U.S. patent application number 15/315837 was filed with the patent office on 2017-06-29 for method for producing an array of planar microparticles with surface molecular multiplexing, resulting array and use thereof.
The applicant listed for this patent is CONSEJO SUPERIOR DE INVESTIGACIONES CIENT FICAS (CSIC), UNIVERSITAT DE BARCELONA. Invention is credited to Juan Pablo Agusil Antonoff, Marta Duch Llobera, Jaume Esteve Tinto, Maria Luisa Perez Garcia, Jose Antonio Plaza Plaza, N ria Torras Andres.
Application Number | 20170184576 15/315837 |
Document ID | / |
Family ID | 54766201 |
Filed Date | 2017-06-29 |
United States Patent
Application |
20170184576 |
Kind Code |
A1 |
Esteve Tinto; Jaume ; et
al. |
June 29, 2017 |
Method for Producing an Array of Planar Microparticles with Surface
Molecular Multiplexing, Resulting Array and Use Thereof
Abstract
A method for controlled production of an array of planar
microparticles with the multiplexing of molecules on the surface
thereof, intended to function as molecular sensors and/or actuators
and a matrix (array) of microparticles, the surface thereof being
printed with all of the molecular components required to provide
the surface with functionality. Different molecular elements are
multiplexed on the surface of each particle while they are
supported on a substrate by means of a structural foot engraved
below the particle. These microparticles can be released
mechanically from the support on which they are produced using a
controlled mechanical rupture method which is not chemically
aggressive and therefore does not affect the molecules previously
printed on the surface. The array and the particles contained
therein offer great versatility in both chemical and/or biological
applications.
Inventors: |
Esteve Tinto; Jaume;
(Barcelona, ES) ; Plaza Plaza; Jose Antonio;
(Barcelona, ES) ; Duch Llobera; Marta; (Barcelona,
ES) ; Torras Andres; N ria; (Barcelona, ES) ;
Perez Garcia; Maria Luisa; (Barcelona, ES) ; Agusil
Antonoff; Juan Pablo; (Barcelona, ES) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CONSEJO SUPERIOR DE INVESTIGACIONES CIENT FICAS (CSIC)
UNIVERSITAT DE BARCELONA |
Madrid
Barcelona |
|
ES
ES |
|
|
Family ID: |
54766201 |
Appl. No.: |
15/315837 |
Filed: |
June 3, 2015 |
PCT Filed: |
June 3, 2015 |
PCT NO: |
PCT/ES2015/070439 |
371 Date: |
March 3, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B82Y 30/00 20130101;
G03F 7/0002 20130101; G01N 33/48 20130101; B82Y 40/00 20130101;
B01J 19/0046 20130101; B01J 19/00 20130101; G01N 33/582 20130101;
B01J 2219/00756 20130101; G01N 33/5432 20130101 |
International
Class: |
G01N 33/543 20060101
G01N033/543; G01N 33/58 20060101 G01N033/58; G03F 7/00 20060101
G03F007/00; B01J 19/00 20060101 B01J019/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 5, 2014 |
ES |
P201430864 |
Claims
1. A method for producing an array of planar microparticles with
functionalized surfaces, said method comprising: a) preparing a
structuration layer of a microparticle starting material on top of
a substrate that serves as a support; b) shaping the microparticles
in the structuration layer by using a microelectronic lithography
technique that shapes a geometry and lateral dimensions, and an
engraving technique with which a thickness of the microparticles is
defined; c) forming a foot in an upper part of the substrate that
is found under the structuration layer in order to support each
microparticle using engraving techniques; and d) chemically
functionalizing the surface of the microparticles that are
supported upon the substrate by the feet using one or more
molecular components.
2. The method according to claim 1, wherein the substrate is formed
of a single material, said material being a silicon sheet or by two
materials, including a second material in the form of a layer that
is located in the upper part of the substrate beneath the
microparticle structuration layer.
3. (canceled)
4. The method according to claim 1, wherein the structuration layer
of the microparticles is a material selected from the group
consisting of polycrystalline silicon, silicon oxide, nitride
selected from the group consisting of silicon, gold, platinum,
copper, aluminum, nickel, cobalt, chromium, metal oxides; tantalum,
iron and aluminum silicates; and silicide selected from the group
consisting of tantalum silicide, iron silicide and aluminum
silicide, and wherein the starting material of the structuration
layer and the upper part of the substrate where the feet are
engraved have a relationship between their rupture limits greater
than or equal to 1.
