 is not present. Instead, the phase
detector 162 and a phase shift unit 164 form a quadrature phase
detector. In this arrangement the signal 82 is split into two
components. One passes directly into one port of the phase detector
162 and the second is phase shifted by 90 degrees (at 70 KHz) and
tuned by an appropriate capacitor-inductance-resistor bandpass
filter in phase shift unit 164, before passing into the second port
of the phase detector 162.
[0103] In this arrangement, the phase shift of the phase shift unit
164 is frequency dependent. Therefore, if the signal deviates from
70 KHz then the phase shift will deviate from the basic value (i.e.
the value of the phase shift at 70 KHz). For example, frequencies
greater than 70 KHz could result in a phase shift greater than 90
degrees and frequencies less than 70 KHz could result in a phase
shift of less than 90 degrees. The output signal 86 from the phase
detector 162 is proportional to the phase difference between the
two signal components and hence the level of deviation of the
signal 82 from 70 KHz. The phase detector output signal 86 adjusts
the variable capacitor to bring the frequency of the VCO back to a
frequency of 70 KHz away from the output signal of the crystal
oscillator 74.
[0104] It will be noted, from FIG. 6 for example, that when
measurements are taken as the paramagnetic particles are drawn to
the plate 31/128 by the action of the magnetic field generated by
the magnets 40/46 and 140/146 that there is a step change in output
voltage or frequency. This effect is caused by the proximity of the
magnet 46/146, which is typically of Neodymium, to the electrical
coil 68/131, which causes the inductance of the coil 68/131 to drop
as the coil 68/131 approaches magnetic saturation. This has the
effect of reducing the inductance of the coil 68/131, which tries
to skew the frequency of signal 78.
[0105] The paramagnetic particles, which are typically of Magnetite
or Ferrite, have the effect of increasing the permeability of the
electrical coil 68/131, when in close proximity to the coil. This
effectively increases the inductance of the electrical coil 68/131
and tends to try to lower the frequency of signal 78. Ferrite
ceramics have the same effect on signal 78.
[0106] Thus, if the magnets 40/140 and 46/146 are given a tip made
from Ferrite, or are coated with Ferrite, then the shift in the
resonant frequency of the PLL/FLL circuit can be balanced out to a
large extent. This results in good sensitivity to paramagnetic
particles regardless of whether they are close to the electrical
coil 68/131 or not.
[0107] Although the pole faces 58/160 and 60/160 are shown in FIGS.
2 and 11 as being flat, they may have a rounded profile to give an
evenly distributed magnetic field at the flat surface of the plate
31/128, thus allowing an even layer of paramagnetic particles to
form.
[0108] Although the example given above describes the use of the
apparatus of the invention in performing sandwich assays, it will
be appreciated by those skilled in the art that it can be used in
performing other types of assays. For example, the apparatus could
be used to perform a "displacement assay", in which antigen coated
paramagnetic particles are initially bound to the on the upper
surface of the plate 31 and are displaced, on the introduction of a
sample containing the target antigen into the measurement chamber
24, from the plate 31 due to competitive interaction between the
target antigen and the antigen of the paramagnetic particles,
resulting in a change in the detection signal.
[0109] Alternatively, the apparatus of the invention can be used to
perform a "competitive assay", in which a binding agent is attached
to the upper surface of the plate 31. A first complementary binding
agent, the target antigen, is introduced into the measurement
chamber 24 with the sample, whilst a second complementary binding
agent is attached to paramagnetic particles, and the first and
second complementary binding agents compete to bind to the binding
agent of the plate 31. The greater the concentration of the target
antigen, the fewer paramagnetic particles will bind to the binding
agent of the plate 31, and the detection signal will change
accordingly.
