U.S. patent application number 14/979121 was filed with the patent office on 2016-07-14 for microfluidic devices and/or equipment for microfluidic devices.
This patent application is currently assigned to ELTEK S.P.A.. The applicant listed for this patent is ELTEK S.P.A.. Invention is credited to FULVIO CERUTTI, COSTANZO GADINI.
Application Number | 20160202153 14/979121 |
Document ID | / |
Family ID | 43735515 |
Filed Date | 2016-07-14 |
United States Patent
Application |
20160202153 |
Kind Code |
A1 |
GADINI; COSTANZO ; et
al. |
July 14, 2016 |
MICROFLUIDIC DEVICES AND/OR EQUIPMENT FOR MICROFLUIDIC DEVICES
Abstract
An equipment for controlling operation of a biomedical
microfluidic device includes a system for connection with the
microfluidic device, having at least one of an arrangement for
fluidic connection and an arrangement for electrical connection, a
system for electrical supply or control of an electrical or
electronic arrangement of the microfluidic device, a system for
handling at least one fluid required for operation the microfluidic
device, and a system for signal communication with the microfluidic
device.
Inventors: |
GADINI; COSTANZO;
(FRASSINETO PO (ALESSANDRIA), IT) ; CERUTTI; FULVIO;
(IVREA (TORINO), IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ELTEK S.P.A. |
CASALE MONFERRATO (ALESSAN |
|
IT |
|
|
Assignee: |
ELTEK S.P.A.
CASALE MONFERRATO (ALESSAN
IT
|
Family ID: |
43735515 |
Appl. No.: |
14/979121 |
Filed: |
December 22, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13642596 |
Dec 21, 2012 |
9250163 |
|
|
PCT/IB2011/051733 |
Apr 20, 2011 |
|
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14979121 |
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Current U.S.
Class: |
435/308.1 ;
73/863.23 |
Current CPC
Class: |
B01L 2200/0631 20130101;
G01N 2015/1486 20130101; B01L 2200/0668 20130101; B01L 2400/086
20130101; G01N 1/28 20130101; G01N 1/4077 20130101; G01N 2001/4088
20130101; B01L 3/502761 20130101; B01L 3/502753 20130101; B01L
2300/0681 20130101; B01L 2200/0652 20130101; B01L 2300/0816
20130101; G01N 15/12 20130101; B01L 2300/0877 20130101; B01L
2200/028 20130101; C12M 47/02 20130101 |
International
Class: |
G01N 1/28 20060101
G01N001/28; C12M 1/00 20060101 C12M001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 20, 2010 |
IT |
TO2010U000068 |
Claims
1. An equipment for controlling operation of a biomedical
microfluidic device for separating particles from a fluid, the
microfluidic device having a body in which at least one first
microfluidic path is defined for the fluid, wherein the equipment
comprises at least one of a system for connection with the
microfluidic device, comprising at least one of an arrangement for
fluidic connection and an arrangement for electrical connection, a
system for electrical supply or control of an electrical or
electronic arrangement of the microfluidic device, a system for
handling at least one fluid required for operation of the
microfluidic device, and a system for signal communication with the
microfluidic device.
2. The equipment according to claim 1, further comprising a control
system configured for controlling at least one of the system for
connection, the system for electrical supply or control, the system
for handling and the system for signal communication.
3. The equipment according to claim 1, wherein the system for
handling comprises at least one of an arrangement for supplying the
microfluidic device with a first fluid; an arrangement for
supplying the microfluidic device with a first fluid and an
auxiliary fluid; an arrangement for discharging from the
microfluidic device a first fluid; an arrangement for discharging
from the microfluidic device a mixture comprising a first fluid and
an auxiliary fluid; an arrangement for supplying the microfluidic
device with a fluid containing functionalized particles or beads;
an arrangement for supplying the microfluidic device with a
flushing liquid; an arrangement for collection of a mixture or a
fluid discharged from the microfluidic device;
4. The equipment according to claim 1, wherein the system for
handling comprises at least one of: an arrangement for supplying an
aeriform; an arrangement for subjecting to a pressure, or to a
depression, a fluid contained in, or supplied to, the microfluidic
device; and a source of a compressed aeriform.
5. The equipment according to claim 1, wherein the system for
handling comprises at least one container for a fluid to be
supplied to the microfluidic device, and an arrangement associated
to the container for agitating the fluid and/or functionalized
particles or beads contained in the fluid.
6. The equipment according to claim 1, wherein the system for
handling comprises: an arrangement for supplying the microfluidic
device with a first fluid, including at least one connection
element for hydraulic connection to at least one first inlet of the
microfluidic device; and an arrangement for supplying the
microfluidic device with an auxiliary fluid separately with respect
to the first fluid, including at least one element for hydraulic
connection to at least one second inlet of the microfluidic
device.
7. The equipment according to claim 1, wherein the arrangement for
fluidic connection comprises at least one hydraulic connection
element configured for connection to at least one of an inlet and
an outlet of the microfluidic device.
8. The equipment according to claim 7, wherein the at least one
hydraulic connection element includes one of a valve member, a
fluid-retention member and an arrangement designed for easing
opening of a retention valve of the microfluidic device.
9. The equipment according to claim 1, wherein the system for
handling comprises an arrangement for supplying the microfluidic
device with at least two different fluids at two different
pressures.
10. The equipment according to claim 1, wherein the system for
handling comprises at least one of a pressure regulating
arrangement and a pressure sensing arrangement designed to be
connected in fluid communication with the at least one microfluidic
path of the microfluidic device.
11. The equipment according to claim 1, wherein one of the system
for electrical supply or control and the arrangement for electrical
connection comprises at least one of: an electric connector
arrangement couplable to an electric connector arrangement of the
microfluidic device; an arrangement for inductive supply of the
microfluidic device.
12. The equipment according to claim 1, wherein the system for
signal communication comprises at least one of: an arrangement for
unidirectional or bidirectional communication of data or
information with the microfluidic device; an arrangement for
wireless communication with the microfluidic device; an arrangement
for reading identification data of the microfluidic device; an RFID
arrangement; and a reader of a code carried by the microfluidic
device.
13. The equipment according to claim 1, comprising one or more
disposable parts.
14. The equipment according to claim 13, comprising at least one
connector device couplable to a container for containing a first
fluid, the connector device having a respective body to which there
are associated one first fluidic connection element and one second
fluidic connection element, the first connection element being
connectable to a line for supplying the first fluid from the
container to the microfluidic device and the second connection
element being connectable to a line for supplying the container
with a second pressurized fluid.
15. The equipment according to claim 13, comprising a container for
containing a first fluid, having an outlet prearranged for coupling
with an inlet of the microfluidic device and an inlet prearranged
for connection to a source of a second fluid under pressure.
16. The equipment according to claim 1, comprising an arrangement
for carrying out a final flushing step of the microfluidic
device.
17. The equipment according to claim 1, comprising a protected
and/or thermostatted housing configured for receiving the
microfluidic device.
18. The equipment according to claim 1, wherein the system for
signal communication s configured for writing data in a memory of
the microfluidic device.
19. A control equipment for use in conjunction with a microfluidic
device having a body in which at least one first microfluidic path
for a fluid is defined, wherein the control equipment is
prearranged for connection with the microfluidic device and
comprising at let one of a system for controlling one or more fluid
flows required for operation of the microfluidic device, and a
system for controlling electric or electronic devices of the
microfluidic device.
20. A biomedical microfluidic device for separating particles from
a fluid for use with the equipment of claim 1, the microfluidic
device having a body in which at least one first microfluidic path
for the fluid is defined and comprising at least one of an
arrangement for fluidic connection with the equipment, an
arrangement for electrical connection with the equipment and an
arrangement for signal communication with the equipment.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
13/642,596 filed on Dec. 21, 2012, published as U.S. Publication
No. US2013/0086980A1 on Apr. 11, 2013, which is a 371 National
Phase of PCT/M2011/051733 filed on Apr. 20, 2011, which claims
priority to Italian Utility Model Application No. TO2010U000068
filed on Apr. 20, 2010, entire disclosures of which are
incorporated herein by reference.
[0002] This application is also related to U.S. Ser. No. ______,
filed on Dec. 22, 2015 (Attorney Docket No. 2177.221B (KUS13108),
the entire disclosure of which is incorporated by reference.
FIELD OF THE INVENTION
[0003] The present invention relates in general to microfluidic
devices, preferably of the type generally identified by terms such
as "Lab-On-Chip" or "Lab-On-a-Chip" (LOC) or "Micro Total Analysis
Systems" (.mu.TAS), particularly for medical and/or biological
(referred to hereinafter for simplicity as "biomedical")
applications and/or for diagnostic applications, as well as to
equipment that can be used in combination with microfluidic
devices.
[0004] More in particular, the invention regards a microfluidic
device for separating and/or concentrating a sub-population of
particles from a biological fluid, the device comprising at least
one first microfluidic path having an inlet, for introduction of
the biological fluid into the first path, and an outlet, for
release from the first path of a sample of fluid enriched in the
aforesaid sub-population of particles.
PRIOR ART
[0005] For the purposes of diagnosis and study of certain diseases,
for example of the blood, of the bone marrow, and of the
corresponding tissues and organs, it proves useful to identify and
analyse specific cells or particles present in a biological
fluid.
[0006] Blood is a biological fluid basically made up of a
corpuscular part and a fluid part. The corpusculate part typically
comprises erythrocytes, leukocytes, and platelets, whereas the
fluid part is constituted by plasma. Erythrocytes and leukocytes
are cells, whereas the platelets are cellular fragments. The most
numerous cells are the erythrocytes, or red blood cells, which
basically perform the exchange of oxygen and carbon dioxide between
the lungs and the body tissues. Leukocytes, or white blood cells,
represent the smaller population of blood cells, and basically have
the function of controlling the response of the immune system of
the body and defending it from infectious organisms and foreign
agents, both in the tissues and in the blood stream. The platelets
basically have the function of bringing about coagulation of the
blood.
[0007] The leukocytes population comprises cells of various types,
which play distinct roles in immune response. The leukocytes can be
divided into granulocytes and lymphoid cells. The granulocytes are
in turn distinguished into neutrophils, eosinophils, and basophils.
The lymphoid cells are, instead, distinguished into lymphocytes and
monocytes. In general, erythrocytes and platelets are the particles
of the blood of smallest dimensions. Indicatively, erythrocytes
have a discoidal shape, with a diameter of approximately 6-8 .mu.m
and a thickness of approximately 2 .mu.m, whilst the platelets have
a diameter of approximately 2-4 .mu.m. The cells that constitute
the leukocytes have on average larger dimensions, ranging from 7 to
20 .mu.m. Purely by way of indication, neutrophils have a diameter
of approximately 12-14 .mu.m, eosinophils a diameter of
approximately 10-15 .mu.m, basophils a diameter of approximately
14-16 .mu.m, lymphocytes a diameter of approximately 7-15 .mu.m,
and monocytes a diameter of approximately 16-20 .mu.m. These
dimensions are purely indicative, given that, by way of example,
erythrocytes have been observed with a diameter of more than 9
.mu.m (macrocytes) or less than 6 .mu.m (microcytes). In the same
way, given that blood cells are subject to a certain degree of
deformability, their dimensions may be smaller than the ones
indicated above, on account of their dynamic behaviour in a flow of
liquid. Moreover, cells affected by disease (for example, tumoural
epithelial cells) may on average have dimensions that differ from
those of healthy cells and/or have dimensions larger than 15
.mu.m.
[0008] Leukocytes, as other particles present in the blood, can
furnish important information on various illnesses. The
identification of these illnesses hence involves the identification
and isolation of certain cells or particles in a blood sample.
Isolation may be conducted using non-invasive techniques, employing
separation devices, which isolate the particles of interest on the
basis of their biophysical characteristics, for example, their
dimensions.
[0009] For this purpose, there is known the use of miniaturized
devices, obtained with micromachining techniques, into which a
blood sample is introduced. Certain of these microfluidic devices,
generically known as "Lab-On-Chip" or "Lab-On-a-chip" (LOC) or
"Micro Total Analysis Systems" (.mu.TAS), also have functions of
analysis on the separate particles. These devices are defined as
"microfluidic devices" since they integrate one or more
miniaturized hydraulic components, such as paths, passages, valves,
filters, and obstacles for particles, which have at least one
characteristic dimension (length, width, height, thickness, section
of passage, etc.) of less than 1 mm.
[0010] In combination with the above devices it is known to use
functionalized particles, prearranged for interacting with target
cells or substances. These particles, also known as beads, have in
general an approximately spherical shape and nanometric or
micrometric dimensions, and have a nucleus sensitive to applied
magnetic fields. Typically, these beads bind to a target cell,
giving rise to an aggregate of particles, which, by means of a
magnetic field, can be separated from the rest of the solution
analysed.
SUMMARY OF THE INVENTION
[0011] Miniaturization techniques enable microfluidic devices for
separating cells to be obtained that are on average efficient and
presuppose the use of modest amounts of blood. Known microfluidic
devices, however, are frequently relatively complicated to produce
or far from practical to use, or present low reliability.
[0012] The object of the present invention is in general to provide
microfluidic devices that are simple to produce and/or practical to
use.
[0013] Another object of the present invention is to provide
microfluidic devices and/or corresponding equipment suitable for
improving and/or facilitating the separation and/or enrichment
and/or identification and/or transformation and/or analysis of
particles, where: [0014] "identification of particles" refers both
to the case of a marking of individual target cells or particles,
for example with fluorescent beads, and to the case of a more
generic identification and/or analysis, via suitable means, of a
microfluidic device or a part thereof containing target particles;
[0015] by "transformation of particles" is meant at least the
possible cultivation of cells and/or an extraction of parts of
cell, effected directly in at least one part of the microfluidic
device itself that has previously carried out a separation.
[0016] Another general object of the invention is to provide
equipment that can be used advantageously in combination with
microfluidic devices.
[0017] The above and further objects that will emerge more clearly
hereinafter are achieved according to the invention by devices
having the characteristics indicated in the attached claims. The
claims form an integral part of the technical teaching provided
herein in relation to the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Further purposes, characteristics and advantages of the
present invention will emerge clearly from the ensuing detailed
description and from the annexed drawings, which are provided
purely by way of explanatory and non-limiting example, and in
which:
[0019] FIG. 1 is a schematic representation in plan view of a
microfluidic arrangement, aimed at illustrating the general working
principle of a methodology of micro-filtering or micro-separation
envisaged in a preferential embodiment of the invention;
[0020] FIG. 2 is a schematic representation of some micro-filtering
or micro-separation arrangements according to the known art;
[0021] FIG. 3 is a partial and schematic perspective view of a
microfluidic device according to the invention;
[0022] FIGS. 3A, 3B and 3C are, respectively, a first detail and a
second detail, at an enlarged scale, of the device of FIG. 3, and a
partial perspective view of a variant of the device of FIG. 3;
[0023] FIGS. 4A and 4B are schematic illustrations of examples of
aggregates of particles that can be separated by means of a device
according to the invention;
[0024] FIG. 5 is a partial and schematic perspective view of
another microfluidic device according to the invention;
[0025] FIG. 6 is a view similar to that of FIG. 5, with the
addition of a body for closing the device;
[0026] FIGS. 7-11 are perspective views, at an enlarged scale, of
some details of the device of FIGS. 5 and 6;
[0027] FIGS. 12-14 are perspective views, at an enlarged scale, of
further details of the device of FIGS. 5 and 6;
[0028] FIG. 15 is a perspective and schematic view of a further
microfluidic device according to the invention;
[0029] FIGS. 16 and 17 are perspective views of another
microfluidic device according to the invention, with and without
lid, respectively;
[0030] FIGS. 18 and 19 are perspective views, at an enlarged scale,
of respective details of the device of FIG. 19;
[0031] FIGS. 20 and 21 are perspective views from different angles
of another microfluidic device according to the invention;
[0032] FIG. 22 is a perspective view of the device of FIGS. 20-21,
without a respective lid;
[0033] FIG. 23 is an enlarged perspective view of a portion of the
device of FIG. 22;
[0034] FIGS. 24 and 25 are details at an enlarged scale of FIG.
23;
[0035] FIG. 26 is a perspective view at an enlarged scale of a
portion of the device of FIGS. 20-26;
[0036] FIG. 27 is an enlarged perspective view of a portion of the
device of FIG. 22;
[0037] FIGS. 28 and 29 are details at an enlarged scale of FIG.
27;
[0038] FIGS. 30 and 31 are perspective views from different angles
of a main body according to a variant of the device of FIGS.
20-29;
[0039] FIGS. 32 and 33 are a perspective view and a side view of
another microfluidic device according to the invention;
[0040] FIG. 34 is an enlarged detail of FIG. 32;
[0041] FIG. 35 is a perspective view of a portion of a microfluidic
device according to the invention;
[0042] FIG. 36 is a perspective view of a body of another
microfluidic device according to the invention;
[0043] FIGS. 37, 38 and 39 are details at an enlarged scale of the
body of FIG. 36;
[0044] FIGS. 40 and 41 are perspective views from different angles
of another microfluidic device according to the invention;
[0045] FIGS. 42 and 43 are an exploded view and a partially
sectioned perspective view, respectively, of the device of FIGS. 40
and 41;
[0046] FIG. 44 is a perspective view similar to that of FIG. 41,
with a part of the device removed;
[0047] FIG. 45 is a perspective view of a part of the device of
FIGS. 40-44;
[0048] FIG. 46 is a plan view from beneath of another fluidic
device according to the invention;
[0049] FIGS. 47 and 48 are perspective views from different angles
of another microfluidic device according to the invention;
[0050] FIG. 49 is a partially sectioned perspective view of the
device of FIGS. 47-48;
[0051] FIG. 50 is a perspective view of a part of a microfluidic
device according to the invention, such as the device of FIGS.
47-49;
[0052] FIG. 51 is a plan view, at an enlarged scale, of the part of
device of FIG. 50;
[0053] FIGS. 52 and 53 are perspective views from different angles
of a part of a microfluidic device according to the invention;
[0054] FIG. 54 is an exploded view of the part of microfluidic
device according to FIGS. 52 and 53;
[0055] FIGS. 55 and 56 are perspective views from different angles
of another microfluidic device according to the invention;
[0056] FIGS. 57 and 58 are an exploded view and a partially
sectioned perspective view, respectively, of the device of FIGS. 55
and 56;
[0057] FIGS. 59 and 60 are two schematic sections of another device
according to the invention, in two different operating
conditions;
[0058] FIG. 61 is a cross section, at an enlarged scale, of a
variant of the device of FIGS. 59-60;
[0059] FIG. 62 is a cross section of FIG. 61;
[0060] FIGS. 63 and 64 are a schematic section and a corresponding
enlarged detail, respectively, of another device according to the
invention;
[0061] FIG. 65 is a plan view of the detail of FIG. 64;
[0062] FIGS. 66 and 67 are a plan view and a view in cross section
of a valve means of the device of FIGS. 63-64;
[0063] FIGS. 68 and 69 are perspective views, respectively from
above and from beneath, of a part of a microfluidic device
according to the invention;
[0064] FIG. 70 is a partially sectioned perspective view of the
device of FIG. 68;
[0065] FIG. 71 is a partially sectioned perspective view, at an
enlarged scale, of a part of the device of FIG. 70;
[0066] FIG. 72 is a perspective view of a variant of the part of
FIG. 71;
[0067] FIGS. 73 and 74 are perspective views, from different
angles, of a part like the one illustrated in FIG. 72 coupled to a
support or adapter;
[0068] FIG. 75 is a perspective view of the support or adapter of
FIGS. 73 and 74;
[0069] FIG. 76 is a simplified representation of an apparatus
according to the invention, that can be used together with a
microfluidic device, in particular for circulation and/or
management of flows of fluids and/or control of the device
itself;
[0070] FIGS. 77 and 78 are simplified representations of further
apparatuses of the same type as that of FIG. 76;
[0071] FIG. 79 is a schematic representation of equipment according
to the invention, of a disposable type that can be used in
combination with a microfluidic device;
[0072] FIG. 80 illustrates in schematic form an example of process
of moulding of a microprocessed body forming part of a microfluidic
device according to the invention;
[0073] FIG. 81 is a perspective view of another microfluidic device
according to the invention;
[0074] FIGS. 82 and 83 are a perspective view in partial cross
section and an exploded view, respectively, of the device of FIG.
81;
[0075] FIG. 84 is a partial and schematic perspective view of a
further microfluidic device according to the invention;
[0076] FIG. 85 is a schematic representation in plan view of a
sensor or particle-counter system for a fluidic device according to
the invention;
[0077] FIG. 86 is a schematic representation in plan view of a
system for alignment of particles of a fluidic device according to
the invention, in combination with a sensor or particle-counter
system; and
[0078] FIG. 87 is a schematic representation in plan view of an
acoustic-separation system and/or particle sensor of a fluidic
device according to the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0079] Reference to "an embodiment" or "one embodiment" in the
framework of the present description is meant to indicate that a
particular configuration, structure, or characteristic described in
relation to the embodiment is comprised in at least one embodiment.
Hence, the phrases such as "in an embodiment" or "in one
embodiment" and the like that may be present in different points of
the present description do not necessarily all refer to one and the
same embodiment. In addition, the particular configurations,
structures, or characteristics may be combined in any adequate way
in one or more embodiments. The references used herein are provided
merely for convenience and do not define the sphere of protection
or the scope of the embodiments.