5. (canceled)
6. The method according to claim 1, wherein the preparation of the
structuration layer in stage a) is carried out by depositing or by
growing said structuration layer on top of the substrate through a
microelectronics technique selected from the group consisting of
thermal growth, chemical vapor deposition, sputtering, and
evaporation.
7. The method according to claim 1, wherein the formation of the
foot of each microparticle in stage c) is carried out by partially
engraving the upper part of the substrate located beneath the
microparticles using a microelectronic technique, with a variable
cross-section of the foot having two different parts, where one
part is narrower than the other, or with one constant cross-section
of the foot that is less than the cross-section of the
microparticle.
8. The method according to claim 7, wherein the foot has a constant
cross section that is less than or equal to 50% of the cross
section of the microparticles.
9. The method according to claim 7, wherein the partial engraving
of the foot is carried out via a lateral physical etching or a
lateral chemical etching.
10. The method according to claim 1, wherein the microparticles are
shaped in stage b) using photo-lithographic techniques.
11. The method according to claim 1, wherein the microparticles are
shaped all with the same shape and size or in two or more groups
with different shapes and sizes.
12. (canceled)
13. The method according to claim 1, wherein the functionalization
of stage d) is carried out using a molecule-printing technique
selected from the group consisting of microcontact printing,
dip-pen nanolithography, and polymer-pen lithography technique.
14. The method according to claim 1, wherein the molecular
component is a molecule with chemical and/or biological activity,
selected from the group consisting of organic compounds, polymers,
peptides, proteins, nucleotides, nucleic acids, and any combination
thereof.
15. The method according to claim 1, wherein the surface of each
microparticle is functionalized in stage d) with more than one
different molecular element, or with a single molecular element
more than one time.
16. The method according to claim 1, which further comprises: e)
breaking the feet that support the microparticles by applying
mechanical breaking loads in order to separate said microparticles
from the substrate and individualize them.
17. The method according to claim 16, wherein the controlled
mechanical breaking load of the foot of each microparticle is
applied by means of a technique selected from the group consisting
of rasping, cutting, cryofracturing, and applying an adhesive
material on the already functionalized surface of the
microparticles and subsequently pulling it off, and dissolving the
adhesive in media that do not affect the molecular
functionalization of the microparticle.
18. The method according to claim 16, which further comprises: f)
gathering the individualized microparticles in a suspension
medium.
19. (canceled)
20. (canceled)
21. (canceled)
22. A planar microparticle with surface molecular multiplexing
individualized and released using the method defined in claim
17.
23. A suspension of microparticles with surface molecular
multiplexing obtained using the method defined in claim 18.
24. (canceled)
25. (canceled)
26. (canceled)
27. A method for detecting, analyzing and/or acting upon one or
more parameters of a sample, the parameters being selected from the
group consisting of chemical parameters, biological parameters and
a simultaneous mixture thereof, the method comprising: adding to
the sample one or more microparticles according to claim 22, and
measuring a signal emitted by at least one of the
microparticles.
28. The method according to claim 27, further comprising
transporting drugs or reagents incorporated into the
microparticle.
29. A method for detecting, analyzing and/or acting upon one or
more parameters of a sample, the parameters being selected from the
group consisting of chemical parameters, biological parameters and
a simultaneous mixture thereof, the method comprising: adding to
the sample a microparticle suspension according to claim 23, and
measuring the signal emitted by at least one of the microparticles.
Description
SECTOR AND OBJECT OF THE INVENTION
[0001] The present invention is aimed at the application of new
technologies for the manufacture of arrays of microparticles in the
broad field of engineering. Due to the very nature of the array
produced as well as the microparticles it contains, and which may
be singled out by mechanical means, the area of application of this
invention is very broad, encompassing the sectors of chemistry,
cellular biology, medicine and pharmacology.