Lysing Arrangement
[0110] Referring now to FIG. 13, an apparatus for cell lysis is
shown generally at 200, and comprises a lysing chamber 202 for
holding a liquid 204 containing cells 206 to be lysed. The lysing
chamber 202 also contains a plurality of particles 208 to enhance
lysing. One or more sonicator probes 210 are provided to introduce
energy in the form of sound waves at ultrasound frequencies into
the chamber 202. Alternatively, one or more ultrasonic transducers
may be integrated into the chamber 202 or positioned adjacent the
chamber 122 to introduce the ultrasound energy into the chamber
202. Using particles of a suitable size and at a suitable density,
ultrasound energy introduced into the chamber 202 by the sonicator
probe 210 enables the particles 208 to acquire sufficient kinetic
energy to lyse cells 206 mixed with the particles 208 in the
chamber 202. The sonicator probe 210 may be activated in a
continuous or pulsed fashion for a sufficient time to cause lysing
of the cells 206 to occur.
[0111] Varying degrees of cell lysis can be achieved by adjusting
one or more of the following parameters: the amount of ultrasound
energy imparted, the type of particle 208 used, the concentration
of the particles 208 or the size of the particles 208 used. The
particles 208 should be of a size suitable to cause effective
lysing. Preferably the particles 208 that are used to enhance the
cell lysis are in the range of 0.1 .mu.m-100 .mu.m, or more
preferably between 1.mu.m-20 .mu.m. The particles 208 should be
used in a concentration range suitable to cause effective lysing of
the amount of cells 206 in the chamber 202.
[0112] The particles 208 should be appropriately constructed and/or
formed from material of appropriate density to cause cell lysis.
For example, the particles 208 may be made from metal or a plastics
material, or a combination of metal and a plastics material, or may
be of any other suitable material.
[0113] By controlling the degree of cell lysis, various cell
components, for example proteins and organelles, can be released
from the cells 206. Alternatively, the cells 206 can be greatly
disrupted to release enhanced levels of intracellular protein above
and beyond that released using sonication alone.
[0114] Any type of cell, including mammalian cells, non-mammalian
cells, plant cells, bacteria, yeasts and spores or a mixture
thereof, may be disrupted using the apparatus and method described
above with reference to FIG. 13.
[0115] The apparatus shown in FIG. 13 may be used to identify,
quantify or separate a component of interest from lysed cells. In
this application, the particles 208 are coated with a binding agent
to which intracellular components may bind, so as to capture such
intracellular components. The binding agent may be, for example, an
antibody, a lectin, DNA, RNA, a receptor protein or any other
binding agent or moiety. The intracellular component of interest
may be, for example, a protein or a cell-organelle that binds
specifically to the binding agent. The intracellular component that
binds to the binding agent may be identified, quantified or
separated by using a label or reporter molecule which is associated
with the intracellular component/binding agent complex formed
during binding. For example, the label may be an enzyme which
reacts with a suitable substrate to produce a coloured or
fluorescent product. This reaction product may be used to identify,
quantify or separate the intracellular component, as will be
apparent to those skilled in the art.
[0116] FIG. 14 shows a modified version of the cell lysis apparatus
of FIG. 12 in which means for magnetically detecting a target
component of a cell, such as a protein or cell-organelle, is
provided. Elements common to this embodiment and the embodiment of
FIG. 12 are denoted by like reference numerals. The lysing chamber
202 of this embodiment may form the measurement chamber of a fluid
analysis system as described above with reference to FIGS. 1 to
11.
[0117] In this modified apparatus, the chamber 202 contains liquid
204 comprising a sample of cells 206 to be lysed, and a plurality
of magnetic particles 220. The magnetic particles may be, for
example, ferromagnetic, diamagnetic, paramagnetic or
super-paramagnetic. The magnetic particles 220 are coated with a
binding agent to which a target component, such as a protein or
cell organelle, may bind. A sensor surface 222 is coated with a
similar binding agent 224 to that used to coat the magnetic
particles 220, such that the target component may bind to the
binding agent on the sensor surface 222. A magnetic sensing means
226 is provided beneath the sensor surface. The magnetic sensing
means 226 may be integrated into the lysis chamber 202, or may be
positioned adjacent the lysis chamber 202. The magnetic sensing
means 226 may be a magnetic coil or may be a resonant coil
magnetometer, a magneto-resistive sensor, a micro-machined
cantilever device or a superconducting quantum interference device,
for example.