[0080] Where not otherwise indicated, in the present description
and in the attached [0081] by "bead" is meant functionalized
particles or microparticles and/or generic vectors of ligands, such
as antibodies, in particular prearranged for interacting with
target cells, substances, or particles, said beads preferably
having an approximately spherical shape and/or nanometric or
micrometric dimensions; [0082] by "particle" and derivatives is
meant any solid object not dissolved in a biological fluid, such as
cells, aggregates of cells, beads, aggregates of cells and beads,
etc.; [0083] by "buffer" and the like is meant a liquid substance
designed to dilute the biological fluid or blood and/or facilitate
and/or modify the flow of the biological fluid or blood within a
microfluidic device, or more in general a liquid designed to
interact with the biological fluid for the purposes of the
invention; the buffer may, for example, be distilled water or a
physiological or saline solution, possibly containing an
anticoagulant and/or bead. [0084] The microfluidic devices
described hereinafter are preferably of the type envisaged at least
for concentrating target particles in an organic fluid, or else to
obtain, from an organic fluid at inlet containing a population of
particles, a sample enriched in a sub-population of target
particles, or else devices designed at least to separate particles
of specific interest.
[0085] Examples of target particles are leukocytes, foetal
erythrocytes, carcinogenic cells, infectious organisms, as well as
beads and aggregates of particles, such as for example a bead to
which one or more cells are bound, or a cell to which a number of
beads are bound.
[0086] Preferably, the devices described in what follows include
separation or filtering means, such as means of a substantially
mechanical type, set along a path or between an inlet and an outlet
of a path for the fluid containing the population of particles. In
particular, the separation means are configured so as to withhold
or separate at least the target particles, for example ones having
a certain shape, size or deformability, or given physical
characteristics, whereas other particles are evacuated from the
device. These separation or filtering means are preferably defined
directly in the body of the device, via micromachining and/or
microdeposition techniques, and hence without having to mount or
fix components for said purpose, such as membranes or sieves
configured as additional parts.
[0087] The devices described in what follows may moreover be used
for removing excess fluid from a sample of particles, without this
entailing a substantial loss of target particles in the fluid
removed.
[0088] The invention will be described in what follows with
reference to enrichment of target particles or elements contained
or dispersed in a biological fluid, in particular to enrichment of
tumour cells and/or leukocytes and/or other target cells in a blood
sample. Consider, however, that the invention is in general
applicable also to particles of a different type and nature,
present also in fluids different from biological fluids;
consequently, in the ensuing description, the term "blood" may be
considered, if necessary, as indicating also a generic fluid.
[0089] There follows a description of inventive embodiments of
microfluidic devices and inventive embodiments of equipment that
can be used in combination with microfluidic devices. The
microfluidic devices described are of particularly advantageous use
in combination with the equipment described, but this does not
exclude either use of the aforesaid devices in combination with
equipment different from the one described herein, or use of the
aforesaid equipment in combination with microfluidic devices
different from the ones described herein.
[0090] As will emerge clearly in what follows, in a preferential
embodiment of the invention, the microfluidic device is conceived
for carrying out a micro-filtering or micro-separation of a type
here defined as "mixed flow", which will now be briefly described
with reference to FIG. 1.
[0091] In general terms, in accordance with such an inventive
principle, a first fluid BL, in particular a biological fluid under
analysis, is made to flow in a first path or channel 3, here
defined as "main path", between at least one inlet 4 and at least
one outlet 5. This main path 3 extends at least in part between a
first path or channel and a second path or channel, here defined as
"auxiliary paths" and designated by 8 and 9, which are delimited
with respect to the main path 3 by a first lateral delimitation 10
and a second lateral delimitation 10 and 11, each provided with
respective passageways, where the path 8 has at least one inlet 6
and the path 9 has at least one outlet 7. Preferably, these lateral
delimitations or dikes define the minor dimension of the cross
section of the path 3.
[0092] Introduced through the inlet 6 into the first auxiliary path
8 is an auxiliary fluid LB, such as a liquid buffer (or else a
portion of the biological fluid being analysed itself), which is to
mix with the first fluid BL.
[0093] The second auxiliary path 9 basically performs functions of
discharge channel for a part of the mixture BL+LB of the two
fluids, that in particular contains particles different from the
target particles, with the corresponding second lateral
delimitation 11, provided with the corresponding passageways, that
performs to functions of micro-filtering or micro-separation.
[0094] Via the passageways of the first lateral delimitation 10 the
auxiliary fluid LB penetrates laterally into the main path 3,
mixing with the first fluid BL.
[0095] In this way, in general, the auxiliary fluid LB imparts on
the flow of the first fluid BL and/or on particles contained
therein a component of thrust or movement, preferably towards the
second lateral delimitation 11 and/or towards the outlet 5 of the
main path 3. The particles of dimensions smaller than those of the
passageways of the second lateral delimitation 11 are in this way
induced to pass into the second auxiliary discharge path 9,
together with a part of the mixture, designated by BL+LB, whereas
the particles having dimensions larger than those of the
passageways of the second lateral delimitation 11 remain in the
main path 3 so as to converge towards the outlet 5 in a part of the
mixture that is enriched with said target particles, designated by
LB+BL.
[0096] The passageways of the auxiliary fluid LB from the path 8 to
the path 3 are preferably configured, for example in terms of
dimensions and/or orientation, so as to cause the auxiliary fluid
to bring about an albeit minimal deviation or turbulence in the
flow of the first fluid. In particular, the purpose of this is to
cause the first fluid BL and/or particles contained therein to flow
in the proximity of the passageways of the second lateral
delimitation 11 in order to favour an optimal separation. The modes
of introduction or mixing of the auxiliary fluid LB are hence
preferably such as to change the direction of the threads of the
first fluid BL that flows in the main path--as indicated by the
small arrows within the main path 3--in order to prevent in said
path 3 the flow of the first fluid from being prevalently laminar
or oriented in just the direction of flow of the main path. The
flows in the paths 3 and 8--and consequently in the path 9--are
preferably continuous flows in order to produce continuous
perturbations of the flow in the path 3; said flows could, however,
be at least in part of a pulsed type.
[0097] Hence, basically, in accordance with the methodology of
separation or concentration here proposed:
[0098] a) a first microfluidic channel 3, a second microfluidic
channel 8, and a third microfluidic channel 9 are provided, having
at least respective mutually adjacent parts, in particular adjacent
in length, with a said at least one part of the first channel 3
that is delimited laterally, with respect to a corresponding said
at least one part of the second channel 8 or of the third channel
9, by a first lateral delimitation 10 or a second lateral
delimitation 11, respectively, said lateral delimitations having
respective passageways for connecting the second channel 8 to the
first channel 3 and the third channel 9 to the first channel 3,
respectively; and
[0099] b) introduced into the first channel 3 is a biological fluid
BL and introduced into the second channel 8 is an auxiliary fluid
LB, in such a way that a flow of the auxiliary fluid LB at outlet
from the passageways of the first lateral delimitation 10 forms, in
the first channel 3, a mixture LB+BL with the biological fluid BL,
with a first fraction of this mixture LB+BL that flows in the third
channel 9 through passageways provided at a filtration and/or
separation part of the second lateral delimitation 11a and with a
second fraction of the mixture LB+BL that remains in the first
channel 3. At least part of the auxiliary fluid LB at outlet from
the passageways of the first lateral delimitation 10 is in
particular such as to obtain at least one of the following effects:
[0100] imparting on the flow in the first channel 3 and/or on
particles contained therein at least one from among a component of
thrust or movement, a transverse component, a turbulence, and an
irregular motion; [0101] forcing particles or aggregates of
particles having a dimension smaller than a dimension of the
passageways of the second lateral delimitation 11 to pass into the
third channel 9; and [0102] forcing towards an outlet 5 of the
first channel 3 particles or aggregates of particles constituting a
sub-population of interest.
[0103] The modes of introduction of the fluids into the channels 3
and 8 may be different, and comprise, for example, at least one
from among: [0104] introducing in a continuous way the biological
fluid and the auxiliary fluid; [0105] introducing in a pulsed way
the biological fluid and the auxiliary fluid; [0106] introducing in
an alternating way the biological fluid and the auxiliary fluid;
[0107] introducing in an alternating way the biological fluid and
introducing in a continuous way the auxiliary fluid.
[0108] It should be emphasized that the system and the methodology
of mixed-flow micro-filtering or micro-separation described herein
are not functionally and constructively comparable to known systems
and methods of tangential filtering or separation, some of which
are schematically illustrated in FIG. 2.
[0109] Visible in part A of FIG. 2 is a first typical example of
tangential filter, in which a first channel CH1 is provided,
introduced into which is the fluid Fin to be treated. Designated by
CH2 is a second, discharge, channel, separated from the channel CH1
by filtering means FM, constituted by a wall provided with
passageways Fn. Flowing in the channel CH2 is a flow Fs,
constituted by a reject portion of the fluid being analysed
itself.
[0110] The channel CH1 has to be typically very narrow in order to
possibly cause all the fluid Fin to pass against the filtering wall
FM and in order to prevent the risk of a portion of
fluid--designated by FL--from flowing in the form of laminar flow
in an area at a distance from the wall FM, such as the wall of the
channel CH1 opposite to the wall FM, thus causing a reduced
filtering capacity.
[0111] In order to reduce said risk, according to a known technique
represented in part B of FIG. 2, both of the walls FM that delimit
the channel CH1 laterally can be configured as tangential-filtering
means; in this case, then, two reject channels CH2 are envisaged.
Also in a solution of this sort, however, it is in any case
necessary to provide a very narrow channel CH1 in order to prevent
central areas of laminar flow FL from being created, with the
consequent risk of a part of fluid flowing from the inlet to the
outlet of the channel CH1 without coming into contact with the
filtering means FM, i.e., with the risk of not all the fluid Fin
being treated appropriately.
[0112] Consider that, in said type of microfluidic filter, the
dynamics of the fluid is regulated by known phenomena, such as the
Reynolds number (Re), which substantially identifies ratios between
certain physical quantities of the fluid and of the device (for
example, speed and viscosity of the fluid, section and/or
dimensions of the channel, etc.). For Reynolds numbers Re 2000 a
flow is typically considered as stable or laminar, such as a flow
formed by thin laminas that flow in a direction parallel to the
walls or to the direction of the channel. For Reynolds numbers
Re>2000 but Re<3000 the flow is, instead, considered as being
in a transitional regime, in which small undulations start to form.
For Reynolds numbers Re.gtoreq.3000 the flow is instead considered
as being in a turbulent regime, i.e., characterized by a disorderly
motion of the fluid threads.
[0113] Considering a tangential filter of the type illustrated in
part C of FIG. 2, which is substantially equivalent to that of part
A but with a duct CH1 for the fluid to be treated that is far
wider, it is evident that there is an increased risk of having a
laminar flow FL that passes undisturbed between the inlet and the
outlet, without coming into contact with the filtering means FM,
i.e., without being treated. In said configuration, to prevent said
drawback, the speed of the fluid is typically increased
considerably in order to increase the Reynolds number and obtain a
turbulent motion, which would bring all the fluid into contact with
the means FM. Said solution should, however, be avoided in the
treatment of biological fluids, such as blood, in so far as the
increase in speed brings about an immediate damage to the cells on
account of phenomena of lysis. Moreover, to obtain such an increase
of speed, given the same cross section of the channel CH1, the
pressure of the fluid Fin at inlet should be increased
considerably: also this solution is unadvisable in the presence of
a biological fluid in so far as the high pressure would damage the
cells present in the fluid.
[0114] In an attempt to overcome the aforesaid drawback,
tangential-filtering devices have been proposed of the type
illustrated in part D of FIG. 2, provided with channels made in
which are considerable mechanical alterations of the path of the
fluid Fin, for example, by introducing restrictions and/or widened
areas in the path and/or inserting projections RI that divert the
flow. Also said solutions are, however, unsuited to preventing the
drawbacks referred to above in so far as they determine mechanical
obstructions and/or extensive surfaces against which the cells
strike, or restrictions in which local increases in speed are
brought about that lead to lysis of the cells. Consider, in this
regard that there typically exists the need to be able to treat
relatively large amounts of fluid (for example, 5-10 ml) in
relatively short times (for example, 15-30 minutes); in this case,
the aforesaid restrictions or alterations of the section of passage
bring about a deceleration in the flow of the fluid and hence a
longer treatment time.
[0115] Part E of FIG. 2 illustrates another known technique used in
order to possibly cause all the fluid Fin to pass in contact with
the filtering wall FM, which exploits the centrifugal forces that
are created when the fluid flows in a curved channel CH1, at a
relatively high speed. Also in this case it should be noted that
the aforesaid high speed entails the risk of lysis, due to the
cells that strike against the walls. To obtain said high speed the
pressure of the fluid at inlet Fin must moreover be increased, with
the risk of further damage to the cells. The path of the fluid to
be treated cannot in any case have a significant width or cross
section, and consequently it does not enable a high flow rate of
blood to pass. Conversely, with very wide ducts, the centrifugal
force alone is not sufficient to cause the particles or the threads
of fluid to be displaced until they come into contact with the
filtering means FM.
[0116] In this regard, it should be considered that in microfluidic
circuits there typically exist constraints or difficulties in
providing relatively deep channels, on account of the processes
that typically allow a maximum depth of tens or some hundreds of
microns to be reached. In addition, given that the filtering
elements should be provided along the side walls of the ducts,
according to the known art an increase in the section of flow of
the duct should be obtained principally by widening the duct, with
the consequent drawbacks indicated above, i.e., worsening of the
circulation of the fluid along the filtering walls, and hence
inefficiency or ineffectiveness of filtering or separation.
[0117] As will emerge also more clearly in what follows, the
methodology of mixed-flow micro-filtering or micro-separation
proposed according to the aforesaid preferential embodiment of the
invention overcomes the limits indicated above of the known art,
enabling an efficient and effective treatment of adequate amounts
of the fluid under analysis, in relatively short times, at modest
pressures and with paths for the fluid having a relatively wide
section, and hence with speeds of the flow accordingly reduced, to
the advantage of quality of filtering and/or separation, including
the integrity of the target particles.
[0118] FIGS. 3, 3A, and 3B represent an example of embodiment of a
biomedical microfluidic device according to the invention,
substantially a LOC or a .mu.TAS, which is configured for
separating a sub-population of particles from a biological fluid.
The device in question, designated as a whole by MD, is represented
in an extremely schematic form, merely by way example of its
working principle, based upon a micro-filtering structure of the
type here defined as "mixed-flow". The device MD is represented
without a top closing body, or lid, thereof, which is preferably
made of transparent material.
[0119] The structure of the device MD includes a first section 1,
having a first body or main body 2, for example made of at least
one from among an elastomeric or silicone material, a vitreous
material, a semiconductor material. In what follows, it will be
assumed that the material in question is a silicone material, for
example polydimethylsiloxane (PDMS), and hence at least slightly
yielding and/or preferably transparent. Defined in the body 2--via
micromachining techniques--are various functional elements on a
nanometric or micrometric scale, comprising at least means for
mechanical separation or filtering of the fluid. These means
include a first microfluidic channel 3 defined in the body 2. In
what follows, also with reference to other embodiments of the
invention, the channels provided in the microfluidic devices will
be also referred to as ducts or paths.
[0120] In one embodiment, at least one inlet portion of the channel
3 has a width greater than 100 .mu.m, preferably between 500 and
20000 .mu.m. Preferably, said inlet portion defines at least one
passageway designed to allow at least one type of target particles
or tumour cells to pass.
[0121] In one embodiment, at least an outlet portion of the channel
3 has a width greater than 20 .mu.m, preferably between 20 and 100
.mu.m. Preferably, said outlet portion defines at least one
passageway designed to allow at least one type of target particles
or tumour cells to pass.
[0122] According to a preferred embodiment, the shape or cross
section of the channel 3 is such as to allow a relatively large
amount of fluid to flow, preferably of between 2 ml and 10 ml, in a
relatively short time, preferably of between 10 and 30 minutes, in
particular without significant increase in pressure and/or speed of
the fluid in order to prevent phenomena of lysis or damage to the
particles or cells.
[0123] In the case exemplified, in the body 2 of the device MD the
aforesaid inlet portion defines a passageway or first inlet 4 for
the introduction of a biological fluid, i.e., a first fluid, into
the channel or path 3, and an outlet portion, defining a passageway
or first outlet 5 for release from the path 3 of a sample of fluid
enriched in the sub-population of target particles. As may be seen,
the path 3 extends in length between the portion including the
inlet 4 and the portion including the outlet 5 so as to define at
least one direction of flow.
[0124] In one embodiment, at least one portion of the channel 3 has
a depth or height greater than 20 .mu.m, preferably between 30 and
300 .mu.m.
[0125] Preferably, the aforesaid inlet portion or the at least one
first inlet 4 of the channel 3 has a depth or height greater than
20 .mu.m, preferably between 30 and 100 .mu.m, and the aforesaid
outlet portion or the at least one outlet 5 of the channel 3 has a
depth or height greater than 30 .mu.m, preferably between 50 and
300 .mu.m, in particular in order to enable also flow of target
particles associated to beads in at least a terminal part of the
channel 3.
[0126] In what follows, it will be assumed that the biological
fluid is blood and that the target particles are certain monocytes
M and/or tumour cells TC (FIG. 3A).
[0127] The device MD further includes at least one second inlet 6,
defined at least in part in the body 2, for introduction of a
second liquid, i.e., an auxiliary fluid, that is to mix with the
first fluid, i.e., the blood, in the first path 3. In the
non-limiting example considered, the auxiliary fluid is a liquid
buffer agent LB, preferably transparent, for example constituted by
a physiological or saline solution, that performs the function of
diluting the blood in the path 3 and/or facilitating flow thereof
and/or favouring separation of particles. The buffer can contain an
anticoagulant and/or other substances designed for the purpose.
[0128] The buffer preferably has a predefined conductivity or other
physical, chemical and electrical characteristics also in view of
operation of electrical arrangements of the microfluidic device,
such as a particle counter, a device for alignment of particles,
electrical separation means, etc.
[0129] The device MD has a second outlet 7, which is also defined
at least in part in the body 2, for discharge of a fraction of the
blood, in particular a fraction enriched in particles different
from the target particles, such as erythrocytes, platelets, and
part of the leukocytes (i.e., the leukocytes different from the
target leukocytes). The aforesaid discharge fraction of blood forms
part of a mixture including also the buffer.
[0130] In one embodiment, based upon the aforesaid mixed-flow
filtering or separation technique, the first path 3 is at least in
part defined in the body 2 between a second microfluidic channel or
path and a third microfluidic channel or path, designated by 8 and
9, which are in fluid communication with the inlet 6 and the second
outlet 7, respectively. The paths 8 and 9 are substantially
adjacent, in particular adjacent in length, to at least a
significant part of the path 3. In the example illustrated, the
first path 3 is delimited laterally from the second and third paths
8 and 9 by at least one first separation element or lateral
delimitation and one second separation element or lateral
delimitation, respectively. The three paths and the corresponding
lateral delimitations are hence directly made in or deposited on
one and the same face of the body 2, which defines the bottom of
the paths themselves. Preferably, these lateral delimitations
define the minor dimension of the cross section of the first path
3.
[0131] In one embodiment, at least one portion of the channel or
path 8 and/or of the channel or path 9 has a depth or height
greater than 20 .mu.m, preferably between 30 and 30000 .mu.m.
[0132] In one embodiment, at least one portion of the channel or
path 8 and/or of the channel or path 9 has a width greater than 100
.mu.m, preferably between 200 and 50000 .mu.m.
[0133] The first lateral delimitation, designated by 10, has first
passageways 10a (FIG. 3A), which connect the first path 8 to the
second path 3: in this way, the liquid buffer LB can pass from the
path 8 to the path 3 and mix with the blood, preferably with a
gradual introduction of buffer along the path 3, i.e., in such a
way as to mix the buffer with the blood in a gradual or distributed
way along the entire path 3. As has been seen previously, said
mixing between the fluid of the path 8 (here the buffer LB) with
the fluid of the path 3 (here the blood BL) constitutes an
inventive characteristic underlying operation of mixed-flow
micro-filtering. For this purpose, as has been seen, the aforesaid
mixing occurs preferably in a gradual and/or proportional way
and/or along at least a part of the path 3. For said purpose, the
passageways 10a are distributed along at least a substantial part
of the path 3. Preferably, hence, the liquid buffer is made to
penetrate laterally from the path 8 to the path 3, tending to
traverse it in the direction of its width (i.e., in the major
dimension or major side of the cross section of the path 3).
[0134] The second lateral delimitation, designated by 11, has
second passageways 11a (FIG. 3A), which connect the first path 3 to
the third path 9. Preferably, also the second passageways 11a are
distributed along a substantial part of the path 3. In this way,
part of the blood-buffer mixture can pass from the path 3 to the
path 9 and then be evacuated through the discharge outlet 7.
[0135] In one embodiment, inserted into the microfluidic device MD
are functionalized particles, such as beads, preferably in a
dispersed or non-aggregated form, that are bound to adhere to
target particles. The aforesaid beads are bound to aggregate or fix
to the target particles within the microfluidic device MD. The
beads BE, or rather the ligands that they carry, preferably have a
specific affinity with the targets, such as cells, nucleic acids,
proteins, or other bio-molecules, giving rise to an aggregate of at
least one target particle and at least one functionalized particle.
The beads BE used according to the invention can have at least one
part sensitive to applied electrical fields so as to enable a fast
and effective separation thereof from the rest of the solution
analysed, together with the target particles. Preferably, the beads
are of a fluorescent type or are functionalized in order to enable
a convenient detection, for example of an optical and/or electrical
type.