STATE OF THE ART
[0002] In the field of engineering and material science, a particle
is understood to be any body with a micrometric or nanometric scale
and having a mass, and which may be obtained naturally or
artificially by means of physical and chemical methods. At present,
the different existing techniques for obtaining micro- and
nanoparticles may be classified into two large groups, based on
whether they are produced directly in an individual manner, or
whether they are produced in the form of an ordered matrix or
array. All of the techniques encompassed in the first of these two
groups (individual particle manufacture) are based on two different
types of approaches, called "top-down approach" and "bottom-up
approach". The first of them is based on the production of a
particulate material out of a "bulk" of material or larger
structures, through progressive reductions in size (Dorian A.
Canelas, Kevin P. Herlihy and Joseph M. DeSimone. Wiley Inter. Rev.
Nanomed. Nanobiotechnol. (2009), 1, 4, 391-404). The bottom-up
approach, on the other hand, consists of supramolecular chemical
synthesis, which uses the chemical information contained in the
different individual components (atoms or molecules) to get them to
spontaneously group together into larger complex particles, by
means of "self-assembly" processes (Wei Wang, Baohua Gu, Liyuan
Liang and William Hamilton. J. Phys. Chem. B (2003), 107,
3400-3404). In recent years there has been increased scientific
interest in the development of new polymer-based materials and
compounds. By using chemical synthesis techniques, such diverse
techniques have been developed as "flow-focusing", pulverization or
microemulsion, which, combined with microfluidics, are also being
used to fabricate particles, both simple and compound (K. P. Yuet,
D. K. Hwang, R. Haghgooie and P. S. Doyle. Langmuir (2010), 26, 6,
4281-4287).
[0003] What is common to all of these self-assembly techniques is
the direct production of large quantities of identical particles,
made individually and at a low cost. The main drawback of these
particle-manufacture methods is that strictly chemical methods must
be applied for their functionalization, whereby either a total and
single (identical) functionalization may be obtained for all of
them, or a combination of two or more functionalizations, by using
chemical substances, as long as these do not affect one another. It
is a difficult and highly complex process due to the multiple
incompatibilities that this entails (neither versatility nor
discretization). The particles produced through the methods
described above are usually used in the field of pharmacology and
biomedicine as "drug delivery systems" (Tasciotti E., Liu X W,
Bhavane R., Plant K., Leonard A. D., Price B. K., Cheng M. M. C.,
Decuzzi P., Tour J. M., Robertson F. and Ferrari M. Nature
Nanotechnol. (2008), 3, 3, 151-157) and as "nanocarriers" (D. Peer,
J. M. Karp, S. Hong, O. C. Farokhzad, R. Margalit and R. Langer.
Nat. Nanotechnol. (2007), 2, 751-760); although they are also
widely used, in the case of magnetic particles, as "magnetic
separation bioprocesses", by means of magnetophoresis and
electromagnetophoresis, as well as for research into new materials
and compounds.
[0004] On the other hand, there is the set of
ordered-particle-matrix manufacturing techniques, based on micro-
and nanoelectronic fabrication processes. These techniques, both
those based on conventional photo-lithography processes (M.-H. Wu
and G. M. Whitesides. Appl. Phys. Lett., (2001), 78, 16, 2273-2275)
and those based on processes known as "soft-lithography" (Y. N. Xia
and G. M. Whitesides. Angew. Chem., Int. Ed. (1998), 37, 551-557),
such as micromolding, microcontact printing (MCP), among others,
also allow for the simultaneous production of thousands of units
(the concept of "batch processing"), in a controlled and low-cost
manner, but with high adaptability for the use of materials as
diverse as metals and polymers, including a large number of
biomaterials, and the possibility of making changes between them.
These processes give the designed particles a great deal of
versatility, both in terms of shape and dimensions, and make it
possible to simultaneously produce different types of particles.
Although in some cases the manufacture process of this type of
particles is more complex than those mentioned previously, these
techniques make it possible to obtain particles with multiple
surfaces, opening up the range to a wide variety of possible
applications. Because of their ordered and controlled location on
the surface of the substrate where they are produced, these
particles make it possible to apply different types of
functionalization on a single particle, in a localized manner
within the same. The currently existing techniques for separating
particles obtained through lithographic processes from the produced
substrate, a concept known as particle release or
individualization, require the use of methods based on the use of
chemical agents that may be aggressive for the multiplexed
molecules on their surface, as in the case of the "surface
technique" (E. Fernandez-Rosas, R. Gomez, E. Ibanez, L. Barrios, M.
