Note: Descriptions are shown in the official language in which they were submitted.
CA 02370745 2001-10-17
H264WP3
DESCRIPTION
Method of representing biologically activated inductance-altering particles
and device for carr~ring out the method
The invention concerns a method of representing biologically
activated inductance-altering - in particular ferromagnetic or
superparamagnetic - particles. The invention further concerns a device
for detecting and counting suspended biological microparticles in liquid
samples, in particular for carrying out the specified method.
Hitherto the procedure involved in counting bacteria, blood cells or
cell constituents in aqueous solutions has been effected by means of
through-flow cytometers or Coulter counters. Here the corresponding
particles are colored and identified on the basis of optical signals or
1o counted by capacitive measurement procedures.
In consideration of those factors the inventor set himself the aim of
simplifying such measurement operations.
That object is attained by the teaching of the independent claim;
the appendant claims set forth advantageous developments. In addition
i5 the scope of the invention embraces all combinations comprising at least
two of the features disclosed in the description, the drawing and/or the
claims.
In accordance with the invention monovalent primary antibodies are
mixed with inductance-altering, in particular ferromagnetic or
20 superparamagnetic, particles in multiple excess, which are coated with
secondary antibodies; aggregated particles which comprise a monovalent
primary antibody and antibody-coated ferromagnetic partial particles are
then separated by means of partial sedimentation in a centrifuge. Instead
of primary antibodies it is also possible to use viruses or gene samples,
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CA 02370745 2001-10-17
whose sheathing proteins or spacer molecules are targeted by the
secondary antibodies.
In accordance with a further feature of the invention the detecting
or counting biological particles are immunologically, phagologically or
molecular-biologically joined to aggregated particles which, when
subsequently flowing through a metal coil - in particular the gap of a C-
shaped metal coil with a ferromagnetic core - trigger measurable and
countable alterations in inductance.
It has also proven to be advantageous for inductance-altering
1o particles, before flowing through the metal coil, to be retained by means
of an electromagnet in a plastic capillary and there to be joined to the
biological particles flowing into the capillary, while the sample in which
same were contained is taken out of the capillary. In addition, countable
alterations in the natural oscillation frequency are to be produced by the
~5 metal coil as part of an electronic resonant circuit.
In order to obviate the apparatus expenditure in regard to optical
measurement and to achieve a higher degree of specificity in comparison
with capacitive measurement, a different measurement principle is
therefore used for detection of the individual particle: measurement of the
20 alteration in inductance of a microcoil of metal. As however biological
particles have a permeability constant a of approximately 1, they have to
be previously marked by means of inductance-altering substances for
detection and counting procedures by means of a coil. That marking is
effected by immunological, phagological or molecular-biological coupling of
25 ferromagnetic or superparamagnetic particles which are monovalently
joined either to antibodies, virus docking molecules or gene samples at
spacer molecules.
The scope of the invention includes a device of the kind set forth
above, having a delivery line for a sample to be measured, which is
3o surrounded as a measuring line by a metal coil as a measuring coil which
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in turn is connected to a device for exciting oscillation and measuring
resonance events.
In a particular embodiment that metal coil is laid around a core
which is bent approximately into a C-shape and whose ends delimit a gap;
the measuring line is laid through that gap.
In accordance with a further feature of the invention the delivery
line is connected to a device with capillaries - in particular with Teflon
capillaries - ; the latter are associated with an electromagnet and can be
arranged in a space surrounded by a pole piece.
to Advantageously provided between the electromagnets and a valve
of the delivery line is a branch line for excess sample. In addition at least
one resistor and a capacitor can be arranged in front of each device for
exciting the oscillations and measuring resonance events, towards the
metal coil.
The measuring coil, a piezoelectric pump arranged upstream thereof
and a downstream-arranged resistor and capacitor respectively are to be
parts of a microsystem-technical unit in accordance with the invention.
Therefore coupling of the ferromagnetic markers occurs in the
device which at the same time permits enrichment of the particles to be
2o counted: the markers are retained in the Teflon capillary by means of an
electromagnet as a sorption layer, until the entire sample has been
pumped into the capillary and at the same time the excess sample has run
out of the capillary. Thereupon the magnet is switched off so that the
markers freely diffuse and can saturate the surface of the biological
particles. The capillary content is then pumped by the above-mentioned
piezoelectric pump through the metal coil, in particular through the gap of
the metal coil, which is of a C-shaped configuration, with a ferromagnetic
core. The metal coil is etched in the form of a spiral onto a circuit board
and is connected with capacitor and resistor as a resonant circuit. The
resonant circuit is excited by a frequency corresponding to that natural
resonant frequency which is generated when an averagely marked
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biological microparticle is in the gap or the coil. As a result a resonance
oscillation always occurs in the resonant circuit when a corresponding
microparticle passes through the coil.