[0136] For said purpose, in one embodiment, provided in the body 2
of the device MD is a further inlet, designated by 6', in fluid
communication with a further microfluidic path designated by 8'. In
the example, also the further path 8' is adjacent in length to the
path 3 and is delimited with respect thereto via the lateral
delimitation 10, preferably with characteristics substantially
equivalent to those described with reference to the path or duct 8.
This further path 8' is provided for introduction from the
corresponding inlet 6' of a further buffer agent--which may be
similar to the one already indicated--containing beads BE, i.e.,
particles functionalized so as to adhere to target particles, such
as tumour cells TC, in order to obtain aggregates of particles.
FIG. 4A illustrates for said purpose one such aggregate, as well as
a bead BE, schematically highlighted in which are the respective
ligands AB, in particular of the antibodies AB.
[0137] In the example, the paths 8 and 8' are distinct from one
another and are each provided with a respective inlet 6 and 6';
however, said two paths could be replaced by a single path that
extends adjacent to the path 3, through an inlet of which there can
be introduced indifferently a buffer without beads or a buffer
containing beads.
[0138] Preferably, also with a view to the possible use of beads,
the buffer used is of a type designed to not damage the
corresponding functionalized bonds and/or the antibodies.
Preferably, the buffer has a predefined conductivity and
characteristics such as to not damage said functionalized bonds
and/or antibodies, in particular for the purposes of separation
and/or detection of the particles.
[0139] It should be noted that, according to the known art, the
beads are added preliminarily to a blood sample to be analysed,
i.e., prior to introduction of the blood into a microfluidic
device.
[0140] In the case of use of beads BE, the passageways 10a will
preferably have at least one characteristic dimension--such as the
width (horizontal cross section) or the height (vertical cross
section) greater than the characteristic dimension or diameter of
the beads.
[0141] The passageways 11a have, instead, at least one
characteristic dimension--such as the width or the height--that is
smaller than the characteristic dimension or diameter of the target
particles, whether these be leukocytes, such as for example altered
monocytes M, tumour cells TC, or aggregates of particles,
constituted for example by beads BE and tumour cells TC. Merely by
way of example, the following cases of target particles may arise:
[0142] tumour cells TC, such as epithelial tumour cells, and/or
leukocytes, such as monocytes M, having dimensions greater than the
section of passage of the passageways 11a; [0143] altered
leukocytes and/or tumour cells TC, such as epithelial tumour cells,
having dimensions smaller than the section of passage of the
passageways 11a, in which case the beads BE increase the dimensions
thereof beyond those of the aforesaid section of passage.
[0144] The beads BE used for the purposes of the invention can
present electrical characteristics, or a electrical polarity, in
order to be attracted and/or repelled electrically, in particular
for the purposes of a separation or electrical displacement
thereof, and hence a separation or displacement of the particles or
target cells TC attached to said beads. As will be seen, the beads
BE and/or corresponding target cells TC can be displaced or
separated via an electrical field, for example generated via
electrodes. For said purpose, the beads BE can advantageously be of
an anionic type.
[0145] As has already been explained, in one embodiment, the
passageways 10a are configured for introducing the liquid buffer LB
from the path 8 and/or 8' into the path 3 in such a way as to
create an albeit minimal deviation or turbulence in the biological
fluid that flows in the path 3, in particular in order to cause all
the fluid and/or the particles contained therein to flow in the
proximity of the passageways 11a, for the purposes of an optimal
separation. The introduction or mixing of the buffer LB is
preferably designed to change the direction of the threads of
biological fluid of the path 3 in order to prevent the flow in the
path itself from being prevalently laminar or oriented in just the
direction of the path 3.
[0146] For example, in a preferential embodiment, the passageways
10a and 11a are oriented in a direction generally transverse to the
direction of the flow of the blood-buffer mixture in the path 3,
preferably in such a way that the buffer LB leaving the passageways
10a of the lateral delimitation 10 imparts on the flow itself
and/or on particles contained therein a component of thrust towards
the opposite lateral delimitation 11 and/or towards the outlet 5.
In this way, the particles or possible aggregates of particles of a
dimension smaller than the aforesaid characteristic dimension of
the passageways 11a are forced to pass into the path 9. Conversely,
the particles or aggregates of particles having a dimension larger
than the aforesaid characteristic dimension of the passageways 11a,
which constitute a sub-population of target particles, remain in
the path 3, preferably entrained by the buffer and/or by the
blood-buffer mixture along the duct 3, and/or towards the outlet 5
that constitutes in itself a area of concentration of the target
particles.
[0147] It is to be noted in this regard that the passageways 10a
are preferably distributed along the path 3 and are at least in
part oriented so as to have each a direction transverse or angled
with respect to the portion of the path 3 into which the single
passageway 10a gives. The angle between the single passageway 10a
and the portion of duct 3 into which said passageway gives is
preferably between 1.degree. and 89.degree., in particular between
5.degree. and 45.degree..
[0148] Consider moreover that the path 3 preferably has a section
of passage that narrows towards the outlet, in particular in order
to compensate for the progressive reduction in the flow rate in the
path 3 due to the fluid that exits from the discharge path 9,
and/or in order to facilitate passage of a part of the mixture
present in the path 3 towards the passageways 11a, i.e., towards
the discharge path 9.
[0149] Likewise consider that the passageways 11a and/or the third
discharge channel or path 9 are preferably shaped in such a way as
to prevent any return of the fluid mixture or reject particles from
the path 9 to the path 3, i.e., preferably shaped in such a way as
to facilitate the discharge flow towards the outlet 7.
[0150] In a preferred embodiment, the device MD includes at least
one unit or second section, for collecting or concentrating the
sample of target particles, designated as a whole by 20, visible in
FIGS. 3 and 3B.
[0151] This section has a casing or collection body of its own,
designated by 21. The collection body 21 can be configured as a
distinct part appropriately coupled, in particular mechanically and
hydraulically, to the main body 2, or else it may be constituted by
a portion of the same body 2.
[0152] The body 21 of the section 20 defines at least one cavity
21a with at least one inlet 22, in fluid communication with the
outlet 5 of the first path 3. The outlet of the path 3 can--as in
the example--correspond to the inlet 22 of the section 20, and vice
versa. The body 21 has also a discharge outlet 23, where between
the inlet 22 and the outlet 23 separation or filtering means 24 are
provided, i.e., means for withholding within the section 20 a
sub-population of target particles. In the example, the means 24
basically consist of a filter or sieve, formed by a series of
barriers or obstacles 25, which rise from a bottom of the cavity
21a of the section 21. In addition and/or as an alternative there
may be envisaged other separation means, such as separation means
of an electrical type, for example in the form of electrodes for a
electrophoresis or a dielectrophoresis (DEP) or for the already
mentioned separation via electrical attraction and/or repulsion of
the beads BE. In the case illustrated, the means 24 extend
longitudinally substantially parallel to the direction of the
flow.
[0153] The obstacles 25 define between them passageways, designated
by 24a in FIG. 3B, having a characteristic dimension smaller than
the characteristic dimension or diameter of the target particles.
In this way, the target particles remain withheld within the
section 20, in its part upstream of the separation means 24; at
least part of the blood-buffer solution that penetrates into the
body 21 can flow out from the outlet 23, with the possible
particles not of interest contained therein.
[0154] The bottom of the cavity 21a can be defined by the body 21
or else by an additional element, such as a substrate made of
glass, plastic, or semiconductor material, preferably at least in
part transparent. In such a case, underneath the aforesaid
substrate there can be provided and/or integrated a lighting source
or an optical sensor, designed to irradiate the target particles to
facilitate optical analysis thereof; in the case of a semiconductor
material, the lighting source can be at least in part integrated or
associated to said semiconductor material.
[0155] Obviously, in the device MD, i.e., in the section 1 and/or
in the section 20, there can be provided also other electrical
devices, such as sensor means and/or actuator means and/or
electrical separation means, for example, obtained with MEMS
(microelectromechanical systems) technology or NEMS
(nanoelectromechanical systems) technology, as will emerge from
what follows: for such cases, the section 20 can be conveniently
provided with electrical-interconnection means. As already
mentioned, in one embodiment, at least one part of the section 20,
such as its bottom, can be constituted by a substrate made of
semiconductor material, for example silicon. Such a substrate can
integrate, in addition to the means 24, also devices obtained with
MEMS or NEMS technology.
[0156] In one embodiment the device, the section 1 and/or the
section 20, comprises a sensor or a device--hereinafter referred to
for reasons of brevity as "particle sensor" or "particle
counter"--configured for detecting and/or counting particles, such
as the target particles.
[0157] A particle counter of this sort may be of the type
comprising electrodes, in particular arranged along at least one
part of the path 3 and/or in substantial correspondence or
proximity of the outlet 5 and/or of at least part of the section
20, preferably electrodes in the proximity of or in contact with
the fluid. In one embodiment the particle counter comprises an
electrical circuit designed to detect electrical variations or
perturbations, such as variations of impedance or capacitance,
caused by the presence or passage of particles or cells in the
proximity of an active part of the electrical circuit, represented,
for example, by two detection electrodes. The particle counter can
possibly be of an optical type, for example provided with a
transmitter and a receiver of light radiation, in particular of the
type designed to detect an optical variation caused by the presence
or passage of at least one cell or particle. The particle sensor
could in any case be of some other type and/or designed to detect
also other physical quantities, in particular characteristics of
the particles, such as dimensions and/or shape.
[0158] Preferably, the means of the particle counter that are
designed to detect the passage of particles are located in an area
of passage or in a duct of capillary dimensions, indicatively
comprised between 2 and 100 .mu.m, in particular between 10 and 50
.mu.m, such as, for example, the terminal part of the path 3 or the
outlet 5 and/or the inlet portion or duct of the section 20. The
provision of a capillary passage or duct may prove useful for
aligning or setting in a row the target particles in order to
facilitate their detection.
[0159] In a preferred embodiment, as represented in FIG. 3, the
passageways 10a and 11a are substantially distributed in a
continuous way for at least a significant part of the entire length
of the respective lateral delimitations 10 and 11.
[0160] In the example illustrated, the lateral delimitation 10 is
basically constituted by a series of barriers or obstacles,
designated by 12 in FIG. 3A, separated from one another by the
passageways 10a. The shape of the obstacles 12 can be chosen
according to the shape to be assigned to the passageways 10a. For
example, in a particularly advantageous embodiment, the passageways
10a have at least one terminal stretch inclined in such a way that
the buffer leaving said passageways imparts on the flow in the
first path 3 a component of thrust that facilitates flow thereof
towards the outlet 5. Preferably, the passageways 10a have the
aforesaid terminal stretch inclined in such a way that the buffer
leaving said passageways imparts on the flow in the first path 3
also a component of thrust and/or a transverse component and/or a
turbulence and/or an irregular motion that will facilitate at least
in part flow thereof towards the separation means that include the
passageways 11a.
[0161] An example of said conformation is clearly visible, for
example, in FIG. 3A, where the obstacles 12 of the lateral
delimitation 10 are shaped in such a way as to define a terminal
stretch of the passageways 10a that is at least approximately
tangential, i.e., almost parallel or slightly inclined, with
respect to the main direction of flow in the path 3.
[0162] Preferably, the path 3 is without significant obstacles, in
order to facilitate flow of the blood from the inlet 4 towards the
outlet 5, albeit with relatively low speeds and pressures. The
mixed-flow microseparation device MD is hence preferably designed
to allow the blood to flow in a gentle way and without any
mechanical stresses at least in the path 3 and/or in the means
designed to separate and/or withhold the target particles.
[0163] As has been said, also in the case of a relatively large
width of the path 3, the passageways 10a of the mixing fluid, here
represented by the buffer, considerably reduces the risk of causing
passage of a fair amount of the biological fluid from the inlet 4
towards the outlet 5, without this reaching the separation means or
passageways 11a.
[0164] Also the lateral delimitation 11, in the example
represented, is constituted by a series of obstacles, designated by
13 in FIG. 3A, basically configured as vertical projections or
appendages, which define between them the passageways 11a. The
lateral delimitation 11 can hence be configured basically as a wall
provided with ducts or passages of predefined or calibrated
section.
[0165] In one embodiment, at least the passageways 11a comprise a
number of passageways that are differentiated from one another as
regards the dimensions of section or width, in particular a cross
section that increases from the inlet 4 to the outlet 5 of the
first path 3, as may be clearly seen, for example, in FIGS. 3 and
3A.
[0166] The more or less narrow width of the passageways 11a of the
lateral delimitation 11 depends upon the distance between the
obstacles 13 in a direction substantially parallel or inclined with
respect to the flow of the path 3.
[0167] Merely by way of indication, the distance between the
obstacles 13 of at least one stretch of the path 3, i.e., the width
of at least some passageways 11a, can be of between 2 and 8 .mu.m,
in order to enable outlet of erythrocytes when they are oriented
horizontally and/or vertically.
[0168] In one embodiment, at least the lateral delimitation 11
comprises a number of arrays of obstacles 13 and/or of passageways
11a, preferably substantially parallel to one another, as may be
clearly seen in FIGS. 3 and 3A. As has been said, the obstacles 13
define between them--in a direction substantially parallel or
inclined with respect to the flow of the path 3--the passageways
11a. Defined, instead, between the various arrays of obstacles 13
are intermediate channels, one of which is designated by 11b in
FIG. 3A. The lateral delimitation 11 may in each case comprise just
one array of obstacles 13, i.e., be without the aforesaid
intermediate channels 11b.
[0169] From FIGS. 3 and 3A it may likewise be noted that, in one
embodiment, the number of the arrays of obstacles 13 differs in
different portions of the lateral delimitation 11. In the example
illustrated, the lateral delimitation 11 has four portions (not
indicated). The first three portions starting from the left (with
reference to the figure) consist of three arrays of obstacles 13,
whereas the terminal portion, which is closest to the outlet 5,
consists of just two arrays of obstacles 13. The distance between
the obstacles 13 and between the arrays of obstacles of the various
portions increases, in the example, from the inlet 4 to the outlet
5.
[0170] Of course, the dimensions of the obstacles 12 and 13 and of
the passageways 10a and 11a, as well as the distances between to
the obstacles 12, 13 is chosen as a function of the dimensions of
the target particles that are to be withheld within the path 3
and/or according to the dimensions suitable for bestowing an
appropriate mechanical strength on the obstacles (in order to
confer a higher mechanical strength on the obstacles, these could,
for example, be wider than the ones represented, albeit given the
same width of the passageways, i.e., maintaining the same distance
between the obstacles).
[0171] It should moreover be noted that it is possible to provide
lateral delimitations 10 and/or 11 that are involved in just a part
thereof by respective passageways 10a and/or 11a, or else lateral
delimitations 10 and/or 11 that comprise sets of respective
passageways 10a and/or 11a distributed along the path 3, but set at
a distance from one another (i.e., lateral delimitations 10 and/or
11 that have, for example, at least one part without passageways
set between two parts provided with passageways). In embodiments of
this sort, the stretch or stretches of a lateral delimitation
provided with passageways may also be more or less staggered with
respect to the stretch or stretches of the opposite lateral
delimitation provided with passageways, and/or one or more
stretches of a lateral delimitation provided with passageways can
have a development in length different from that of one or more
stretches of the opposite lateral delimitation provided with
passageways.
[0172] Operation of the device MD, as regards separation of the
particles, is very simple and occurs according to the principle of
the mixed-flow separation already explained previously.
[0173] Delivered to the inlet 4, preferably in a continuous way, or
possibly in a pulsed way, is a sample of fluid to be analysed, such
as a blood sample already partially diluted with a buffer solution
and/or an anticoagulant, preferably with the fluid or blood under
slight pressure. Likewise, delivered to the inlet 6 is the buffer
LB, preferably in a continuous way, or possibly in a pulsed way,
and preferably at a pressure slightly higher than that of the
blood. If the use of beads BE is envisaged--as in the example--also
delivered to the inlet 6' is the corresponding buffer with beads,
preferably at a pressure similar to that of the buffer introduced
through the inlet 6.
[0174] The buffer at outlet from the passageways 10a forces the
flow of the biological fluid into the path 3, in part towards the
outlet 5 and in part towards the lateral delimitation 11. As has
been said, in this way, induced in the flow of the biological fluid
in the path 3 is a component of thrust and/or a transverse
component and/or a turbulence and/or an irregular motion of the
fluid itself.
[0175] Possibly the shapes and dimensions of the passageways 10a
and/or the pressure of the buffer are such as to induce also a
component of suction of the flow of the buffer entering the channel
3 due to the Venturi effect.
[0176] The buffer at outlet from the passageways 10a forces the
biological fluid and the particles contained in the flow of the
path 3 in part towards the lateral delimitation 11, so as to cause
particles other than the target particles to penetrate into the
passageways 11a and to pass into the path 9, and in part towards
the outlet 5, to cause the target particles to arrive at their
destination. In other words, the buffer forces the particles and/or
the fluid of the path 3 in such a way as to cause them to pass in
the proximity of the separation means represented by the
passageways 11a.
[0177] The fact that the section of the passageways 11a increases
along the development of the path 3 means that--tendentially--from
the flow passing in the path 3 there will be first evacuated the
particles of smaller dimensions and, proceeding along the path 3,
there will be evacuated particles of progressively larger
dimensions, but in any case of dimensions smaller than those of the
target particles.
[0178] The residual part of the blood-buffer solution not
eliminated through the outlet 7 reaches the inside of the section
20. This residual part is evidently enriched in target particles,
even though it can still contain particles that are not of
interest. The preponderant part of the solution hence exits from
the outlet 23 of the section 20, entraining along with it the
particles not withheld by the separation means 24, in particular,
those having dimensions smaller than the ones defined by the
passageways 24a. The target particles remain, instead, withheld
within the section 20, thanks to the separation means 24. This
sample enriched in the sub-population of target particles can
subsequently be subjected to analysis, according to techniques in
themselves known, even without having to extract the particles from
the device (for example, for an analysis of an optical type).
[0179] As mentioned previously, the channels or paths 3, 8 and 9
are formed in one and the same face of the body 2, which also
defines the corresponding bottom surfaces thereof. In one
embodiment, at least one of the aforesaid paths has at least two
stretches of different depth.
[0180] FIG. 3C illustrates such a variant in relation to the first
path 3 and to the third path 9. In said figure, it may in
particular be noted how a terminal portion of the bottom of the two
paths in question is lowered with respect to a corresponding
portion of bottom upstream. This stretch of bottom that is lowered
or with increased depth, which is substantially in common between
the two paths 3 and 9, is designated by 14. Its depth is preferably
greater than 30 .mu.m, more preferably, at least 100 .mu.m.
[0181] This measurement reduces the risk of clogging of the device,
considering the fact that, in the terminal stretch of the path 3,
the flow has a increased concentration of particles of larger
dimensions: in said area, in fact, there converge the beads BE,
which tend to bind to the cells or target particles TC, thus
increasing the dimensions thereof.
[0182] Moreover, a number of aggregates of particles--for example,
each comprising a tumour cell TC and a number of beads--could bind
to one another via one or more beads that function as "bridge", as
exemplified in FIG. 4B. In this situation there may form a sort of
lump, with consequent clogging of the device. The larger section of
passage determined by the presence of the stretch 14 having a
greater depth or lowered bottom limits the risks of clogging,
enabling outflow of lumps and preventing accumulation of particles.
To prevent formation of lumps also along the path it is
advantageous, as in the case represented in FIG. 1, to inject the
beads in a terminal part of the path 3.
[0183] It should be noted that the portion 14 with increased depth
preferably corresponds to a portion provided with passageways 11a
having a greater width, such as passageways 11a having a width
equal to or greater than 10 .mu.m. This entails the advantage of
enabling the use of a stronger mould, such as a mould with stronger
projections, i.e., the projections corresponding to the aforesaid
passageways 11a, given that said projections of the mould have
dimensions corresponding to the width and depth of the passageways
11a (hence a height greater than 30 .mu.m, but also a width equal
to or greater than 10 .mu.m). To a greater height of the
projections of the mould corresponds a larger width, and hence a
greater strength, preventing the risk of damage to the mould during
moulding of the body of the device.
[0184] As has been seen, according to one embodiment, the device MD
is configured for enabling introduction and/or passage of beads BE,
and envisages for the purpose a corresponding dedicated inlet 6'.
As has been said, in any case, the beads BE can be introduced
within the device MD exploiting the inlet 6 for the buffer, and
consequently the prevision of a dedicated inlet 6' and a dedicated
duct 8' is not indispensable. It will likewise be appreciated that
the beads BE--as likewise the buffer that constitutes the auxiliary
fluid--are introduced into the device MD separately from the flow
introduced into the first path 3, here the flow of blood,
preferably dispersed in another flow, here the flow of the buffer,
and that these bind to particles or target cells within the device
MD. As has been said, moreover, the device MD and the beads BE can
be configured in such a way that the beads can be subjected to
forces of attraction or repulsion by means of electrical fields in
order to separate particles.
[0185] According to a feature that constitutes an autonomous aspect
of the invention, the beads BE may be used in order to improve
mixing and/or the agitation of the fluid, such as the blood that
flows in the path 3. For example, the beads BE can be subjected to
forces of attraction or repulsion, designed to determine
displacements thereof in the fluid, with consequent agitation or
turbulence in the fluid itself, which--as has been
said--facilitates separation. It should be noted that the beads
aimed at such a use can also being without ligands or
antibodies.
[0186] FIGS. 5-11 are schematic representations of a biomedical
microfluidic device according to the invention, of a general
configuration slightly different from those of FIGS. 3, 3A, 3B, 3C,
but based upon the same principle of mixed-flow separation, of a
continuous or pulsed type. In FIGS. 5-11 there are hence used the
same reference numbers as those used in the previous figures to
indicate elements that are technically equivalent to the ones
already described. In these FIGS. 5-11 the collection section in
communication with the outlet 5 is not represented, as neither are
connector members at the inlets and outlets of the device.