Duch, J. Esteve, C. Nogues and J. A. Plaza. Small (2009), 5, 21,
2433-2439), where a sacrificial layer located between the particle
and the substrate is etched with chemicals.
[0005] Multiplexed chips (understood as those chips that bring
together in a single substrate--particle--various types of channels
that can provide and receive different information, by means of
each one of the functionalizations printed on their surface) made
up of an ordered matrix of molecular elements, and with dimensions
on the order of centimeters, for example "DNA chips", have been
widely used in fields such as medicine and biology to identify,
quantify and determine the workings of certain molecules (S. F.
Kingsmore. Nat. Rev. Drug. Discov (2006), 5, 310-320). For cases in
which small volumes of samples must be analyzed, the current
technological solution is to produce particle suspensions in which
each element is comprised by a sub-population of particles that
differ from the other groups in that they have different
anisotropic attributes (shape, dimensions, color, etc.). The lack
of multiplexing on a single particle of the ones containing these
suspensions, a characteristic multiplexed chips do offer,
constitutes a significant limitation when it comes to analyzing
small volumes (for example, the inside of a cell).
[0006] The present invention presents a new proposal based on the
manufacture of an ordered matrix of planar microparticles with
surface molecular multiplexing, whose geometry and dimensions may
vary depending on their final application. The functionalization of
its surface may be carried out in a controlled and ordered manner
with a wide variety of molecules simultaneously, for example
proteins and DNA. The microparticles are molded and prepared on a
substrate that they are held on by a securing foot engraved below
them, whereby they may be separated (released) through a method of
controlled mechanical rupture without any chemical release
techniques, as a result of the formation of this new structural
element called a "foot" of the microparticle in the array.
[0007] This foot, which is similar to a column or pillar, acts as
an element for securing these microparticles that make up the array
or matrix to the substrate during the processes of molecular
shaping and multiplexing on their surface, and likewise acts as an
element that concentrates mechanical stress, making it the weakest
point in the entire assembly. This makes it possible, should one
wish to free the particles, to apply directed mechanical stress in
order to break the foot in a controlled manner, without damaging
the microparticles or the multiplexed molecules, since no chemical
releasing agents are used which might affect the structure or
function of said molecules. In other words, apart from being able
to produce surface-functionalized microparticles, here, as opposed
to in other known methods, it is possible to individualize the
microparticles in the array by means of a chemically non-aggressive
method, i.e. one which does not require the use of chemical agents
that might damage the integrity of the microparticle or affect its
prior molecular functionalization. Once released, said
microparticles, as with the array, are able to act as sensors or
actuators of different activities, both chemical and biological,
brought about in the medium where they are found, for instance
certain chemical reactions or variations in physical parameters
such as temperature and pH, among others.
SUMMARY OF THE INVENTION
[0008] The invention described herein relates to an array of planar
microparticles with surface molecular multiplexing prepared from a
starting material deposited or grown on a substrate acting as a
support, whose mission is to act as a molecular detector, sensor
and/or actuator in a sample medium which may be chemical or
biological. Specifically, the array is manufactured with
microelectronic technology, and the functionalized microparticles
that it contains are characterized in that they have dimensions
which may be comprised in the range of 1 .mu.m to 100 .mu.m, as
required by their final application, it being possible to prepare
them on the substrate in large quantities, with well-defined shapes
and dimensions. Due to its size (micrometric) and its surface
functionalization, the microparticle array which may be obtained
using this method offers great versatility with regards to specific
applications, and may be used both in chemical and biological
media, always for scientific and technical purposes.
[0009] The foot that is engraved under the starting material of the
microparticle (which corresponds to the upper portion of the
support) acts as a structural element, individually joining each
microparticle to the substrate during the manufacturing and
molecular multiplexing processes, and allowing the microparticles
to be placed on the substrate in an ordered manner. Moreover, as a
result of the formation of this new structural element, the
microparticles may be separated from the substrate and
individualized in a controlled manner by applying directed
mechanical stress, for instance transverse mechanical stress, a
chemically non-aggressive method which therefore respects the
integrity of the surface-multiplexed molecules. Thus, all of the
prior functionalization carried out is preserved unaltered,
allowing the microparticles to be released subsequent to
functionalization.