An example of the use of that method is the detection of coli
bacteria in water samples. For that purpose monovalent primary E.-coli
specific antibodies are conjugated with secondary antibodies coupled to
magnetic beads. The suspension of those conjugates is pumped into the
Teflon capillary and fixed there by means of an electromagnet. When the
water sample to be investigated flows through the capillary, coli bacteria
1o are retained to the conjugates by way of the primary antibodies. After the
magnet is switched off the suspension of magnetically marked coli bacteria
can be pumped through the measuring coil or the gap of the metal coil.
The number of resonance events in the connected resonant circuit
corresponds to the number of coli bacteria in the original water sample.
By virtue of the use of that arrangement and the corresponding
conjugates, it is possible to automatically count bacteria without the
expensive use of through-flow cytometry. Furthermore it is possible with
that measuring method to achieve miniaturization of the detection
arrangement.
The described procedure is used for detecting and counting particles
such as bacteria, cells or cell constituents in aqueous solutions. That
procedure permits miniaturization of the automatic particle counting
method. For that purpose the particles are marked prior to the
measurement procedure by the reaction with monovalent antibody-coated
or virus-coated ferromagnetic particles. Inductive measurement is based
on passage of the ferromagnetic particles aggregated with the biological
particles through the microcoil, designed in the above-described manner,
of an electronic resonant circuit. The resonance events which occur upon
such particle passage are counted.
The device according to the invention can be used in medicine,
microbiology and hygiene, for example for counting out blood cells; it is
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possible to count out ecologically relevant micro-organisms or detect
pathogenic germs.
Further advantages, features and details of the invention will be
apparent from the description hereinafter of a preferred embodiment and
with reference to the drawing in which:
Figures 1 and 3 each show a diagrammatic view relating to a
method according to the invention, and
Figure 2 is a diagrammatic perspective view of a detail from Figures
1 and 3.
1o Prior to a method of detecting coli bacteria in a water sample Z
supplied through a line 10 monovalent primary E.-coli-specific antibodies
are conjugated to secondary antibodies coupled to magnetic beads. The
line for the monovalent magnetic particles F is denoted by reference 12.
Both lines 10, 12 include hose pumps 14 and downstream of same are
combined to form a common delivery line 16.
The reagent with ferromagnetic, biologically activated particles is
pumped by way of the lines 12 and 16 into a Teflon capillary 20 and is
fixed there by means of an electromagnet 22 whose magnetic coil is
identified by reference numeral 24 and with which there is associated the
2o Teflon capillary 20 which is wound on in a z-shape, in a concentric pole
piece 26. The latter with a pole pin 28 surrounded thereby at a radial
spacing defines an annular space 30 for the Teflon capillary.
When the water sample Z to be investigated flows through the
capillary 20 coli bacteria as biological particles to be counted are retained
2s by way of the primary antibodies to the ferromagnetic conjugates. After
the electromagnet 22 is switched off the suspension of magnetically
marked coli bacteria can be transported by virtue of a piezoelectric pump
32 in a measuring line 34 through an etched metal coil as a measuring coil
36 of a microsystem-technical unit 40. The counted particles are
3o discharged therefrom in the direction indicated by the arrow X.
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In the embodiment of Figure 3 the suspension is transported in the
measuring line 35 through the gap 52 of a ferromagnetic core 50 of a
measuring coil 36a, the core 50 being curved in a C-shape.
The free ends 38, 38a of the measuring coil 36, 36a - downstream
of a resistor 42 and a capacitor 44 - are connected to a device 46 for
exciting the oscillation and for measuring resonance events; there
conversion into counting pulses takes place.
The number of resonance events in the connected resonant circuit
corresponds to the number of coli bacteria in the original water sample Z.
l0 Provided between the Teflon capillary 20 and the piezoelectric pump
32 is a line branch 18 - which includes a valve 48 - for excess sample
portions Q, with a valve 48 being connected downstream thereof in the
delivery line 16.
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