[0187] As may be seen for example in FIG. 5, in this device the
three paths 3, 8 and 9, as the corresponding lateral delimitations
10 and 11, present a substantially rectilinear development, as in
the embodiment of FIGS. 3, 3A, 3B, 3C. Also in this case, the paths
and the lateral delimitations are formed in one and the same face
of the body 2. In the specific case, at said face, the body 2 has a
generally hollowed region, delimited laterally by two walls 2a, and
defined within said hollowed region are the paths 3, 8 and 9 with
the corresponding lateral delimitations 10 and 11.
[0188] Designated by 3a, 8a and 9a in FIG. 5 are the bottom
surfaces of the paths 3, 8 and 9. In a possible embodiment the
bottom 8a, 9a of at least one of the second and third paths 8, 9
has at least one stretch lowered with respect to the bottom 3a of
the path 3. At least one portion of the duct 8 and/or 9 can have,
for example, a depth or height greater than 100 .mu.m, such as a
height comprised between 500 .mu.m and 30000 .mu.m.
[0189] FIG. 5 highlights the specific case in which both the bottom
8a and the bottom 9a are lowered, throughout their development,
with respect to the bottom 3a, with the paths 8 and 9 that are
hence deeper than the path 3. This feature enables increase in the
flow rate of the buffer fluid and of the discharge fluid, at the
same time maintaining a contained width of the device.
[0190] In the example, the paths 8 and 9 have constant depth.
However, a variable depth is not ruled out, for example a path 8
for the buffer with a greater depth towards the inlet 6 and a
smaller depth towards the opposite end, and/or a discharge path 9
with a greater depth towards the outlet 7 and a smaller depth
towards the opposite end.
[0191] In one embodiment, at least a longitudinal portion of the
first path 3 has a decreasing cross section and is adjacent in
length to at least one of a longitudinal portion with decreasing
cross section of the second path 8 and a longitudinal portion with
increasing cross section of the third path 9. FIG. 5 highlights the
specific case of a path 3 that has a decreasing cross section
throughout its development, of a path 8 with a decreasing cross
section throughout its development, and of a third path 9 with an
increasing cross section throughout its development.
[0192] This embodiment, which must be understood as alternative or
complementary to the one for the lowered portion of the bottom
surfaces 8a and 9a, is useful for a better distribution of the
flows and/or of the corresponding pressures. Very schematically,
the particular decreasing shape of the path 8 tends to guarantee an
optimal flow rate and/or pressure in all the first passageways 10a;
also the particular decreasing shape of the path 3 tends to
guarantee an optimal flow rate and/or pressure in all the second
passageways 11a. The increasing shape of the path 9 tends, instead,
to facilitate outflow of the discharge substances and/or to prevent
anomalous distributions or increases in the pressure at outlet.
[0193] The shape of the path 9 could, however, be different, in the
perspective of facilitating outflow of the discharge substances and
preventing anomalous distributions or increases in the pressure at
outlet, in particular to prevent any return of fluid from the path
9 to the path 3.
[0194] Consider that, preferably, at least the paths 3 and 9 are
designed to enable maintenance of a pressure in the path 3 that is
slightly higher than the pressure of the discharge duct 9, in
particular in order to have a minimal difference of pressure, i.e.,
such as not to induce a damage to the cells or particles, at the
same time preventing a return of fluid from the path 9 to the path
3: this is made possible by using the mixed-flow micro-separation
system, which, as has already been said, enables use of low fluid
pressures.
[0195] The decreasing or narrowing shape of the path 3 is moreover
suited for the aforesaid integration of a particle counter or
particle sensor, i.e., for the definition thereon of appropriate
capillary ducts, preferably in the stretch in the proximity of the
outlet 5 or corresponding thereto.
[0196] Once again in FIG. 5, designated by 111, 112, 113 and 114
are different portions of the lateral delimitation 11, each
distinguished by a different cross section of the respective
passageways 11a and/or by a different number of arrays of obstacles
13 (see also FIGS. 7-9).
[0197] As has been explained previously, in fact, the lateral
delimitation 11, or its portions, can be formed by a number of
arrays of obstacles 13. In the case represented in FIGS. 5-11, for
example, the portions of lateral delimitation designated by 111,
112 and 113 (FIG. 7) and 114 (FIG. 11) consist respectively of four
arrays, three arrays, two arrays, and one array of obstacles 13, it
being possible, however, for each portion of lateral delimitation
111, 112, 113 and 114 to comprise even just one array of obstacles
13. As has been said, moreover, the cross section of the
passageways 11a can decrease from the inlet 3 to the outlet 5 of
the path 3.
[0198] In one embodiment, at least one first portion of lateral
delimitation, for example the portion 111, defines respective
passageways 11a, having a calibrated section of a width preferably
of between 2 and 8 .mu.m, where in particular said first portion of
lateral delimitation extends for at least one third of the length
of the entire duct 3 for the fluid; preferably, said first portion
of lateral delimitation defines respective passageways 11a designed
to allow erythrocytes to pass.
[0199] In one embodiment, at least one second portion of lateral
delimitation, for example one of the portions 112-114, defines
respective passageways 11a, having a calibrated section of a width
preferably of between 2.1 and 25 in particular between 3 and 13
Preferably, said second portion of lateral delimitation defines
respective passageways 11a designed to allow at least one type of
leukocytes to pass.
[0200] In one embodiment, at least one third portion of lateral
delimitation, for example one of the portions 113-114, defines
respective passageways 11a, having a calibrated section of a width
preferably larger than 5 .mu.m, in particular between 6 and 25
.mu.m. Preferably, said third portion of lateral delimitation
defines respective passageways 11a, 5 designed to allow at least
one type of target particles or tumour cells to pass.
[0201] Also visible in FIG. 9 are the intermediate channels 11b
defined between various arrays of obstacles 13. The presence of
intermediate channels 11a can prove useful to bring back into the
path 3 any possible particles of interest that have accidentally
passed beyond the first array of obstacles 13, but not the
subsequent array or arrays.
[0202] Once again in FIG. 9, it may be noted how, in one
embodiment, obstacles 13 of the lateral delimitation 11 can have a
lateral profile that narrows from the first path 3 to the third
path 9. In this way, the passageways 11a have a generally flared or
divergent shape in the direction the channel 9, and this reduces
the risk of adhesion of particles, i.e., the risk of clogging. In
said embodiment, it may be noted that the obstacles 13 of the
lateral delimitation 11 can have a substantially trapezial shape,
i.e., side walls inclined with respect to one another, in
particular in order to provide passageways 11a having a variable
cross section. Preferably, the second passageways 11a have a first
inlet section, substantially facing the path 3, having a predefined
or calibrated section in order to enable passage or otherwise of
given particles or cells present in the fluid. The opposite end of
said second passageways 11a, i.e., the end facing the discharge
channel 9, has, instead, a larger cross section, in particular in
order to facilitate the outflow of the particles towards the
discharge and/or to prevent risks of clogging of the passageways
11a. The passageways 11a preferably have a rounded profile or edges
at least in the aforesaid first inlet section, substantially facing
the path 3, in particular in order to prevent damage to the cells
or particles that circulate in the path 3 itself.
[0203] Partially visible in FIG. 6 is an upper closing body or lid
26 of the device MD, designed to delimit the paths 3, 8 and 9 at
the top. In the example illustrated, the lid is substantially
planar, preferably made of a transparent material, for example a
plastic or glass, and is applied in a fluid-tight way, for example
via gluing or sealing, on the upper surface of the walls 2a (FIG.
6). In one embodiment (not illustrated), the lid 26 can be of an
openable or removable type.
[0204] In one embodiment, at least one of the lateral delimitations
10 and 11 integrates sealing elements, which are bound to
co-operate with the lid 26. Advantageously, at least some of these
sealing means are provided integral with the lateral delimitations
10 and 11, and more in particular, with the obstacles 12 and 13
that constitute them. Preferably, the sealing means referred to
above have a shape or are made of a material at least in part
yielding or deformable. Even more preferably, at least part of the
body of the device according to the invention, for example at least
the lateral delimitations 10 and 11 and/or the aforesaid sealing
means, are made of elastic or elastomer material, such as a
silicone or a PDMS (polydimethylsiloxane) or a polysiloxane. In
particular, the aforesaid sealing means are made of a single piece
or integrated with at least part of the body of the device and/or
are made of the same material, preferably integrated or made of a
single piece with the lateral delimitations 10 and 11.
Additionally, as is visible, the lateral dikes 10 and/or 11 are
preferably formed integrally or as a single piece with at least
part of the body of the device and/or made of the same
material.
[0205] From FIG. 9 it may be noted how, in one embodiment, the
obstacles 13 are provided at the top with appendages 13a,
preferably having a progressively decreasing thickness, such as a
substantially triangular or trapezial or semicircular cross
section, i.e., having a shape designed to provide respective
predefined-deformation seal lips, which are designed to co-operate
with the inner surface of the lid 26. Likewise, as may be seen in
FIG. 10, in the obstacles 12 that constitute the lateral
delimitation 10 there may be integrally made respective upper lips
12a, preferably predefined-deformation ones, in particular having a
progressively decreasing thickness, such as a substantially
triangular or trapezial or semicircular cross section, that are
designed to co-operate in a fluid-tight way with the lid 26.
[0206] The seal lips or projections 12a and 13a preferably have a
shape such as to be mouldable with movements of the mould in a
single direction, i.e., without any undercut, said direction being
preferably the same as direction of moulding of the lateral
delimitations 10 and 11 and/or of the body 2. The sealing elements
12a guarantee that the flow of the buffer fluid will be oriented in
the desired direction, preventing any leakage from the sides.
[0207] The obstacles 12 and 13 preferably have a cross section or
dimensions larger than those of the sealing elements 12a and 13a,
in particular in order to enable the aforesaid calibrated
deformation of the sealing elements 12a, 13a without causing any
significant deformation of the respective obstacles 12, 13 and
hence prevent alterations or deformations of the calibrated
passageways 10a and/or 11a. It follows that, even in the presence
of stresses and deformations of a part of the aforesaid obstacles,
such as the upper sealing portion integrated therein, the
calibrated width of the passageways, and in particular of the
passageways 11a, remains in any case intact. As may be noted for
example in FIG. 9, each fluid-tight element 13a extends between two
adjacent passageways 11a and comprises two lateral end edges
substantially coinciding with the walls of the passageways 11a
defined by the obstacles 13; the lateral edges of two adjacent
elements 13a are preferably set at a distance apart as are the
obstacles 13. During compressive deformation of the sealing
elements 13a, the aforesaid lateral edges could undergo
deformation, but the calibrated section of passage of the
passageways 11a remains in any case guaranteed thanks to the main
body of the underlying obstacles 13, which have a structure such as
to not allow deformation in the course of the aforesaid
compression.
[0208] In the example of FIGS. 5-11, the sealing elements 13a are
substantially oriented in the direction of the path 3; i.e., they
are substantially transverse to the passageways 11a. Likewise, the
sealing elements 12a are at least in part substantially oriented in
the direction of the path 3 and/or of the path 8, and in part set
alongside the passageways 10a, preferably extending at least along
a side of a fair share of the respective passageway 10a.
[0209] Sealing elements made in a way similar to the ones
previously described are preferably present also in other parts of
the device, such as the outer perimeter of the body 2 or the
perimeter of the various channels or paths, preferably made of a
single piece or integrated in the body 2 of the section 1. Also
these further sealing elements are configured to prevent any
leakage of fluid towards the outside of the device and/or between
the different paths. Elements of this type, configured preferably
as seal lips or projections, are designated by SM, for example, in
FIGS. 8 and 9.
[0210] In a preferred embodiment, in particular in a device with a
mixed-flow separation structure, the sections of passage of the
paths 3, 8 and 9 and of the passageways 10a and 11a are sized in
such a way that: [0211] given the same pressure, the flow rate of
the fluid of the path 8 is higher than the sum of the individual
flow rates of the passageways 10a, and/or [0212] given the same
pressure, the sum of the individual flow rates of the passageways
10a is greater than the sum of the individual flow rates of the
passageways 11a, and/or [0213] given the same pressure, the sum of
the individual flow rates of the passageways 10a and of the flow
rate of the fluid in the path 3 is higher than the sum of the
individual flow rates of the passageways 11a.
[0214] To clarify the above concept, FIGS. 12-14 illustrate
portions already highlighted above of the device MD of FIGS. 5-11,
with the further indication of some of the sections of passage of
interest. In particular, in said figures, Ss is the section of the
path 3, Sb is the section of the path 8, Sb1, Sb2, Sb3 . . . are
the sections of the passageways 10a, Su is the section of the path
9 and Ss1, Ss2, Ss3 . . . are the sections of the passageways
11a.
[0215] In order to prevent any reflux from the path 3 towards the
path 8, i.e., to guarantee a uniform flow rate in the passageways
10a, it is preferable for the section of the path 8 to be larger
than the summation of the sections of the passageways 10a, i.e.:
Sb>Sb1+Sb2+Sb3+Sbn.
[0216] Likewise, in order to prevent any reflux from the path 9 to
the path 3, i.e., to guarantee a uniform flow rate in the
passageways 11a, it is preferable for the sum of the cross section
of the path 3 and of the passageways 10a to be greater than the
summation of the sections of the passageways 11a, i.e.:
(Ss+(Sb1+Sb2+Sb3+ . . . Sbn))>Ss1+Ss2+Ss3+ . . . Ssn.
[0217] The above considerations apply in general, given the same
pressure, since in this case what carries the most weight is the
section of the paths and of the passageways: formulas similar to
the ones indicated could, on the other hand, refer also to the flow
rates of the paths and of the passageways.
[0218] Preferably, in any case, the pressure (P8) in the path 8
will be slightly higher than the pressure (P3) in the path 3, which
in turn will be slightly higher than the pressure P9 in the
discharge path 9, i.e.: P8>P3>P9. In this way, it is possible
to prevent any fluid present in the path 3 from flowing back into
the path 8 and, in the same way, prevent any fluid present in the
path 9 from flowing back into the path 3. Said formulas or
preferential conditions may be preferably referred also to portions
or sections of the device described, such as for example a first
half or a second half of the device; this is in particular obtained
via the use of said variable dimensions of the paths and
passageways.
[0219] FIG. 15 regards an embodiment in which the body 2 is set
between a lid 26 and a lower body 30. The presence of the lower
body 30 can be useful for stiffening the device as a whole--in
particular when the body 2 is made of silicone material or some
other elastomer--and bestowing a precise planarity for requirements
of fluid-tightness. The body 30 is preferably made of a relatively
rigid material, such as a plastic material, a metal material, a
glass-reinforced plastic, a ceramic material, etc.
[0220] In one embodiment, associated to the lower body 30 are at
least two electrodes, and in particular a cathode 31 and an anode
32, via which it is possible to apply an electrical field to the
fluid that flows in the path 3, which is designed to induce a
displacement of particles of the blood and/or of beads BE present
in the flow. The electrodes 31, 32, which in the case exemplified
are rectilinear, can be used for example to facilitate displacement
of particles from the path 3 to the path 9 via techniques of
electrophoresis of an insulated type (without contact with the
fluid) or of electrophoresis or of the aforesaid attraction and/or
repulsion of beads BE, such as beads BE associated to particles or
cells TC. As has been said, an attraction or repulsion of beads,
performed via appropriate excitation means, can also be used for
inducing agitation or turbulence aimed at improving displacement of
the fluid towards the lateral delimitation 11 provided with the
passageways 11a.
[0221] In the embodiment illustrated, in the assembled condition of
the bodies 2 and 30, the electrodes 31 and 32 are set substantially
corresponding to the lateral delimitations 10 and 11, respectively.
Preferably, the electrodes extend parallel to the lateral
delimitations, but with the electrode 32 lying underneath the
bottom 3a of the path 3 and the electrode 31 lying underneath at
least part of the bottom of the path 9.
[0222] It should be noted that the electrodes could involve only
part of the path 3 and of the path 9, for example a terminal
portion thereof. Possibly, electrodes for electrophoresis or
dielectrophoresis or separation of beads BE could be provided in
the collection section 20.
[0223] FIGS. 16-19 are schematic illustrations of a biomedical
microfluidic device according to the invention, of a general
configuration similar to that of FIGS. 5-11 and provided with the
collection section 20. Hence, also in FIGS. 16-19 the same
reference numbers are used as those of the previous figures to
designate elements that are technically equivalent to the ones
already described.
[0224] From FIGS. 16 and 17 it may be noted how, in one embodiment,
the lid 26 can include a respective portion 26a that functions as
top closing also for the collection section. In said embodiment,
the body of the lid 26 is preferably made of transparent material,
such as glass or transparent rigid plastic. It should be noted that
the lid 26a can also be configured as a distinct part with respect
to the lid 26, possibly of an openable or removable type, for
example for taking out target particles and/or for introducing into
the section 20 a culture medium.
[0225] In one embodiment, the body 2 has a first face and a second
face, defining a thickness of the body itself, and at least one of
the inlets and the outlets of the device includes a duct that
extends between the two aforesaid faces. FIGS. 16 and 17 represent
the specific case in which the inlet 4, the at least one inlet 6,
6', and the at least one outlet 7 are configured as through holes
or ducts of the body 2, which open at the top end and,
respectively, at the bottom 3a, 8a and 9a of the paths 3, 8 and 9.
In the specific case, moreover, also the outlet 23 of the section
20 includes a similar through hole or duct formed in the collection
body 21. In the exemplified embodiment, moreover, the aforesaid
inlets and/or outlets are in communication with corresponding
hydraulic attachments or connectors 4b, 6b, 23b, which can be
conveniently formed integral with the body 2 and/or 21; also the
outlet 7 is conveniently connected to a similar connector, not
visible in the figure.
[0226] The presence of the aforesaid connectors, preferably in a
lower position, facilitates connection of the device MD, for
example on a laboratory apparatus designed for circulation of the
fluids and/or analysis of the target particles in the section 21,
as well as enabling containment of the lateral overall dimensions
of the device itself.
[0227] In the embodiment of FIGS. 16 and 17 end walls 2a' are
provided (see FIG. 17) that close the paths 3, 8 and 9 at the
longitudinal ends.
[0228] In one embodiment, a number of inlets 6, 6' for the liquid
buffer are provided, positioned in different points along the
development of the corresponding path 8. FIG. 17 illustrates the
specific case of three inlets set along the path, of which one
inlet 6 is in its initial area, one inlet 6 is in its intermediate
area, and one inlet 6' is in its terminal area. This arrangement is
useful in the case of a relatively narrow path 8, that is shallow
and long, i.e., with as small section, in order to guarantee a
uniform flow rate of the buffer in the path itself and to prevent
areas of negative pressure or non-uniform flow rate, which might
cause possible reflux of blood into the path 8. FIG. 17 moreover
illustrates the case of a path 8 for the buffer having a
substantially constant cross section. As has been explained
previously, it is moreover preferable to introduce buffer
containing beads in a terminal stretch of the path 3, whence the
positioning the inlet 6' in the terminal stretch of the path 8 as
represented in FIG. 17. In this configuration, from the inlets 6
there could be introduced buffer, whereas from the inlet 6' there
could be introduced a buffer added with beads.
[0229] Visible in greater detail in FIGS. 18 and 19 is the
collection section 20, with the corresponding area of connection to
the path 3. In this case, the mechanical-separation means 24 extend
substantially orthogonal to the flow. Also in this embodiment the
obstacles 25 preferably integrate sealing elements in the form of
upper lips or projections, having functions similar to the lips
previously designated by 12a and 13a; moreover visible are
stretches of elements or lips of the type already designated
previously by SM.
[0230] FIGS. 20-28 are schematic illustrations of another
biomedical microfluidic device according to the invention, having a
layout of the corresponding paths different from that of the
embodiments of the previous figures, but based in any case on the
same working principle. Also in FIGS. 20-28 the same reference
numbers are used as those used in the previous figures to designate
elements that are technically equivalent to the ones already
described.
[0231] Also in this embodiment, the section 1 of the device
preferably comprises a main body 2, defined in which are the
microfluidic paths, as well as a lid 26. The device MD comprises a
collection section 20, with the corresponding body 21 integral to
the body 2 or configured as distinct part with respect thereto.
Likewise, the lid 26a of the section 20 can be constituted by a
portion of the lid 26 or be distinct with respect thereto.
[0232] In one embodiment, the first path 3, the second path 8, and
the third path 9 present a development that is at least in part
substantially spiral-shaped or wound on itself, or in any case
preferably have at least in part a curved development. Said shape
or development of the paths is principally aimed at containing the
overall dimensions of the device.
[0233] FIG. 22 illustrates a configuration of the above sort, where
the paths in question are defined on a face of the body 2, with the
inlets 4, 6 and the outlet 7 that include a through duct or hole
between the two faces of the body 2. Likewise, the outlet 23
includes a through hole of the body 21 of the section 20. Also in
this embodiment lower hydraulic attachments or connectors 4b, 6b,
23b are envisaged, which can be conveniently formed integral with
the body 2 and/or 21. In FIGS. 20-22 a similar connector 7b is also
visible, in communication with the outlet 7.
[0234] In one embodiment, moreover, the three paths 3, 8 and 9 have
a main stretch that is substantially spiral-shaped or wound on
itself, or at least in part curved, and at least one of them has a
substantially rectilinear stretch. Also said shape or development
of the paths is basically provided for containing the overall
dimensions of the device.