[0010] The great industrial advantage of this proposed method
resides in the possibility of manufacturing surface-functionalized
microparticles in series on a substrate, specifically on a foot
that acts as a support and enables the use of parallel printing
methods for the simultaneous molecular functionalization of all of
the microparticles, for example in the same way. Moreover, in a
preferred embodiment of the invention, the foot formed under the
starting material that gives rise to the microparticles during the
manufacturing process enables them to be released or individualized
by mechanical separation from the foot by breaking the latter. Due
to its own design and geometry, which is similar to a column or
pillar that sustains the microparticle, this foot becomes the most
fragile area in the structure, concentrating the mechanical stress
and ensuring the fracture area should one wish to release the
microparticles for individual use.
[0011] Ideally the foot should have a variable (non-constant)
cross-section, i.e. with two different areas such that one of them
is narrower, the narrowest area of the cross-section preferably
being in the center. Thus, the foot offers the resistance needed to
sustain the microparticle during the functionalization process, and
at the same time has a narrower area where the majority of the
mechanical stress is concentrated in the case of controlled
release. In a particular embodiment, the foot may also have a
constant cross-section, so long as said cross-section is smaller
than the cross-section of the microparticle itself, preferably less
than or equal to 50% of the size of the cross-section of the
microparticle.
[0012] Due to its design and geometry, both the foot and the
microparticles in the array may be formed out of the same material,
although it is preferable for the foot to be made with fragile,
non-ductile materials, thereby further facilitating the controlled
rupture thereof.
[0013] As such, the first object of the present invention is the
method itself for producing an array of planar micrometric
particles with functionalized surface, said method comprising the
following stages: [0014] a) preparing a layer of a microparticle
starting material (also called structuration layer) on top of a
substrate that serves as a support, typically a silicon sheet,
although it may be any other material that is equally suitable for
this purpose; said layer of microparticle starting material may be
of a typical microelectronics material, such as polycrystaline
silicon, silicon oxide, silicon nitride, gold, platinum, aluminum,
etc.; [0015] b) shaping the microparticles in the structuration
layer previously prepared on top of the support by means of common
lithography-based microelectronics techniques, with which the
geometry and lateral dimensions are defined, and engraving
techniques with which the thickness is defined after preparing the
layer; [0016] c) the key point in the method is the formation of a
foot in the upper part of the substrate that is found below the
previously molded microparticles, such that each foot sustains a
microparticle. The foot of each microparticle is formed by
engraving said upper part of the substrate, wherein said engraving
may be carried out by means of common microelectronics techniques.
In this way, the feet of the microparticles are prepared to be
sufficiently stable from a mechanical point of view in order to
sustain them during the subsequent stage of molecular
functionalization on the surface of the microparticles, but also
fragile enough to allow them, if so desired, to be broken in a
controlled manner by applying directed mechanical forces, thus
allowing the foot to be broken and the microparticle to be released
from the array. This is why it is advisable for the foot to be
produced from a fragile material such as silicon; and [0017] d)
functionalizing the surface of the microparticles that are
supported by the feet by means of at least one molecular component,
preferably by means of a method that makes it possible to
functionalize a large number of microparticles in parallel, by way
of example, soft-lithography techniques such as microcontact
printing (MCP), dip-pen nanolithography (DPN), polymer-pen
lithography (PPN) or nano-imprint lithography (NIL).
[0018] Basically, the substrate that acts as a support for the
manufacture of the microparticles is covered in a layer that
defines the original material of said microparticles that will
subsequently be molded, and which may be selected from the group of
materials consisting of: silicon and derivatives thereof (silicon
nitride or silicon oxide, polycrystalline silicon), gold, platinum,
aluminum, copper, nickel, cobalt or chromium; metal oxides; and
silicates or silicides of compatible metals such as tantalum, iron
or aluminum. This layer is structured or molded upon the substrate
to define the desired shape of the microparticles, and subsequently
the foot located below them is structured or defined.