[0118] In use of the apparatus of FIG. 14, liquid containing the
cells 206 to be lysed is placed in the chamber 202 with the
magnetic particles 220 and a sonicator probe 210 is activated
either continuously or in a pulsed manner for a time sufficient for
lysis of the cells 206 to occur.
[0119] FIG. 15 shows the apparatus of FIG. 14 after lysis has
occurred. Elements common to this Figure and FIGS. 13 and 14 are
denoted by like reference numerals.
[0120] Lysing of the cells 206 by continuous or pulsed activation
of the sonicator probe 210 produces lysed cells 230 and causes the
target components such as protein(s) and/or cell organelle(s) to
bind to the binding agent that is used to coat the magnetic
particles 220, to form a bound complex comprising the component
(e.g. protein(s) or cell organelle(s)) of interest and magnetic
particles, hereinafter referred to as "bound particles" 232.
[0121] FIG. 16 shows the apparatus of FIGS. 14 and 15 when an
external magnetic force is used to manipulate the magnetic
particles 220 after lysis has taken place. Again, common elements
are denoted by like reference numerals. The external magnetic force
may be provided by one or more permanent magnets, or by adjusting
one or more electromagnets, for example. The magnets may be mounted
externally of the chamber 202 or may be integrated into the chamber
202.
[0122] The externally applied magnetic force acts in the direction
of the arrow 240 and is used to pull the bound particles 232 and
the magnetic particles 220 towards the sensor surface 222, where
the bound particles 232 bind to the binding agent 224 on the sensor
surface 222. The bound particles 232 become cross-linked to the
sensor surface 222, causing them to be immobilised on the sensor
surface 222.
[0123] FIG. 17 shows the apparatus of FIG. 16 with the external
magnetic force applied in the direction of the arrow 250 (i.e. the
direction of the external magnetic field is reversed), and used to
pull unbound magnetic particles 220 away from the sensor surface
222, thus leaving just the bound particles 232 attached to the
binding agent 224 on the sensor surface 222, which allows the
sensing means 226 to quantify the amount of bound particles 232
present. The amount of bound particles 232 detected by the sensing
means 226 can then be used to determine the amount of the target
components such as protein(s) and/or cell organelle(s) present.
[0124] Using the method and apparatus described above with
reference to FIGS. 14 to 17, lysis of cells and identification,
quantification or separation of intracellular components such as
proteins and cell-organelles to be performed in the same
vessel.
[0125] Experiments carried out in relation to lysis of cells using
particles will now be described.
Further Results
[0126] FIG. 18 plots the amount of total protein released after
sonication of Jurkat cells in the lysis chamber when no particles
are present, when particles of 2.8 .mu.m diameter are present and
when particles of 1 .mu.m diameter are present.
[0127] FIG. 19 shows a plot of the dose response of a
magneto-immunoassay to prostatic specific antigen (PSA) released
from LNCAP cells by particle enhanced sonication.
[0128] FIG. 20 shows a Scanning Electron Microscope (SEM) image of
Jurkat cells sonicated with no paramagnetic particles present.
[0129] FIG. 21 shows a SEM photo of Jurkat cells sonicated in the
presence of 2.8 .mu.m particles.
[0130] FIG. 22 shows a SEM photo of Jurkat cells sonicated in the
presence of 1 .mu.m particles.
[0131] In a specific example using the apparatus shown in FIG. 13,
Jurkat cells were lysed using different sized particles in
conjunction with ultrasound.