[0235] This specific case is illustrated in FIG. 22, where all
three paths have at least one respective substantially rectilinear
stretch at the corresponding initial and/or terminal portions.
[0236] With reference to a preferred embodiment, the first inlet 4,
for the blood, and the second inlet 6, for the buffer, are located
in a region of the body 2 around which the first, second, and third
ducts 3, 8 and 9 develop in a spiral, i.e., are wound along a
curved path, preferably number of times on themselves. As may be
noted, the example of embodiment of FIG. 22 also presents said
preferential characteristic, which enables simplification and
reduction of the dimensions of the device MD as a whole, in
particular enabling reduction of the overall dimensions of
relatively long paths, albeit maintaining a relatively constant
profile along the entire path. Consider, however that the paths of
the device according to the invention could also have a different
development, albeit suited for reducing the overall dimensions
thereof, such as a substantially S-shaped development, or else a
development wound along a substantially square or rectangular path,
preferably with rounded corners. In this case, the development of
the paths would present respective alternations of rectilinear
stretches and curved stretches.
[0237] It should be emphasized that in the devices considered
herein the curved or spiral development of the paths is not
exploited for inducing phenomena of separation by centrifugal
force. As explained previously, in fact, in the case of the present
invention, flows at low speed are preferably adopted to prevent
damage to the particles.
[0238] The device according to the invention preferably envisages
relatively long paths. In particular, a preferred length of the
paths 3, 8, 9 is at least 50 times the average width of the path 3;
more preferably, the length of the paths 3, 8, 9 is more than 200
times the average width of the path 3.
[0239] In other embodiments, once again in the case of a spiral or
wound development of the paths, the inlets for the fluids, such as
the inlets 4 and/or 6, can be made on the periphery of the spiral
(for example, in positions the like the ones of the outlets 5 and 7
of FIG. 22), whereas the corresponding outlets, such as the outlets
5 and/or 7 can be made in the central region of the spiral (for
example, in positions like the ones of the inlets 3 and 4 of FIG.
22). In said perspective, it is also possible to provide a
configuration with the inlet 6 or 6' for the buffer "opposed" to
the inlet 4 for the blood, i.e., with an inlet 4 at the centre of
the spiral and an inlet 6 or 6' on the outer perimeter.
[0240] Visible in the details represented in FIGS. 23-26 are the
various paths, where the path 3 is adjacent in length to the paths
8 and 9 and is laterally delimited with respect to them by the
lateral delimitations 10 and 11, with the corresponding passageways
10a and 11a and the obstacles 12 and 13. In this embodiment, walls
2a'' are provided that delimit laterally the ensemble of the three
paths and that have a development that is for the most part
spiral-shaped. Also the walls 2a'' can be provided at the top with
seal elements or lips having functions similar to those of the
elements previously designated by SM.
[0241] From FIG. 25 it may be noted in particular how the path 8 of
the buffer is delimited in length between a wall 2a'' and the
lateral delimitation 10, whilst the discharge path 9 is delimited
in length between the lateral delimitation 11 and a wall 2a'' wound
in a spiral.
[0242] Also in this embodiment the sections of the various paths
are preferably variable, and in particular the cross sections of
the paths 3 and 8 narrow from the inlet 4 to the outlet 5, whereas
the cross section of the path 9 widens progressively.
[0243] Shown in the non-limiting example illustrated is a spiral
configuration with uniform development, i.e., with a constant total
width given by the set of the three paths 3, 8, 9. In possible
embodiments, not shown, however, there may be envisaged windings in
a spiral of a different type, in particular with non-uniform
development, for example similar to a hyperbolic or logarithmic
spiral, or to the case where paths with variable sections are wound
in a spiral (like the paths 3, 8 and 9 of a device of the type
shown in FIG. 17, where the total width of the sum of the paths 3,
8 and 9 is not constant, and in fact increases from the end having
the inlet 4 to the end having the outlet 5).
[0244] The distance between the obstacles 13 can likewise increase
in order to define passageways 11a of cross section increasing from
the inlet 4 to the outlet 5.
[0245] The solution with paths wound in a spiral enables
containment of the dimensions of the device MD, at the same time
guaranteeing the presence of sufficiently long paths for treatment
(i.e., it enables, also given the same dimensions with respect to a
body configured as in FIGS. 3 to 19, paths 3, 8 and 9 that are
decidedly longer to be provided).
[0246] The at least in part curved development of the paths tends
to keep the larger particles along the path 3, preventing them from
being pushed excessively towards the lateral delimitation 11, with
the risk of getting stuck in the passageways 11a. In fact, the at
least in part curved development of the paths, together with the
fact that the obstacles 12 of the lateral delimitation 10 are
shaped so as to define a terminal stretch of the passageways 10a
that is at least approximately tangential or slightly inclined with
respect to the main direction of the flow in the path 3, enables a
thrust to be impressed on the particles in a direction that is
preferably substantially tangential to the lateral delimitation 11,
hence facilitating flow of the larger particles along the duct 3
also in the proximity of the passageways 11a, as shown
schematically in FIG. 26, where it may be noted that the buffer LB
that exits from the ducts 10a, tends to push the particles towards
the lateral delimitation 11, albeit in a direction substantially
tangential or very inclined with respect thereto.
[0247] With this configuration, certain particles (the smaller
ones) pass more easily in the passageways 11a, which have a width
such as to let them through, whereas other particles (the larger
ones) proceed along the path 3 just as easily, being kept along the
stretches of the lateral delimitation 11 provided with passageways
11a that have a width smaller than in the previous stretching, it
thus being possible for these particles to flow more easily along
the path 3 towards the outlet 5.
[0248] Consider in this regard, that, in the example of embodiment
exemplified, the passageways 11a are set substantially facing the
inside of the spiral, i.e., the flow containing the particles or
cells to be rejected moves from the channel 3 to the channel 9 in a
direction substantially from the outside to the inside of the
spiral, and hence in contrast with the centrifugal force. For this
purpose, in the embodiment exemplified, the flows preferably have a
speed such as to prevent or contain effects due to centrifugal
forces. As in the previous examples, the particles and/or the flow
of the first fluid (blood) in the channel 3 are/is subject to the
action of the auxiliary fluid (buffer) of the channel 8, which
imparts on the flow in the channel 3 also a component of thrust
and/or a transverse component and/or a turbulence and/or an
irregular motion that facilitates at least in part flow or passage
thereof towards the separation means represented by the passageways
11a.
[0249] Visible in FIGS. 27-29 is the region of union between the
body 2 and the body 21. In FIG. 29 it may be noted in particular
how, in one embodiment, both the path 3 of the blood and the path 8
of the buffer are in communication with the inlet 22 of the
collection section 20. In this figure it may moreover be noted how
the lateral delimitation 10 and the path 8 can extend as far as
within the section 20. In this way, the buffer can be used to
improve the movement of the flow of blood and/or of the target
particles also for the purposes of final filtering or separation,
via the separation means 24, of the blood that arrives in the
section 20 through the path 3. In FIG. 27 there may moreover be
noted a terminal portion of the path 3 having a capillary section
of passage, in particular in order to improve the flow of cells or
target particles and/or enable improved implementation of the
possible means for detection of the particles, such as a particle
counter and/or a device for aligning or displacing the
particles.
[0250] FIGS. 30 and 31 are perspective views of a body 2 according
to a variant of the device of FIGS. 20-29. The variant in question
is basically distinguished by the presence of the inlet 6' for a
buffer containing beads, as described above with reference to
previous embodiments. In this case, also the inlet 6' is basically
configured as through duct or hole between the two main faces of
the body 2, with a corresponding hydraulic connector 6b'. It should
be noted, in particular from FIG. 31, how in this case also the
path 8' for the buffer containing the beads will have an initial
stretch that is substantially rectilinear, a curved stretch
adjacent in length to the path 3 of the blood, and a final stretch,
once again rectilinear, which extends as far as within the
collection section 20. The path 8, for just the buffer, terminates
substantially in an area corresponding to the aforesaid initial
rectilinear stretch of the path 8'. In particular, the flow of
buffer in the path 8' is substantially the continuation of the flow
of buffer in the path 8, i.e., the two flows of buffer in the paths
8 and 8' are in series with respect to one another or continuous in
order to maintain a uniform flow or action or thrust on the flow of
liquid or particles in the path 3.
[0251] FIGS. 32-34 are schematic representations of another example
of microfluidic device according to the invention, having a
configuration substantially similar to that of the devices of FIGS.
20-29 and 30-31.
[0252] In one embodiment, the collection section 20 is configured
as element that can be separated from the body 2 in which the path
3 is defined.
[0253] As already mentioned, the body 21 of the section 20 can be
configured as a component distinct from the body 2, mechanically
and hydraulically coupled thereto in a separable way, or else be
constituted by a portion of the body 22, which is formed integral
with a second portion of the body 22.
[0254] The device MD of FIGS. 32-34 is obtained according to the
second case referred to above, with the body 22 that is
pre-arranged for enabling separation between the two aforesaid
portions of body, in particular via breaking or cutting.
[0255] From the figures it may be noted how, in the example shown,
the region "I" of interface or union between the main portion of
the body 22 and its auxiliary portion that defines the body 21 of
the section 20, has weakening, or pre-breaking, means 2b such as a
thinned region or a score line. In the example shown, similar
weakening means 2b are envisaged, such as a thinned region or a
score line, also in the region of interface or union between the
body of the lid 26 and its portion that defines the lid 26a of the
collection section 20.
[0256] In this way, as may be appreciated, the entire section 20
can be easily separated from the rest of the body 2 and of the lid
26, for example by breaking or cutting, in particular for the
purposes of subsequent use in analysis or laboratory activities,
such as a use on an apparatus of analysis (for example, a
microscope or a optical detection system), or else for a culture of
the target cells thus separated. This arrangement or structure,
then, in addition to simplifying production of the device,
facilitates practical use thereof.
[0257] In one embodiment, the body 2 and/or the collection section
20 have capillary ducts or of a reduced cross section in the
cutting or breaking area, it being possible for said ducts to be
closed mechanically via a deformation of the material of the body 2
or 21, for example made of thermoplastic material. In the aforesaid
area these ducts can for example be configured for being squeezed
or deformed following upon cutting or breaking in order to
substantially close the ducts themselves or else reduce the port
thereof, forming a sort of lemniscate, and thus prevent leakage
thanks to the adhesion between the molecules of the fluid and those
of the material constituting the body 2 or 21.
[0258] In one embodiment, the microfluidic device according to the
invention comprises identification means. These identification
means can, for example, be provided on the main body 2, or on the
body 21 of the section 20, or else again on the corresponding lids
25 and/or 25a, or on the lower body 30 (FIG. 15), for example in
the form of labels, or else be printed with a suitable ink, or else
be formed integrally in said bodies and/or lids. FIG. 35 regards
this second case, and visible therein is a portion of the body 2,
integrally defined in which is a barcode BC. A similar barcode can
be provided, as has been said, also in the body 21 and/or in the
lid 26 and/or in the lid 26a.
[0259] The barcode may be obtained for example via printing,
deformation, or engraving on at least a part of the body of the
device MD, such as the body 2, the body 21, the lid 26, the lid
26a. In this case, the barcode BC can be obtained by providing
recesses and/or reliefs on the aforesaid part of body, where each
recess and/or relief has a shape corresponding to a corresponding
bar of the barcode BC.
[0260] In one embodiment, the aforesaid body has, in a point
corresponding to a bar of the barcode BC, a thickness smaller than
the thickness of areas adjacent to the bars, in such a way that the
material that forms the bars can be more easily traversed by an
optical ray (basically areas that are transparent and
non-transparent to the optical ray are obtained), for the purposes
of detection of the barcode BC. In another embodiment, the recesses
and/or reliefs are rendered reflecting, for example via reflecting
paint or metal inserts, or else are coloured and rendered opaque,
with respect to the adjacent areas of the body, which could instead
be transparent or reflecting.
[0261] In other embodiments, the identification means can comprise
an electronic device provided with memory means, such as an RFID
device, in particular a device designed to receive and transmit
signals and/or data in wired or wireless mode, of the type
comprising at least one antenna and/or an appropriate electronic
circuit (an example of such an embodiment will be described
hereinafter with reference to FIG. 77).
[0262] Irrespective of practical implementation, in one embodiment,
the identification means contain calibration data or parameters of
an apparatus of analysis, on which the device MD or the section 20
are used, such as data or parameters predefined during production
of the device according to the invention, in particular designed to
enable recognition of the device and/or to provide information to
the apparatus of analysis.
[0263] In addition or as an alternative, the identification means
can be of a type designed to enable modification of the aforesaid
data or parameters during at least one step of the cycle of use of
the device, for example, via writing of data in a memory of the
device, and in particular can contain information identifying the
subject to whom the biological fluid belongs and/or the type of
sample of fluid and/or the type of analysis to which the sample is
to be subjected.
[0264] The identification means can then serve to identify the
device MD on the basis of the type of measurement for which it is
pre-arranged. In this perspective, the apparatus of analysis can
advantageously be configured for detecting automatically the
identification information, for the purposes of self-calibration
and/or for pre-selecting automatically the operation of the
apparatus according to the type of device MD (considering the fact
that there could exist a number of types of devices MD, for
example, ones optimized in terms of shape on the basis of the type
of cell to be selected). Such an identification system also enables
the control system of the apparatus of analysis to signal a
possible erroneous mounting of a device MD, for example, not
corresponding to the type of process selected on the apparatus, as
in the case of a test selected by the user via a PC program not
corresponding to the type of device mounted on the instrument of
analysis.
[0265] FIGS. 36-39 are schematic representations of another example
of microfluidic device according to the invention, of a
configuration substantially similar to that of the devices of FIGS.
20-29 and 20-31, in particular a device with mixed-flow separation
structure. Also in these figures the same reference numbers are
used as the ones already used above to designate elements that are
technically equivalent.
[0266] According to the embodiment of FIGS. 36-39 (where the lid of
the device has not been represented), the body 2 has a
substantially circular shape. The collection section 20 is moreover
directly integrated in the body 2.
[0267] This configuration of the body 2, which could in any case
have a shape different from the circular shape, enables placing of
the device, possibly without the corresponding lid, on standard
supports of analysis used in the bio-technological sector, such as
in particular supports of the type known as "Petri dishes" and the
like, typically having a cylindrical shape. Alternatively, the
device according to the invention can be shaped so that it can be
housed in devices or on supports having some other shape, such as
Petri dishes of some other type, for example of a squared shape.
According to a preferential variant, then, at least a part of the
device according to the invention can have a shape similar to or
congruent with standard supports of analysis used in the
bio-technological sector, such as in particular supports of the
Petri-dish type and the like.
[0268] This feature facilitates certain operations, such as
operations of analysis using standard apparatuses already
pre-arranged for the aforesaid supports, such as optical viewing
devices (microscopes), or operations of storage, for example using
apparatuses already prearranged for said supports.
[0269] Said configuration can be particularly advantageous also for
the purposes of possible use of the device MD for cell cultures. In
such a case the device will be provided, preferably in its section
20, with an inlet or attachment for introduction of a culture
medium, as well as at least one aeration duct (not shown).
[0270] In the case where an aeration opening is necessary, this
will preferably be provided with sealing means that, whilst
guaranteeing passage of air, oppose infiltration of foreign
substances, such as bacteria. These sealing means can include an
air-permeable membrane with calibrated porosity, for example made
of Goretex.RTM. or similar microporous material. Furthermore, as
will emerge hereinafter, the device according to the invention may
be provided with valve means between the body 2 and the section 20,
and said valve means can advantageously be exploited for the
introduction of the aforesaid culture medium. The device according
to the invention can also be provided with valve means on the
inlets of the body 2, and said valve means can advantageously be
exploited to prevent any reflux and/or to enable introduction of
fluids--also in a pulsed and/or alternating way--without any reflux
by the body 2.
[0271] FIG. 36 illustrates the case of a device MD provided with an
inlet 6' and a path 8' for a buffer containing beads, but said
elements could of course be omitted, in so far as they are not
essential.
[0272] In one embodiment, the collection section 20 comprises a
terminal stretch of the path 3, defined in the main body 2. This
characteristic is included, for example, in the device of FIG. 36
and is visible in particular in the details represented in FIGS.
37-39. In such an embodiment, the section 20 is not separable or
distinct from the body 2, but it nevertheless proves useful, both
when it is envisaged in devices of a circular configuration (for
example, for positioning in Petri dishes), or also in devices of
another shape (for example, rectangular), to enable a more
convenient positioning on instruments of analysis, such as a
microscope.
[0273] In particular from FIG. 39 it emerges how, also in this
case, the path 3 gives out inside the collection section 20, with
the lateral delimitation 10 and the path 8 that extend within the
section itself.
[0274] In one embodiment, or in any case according to a feature
that constitutes an autonomous aspect of the invention, at least
one portion of the collection section 20 is shaped in such a way as
to convey the target cells or particles into a restricted and/or
predefined area, in particular in order to improve the
concentration thereof and/or facilitate identification thereof. For
this purpose, with reference for example to FIG. 38, designated by
24b is such an area for collection of the target particles, which,
in this example, is represented by a terminal or restricted portion
defined between the separation element 24 and the side wall 2a''.
As may be seen, the aforesaid collection area can be defined by
means of an inclined arrangement of the separation means 24 with
respect to the main direction of the flow and/or to a side wall of
the collection section, preferably to define substantially an acute
angle.
[0275] FIGS. 40-45 illustrate a further microfluidic device
according to the invention, in particular with mixed-flow
structure. Also in these figures the same reference numbers as
those of the previous figures are used to designate elements that
are technically equivalent to the ones described previously.
[0276] In this version of device, and as may be clearly seen from
FIGS. 40 and 41, the body 2 is set between an upper closing body or
lid 26 and a lower supporting body 30. The body 2 has a general
configuration similar to that of the device MD of FIG. 36, even
though this is not indispensable.
[0277] In one embodiment, the collection section 20 is mounted or
at least partially integrated in the lower body 30, with the lower
body 30 that is possibly separable from the body 2. FIG. 42 refers
specifically to the first case considered, where the body 21 of the
section 20--made, for example, of plastic material--is mounted on
the lower body 30, in particular but not necessarily in a separable
way. It will, however, be appreciated that the body 21 may also be
defined integrally in the lower body 30, which is also, for
example, made of plastic material or of glass-reinforced plastic.
In the non-limiting example illustrated, the lower body 30 is
provided with holes, through which respective lower inlet or outlet
connectors of the body 2 are to be inserted. These through holes
are designated in FIG. 42 by 4c, 6c, 6c' and 7c, respectively, for
the connectors designated by 4b, 6b, 6b' and 7b in FIG. 41.
[0278] In one embodiment, the collection section 20 is operatively
coupled to the lower face of the body 2, and the outlet 5 of the
path 3 includes a passage or through hole of the body itself. In
one embodiment, the body 2 defines a housing in which the
collection section 20 is at least partially received.
[0279] The device MD of FIGS. 40-45 includes both of these
characteristics, as for example is clearly visible in FIG. 44,
where designated by 5 is the through hole of the body 2 that
provides the outlet of the path 3, whilst designated by 33 is a
housing in which part of the body 21 of the collection section is
designed to be received, in particular but not necessarily in a
removable way. In this embodiment, the section 20 does not require
a respective lid. In this embodiment, the outlet 5 gives out into
the housing 33.
[0280] The collection section 20 is here configured as a distinct
part both with respect to the body 2 and with respect to the lower
body 30 and has a respective collection body 21. The bottom of the
cavity 21a of the body 20, integrating the separation means 24, can
be formed integrally by the body 21 or, as in the case shown, be
constituted by a respective substrate 40, for example made of glass
or semiconductor material, defined on which are the means 24. In
the case of use of a semiconductor material, in the aforesaid
substrate there can be integrated electrical and/or electronic
and/or electromechanical devices, such as for example lighting
means, optical sensor means, electrodes for electrophoresis or
separation of particles or cells, electrodes for attraction and/or
repulsion of beads BE, electrodes for detection and/or counting
and/or alignment of particles or cells, solenoid valves and/or
miniaturized electric pumps, etc., preferably obtained with MEMS or
NEMS technology. In the case of a glass substrate, electrical
and/or electronic devices can be positioned in the body 21
underneath the aforesaid substrate. In the case where the section
20 includes also sensor means for the analysis of the sample of
target particles, as mentioned previously, it is not indispensable
to envisage the possibility of removal of the section 20 from the
section 1.
[0281] In one embodiment, such as the one represented in FIGS.
40-45, the section 20 is provided with a respective lower outlet
connector. This outlet connector, which is preferably formed
integral with the body 21, is clearly visible in FIG. 45, where it
is designated by 23b, and is in fluid communication with the outlet
23 of the section 20. In the case where the body 21 of the section
20 is defined integrally in the lower body 30, also the connector
23b will be preferably made of a single piece with the lower body.
Furthermore, in the case where the bottom bearing the separation
means 24 is configured as substrate apart with respect to the body
21, said substrate will be provided with a suitable passage or
through hole for discharge of the fluid in excess from the sample
of target particles, with said hole that will come to correspond
to, or in any case be in fluid communication with, the outlet 23
formed in the body 21.
[0282] As may be appreciated in particular from FIGS. 41 and 42, in
the assembled condition of the device, the body 21 of the section
20 is coupled to the lower body 30 with the outlet connector 23b of
the section 20 that is fitted through the hole designated by 7c
(FIGS. 41 and 42) of the body 30, whilst the top part of the body
21 is received at least in part in the housing 33 (see in
particular FIGS. 43 and 44).