[0019] The foot carries out a dual function. On the one hand, it
keeps the microparticle joined to the substrate throughout their
manufacturing process, as well as during the subsequent
functionalization steps, ensuring its position at all times. On the
other hand, since it is the weakest and most fragile part of the
whole structure, it acts as a stress-concentrating element,
enabling it to break should one wish to separate or release the
microparticles from the matrix.
[0020] After shaping or molding the foot of the microparticles by
means of partial engraving (i.e. only in part and not the entire
cross-section constantly, because a column or pillar shape is
preferable), the surface of said microparticles is functionalized
with the various molecular elements that have been selected (such
as organic compounds, polymer chains, proteins, DNA, etc.). This
action is carried out in parallel, although given that the surface
of the substrate contains various microparticles, the
functionalization in parallel may be repeated in series in order to
endow the particles with more than one functionalization; or, it is
also possible to repeat the printing of the same substance several
times. Thus, thanks to functionalization, microparticle
multiplexing is achieved in such a way that a single molecular
element printed more than once or more than one different molecular
element is located in an orderly way on the surface of each one of
the microparticles, unlike the planar chips of molecular matrices,
with dimensions on the order of centimeters, that do not analyze
small volumes, which could be, for example, a cell of an ex vivo
and in vitro sample, and unlike the suspensions of known
micro-nanoparticle sub-populations, where each sub-population has a
single molecular element but does not allow for the multiplexed
analysis of a same microparticle. The embodiment of an array of
molecularly multi-functionalized microparticles on the order of
microns, as is the case of the present invention, also allows for
the multiplexed molecular analysis in small volumes.
[0021] The array of microparticles, after its functionalization,
can be stored in a dry place.
[0022] A second object of the present invention relates to the
array of planar microparticles with functionalized surface itself
obtainable by using the method described above, as well as the
microparticles themselves which can be separated from the matrix by
means of controlled mechanical disruption of the foot. Preferably,
a suspension of these microparticles can be prepared.
[0023] The array of microparticles, as well as the microparticles
themselves that are functionalized and released from the support,
and the suspension that can be prepared with the microparticles can
act thanks to their properties as molecular detectors, sensors
and/or actuators. In other words, the invention also has a third
object which covers the use of these products as sensors, actuators
or detectors of physical, chemical and biological parameters
simultaneously or separately in a medium or sample.
[0024] The present invention is based on the relative observation
of the array of microparticles, which are manufactured on the
support with micrometric dimensions (physical lateral dimensions
preferably comprised between 1 .mu.m and 100 .mu.m, and preferably
with a thickness of between 20 nm and 5 .mu.m) and duly
functionalized molecularly through certain features outlined in a
selective and controlled way on its surface.
DESCRIPTION OF THE FIGURES
[0025] FIG. 1: Representation of two possible configurations of
microparticles in the array obtained through the method described,
one in the shape of a parallelepiped with a width A, length L and
thickness E, and the other in the shape of a circle or a disk, with
a diameter D and thickness E, according to the invention, where (1)
represents the material that makes the foot, (2) the microparticle
and the substrate (4). 1.A shows perspective views and 1.B shows a
cross-sectional view.
[0026] FIG. 2: Representation of a possible microparticle
configuration on the support in the array, where its upper surface
has been coated, in a localized manner, with different molecular
elements designed for its functionalization (3). 2.A shows a
cross-sectional view and 2.B shows a perspective view.
[0027] FIG. 3: Diagram of a preferred manufacturing process of the
array of microparticles based on microelectronic technology,
photo-lithographic processes and layer etching, according to
Example 1, wherein after producing the microparticle matrix, these
microparticles are released and prepared in suspension. A straight
section that shows the manufacturing process of a microparticle,
with a substrate that is mainly a silicon sheet (4) that acts as a
support, upon which there is a layer (5), typically made up of
polycrystalline silicon, silicon oxide, silicon nitride, gold,
platinum, aluminum or chromium, which constituted the original
material that became the microparticles (2) (FIG. 3.A). To define
the microparticles, a photo resin layer (6) was deposited on said
layer of original material of the microparticles (FIG. 3.B), which
was then partially eliminated from specific areas (7), forming a
structured photo resin layer (8) that contained the geometry and
lateral dimensions of the microparticles to be produced (FIG. 3.C).