[0132] Jurkat cells were cultured in 75 mm.sup.2 tissue culture
flasks in sterile penicillin/streptomycin supplemented RPMI-1640
containing 10% newborn calf serum and L-glutamine and incubated in
a humidified atmosphere at 37.5.degree. C. with CO.sub.2. The cells
were routinely passaged 1:4 (1 part cells: 4 parts growth medium)
every 2 to 3 days. At 3 days post-passage, the cells were
centrifuged for 5 minutes at 21.degree. C. at 1500 rpm. The cells
were then re-suspended in 1 ml penicillin/streptomycin-supplemented
RMPI (50 ml of FBS +5 ml L-Glutamine+5 ml Penicillin &
Streptomycin to 500 ml of RPMI 1640). The cells were counted by the
Trypan Blue exclusion method, in which a 20 .mu.l sample of the
cell suspension was mixed with 20 .mu.l Trypan Blue stain (0.2% w/v
Trypan blue dissolved in PBS and stored at 4.degree. C.). The
suspension was gently vortexed and 10 .mu.l of the stained cells
were counted using a haemocytometer.
[0133] To demonstrate the effect of particle size on the efficiency
of cell lysis Jurkat cells were centrifuged at 200 g for 10 minutes
and the supernatant was discarded. The pellets obtained were
re-suspended in 1 ml phosphate buffer saline in the chamber, and 5
.mu.l of 2.8 .mu.m or 1 .mu.m paramagnetic particles
(Dynabeads)were added. The mixture was then treated with a
sonicator probe for 1 minute. To prevent excessive heat generated
by the probe, the sample was immersed in an ice bath and the
ultrasound was applied in multiple short bursts. The effect of the
sonication with and without particles was quantified by measuring
total protein released into the supernatant and the physical effect
on the cells was studied using SEM (Scanning Electron
Microscopy).
[0134] The addition of paramagnetic particles to the cells prior to
sonication enhanced the amount of protein released from the cells
in a given time. Moreover, paramagnetic particles of different
sizes enhanced the protein released from the cells to different
extents. Without paramagnetic particles the sonication process
released 4 .mu.g protein/10.sup.6 cells, with the addition of 2.8
.mu.m particles (Dynabeads), twice as much protein was released
from the cells (8 .mu.g/10.sup.6) and 1.0 .mu.m particles
(Dynabeads) released approximately three and half times as much
protein (14 .mu.g/10.sup.6), as is shown in FIG. 16. A significant
difference was observed in the concentration of protein released by
the sonication probe alone and combined with paramagnetic particles
as shown by the total protein measurement (P<0.001).
[0135] Scanning Electron Microscopy (SEM) was used to evaluate the
effect of sonication on cell morphology with and without the
paramagnetic particles (see FIGS. 18, 19 and 20). Surprisingly, the
different size of particles had a very different effect on the
cells. 2.8 .mu.m particles appeared to cause coagulation of the
intracellular proteins whereas 1.0 .mu.m particles induced the
formation of membranous like structures.
[0136] In a second example, the apparatus shown in FIGS. 14-17 was
used to demonstrate the magnetic detection of intracellular
prostatic specific antigen. In this case LNCAP cells were lysed in
the presence of 1 .mu.m paramagnetic particles which were
previously coated with anti-PSA. The floor of the lysis chamber
incorporated the magnetic sensor which had a second anti-PSA
antibody immobilised on its surface. Following sonication in the
presence of the paramagnetic particles, an external magnetic field
was applied to all the paramagnetic particles down to the sensor
surface. Particles which had PSA bound to the surface by the
antibody interaction were cross-linked to the sensor surface by the
binding of the second antibody immobilised on the sensor surface to
the captured PSA molecule on the paramagnetic particle. The
captured PSA acted as a biological bridge holding the particle on
the surface through the immunological linkage. A second external
magnetic field was applied to remove unbound paramagnetic particles
prior to measurement. A resonant coil magnetometer, lying
underneath the sensor surface, was used to detect the presence of
paramagnetic particles attached to the sensor surface. FIG. 18
shows the dose response of a magneto-immunoassay for PSA released
from LNCAP cells by particle enhance lysis.
[0137] These examples demonstrate that paramagnetic particles used
in magneto-biosensors can be used to enhance the release of
intracellular proteins from the cells, as part of an integrated
measuring system for the rapid measurement of intracellular
proteins.
* * * * *