[0283] In one embodiment, the collection section 20 is mechanically
and hydraulically coupled to the body 2. Said characteristic is,
for example, present in the case of the device MD of FIGS. 40-45,
where the housing 33 enables both a hydraulic connection and a
mechanical connection of the body 21 to the body 2. In a preferred
embodiment, as has been said, the body 2 is made of an at least
slightly elastic material, such as a silicone material, and this
facilitates precise coupling in a fluid-tight way of the section 20
to the body 2; obviously, there is nothing to rule out providing
specific sealing means, for example one or more gaskets, which
operate along the periphery of the body 21 at a part thereof
operatively inserted in the housing 33.
[0284] In the assembled configuration (see FIG. 43), the section 20
is set underneath the outlet hole 5 of the path 3. It may be noted
that, as has been explained previously, in the hole 5 there may
possibly converge also the duct 8 or 8' for the buffer. In
particular, underneath said hole 5 there will be located the
portion of the section 21 that is set upstream of the separation
means 24.
[0285] In one embodiment, mounted on the lower body 30 are
electrical and/or electronic components. This is, for example, the
case of the device of FIGS. 40-45, in which, in addition to the
section 20, mounted on the lower body 30 are electrical components,
comprising the electrodes 31, 32 for carrying out a
dielectrophoresis along at least one stretch of the path 3, as well
as terminals 34.
[0286] In one embodiment, at least one of the upper body 26 and the
lower body 30 is configured at least in part for performing
printed-circuit functions. Also said characteristic is present in
the case of the device of FIGS. 40-45. As may be clearly seen from
FIG. 42, the electrodes 31 and 32 are here configured as
electrically conductive paths deposited or in any case directly
formed (for example, via serigraphy or etching) on the face of the
lower body 30 facing the body 2. The electrodes 31 and 32 are
electrically connected to the terminals 34 of a connector, which
provide means for electrical interconnection of the device MD, for
example, to an analysis or control apparatus. The terminals 34,
which are substantially of a pin type, pass right through the
thickness of the lower body 30, with respective parts projecting
from both faces of said body 30. Hence, preferably provided in the
lower face in the body 2 is at least one housing designed to
receive partially also components other than the section 20,
mounted on the body 30. A housing of this sort, which receives part
of the terminals 34, is designated by 35 in FIG. 44. Obviously, in
the case where on the lower body 30 other electrical and/or
electronic components are mounted, such as for example an
integrated circuit, the housing 35 can have dimensions larger than
the ones exemplified, or there may be envisaged a plurality of
housings having a shape and dimensions adequate for the components
to be partially received.
[0287] On the opposite side of the lower body 30 the terminals 34
project, to a significant extent, within a connector body,
designated by 36 in FIGS. 41-43. The connector body 36, here having
a quadrangular section, can be formed integral with the lower body
30 or else be configured as a distinct piece.
[0288] In the case where the collection section 20 integrates
electrical and/or electronic components, on the face of the lower
body 30 having functions of printed circuit there can also be
provided connection paths, for electrical interconnection of the
unit 20. This embodiment is also illustrated in FIG. 42, where the
aforesaid connection paths are designated by 37. In this
embodiment, the body 21 has respective exposed terminals or contact
elements (not visible), which, in the assembled condition, are in
electrical contact with the paths 37, for the necessary supply
and/or for conveying signals to and from the electrical and/or
electronic means present in the section 20.
[0289] It should be noted that a printed circuit or PCB can be
configured as additional component, for example made in a lower
body 30, or in a lid 26, or else can be made directly on the body
2, when this is made of a suitable material, such as, for example,
glass or silicon. Also in this latter case, the conductive paths
can be obtained, for example, with serigraphic technique directly
on the body 2, possibly provided at least in part with an
appropriate insulating layer.
[0290] Also the lid 26 may possibly be configured at least in part
for performing printed-circuit functions, as has been described for
the lower body 30, with electrical and/or electronic components
mounted thereon.
[0291] In one embodiment, between the body 2 and the body 21, i.e.,
the collection section 20, there may be provided an electrical
interconnection. For example, with reference to embodiments like
the ones of FIG. 16-17 or 20-21, the device MD can comprise a
printed circuit or PCB with a portion that extends astride of the
body 2 and of the body 21, said PCB performing functions of
interconnection between electrical/electronic parts belonging to
the body 2 and electrical/electronic parts belonging to the body
21. In such a case, possibly, in the area of interface between the
bodies 2 and 21 the aforesaid printed circuit can be pre-arranged
for enabling breaking or cutting, as has been described previously
with reference to FIGS. 32-34 for the bodies 2 and 21.
[0292] Of course, also with reference to other embodiments
illustrated, an interconnection between electrical/electronic parts
of the body 2 and electrical/electronic parts of the body 21 could
be obtained via a corresponding connection system, for example of
the male-female type.
[0293] In one embodiment, the device according to the invention
comprises at least one of an electric heater and a temperature
sensor. Said means can be useful for keeping the biological fluid
at a pre-set temperature (for example, approximately 37.degree. C.)
during analysis.
[0294] The heater and/or the temperature sensor can be mounted
directly on the body 2 or, as in the case exemplified in FIG. 46,
in a lower body 30 of the device. FIG. 46 regards precisely the
case of positioning of a temperature sensor TS, for example, of a
PTC or NTC type, and of an electric heater EH, for example in the
form of a serigraphed resistor, positioned on a lower body 30 like
the one described previously with reference to FIGS. 40-45, for
example made of glass. It should be noted that also the heater HE
could be of a PTC type, which can also be possibly obtained with
the serigraphic technique. In the case of a PTC heater, it is not
necessary to purposely provide a temperature sensor TS, owing to
the characteristics of self-regulation in temperature of a
positive-temperature-coefficient resistor. The temperature sensor
TS could in any case being useful also for other functions and/or
for a more correct processing of the data.
[0295] FIGS. 47-49 illustrate another microfluidic device according
to the invention, in particular a with mixed-flow microseparation
structure. Also in these figures the same reference numbers as
those of the previous figures are used to designate elements that
are technically equivalent to the ones described previously.
[0296] As has been seen previously, in the device of FIGS. 40-45
the body 21 of the collection section 20 is configured as component
mounted or made at least in part on the upper face of the body 30,
i.e., the one facing the body 2. FIGS. 47-49 refer to a device
conceptually similar to that of FIGS. 40-45, but in the case of
mounting or integration of the body 21, and hence of the section
20, on the bottom face of the body 30.
[0297] As may be seen in FIG. 48, the bottom face of the body 30
may possibly be configured as a printed circuit, mounted on which
are, in addition to the section 20, also other electrical and/or
electronic components, such as the terminals 34 of the connector 36
and an integrated circuit 38. It should be noted that in FIG. 48
the electrically conductive paths for the connection of the various
electrical and/or electronic components have not been
represented.
[0298] It should be noted, in particular in FIGS. 48 and 49, how
projecting from the body 21 of the collection section 20 are
terminals or contact elements 39 for connection with corresponding
paths made on the bottom face of the body 30, said collection
section 20 being substantially configured as an electronic
component of the surface-mount type. Also in this embodiment, the
bottom body 30 has a respective connector body 36, projecting
within which are the interconnection terminals 34 of the device MD
towards the outside world, for example, towards an apparatus of
analysis. The body 30 has a passage or through hole 5b that, in the
assembled condition of the device MD, occupies a position
corresponding to the outlet hole 5 of the path 3; via said hole 5b
the fluid reaches the section 20.
[0299] In one embodiment, the collection section of the
microfluidic device according to the invention includes a substrate
made of semiconductor material, in particular silicon, integrating
at least one of a fluidic device, an electro-mechanical device, an
electrical device, an electronic device in miniaturized form, a
device for transmitting and/or receiving data, MEMS or NEMS
devices, as already mentioned previously.
[0300] Such a case is exemplified in FIGS. 50 and 51, which regard
a collection section 20 that can be used, for example in
combination with the devices MD of FIGS. 40-45 or of FIGS. 47-49.
The idea of providing a substrate made of semiconductor material,
and in particular silicon, integrating electrical and/or electronic
and/or electromechanical devices can in any case be applied also to
other embodiments of the invention, by providing the section 1 or
the section 20 with electrical-interconnection means.
[0301] In the embodiment of FIGS. 50 and 51, the section 20 has a
respective collection body or casing 21, made, for example, of
plastic material, which defines at least in part the cavity 21a.
The bottom of said cavity is, in the example, formed from a silicon
substrate, designated as a whole by 40. The body 21 that houses the
substrate 40 integrates at least partially hydraulic ducts, such as
the outlet hole 23 and the corresponding connector 23b. Moreover
associated to the body 21 are the electrical-connection means 39,
connected to the substrate 40, for example via wire bonding, in an
area protected from the flow of fluid.
[0302] In the example, the substrate 40 is then microprocessed to
define one or more miniaturized devices. In general terms, these
devices can comprise a filter or separation means, an integrated
circuit, a device for measuring and/or processing information
and/or physical quantities, a lighting device, an optical detection
device, a storage device, a device for counting and/or alignment
and/or detection of characteristics and/or of the type of
particles, or a sensor device in general.
[0303] In the specific case represented, in addition to the means
24, in the substrate 40 there may be identified two MEMS
micro-valves 40a, walls 40b that form a microfluidic channel, a
pair of electrodes 40c for electrophoresis and/or cell detection, a
processing and/or sensor part 40d, which can hence integrate
microcontroller and memory functions, as well as lighting means and
sensor means. In the example, this unit is represented
schematically upstream of the means 24, but the same unit, or a
similar unit, may be provided downstream of the means 24. In one
embodiment, the part 40d comprises lighting or excitation means
and/or means designed to highlight the cells or particles, present
in particular in the chamber 40e that is comprised between the
microvalve 40a and the separation means 24. The aforesaid lighting
means, for example comprising one or more light-emitter elements of
a LED or solid-state type, are preferably designed to generate at
least one wavelength of the electromagnetic spectrum suited for the
purpose, such as a wavelength designed to excite appropriately
beads BE attached to cells TC, or designed to highlight predefined
characteristics of the particles or cells, such as the shape.
[0304] In the part 40d--which can be obtained according to
techniques known in the sector of manufacture of silicon-wafer
microcontrollers--may be in part insulated and in part in contact
and/or in a position corresponding to the fluid, preferably by
integrating transmitter and/or receiver means for exchange of
information with an external transmitter and/or receiver device,
for example provided on an apparatus of analysis. In such an
embodiment, then, the identification means of the device previously
described and exemplified in the form of a barcode, can be of an
electronic type and include means for transmitting (and possibly
receiving) signals, for example for sending data identifying the
device MD and/or results of measurements/analyses conducted within
the section 20 and/or other information or data, and for possibly
receiving identification information, configuration and/or setting
parameters, or specific commands for electrical/electronic
components internal to the same section 20.
[0305] Given that in the silicon substrate 40 there may
conveniently be provided nonvolatile-memory means, in the substrate
itself there may also be advantageously implemented an RFID device,
for example of the type mentioned previously, preferably containing
identification data, regarding, for example, information
representing the sample under analysis, the type of analysis to
which the sample is to subjected, data or parameters for
calibration of an apparatus of analysis, data identifying the
subject to whom the biological fluid belongs, etc.
[0306] The sensors integrated in the silicon substrate 40 may
comprise means for counting particles, for example obtained with
electrodes that change their behaviour, such as the electrical
resistance or the capacitance or the oscillation, in proportion to
the cells with which they come into contact. There could
advantageously be integrated in the substrate 40 also microfluidic
devices or miniaturized sensors designed to control the flow rate
and/or pressures of the circulating fluids, as also the
corresponding temperature and/or other relevant physical
parameters.
[0307] In one embodiment, the collection section 20 comprises a
casing body 21 configured as a distinct part with respect to the
body 2, which integrates at least partially hydraulic ducts and
electrical-interconnection means. Such an embodiment is, for
example, represented in FIGS. 52-54, where the same reference
numbers are used as those of the previous figures.
[0308] In this embodiment, the section 20 has a respective body 21,
for example made of plastic material, which defines both a lower
outlet connector 23b and a connector body 41, within which
respective electrical terminals 42 project. These terminals 42 are
connected to electrical and/or electronic components present within
the body 21, for example integrated in miniaturized form in a
silicon substrate like the one previously designated by 40, as may
be seen for example in the exploded view of FIG. 54, provided with
the corresponding outlet hole 23 in fluid communication with the
outlet connector 23b. Alternatively, the bottom of the cavity of
the body 21 can be defined by a glass substrate, in which case the
electrical and/or electronic components of the section 20 may at
least in part be positioned underneath said glass substrate (see,
for example, the foregoing part of description in relation to the
collection section of FIG. 45).
[0309] In one embodiment, such as the one represented in FIGS.
52-54, the body of the section 20 can also include a respective
closing element, which integrates hydraulic-connection means. Also
such an embodiment may be seen in FIGS. 52 and 54, where it may be
noted how the lid 26a of the section 20 defines an upper connector
22b, which provides the inlet 22 of the section 20. The connectors
of the section 20 could be arranged differently, just as the
connector 41, and be in a number different from the one
exemplified.
[0310] In one embodiment, the entire collection section 20 is
configured as a unit that can be separated from the body 2 and is
operatively coupled to the face of the body 2 opposite to the one
in which the path 3 is defined. In such an embodiment, the outlet
of the path 3 is preferably configured as through hole 5 of the
body 2 and/or as hydraulic connector with respect to the connector
22b. In one embodiment, the section 20 has mechanical and hydraulic
interconnection means, which can be coupled in a separable way with
mechanical and hydraulic interconnection means of the first body
2.
[0311] The device represented in FIGS. 55-58 highlights both of
these characteristics, which, on the other hand, do not necessarily
coexist. In FIGS. 55-58 the same reference numbers as those already
used previously are used to designate elements that are technically
equivalent to the ones already described.
[0312] In the embodiment exemplified, the device MD does not
comprise the lower body 30, and the body 2 has a general
configuration similar to that of the body 2 of FIG. 36, but without
the lower outlet connector 23b. In this case, defined in the face
of the body 2 opposite to the one in which the path 3 is defined is
a housing 33', in which there can be fitted at least partially, in
a removable way, the collection section 20, as may be clearly seen
from FIG. 56. In the example illustrated, the section 20 has a
configuration similar to the one described with reference to FIGS.
52 and 53.
[0313] Also in this embodiment, preferably, the body 2 is made of a
material that is at least in part elastic, with the section 20 that
can then be mechanically and hydraulically coupled, in a
fluid-tight way, with the body 2, in particular by exploiting the
slight yieldingness or elasticity of the latter. As may be noted,
in particular from FIG. 58, in the assembled condition of the
device, the inlet connector 22b is fitted in the through hole 5 of
the body 2, which provides at the same time both the outlet of the
path 3 and a connector means co-operating with the connector 22b.
As has been explained previously, in the hole 5 there may possibly
converge also a duct 8 or 8' for the buffer, or else there could be
provided respective further connectors in the body 2 and in the
section 20.
[0314] In one embodiment of the invention, at least one of the
inlets and the outlets of the microfluidic device has valve means,
such as one-way valves and/or retention valves and/or anti-reflux
valves. These valve means may be conveniently made integral in a
body of the device MD, such as for example the body 2, or else be
configured as additional components. FIGS. 59-62 and 63-67
illustrate these two possibilities.
[0315] In the case of the embodiment of FIGS. 59-60 the body 2,
represented only schematically in cross section, with the
corresponding lid 26, has lower connectors, such as the connectors
4b, 6b and 7b, defined within which is a membrane 45 having a
transverse cut so as to define two opposite elastically deformable
lips 45a, which mate in a fluid-tight way with one another.
Membranes 45 of this sort may be provided also in the connectors
6b' and 23b, where envisaged.
[0316] The deformation in opening or closing of the lips 45a can
be, for example, induced by the positive or negative pressure of
the fluid entering the device (blood and/or buffer), or else of the
outlet fluid (fluid with target particles and/or reject fluid, such
as a mixture of reject blood and buffer).
[0317] Once said pressure ceases, or in the case of a negative
pressure or counterpressure, the lips 45a tend to reclose
automatically, also preventing exit of fluid. The sensitivity of
said valves or lips can be predefined on the basis of the type of
material and/or thickness of the membrane.
[0318] In another embodiment, the membrane 45 can also be without
the aforesaid transverse cut, where preferably the membrane 45 has
a pre-cut line or area and/or a perforation area. In a further
embodiment, as highlighted in FIGS. 61 and 62, the apparatus of
analysis, on which the device MD is to be set, may have fluid
connectors 46, provided inside with suitable projections or pins
46a, preferably axial ones, having a thin section or in any case
one such as to not prevent the flow of the fluid. The aforesaid
pins 46a are designed to project inside the connectors 4b, 6b, 7b
of the device MD until they bring about breaking or cutting of the
membrane 45, with consequent formation and opening of the lips 45a,
or else the pins 46a are designed to penetrate between the two lips
with pre-formed cut, causing deformation thereof in opening.
[0319] In one such embodiment, the removal of the device MD from
the apparatus then brings about closing of the lips 45a.
[0320] According to the aforesaid variant with breaking or cutting
of the membrane 45, the pin 46a could advantageously be configured
as a needle, in particular designed to perforate the membrane 45,
which automatically recloses upon removal of the pin.
[0321] In the case of a use of the aforesaid valves as anti-reflux
valves, i.e., ones designed to prevent a reverse flow, the fluid
connectors 46 can be without the projections or pins 46a, and/or
further valves can be provided, both for retention purposes and for
anti-reflux purposes.
[0322] The embodiment of FIGS. 59-62 is, for example, advantageous
in the case of a body 2 made of elastically deformable material,
such as a silicone material. The valves prevent any leakage of
fluid following upon removal of the device, after a fluid has been
made to pass and/or prevent circulation of fluids in undesirable
areas or directions.
[0323] FIGS. 63-67 refer, instead, to the case of valves, such as
one-way valves and/or retention valves and/or anti-reflux valves,
configured as additional components, applied in positions
corresponding to lower connectors of the device MD. In this
embodiment, the aforesaid valves, designated by 45' have a body
made of elastic material, for example, elastomer material, with a
flange-like base part, rising from which is a basically conical or
frustoconical part, with a flattened top end and provided with a
cut 45b (FIGS. 65-67) or with an area of predefined breaking, or
cutting, or perforation. Also in such an embodiment, the
divarication or closing of the flaps or portions of the membrane
can be obtained by exploiting the pressure of the fluid and/or by
inserting a pin or a needle.
[0324] It should be noted that also the valve means 45' can
possibly be obtained integrally by moulding in the body 2.
[0325] The embodiments represented in FIGS. 59-62 and 63-67 regard
integration of valves in lower inlets and/or outlets of a device
MD, but it will be appreciated that the solution can be applied
also to the case of a device MD provided with lateral and/or upper
inlets and/or outlets.
[0326] Valve means are useful in order to prevent any leakage from
the device MD when this is removed from an apparatus of analysis.
Considering in fact that a biological fluid is made to flow in the
device MD, it is preferable for the device itself not to leak
during handling, the reason for this also being to maintain an
isolated environment within the device, for example in the
perspective of preserving the cells or of providing cultures within
the section 20. Of course, the specific configuration of the valve
means may be different from the one, albeit advantageous,
exemplified herein.
[0327] Non-return valves or one-way valves can also serve on the
inlets of the ducts 4 and 8 to prevent any reflux, for example in
embodiments in which flows of blood and buffer are introduced into
the device in an alternating way. In said perspective, even if
first a part of blood is introduced and then the buffer, the buffer
itself cannot "push" the blood backwards, towards tubes and supply
reservoirs of the device, owing to the presence of the one-way
valve means, for example of the type already described above.
[0328] In one embodiment, the collection section 20 is mechanically
and hydraulically coupled in a separable way to the body 2 and has
valve means, such as one-way valves and/or retention valves,
preferably configured at least for preventing exit of material of
the target sample from the corresponding inlet 22 and/or from the
corresponding outlet 23, following upon removal and/or separation
of the section itself from the body 2.
[0329] An example of such an embodiment is illustrated in FIGS.
68-71, where the same reference numbers as those of the previous
figures are used to designate elements that are technically
equivalent to elements already described above.
[0330] In this embodiment, the device includes a body 2 and a lid
26, of a configuration generally similar to that of the device MD
of FIGS. 55 and 56. In this case, however, the outlet of the path 3
does not pass right through the thickness of the body 2, but
consists, instead, of a duct 5 at least in part made in an internal
area of the body 2; i.e., it extends between the upper face of the
body 2, where the path 3 is defined, and the peripheral face of the
body 2 itself.
[0331] Preferably, but not necessarily, defined in a position
corresponding to the peripheral face of the body 2 in which the
duct 5 opens is a positioning or housing seat, such as a flat. It
should be noted, in any case, that the shape of the body 2 does not
necessarily have to be circular, as in the case exemplified.
[0332] The body 21 of the collection section 20 has, by way of
example, a shape similar to that of the section 20 of FIGS. 52 and
53, but with an inlet connector 22b that projects laterally,
instead of at the top, and that is designed to fit into a connector
or widened portion (see FIG. 70) defined at the end of the duct 5.
The seal between the two coupled parts can be obtained
advantageously exploiting the elasticity of the material
constituting the body 2, when this is, for example, an elastomer or
silicone material. There may in each case be envisaged purposely
provided sealing means, such as a gasket.
[0333] It will hence be appreciated how, in one embodiment, the
collection section 20 can be configured as component separable from
the body 2, and have mechanical and hydraulic interconnection
means, which can be coupled in a separable way with mechanical and
hydraulic interconnection means of the body 2.