Said microparticles were formed (structured) by etching the layer
of their starting material (5) in the uncovered areas (7) where the
layer of photo resin (6) had previously been removed, thereby
forming the body of the microparticle(s) (2) (FIG. 3.D). Afterwards
the silicon substrate (4) was engraved immediately below the
microparticles (2) to form the foot (1) (FIG. 3.E). The remaining
photoresin (8) was removed in the upper part of the microparticles
(2). After this the molecular functionalization of the
microparticles was carried out with more than one molecular element
(3) (FIG. 3.F). Lastly, the functionalized microparticles (2) were
released from the substrate (4) by means of controlled rupture, and
were gathered in an aqueous medium, in this example, previously
filtered de-mineralized water, thus producing the microparticle
suspension (9).
[0028] FIG. 4: Scanning electron microscope image showing an
example of producing the array of parallelepiped-shaped silicon
oxide microparticles (dimensions of 3 .mu.m.times.3 .mu.m.times.1
.mu.m) in accordance with Example 1--Section A, wherein one may
easily make out the microparticles arranged in array and anchored
to the substrate by means of a foot. Scale bar=5 .mu.m.
[0029] FIG. 5: Optical fluorescence image showing an example of
surface functionalization of the microparticles by means of the
polymer-pen lithography technique, in accordance with Example
1--Section B. In this case, multiple printings with two different
inks are shown, namely WGA lectin conjugated with the fluorophore
Streptavidin Texas Red.RTM. (red, ink marked with the letter B in
the figure and visualized with darker gray shading on a black
background) and the protein BSA conjugated with the fluorophore
Neutravidin Oregon Green.RTM. (green, ink marked with the letter A
in the figure and visualized with lighter gray shading) prior to
being functionalized with the antibody Goat Anti-Rabbit IgG
conjugated with the marker AMCA and a third ink (blue, not shown in
the figure). In this case, the printed pattern was dots. Scale
bar=10 .mu.m.
[0030] FIG. 6: Scanning electron microscope image showing several
microparticles following their mechanical release from the
substrate by means of controlled mechanical fracture of the feet in
accordance with Example 1--Section C. Scale bar=5 .mu.m.
DETAILED DESCRIPTION AND EMBODIMENT OF THE INVENTION
[0031] As mentioned in the foregoing section, the foot is produced
through molding by engraving the upper part of the substrate, which
is in direct contact with the lower part of the layer of
microparticle starting material. In a particular embodiment of the
invention, the substrate is formed by a single material, most
preferably a silicon sheet, although it may be another suitable
type of substrate that offers mechanical support for the
fabrication of the microparticles, for example borosilicate glass
(commonly known by its commercial name, Pyrex or Duran) or
soda-lime-silica glass, among others. Nevertheless, the invention
is not limited just to these support materials, since any person
skilled in the art will know what types of materials are suitable
and may fulfill the intended function; basically, this includes all
materials that meet the following conditions: [0032] be resistant
to the thermal processes of depositing, evaporating and growing
layers; [0033] be stable at ambient temperature; [0034] be
resistant to certain chemical agents (compounds in liquid or gas
phase) in order to enable the structuration/engraving/processing in
general of the layers in them without affecting the substrate (even
though sometimes they need to be protected, since the chemical
agents tend to be quite aggressive); and [0035] having the ability
to itself be structured/engraved (wholly or partially). This would
be the case in which the foot of the microparticles is produced out
of the substrate itself.
[0036] Thus, the substrate must first and foremost act as a
support, and therefore must be rigid enough to support the
structures, and, while these structures are being processed, it
must maintain its integrity without breaking. Furthermore, in this
preferred embodiment, it must also allow for the foot to be formed
on its upper part, which is in contact with the microparticle
starting material. In a particular embodiment of the invention the
support or substrate may be made of the same material used for the
microparticle starting layer. For example, microparticles can be
manufactured on a substrate that is a silicon sheet with a silicon
foot (i.e. it is engraved into the substrate itself), wherein the
particles have been molded in a polysilicon layer; this enables the
possibility of subsequently carrying out thermal doping processes
to provide the microparticles with charges.