[0334] The section 20 is provided, at the inlet connector 22b, with
valve means, preferably of the type previously designated by 45' in
FIGS. 63-67. Similar valve means are envisaged at the outlet
connector 23b of the section 20. Obviously, the valve means can be
of a different type from the one exemplified.
[0335] It will be appreciated that, in this embodiment, once the
sample of target particles has been obtained, the device MD can be
removed from the laboratory apparatus that manages the flows
(blood, buffer, discharge) and/or controls the device MD, and the
section 20 can then be removed from the device MD, i.e., separated
from the section 1, for subsequent use, for example analysis to be
conducted with other equipment. The presence of the valve means 45'
guarantees that, upon removal of the section 20 from the body 2, no
material of the sample will be dispersed from the inlet connector
22b and outlet connector 23b, such as possible residue of the
blood-buffer mixture with corresponding target particles. The
reduced dimensions of the collection section 20 facilitate handling
thereof during analysis.
[0336] In one embodiment the collection section 20 has at least one
of an aeration passage, preferably mounted at which is an
air-permeable membrane having calibrated porosity, and an inlet for
introduction of a culture medium.
[0337] Also said embodiment may be seen in FIGS. 68-71, where the
aforesaid aeration passage is designated by 47, preferably but not
necessarily defined on the lid 26a, mounted at which is the
aforesaid membrane, designated by 47a, for example made of
Goretex.RTM.. In this embodiment, as inlet for the introduction of
a culture medium there may advantageously be exploited the
connector 22b provided with the valve means 45'. Obviously, there
is nothing to rule out providing a specific inlet for the culture
medium, for example, on the lid 26a, provided with suitable valve
means, which are also of the type designated previously by 45.
[0338] FIG. 72 illustrates a variant of the collection section in
which the aeration passage 47 is formed directly in the body 21, in
a lateral position side, instead of on the lid 26a.
[0339] In one embodiment, which is particularly useful for the
cases where the collection section of the device is
separable/removable from or independent of the body 2, there may be
provided a support or adapter, aimed at facilitating use and/or
handling of the section itself.
[0340] FIGS. 73-75 illustrate an example of support or adapter that
can be used in combination with a separable collection section,
such as the section 20 of FIG. 72. The support, designated as a
whole by 50 has a body, made for example of plastic material,
defined in which is a through cavity 51 having a peripheral profile
congruent with that of the section 20 in question, i.e., a seat 51
designed to at least in part receive or house the section 20.
Preferably, defined at an end of the cavity 51 is a step or
undercut 52, on which a peripheral region of the lid 26a of the
section 20 is designed to bear. In the case of sections 20 having
one or more parts projecting laterally, such as the inlet connector
22b and the aeration passage 47, the cavity 51 is provided with
lateral impressions 53 of a suitable shape, for receiving at least
partially the aforesaid projecting parts. As may be appreciated
from FIGS. 73 and 74, in the coupled condition of the components,
the lid 26a of the section 20 is perfectly visible through the
through opening 51 of the body of the support 50. On the opposite
side, the connector body 41 with the terminals 42 is
accessible.
[0341] The support 50 can conveniently have a circular outer shape,
for its introduction in Petri dishes, or else the support 50 can
have a shape designed to be mounted directly in apparatuses of the
biomedical sector, such as a shape similar to that of a Petri dish.
As may be appreciated, the use of the support 50 can prove in any
case advantageous for simplifying the use and handling of the
section 20, for example, on apparatus or instrument of
analysis.
[0342] By way of indication, the section 1 (i.e., the body 2 when
it is without the section 20) and/or the section 20 and/or the
support 50 can have shapes in plan view and/or dimensions
corresponding or close to at least one of the following: [0343] a
generally circular shape, with diameters comprised between
approximately 35 and approximately 180 mm, such as for example one
of the following diameters: 35 mm, 50 mm, 60 mm, 80 mm, 90 mm, 94
mm, 100 mm, 120 mm, 140 mm, 145 mm, 150 mm, 180 mm; [0344] a
generally quadrangular shape, with sides comprised between
approximately 25 and approximately 120 mm, such as for example one
of the following (side.times.side) 26 mm.times.76 mm, 100
mm.times.100 mm, 120 mm.times.120 mm.
[0345] It will be appreciated that, in various embodiments, the
collection section 20--whether mounted on a support or
not--constitutes in itself a microfluidic device, which can be used
in itself, once separated from the body 2. This is in particular
the case of collection sections 20 that integrate electrical and/or
electronic and/or electromechanical devices of the type indicated
previously, with corresponding interconnection and/or
electrical-supply means (whether wired or wireless) and hydraulic
connectors, as well as possible mechanical and/or electrical
separation means. Hence, said sections 20, in addition to being
usable as "slides" for visual analyses, for example under the
microscope, may possibly be used on apparatuses of analysis
provided with electrical and/or hydraulic interconnections that can
be coupled with those of said sections.
[0346] FIG. 76 represents a principle diagram of a laboratory
apparatus according to the invention that can be used in
combination with a microfluidic device, for example a device having
a mixed-flow separation structure or a device MD of the type
described previously, in particular for circulation and/or handling
of the flows of fluids and/or for control of the device itself.
[0347] The apparatus of FIG. 76, designated by EQ, is connected to
a source of a fluid, preferably an aeriform AC such as compressed
air, not represented, for supplying a first branch 61 and a second
branch 71, operative along which are pressure-regulating devices
suitable for the application, designated by 62 and 72, of a
mechanical and/or electrical and/or electronic type, preferably
precision pressure regulators, possibly programmable ones. In
particular, said pressure regulators can be regulated in a pressure
range comprised between 1 mbar and 1 bar, preferably between 10
mbar and 200 mbar.
[0348] According to one variant, operatively associated to the
pressure regulators 62 and 72 are respective pressure sensors, not
shown, for example in fluid communication with ducts or paths of
the device MD, in particular in order to obtain a feedback and/or
regulation of the pressure.
[0349] The compressed air AC at outlet from the regulators 62 and
72, to pressures P3 and P8, respectively, reaches respective
hermetic containers 63 and 73, contained in which are a biological
fluid, such as blood, and an auxiliary fluid, such as the liquid
buffer; the pressures P3 and P8 are preferably comprised in a
pressure range between 1 mbar and 1 bar, for example, a pressure
range between 10 mbar and 200 mbar.
[0350] Preferably, but not necessarily, associated to the
containers 63 and 73 are respective agitator means, in particular
to maintain in suspension and/or in uniform distribution the
particles and/or the possible beads, which may comprise, for
example, a magnetic agitator 64 and 74, for example, designed to
produce movement of at least one magnetic element or magnetic
capsule 64a and 74a set in the containers 63 and 73,
respectively.
[0351] An agitation or movement of the fluid could possibly be
obtained via an appropriate excitation and/or movement of the beads
dispersed in the fluid, obtained via appropriate excitation means,
such as means designed to induce an electrical field.
[0352] The containers 63 and 73 have a respective outlet connected
via a corresponding duct 65 and 75 to the respective inlet
connectors 4b and 6b (or 6b') of the device MD. The outlet
connector 7b of the device MD is connected to a discharge duct
DR.
[0353] The containers 63 and 73 preferably comprise a respective
casing 63a, 73a and a lid 63b, 73b coupled to one another in a
fluid-tight way, possibly via appropriate additional sealing means.
The lids 63b, 73b are preferably associated in a fluid-tight way to
the respective ducts 61, 65 and 71, 75. Preferably, the inlet ducts
61, 71 extend in the respective casing 63a, 73a for a stretch or
length shorter than the outlet ducts 65, 75. In particular, the
ducts 61, 71 terminate at a higher level, close to the lower part
of the lids 63b, 73b, whereas the ducts 65, 75 terminate at a lower
level close to the lower part of the containers 63, 73, even if
slightly raised to allow the fluid contained therein to flow away,
or else are provided with appropriate openings close to said lower
level. The ducts 61, 71 preferably terminate at the aforesaid
higher level in order to prevent the compressed air AC from
possibly forming bubbles or froth in the fluid contained in the
corresponding container 63 or 73 and/or to prevent the risk of any
reflux of fluid into the ducts for the compressed air. The
apparatus could, however, function also with ducts 61, 71 arranged
or terminating at different levels, i.e., longer inside the
containers 63, 73.
[0354] In a normal operating cycle, the regulators 62 and 72 allow
entry into the containers 63 and 73 of compressed air AC, at the
pressures P3 and P8, so that the blood and the buffer,
respectively, are introduced under pressure within the device
MD.
[0355] As has been explained previously, preferably, the pressure
P8 is slightly higher than the pressure P3, in order to prevent any
reflux of blood from the duct 3 of the device to the duct 8. As
already said, in any case, the pressures of the fluids at inlet to
the device 1, such as the pressures P3 and P8, are preferably
relatively low, i.e., of a value such as to reduce the risk of
damage to or lysis of the particles or cells.
[0356] It should be noted that FIG. 76 presents a schematic
configuration that can apply to any one of the devices MD, 1 and 20
previously described; it follows that the number of the ducts
and/or of the attachments and/or of the containers and/or of the
pressure regulators could vary, and the buffer could contain beads
or be without them.
[0357] Consider moreover that what has been described for brevity
with reference to a system designed to generate pressures could
also refer to a system designed to generate negative pressures
and/or differences of pressure, in particular between at least one
inlet and at least one outlet of the device according to the
invention. Likewise, what has been described for brevity with
reference to a device functioning at positive pressures could also
refer to a device operating with negative pressures and/or
differences of pressure.
[0358] FIG. 77 illustrates another example of apparatus EQ
according to the invention, of a type similar to the one shown in
FIG. 76. In this schematic example (where for simplicity the source
of compressed air and the corresponding pressure regulators have
been omitted), the device MD is of the type with lateral
connectors, provided with valve means of the type described with
reference to FIGS. 59-60, and the apparatus EQ has connectors 46 of
the type illustrated in FIGS. 61-62. It should be noted that also
the hydraulic attachments 46 of the apparatus of FIG. 77 may
possibly be provided with retention valves or valves for closing
the ducts in the absence of the microfluidic device, for example to
prevent any contamination or dispersion of fluid.
[0359] FIG. 77 highlights for simplicity just the connectors 4b for
inlet of the blood and 7b for discharge of the reject mixture. The
apparatus EQ is in any case conveniently provided with at least one
further connector for the introduction of the auxiliary fluid (the
buffer or the buffer with beads) into the device MD, provided for
this purpose with the corresponding inlet connector, similar to the
ones highlighted.
[0360] FIG. 77 represents schematically also a control system of
the apparatus EQ, designated by 80, which can be connected to an
external processor, such as a personal computer 81, for receiving
and/or transmitting data, such as sending of data of configuration
to the system 80 and/or for receiving from the system 80 results of
analyses effected on the sample of target particles. The control
system 80 supervises general operation of the apparatus, including
management of the corresponding pressure regulators.
[0361] In the specific case represented, it may be noted how, in
addition to the container 63 for the blood and a container RD for
the discharge fluid, an additional container 73' is provided for a
liquid, such as the aforesaid buffer or a further flushing buffer,
both said containers being connected--by way of valves 82 and
83--to one and the same line 84 that supplies the connector 46 for
inlet of the blood. This configuration is useful for the purposes
of performing a step of flushing just with buffer of the device MD,
at the end of collection of the sample of target particles in order
to prevent any stagnation of blood inside the device itself and/or
to push all the blood in circulation and cause it pass through the
device MD. In practice, then, for the purposes of
separation/collection of the sample of target particles just the
valve 82, for delivery of the blood, is opened. At the end of said
operating step, the valve 82 is closed, and just the valve 83 is
opened to carry out a final flushing of the device MD with just
buffer.
[0362] Of course, the hydraulic configuration of the apparatus EQ
may differ from the one exemplified in FIG. 77; for example, it may
be provided with a single container for the buffer and with
suitable deviator means controlled by the system 80 for initially
directing the buffer to the inlet 6, 6b of the path 8 of the device
MD and, when the final flushing is to be performed, for directing
the buffer, instead of the blood, to the inlet 4, 4b of the path 3.
As may be appreciated, in this way, when the device MD is removed
from the apparatus, absence inside it of deposits of blood is
guaranteed and/or treatment of all the blood set in circulation is
likewise guaranteed.
[0363] FIG. 77 moreover exemplifies a wireless communication and/or
supply system set between the apparatus EQ and the device MD, for
example for identification purposes, as has been explained
previously. Designated by C1 is a winding and/or an antenna,
forming part of the control circuit 80 of the apparatus EQ, and
designated by C2 is a winding and/or an antenna integrated in the
device MD, for example in its collection section (not represented
in the figure), where said windings and/or antennas C1 and C2
belong to an arrangement of an RFID type. As is known, certain RFID
arrangements include a passive device, i.e., one without battery or
electrical supply (typically having transmitter or transceiver
functions), bearing data and designed to react to a specific
inductive electromagnetic field, produced by a respective reader,
which supplies in response a modulated radiofrequency representing
data. Since it does not have any internal source of energy, the
aforesaid passive device derives its own supply from the
electromagnetic field itself generated by the reader. Hence, in the
case exemplified, following upon positioning of the device MD on
the apparatus EQ, it is possible to set up a communication between
them, without the need for any wiring and without having to provide
the device MD with a battery of its own. According to a variant,
the part of the RFID arrangement carried by the microfluidic device
could, however, comprise a battery or a different electrical
supply.
[0364] It should be noted that a circuit arrangement of the type
indicated above can also be used for electrical supply and/or
writing of data or information in suitable storage means present in
the device MD, for example, data or information deriving or
resulting from the analyses or tests effected on the laboratory
apparatus.
[0365] FIG. 78 highlights an apparatus EQ according to the
invention, altogether similar to that of FIG. 77, used in
combination with a device MD having a different configuration of
the microfluidic paths, which here extend on two opposite faces of
the body 2.
[0366] FIG. 78 moreover highlights the presence of a cover,
designated by 85, for enclosing the device MD in a protected and
thermostatted environment TA, i.e., one designed to be kept at a
substantially pre-set temperature (for example, by exploiting the
means of the device MD itself previously designated by TS and/or EH
in FIG. 46). Preferably, also the area of the apparatus EQ in which
the containers 63, 73, 73', RD and the corresponding ducts are
present is protected and/or thermostatted.
[0367] According to a variant, the device MD of FIG. 78 has a
configuration in which the ducts and/or microfluidic paths are
obtained on at least two opposed sides or faces of the device MD
and/or of the body 2, preferably set in communication by through
ducts or holes, provided with respective covers 26 (or 30).
[0368] FIG. 79 is a schematic illustration of an equipment EQ
according to the invention, substantially configured as kit of
parts, for handling the flows in a microfluidic device, for example
a device MD of the type described previously, having purposes
substantially similar to the ones of the equipment of FIG. 76, but
of simplified construction and use. The figure represents a kit
comprising: [0369] a flexible tube 90; [0370] a first connector 91
with a needle for connection of the tube 90 to the device MD;
[0371] a second connector 92 with two needles, for connection both
of the tube 90 to a container or test tube 93 containing the blood
(or the buffer) and for connection of the test tube 93 to a further
flexible tube 94 for delivery of a fluid or compressed air; and
[0372] a third connector 95 for connection of the tube 94 to a
source of a fluid or compressed air 96 at a suitable pressure.
[0373] Preferably, at least part of the kit EQ of FIG. 79 is of a
disposable type in order to prevent any contamination and/or for a
greater practicality of use.
[0374] In the example illustrated, the connector 92 comprises two
needles 92a and 92b, which are introduced into the test tube 93, in
particular perforating an elastomer plug 93a thereof in order to
enable a convenient connection free from any contamination. In the
assembled condition, the two needles are set staggered with respect
to the central axis of the test tube 93, and connected to the two
respective hoses 90 and 94.
[0375] The test tube 93 is preferably--but not necessarily--a test
tube of a standard type used in the sector, such as a test tube
having an outer diameter of approximately 12.3 mm or 15.2 mm and an
internal diameter of approximately 10.7 mm or 13.3 mm,
respectively, the external diameter of the plug 93a being of
approximately 15-16 mm and 16.5-17.5 mm, respectively.
[0376] According to an advantageous aspect, the connector 92 has
dimensions such that it can be fitted or pressed on the test tube
93 and/or on the plug 93a via the two needles 92a, 92b. Preferably,
the body of the connector 92 has a perimetral portion that
surrounds at least in part the edge of the test tube 93 and/or of
its plug 93a. In said perspective, for example, the internal
diameter of the connector 92 can be equal to or slightly larger
than approximately 12.3 mm or 15.2 mm, or approximately 15-16 mm,
or approximately 16.5-17.5 mm, respectively. Preferably, in order
to facilitate perforation of the plug 93 with the two needles of
the connector 92, the plug itself has a central portion with
reduced cross section.
[0377] In a way similar to what has been described with reference
to FIG. 76, the flow of compressed air generated by the source 96
pushes on the blood into the test tube 93, forcing it into the
device MD via the tube 90. A similar operation occurs obviously in
the case of a container 93 containing the liquid buffer to be
introduced into the device MD in parallel with the blood.
[0378] Considering the fact that, in the devices MD described
herein with mixed-flow separation, fluids at a relatively low
pressure are preferably used, the seals can be obtained easily
and/or low-cost components or materials can be used. This applies,
for example, for the seal between the needles 92a and 92b and the
elastomer plug 93a of the test tube 93 and/or the seal between the
elastomer plug 93a and the test tube 93 and/or the seal of the
various connectors 91, 92, 95, and/or for the use of components of
the kit made of plastic material.
[0379] In a variant, the tube 94 can be connected, for example at
the end of the step of collection of the sample of target particles
or an intermediate treatment step, to a source under pressure of a
liquid buffer, instead of a source of compressed air, both in order
to empty the test tube 93 completely from the blood and in order to
eliminate the final residue of blood that might have remained
within the device MD. In this way, there is the possibility of
treating all or almost all of the blood introduced into the test
tube 93. At the same time a flushing is obtained that, even though
the device MD, the tubes 90, 94, the connectors 91, 92, and the
test tube 93 are of a disposable type, enables a safer and cleaner
handling and subsequent disposal of said components.
[0380] It should be noted that a flushing of the device MD of the
type indicated previously may possibly be obtained by envisaging a
purposely provided flushing accessory, to which the device itself
is to be connected after analysis and/or after taking the sample of
target particles out of the section 20.
[0381] As has been explained previously, the body 2 of the device
MD according to the invention can be made at least of one of
various materials, such as an elastomer or silicone material, a
thermoplastic material, a glass, a semiconductor such as
silicon.
[0382] Use of glass enables an excellent planarity to be obtained,
as compared to an elastomer micromoulded item. On the other hand,
as has been said, also in the case of a microprocessed elastomer
body 2, the presence of the lid 25 and/or of the lower body 30
fixed--for example via gluing or bonding--to the body 2 enables the
necessary planarity to be guaranteed.
[0383] In the case of use of glass an initial machining step is
preferably envisaged (for example, lapping), in order to obtain a
perfectly plane plate, on which to provide the microprocessed
elements by etching (chemical etching, abrasion, laser etching,
plasma etching, etc.), or possibly deposition, in order to form the
walls and/or lateral delimitations of the paths 3, 8, 9.
[0384] In an example of said process, both the glass and the
silicon can be machined as plane surfaces, on which a layer of
protective material (such as a metal mask or a polymeric
photoresist mask) is then positioned or deposited, which leaves
free the paths to be obtained in the underlying material (glass or
silicon), which is dug and removed via appropriate known processes,
for example, chemical etching or microabrasion. The material in
question, whether glass or silicon, can also be dug in other ways,
for example, via laser or plasma etching.
[0385] In order to define the paths 3, 8, 9 and/or the lateral
delimitations 10, 11 deposition techniques may also be used, for
example, by depositing a material designed to form the walls and
the lateral delimitations that delimit the various paths. Said
deposition can be obtained with multiple known technologies, such
as for example serigraphy or ink-jet printing, considering that
there exist inks of various types (metal-based, NPs, dielectric,
doped and otherwise, semiconductor, using biological substances,
etc.).
[0386] Amongst the various techniques that can be employed for
producing a device MD wafer-bonding may be mentioned, which is
suited for embodiments in which a first microfluidic circuit made
of a first material (for example silicone or glass, for the body 2
or the body 21) is associated to a second microfluidic circuit made
of a second material (for example silicon, for the substrate 40 of
the section 20). The body 2 can also be obtained with a moulding
process, including one or more steps of moulding and/or
forming.
[0387] FIG. 80 is a merely schematic illustration of an example of
a process for moulding a device according to the invention, such as
a mixed-flow separation device. The example regards a moulding of a
sub-micrometric imprinting type of a body 2 made of elastomer or
silicone material, such as PDMS, or thermoplastic material, such as
PMMA (polymethylmetacrylate).
[0388] Part A of FIG. 80 highlights the use of a first mould,
including two half-moulds 90 and 91, having respective impressions
90a, 91a for definition of details of the body 2, preferably ones
not having micrometric dimensions, such as for example the ducts 8,
9 and/or the corresponding hydraulic connectors 6b, 7b (see part B
of Figure) and the seal elements or lips, such as of the perimetral
lips S.
[0389] In the example, the impression 90a of the top half-mould 90
is also configured for providing at least one element in relief R,
shaped in said first moulding step, aimed at providing a
semifinished product, designated by T in part B of FIG. 80.
Performed on the element in relief R are the micromouldings in a
second moulding step, in particular using the imprinting
technique.
[0390] The fluid-tight element S can be configured as a lip running
along the perimeter of the body 2, for example for providing seal
towards the outside with respect to a lid 26, such as a seal from
inside, for example in regard to the hydraulic ducts, and/or a seal
from outside, for example in regard to any infiltration of dirt
into the device. Possibly, the element S and/or the element in
relief R can undergo micromoulding processes in the aforesaid
second moulding step, for example to change at least in part the
shape thereof.
[0391] Once the semifinished product T, for example made silicone
or thermoplastic material, has been obtained, this is introduced
into further moulding or forming equipment, as highlighted in parts
C and D of FIG. 80. This forming or imprinting equipment includes a
top mould 95 and a countermould or bottom support 96. The mould 95
is provided with impressions 96, 99b that form at least the
micro-mouldings of the upper surface or face of the semifinished
product T. It should be noted that the equipment could be
configured to enable simultaneous forming or imprinting on at least
two different surfaces, such as two opposite surfaces; hence, with
reference to the example illustrated, also the support 96 could be
provided with imprinting impressions.
[0392] Preferably, the second mould 95 is provided with seats or
cavities 97 designed to receive the element or elements formed in
the first moulding step, such as the elements S and/or R and/or the
ducts 4, 6 and/or the connectors 4b, 6b, so as not to damage
them.
[0393] Once again preferably, the mould 95 necessary for forming
the micromouldings has both projections 98 in the parts coinciding
with "empty" regions of the piece T, and lateral shoulders 99a for
containing the material during the second moulding step. The
presence of the aforesaid projections and lateral shoulders
prevents the material on which the micromouldings are obtained from
deforming laterally, without offering a due mechanical resistance
to compression and/or axial deformation exerted by the mould 95, in
particular in the direction or along the axis of movement of at
least part of the mould. In other words, at the moment of descent
or axial movement of the mould 95 on the piece T, in particular for
impressing the shape of the microfluidic paths, the material
appropriately heated could yield elastically downwards and/or
outwards, and in this case the mould 95 might be unable to impress
adequately its own shape. The presence of the aforesaid shoulders
and projections prevents this eventuality. Obviously, the extension
downwards of the aforesaid projections and shoulders can be greater
than the one exemplified in the figure.
[0394] Preferably, the mould 95 is provided with impressions 96, in
particular ones of a smaller width and height, and impressions 99b,
in particular ones of a larger width and height, which provide the
micro-mouldings of the upper surface or face of the semifinished
product T, such as respective microchannels of a smaller width and
depth and microchannels of a larger width and depth, respectively
(see, for example, what is described with reference to FIG.
3C).
[0395] By way of indication, the maximum width and height of the
projections of the mould (and hence the depth and width of the
paths/channels of the device) preferably fall within a ratio of
approximately 30.
[0396] Consider that the various shapes or characteristics
described with reference to the device of FIG. 80, performed in two
process steps, could also be obtained with just a single process
step, such as a step of moulding or a step of imprinting; for
example, a body 2 as in part D of FIG. 80 could be obtained just
with moulding or injection of material in a mould, in particular a
mould provided with micrometric machinings or seats.
[0397] FIGS. 81-83 illustrate a further embodiment of a
microfluidic device according to the invention. In said figures the
same reference numbers as those of the previous figures are used to
designate elements that are technically equivalent to the ones
already described.
[0398] The device MD of FIGS. 81-83 is of a general conception
similar to that of the devices described previously with a spiral
configuration of the paths 3, 8 and 9 (see, for example, FIG. 22 or
36), but the corresponding idea may be applied also to devices MD
having a different configuration. In this solution, the inlet 4 is
made at the top of the body 2, and more in particular in the lid
26. In this embodiment, the inlet 4 is basically constituted by a
hollow coupling provided in a region of the lid 26, which, in the
assembled condition of the device, has its lower end facing an
initial region of the path 3, delimited by a wall 2a''.
[0399] On said upper inlet 4 there can be fitted a container or
reservoir 100 containing, for example, blood. For this purpose, the
container 100 has a body 101 provided with a lower hydraulic
attachment 102, preferably equipped with the retention means or
valves of the type already described (not shown), or else with a
closure designed to be perforated when it is fitted on the device
MD. For this purpose, the connector of the device MD that provides
the inlet 4 is shaped, in the example, like a needle.
[0400] The container 100 includes a top lid or plug 103, with a
corresponding connector or attachment 104, preferably provided with
said retention means or valves (not shown), in order to inject a
fluid under pressure aimed at pushing the blood into the device MD.
The thrust fluid can be, for example, compressed air and/or a
liquid buffer. Once again by way of example, in a first step air
can be injected and then, when all the blood has left the container
100, a liquid buffer can be injected. Possibly, for this purpose,
level-sensor means can be associated to the container, for example,
ones of an optical type, interfaced with an apparatus for analysis
or management of supply of the flows to the device MD. Similar
level-sensor means can be provided also for the further containers
63, 73, 93 described previously.
[0401] This solution eliminates or in any case considerably reduces
the length of the path that the blood has to follow between a
corresponding container and the device MD, in particular without
the need for connection tubes. In this way, the amount of blood
necessary for testing via the device MD may be reduced. The
solution also prevents any stagnation of blood in the connection
tubes that are typically envisaged for supplying known microfluidic
devices, with the consequent possible errors in tests that require
an analysis on predefined amounts of blood. There is moreover
prevented the need to throw away each time also the tubes that are
soiled with blood or other fluid being examined.
[0402] The device MD according to the embodiment proposed enables
in fact the container 100 to be filled with a predefined amount of
blood or other fluid, that is smaller if compared to the known art,
and to cause it then to flow completely into the device MD, without
any risk of stagnation.
[0403] In the example of embodiment, the container 100 is
represented as a separate container, which can advantageously be
filled apart and then fitted on the device MD. It will be
appreciated, however, that, according to a possible variant, the
container 100 can be directly associated to or integrated or made
of a single piece in the device MD, in particular in its lid 26,
possibly as reservoir obtained apart and then fixed or bonded or
glued to the device MD.
[0404] A container having the same functions as the one previously
designated by 100 can be advantageously integrated in the device
MD, for example in the body 2 or in the lid 26, when it is made of
elastomer. In an integrated container of this sort the blood can be
introduced or injected (through a door or plug, or else injected by
perforating a thin elastic closing wall, or via a retention valve
of the type already described). Likewise, the air could then be
injected into the container through an appropriate opening or a
needle driven into said purposely provided perforatable wall.
Considering that the pressures of thrust on the blood are
relatively low, the elasticity itself of the elastomer material
could guarantee the appropriate seals also during
pressurization.
[0405] It will be appreciated that the solution of associating or
integrating a container or reservoir to the device MD according to
what has been described above applies also to the case where the
same or a similar container is in fluid communication with a
discharge outlet of the device itself, possibly even an outlet of
the section 20. Said solution makes it possible, for example, to
collect the reject liquid, and to dispose of it subsequently along
with the body 2 or the section 20 (in case of integration), or else
to have a separable collection reservoir so as to be able to
dispose of said reservoir separately from the body 2 or the section
20.
[0406] FIG. 84 illustrates a further embodiment of the invention.
The device MD of this figure is illustrated, merely by way of
example, as having a structure similar to that of the device of
FIG. 1, but the solution described in what follows may evidently be
applied also to other configurations of microfluidic device.
[0407] In this embodiment, the device MD is provided with two
collection sections 20 and 20', each of which is in fluid
communication with a respective outlet 5 and 5' of the path 3
and/or of the device MD.
[0408] In the non-limiting example illustrated, the lateral
delimitation 11, with the corresponding passageways, is configured
for eliminating--as the fluid advances along the path 3--particles
of progressively increasing dimensions and at the end isolate in
the two sections 20, 20' larger particles, separated by size on the
basis of the width of the passageways 11a of the last section of
the lateral delimitation 11. In the example, the section 20
represented at the top is designed to collect tumour cells TC,
whilst the section 20' represented at the bottom is designed to
collect monocytes M.
[0409] Hence, in an embodiment of this type, the passageways 11a of
the lateral delimitation 11 can be used as means for separation of
target particles, for example tumour cells of different dimensions
(see what has been said previously with reference to the preferred
sizings of the passageways 11a, in the various sections or portions
of the lateral delimitation 11).
[0410] Highlighted in the example are just two collection sections,
but there is nothing to rule out adopting other configurations,
with a greater number of sections 20, 20'. In addition, collection
sections can be provided in various stretches of the lateral
delimitation 11 distinguished by passageways having different
dimensions, as has been explained previously. As may be noted in
FIG. 84 (as likewise in FIG. 1), corresponding to each aforesaid
stretch of the lateral delimitation 11 is a respective branch 9b
for connection to the path 9. Unlike the case illustrated--where
the branches 9b converge in a single channel--at the end of each of
the branches 9b there could be connected in fluid communication a
respective section 20 for collecting particles of different
dimensions (possibly also rejecting the larger particles, in which
case the outlet 5, or each outlet 5', would be an reject outlet of
the device MID, not necessarily connected to a collection
section).
[0411] The branches 9b and/or at least part of the passageways 11a
can be devised as tortuous stretches or channels, prearranged for
preventing a return flow from the path 9 to the path 3. In another
embodiment, not represented, specific non-return means may be
provided, such as one-way valve means, for example ones comprising
membranes that bend in opening under the thrust of the flow from
the duct 3 to the duct 9 and bend in closing in the presence of a
thrust in the opposite direction. Said membranes may possibly be
obtained by moulding and/or from the body 2, in the case where the
body 2 is made of elastomeric or silicone material.
[0412] The case of FIG. 84 regards a configuration in which the two
sections 20, 20' are connected to the path 3 substantially in
parallel. It will, however, be appreciated that it is also possible
to envisage a configuration with at least two sections connected in
series one after another, i.e., with the outlet of a first section
that is connected to the inlet of a second section. Obviously, in
such a case, the passages of the separation means 24 of the first
section will be wider than those of the second section.
[0413] The embodiment of FIG. 84 moreover highlights how, in one
embodiment, the body 2 can be provided with just one duct 8, which
can be used with a buffer with or without beads BE. Consequently,
in the latter case, the separation of the particles will occur on
the basis of just the size of the particles themselves, without the
aid of beads.
[0414] It should moreover be noted that, in the example of FIG. 84,
the section 20' corresponds to an "intermediate" outlet, with the
larger particles that remain in the duct 3 and do not necessarily
constitute the target particles. In the case where the use of the
device MD is aimed at the collection of particles having an
intermediate size (and not the larger ones), the outlet 5 of the
duct 3 can be used as reject outlet (and hence without necessarily
having to provide the section 20), with the outlet 5' that is,
instead, connected to the section 20' for collection of the
particles of interest.
[0415] As has been explained previously, the device MD according to
the invention can be provided with a detection device, such as a
particle counter, associated to the body 2 or to the section
20.
[0416] A device of this sort can be of an electrical type, i.e.,
with electrodes, or else of an optical type, i.e., with a
transmitter and a receiver of light radiation, or again of an
acoustic type, for example based upon the Doppler effect or the
like. A principle diagram of such a particle counter is highlighted
in FIG. 85.
[0417] In the example of FIG. 85 the particles, including the
target particles which here are assumed as being monocytes M, are
made to pass individually in succession in a section that included
a calibrated or capillary passage, on the two sides of which two
electrodes 120 and 121 are located in opposed positions, in the
case of an electrical particle counter. The section including the
aforesaid calibrated passage can be, as in the example shown, a
terminal stretch of the path 3, appropriately sized for enabling
passage in succession of the target particles. A calibrated passage
of this sort can in any case be defined along the path 3 or also
within the section 20.
[0418] In operation, between the two electrodes 120, 121 a
preferably constant electric current is made to flow. The passage
of a particle between the electrodes 120 and 121 causes an
alteration of the electric current that flows between the two
electrodes, i.e., a variation of electrical resistance, enabling
corresponding counting thereof. In general, the variation of
current, or of resistance, will be proportional to the size of the
particle, thus enabling detection of what type it is. The aforesaid
variation can then be converted into a corresponding voltage pulse
for each particle that has passed. Via a control logic the voltage
amplitude of each pulse is then stored, where the different
amplitude corresponds to a different size of the particle, so that
finally the number of the cells differentiated on the basis of the
size (i.e., the type) can be counted. An implementation of this
sort can be facilitated if the conductivity of the circulating
fluid is known. Conductivity ranges may be experimentally derived
and stored in a control logic of the device MD. In any case,
considering that in the terminal stretch of the path 3 the
particles are for the most part dispersed in just the buffer, the
characteristics of conductivity of the latter can be easily known
beforehand. In said perspective, preferably, the buffer used has
predefined physical and/or electrical characteristics, such as a
known electrical conductivity. Preferably, the buffer has a
predefined conductivity and characteristics such as to not damage
any possible functionalized bonds and/or antibodies, in particular
for the purposes of separation and/or detection of the
particles.
[0419] It should be noted that the principle scheme of FIG. 85 can
be applied also for the case of an optical detection device, in
which case the elements designated by 120 and 121 will be,
respectively, an emitter and a receiver of light radiation. In this
case the buffer preferably has a good transparency to the optical
signal.
[0420] In the case of an acoustic detection device, the elements
120-121 may consist of two transducers or else be replaced by a
single transducer. For example, a single transducer can be devised
for directing in the fluid sound energy, preferably wide-band
ultrasound energy, operating both as transmitter and as receiver. A
particle that comes to occupy the focal region of the transducer
reflects the energy, generating an echo pulse proportional to the
size of the particle.
[0421] The device according to the invention can further be
provided with an arrangement or section configured for aligning the
particles, i.e., arranging them substantially in a row one after
another, for example for the purposes of a subsequent count via a
particle counter.
[0422] A schematic example of this type is illustrated in FIG. 86,
in accordance with which the device MD has a section 130-131 for
acoustic alignment of the particles, upstream of a detector device
120-121, which here is assumed as being a particle counter 120-121.
The particle counter 120-121 can be of the type with (resistive or
capacitive) electrodes, or else an optical counter, or again an
acoustic counter, preferably distinct from the alignment system.
The use of an alignment system, in the body 2 or in the section 20,
enables, for example, counting of the particles also in ducts that
are not necessarily capillary ones, i.e., with a width larger than
that of the cell, in so far as the alignment is not performed by
the duct, but via acoustic means.
[0423] In FIGS. 86, 130 and 131 designate the means designed for
generating the acoustic alignment signal, for example in the form
of opposed electrodes or transducers. In the example, the sections
120-121 and 130-131 are located along the path 3, for example in
the proximity of its terminal portion, or else at the inlet 22 of
the section 20. Preferably, the corresponding stretch of the
lateral delimitation 10 is without passageways, in order to prevent
in this stretch any turbulence that would annul alignment.
Preferably, the buffer has characteristics such as to facilitate
generation and/or propagation and/or detection of the acoustic
signal.
[0424] FIG. 87 illustrates, instead, the case of means for
separating or displacing particles via acoustic waves. These means
comprise, for example, a section including two acoustic transducers
140-141, such as electrodes of an appropriate shape or
interdigitated, the separation system being preferably based upon
the acoustic phoresis technique.
[0425] According to the principle of the acoustic phoresis, the
large particles M are concentrated at the centre of the path 3,
whereas the smaller particles, designated by m, are shifted to the
sides of the path 3 so as to be then discharged in at least one
peripheral duct, here two ducts designated by 9c, which preferably
communicate with the discharge ducts 9 or 23.
[0426] Preferably, the transducers 140-141 are located in a first
area of a microfluidic duct of the device MD, such as the path 3,
whereas the detection means 120-121 are located in a second area of
a microfluidic duct of the device MD, such as the path 3. In
particular, the peripheral duct or ducts 9c are connected to said
microfluidic duct 3 in an area comprised between said first area
and said second area.
[0427] A section of the type illustrated in FIG. 87 can be used as
refinement of the separation, i.e., after an initial separation has
been conducted with a different method, such as a mechanical
separation or filtration. For example, a system of acoustic
phoresis can be used for separating the possible residual particles
(such as erythrocytes or leukocytes that are not of interest) from
the target particles.
[0428] It will be appreciated that, in various embodiments of the
invention, the mixed-flow separation or filtering means including
the corresponding lateral delimitations 10 and 11 with the
corresponding paths 8 and 9 could be replaced by separation or
filtering means according to other technical solutions or according
to the known art, it remaining understood that other
characteristics of the invention remain valid, such as for example
the separability of the section 20, the provision of a collection
section using a substrate made of semiconductor material, the use
of separation and/or counting and/or alignment means of an
electrical/electronic type, and equipment or kits for use in
combination of the device.
[0429] As has been seen previously, the devices according to the
invention, i.e., the device MD, considered as a whole, or its
individual sections 1 and 20 considered separately, can integrate
electrical and/or electronic components or arrangements, and in
this case appropriate electrical-supply means are provided in
regard to an external system, such as, for example, means for
electrical connection to an apparatus of analysis.
[0430] According to an advantageous version of the invention, at
least one of the sections 1 and 20 is provided with
electrical-supply means that operate on the basis of a coupling a
of wireless or pinless type, such as a coupling with galvanic
insulation or with an induction of energy via coupled windings,
comprising for example an antenna or a winding belonging to the
device MD (section 1 and/or section 20) and an antenna or winding
belonging to the aforesaid external system.
[0431] With reference to FIG. 77, a device according to the
invention can be provided with a system comprising windings or
antennas, which can be exploited both for supplying the electrical
energy necessary for performing functions for transmitting and/or
receiving data and for supplying the electrical energy necessary
for carrying out at least one of the further electrical/electronic
functions of the microfluidic device that have been described
previously.
[0432] The configuration of the elements designated by C1 and C2 in
FIG. 77 is suited to describing also the case where the elements C1
and C2 do not belong to a data-transmitter and/or data-receiver
system, but to a power coupling of an inductive type (also known as
"pinless power coupling") without any transmission of data in
radiofrequency, of the type including a primary winding C1 shielded
by a suitable insulating layer, for inductive coupling with a
secondary winding C2.
[0433] Also in such an embodiment, then, the windings C1 and C2
enable supply of the electronic circuitry of the device MD, or of
its sections 1 and/or 20, albeit without transmission or exchange
of data. This solution can prove particularly useful, for example,
for the section 20 of the device, which, also when it is separate
from the body 2, can be supplied electrically in a convenient way.
In this embodiment, then, the section 20 will comprise the antenna
or secondary winding C2 and will receive the energy via the antenna
or primary winding C1. The electrical supply thus provided can be
used for carrying out the various electrical/electronic functions
integrated in the section 20, for example for illuminating or
exciting particles collected in the section itself, via an
integrated optical source, or again for supplying electrical energy
to a heater, for localized heating of the cells, for example for a
cell culture.
[0434] The use of a wireless or pinless electrical-supply system
moreover presents the advantage of enabling a fast and convenient
handling of the device MD or of the sections 1 and/or 20,
preventing the risk of rupture of electrical terminals following
upon erroneous and/or forced insertion. In this regard it should be
considered that the terminals for the connectors may be delicate,
above all when they belong to miniaturized electrical connectors. A
supply of a wireless or pinless type avoids the need for an
electrical connector with the consequent risks of breaking
thereof.
[0435] The antenna or winding C2 can be advantageously obtained on
a printed circuit or PCB with which the section 2 or 20 is
provided, as already exemplified previously, or again on parts made
of insulating material of said sections, for example, glass or
plastic, with techniques in themselves known, for example via
serigraphy or deposition of conductive pastes. The antenna or
winding referred to above can of course be integrated also in a
silicon support of the device.
[0436] It should moreover be noted that a control circuit of the
type previously designated by 80, 81 with reference to FIG. 77 can
belong to a laboratory or analysis apparatus having functions
different from the ones simplified, and hence apparatuses that not
necessarily are designed for circulation of one or more fluids.
Such a control circuit can, for example, be integrated in a viewing
system or in a microscope, for example for detecting particles in
the section 20 after it has been separated from the body 2, or else
be integrated in an apparatus for support or storage of devices MD,
or again be integrated in a cell-culture apparatus.
[0437] The mixed-flow separation technique has been described
previously with reference to mixing of a buffer of the path 8 with
a biological fluid or blood of the path 3. Consider, however, that,
in order to implement the separation technique described, a portion
of the same biological fluid or blood under analysis could be made
to pass in the path 8. Also in such an embodiment, the auxiliary
flow of blood of the path 8 is conveyed via the passageways 10a
into the path 3 in order to mix with the other portion of blood,
for the purposes already described, in particular for determining
components of thrust, forces, or turbulent or irregular motions in
the flow in the path 3. In said modality of use, then, the fluid to
be treated or blood would be injected both into the inlet 4 that
into the inlet 8.
[0438] It is clear that the microfluidic device described by way of
example herein may undergo numerous variations by a person skilled
in the branch, without thereby departing from the scope of the
invention as defined in the ensuing claims.
[0439] The passageways 10a of the lateral delimitation 10 have been
previously described with reference to a shape generally inclined
with respect to the normal direction of flow in the path 3.
However, in other embodiments of mixed-flow separation structures,
the passageways 10a, or at least some of them, can have a different
orientation or shape, for example be orthogonal or inclined in a
direction opposite to the direction of the flow in the duct 3, as
highlighted schematically in the areas designated by Z in FIG. 1.
Also such a different configuration proves suited to inducing
turbulence in the flow of the biological fluid of the duct 3,
following upon introduction therein, via said passageways 10a, of a
flow of an auxiliary fluid, whether this be a buffer or blood.
* * * * *