[0037] In another particular embodiment of the invention that is an
alternative to the preceding one, the substrate is formed by at
least two materials, such that it contains a second material in its
structure that is located in the upper part in the form of a layer,
where it has been deposited or grown. In this way, the foot can be
molded by engraving this second material contained in the upper
part of the substrate. If the substrate contains a second material
in the upper part of its structure, where the feet will be
engraved, this second material may be the same material used to
produce the microparticles themselves, or a different material,
preferably one that is more fragile and less ductile than the
structuration layer so as to guarantee that it behaves correctly
under the subsequent breaking stress, for instance polycrystalline
silicon. For example, it is possible to use a silicon substrate
(sheet) only as the support of the layer that will be used as a
starting material for the foot and for the microparticles, without
intervening in any way in the manufacture of the devices defined in
it. Silicon, though not the only option, is highly recommendable
because it is the microelectronics material par excellence given
its compatibility with most processes and resistance to temperature
changes and chemical agents.
[0038] Likewise, the preparation in stage a) of the structuration
layer, which is the same thing as the layer constituting the
material that gives rise to the microparticles, can be carried out
by depositing it or by growing it on top of the substrate itself.
The materials that the structuration layer can be made of may be
selected from the group consisting of: silicon and derivatives
thereof (silicon nitride or silicon oxide, polycrystalline
silicon), gold, platinum, aluminum, copper, nickel, cobalt or
chromium; metal oxides; and silicates or silicides of compatible
metals such as tantalum, iron or aluminum. This layer can be
deposited or can be grown by any method used in microelectronics:
thermal growth, chemical vapor deposition, sputtering, evaporation,
or other common methods used today. The method selected for this
will be determined by the choice of materials to use.
[0039] To guarantee good mechanical behavior of the entire
structure (microparticle plus foot), it is preferable for the
material chosen for the structuration layer and the material of the
substrate that will be engraved into a foot, whether the substrate
is made of one material or contains a second material in its upper
part, to have a relationship between its rupture limits greater
than or equal to one
(L.sub.rupt.sub._.sub.part/L.sub.rupt.sub._.sub.foot.gtoreq.1).
This not only secures the particles during functionalization, but
also ensures that the foot is more fragile than the microparticle
and is therefore more vulnerable to rupture when mechanical stress
is applied in cases where one wishes to free said microparticles
from the substrate in a controlled manner (facilitate their
release). Nevertheless, if the final application so requires, the
structuration layer and the second material that contains the
substrate in its upper part can be manufactured from the same
material, since their own design and geometry enable this.
[0040] The method of manufacturing the microparticles based on
microelectronic technology makes it possible to define its
dimensions preferably by using photo-lithographic techniques, said
techniques being commonly used in the field of microelectronics.
The use of photo-lithographic techniques makes it possible to form
microparticles into specific shapes and dimensions, chosen with
technical criteria, preferably being identical to one another,
although this technique also allows for the manufacture of groups
of microparticles that are identical to one another but different
from other groups in the same array. The micrometric particles in
the array produced by means of the described method may preferably
have dimensions comprised between 1 .mu.m to 100 .mu.m, both limits
included, on the plane of the microparticle. Also preferably, the
microparticles may have a thickness comprised between 20 nm and 5
.mu.m, both limits included. The microparticles may have varying
geometries, for instance a parallelepiped or circular shape,
although these shapes shall not limit the invention.
[0041] The shape of the foot under the microparticle can be defined
through any engraving technique that allows for partial
elimination, just underneath each microparticle, of the material
forming said foot, whether it is the only material making up the
substrate or the second material that said substrate may contain in
its upper part. In this way said element can be given a preferred
shape of the column or pillar type, with a cross section having two
differentiated portions, one narrower than the other, to force the
mechanical stress to concentrate there, or with a cross section
that is uniform throughout its length but smaller than that of the
microparticle itself, preferably less than or equal to 50% of the
size of the latter's cross section. The technique used to form the
feet should preferably be physical etching (dry reactive etching)
or chemical etching (wet), with lateral etching, depending on the
material or materials present in the structures (in the shape of
both the microparticle and the foot) and which in turn makes it
possible to produce a foot with a constant or varying cross
section, whichever is best for the required application.
[0042] In turn, the microparticles may contain one or more classes
of molecules organized into monolayers in localized areas, which
allow them to have several simultaneous uses, and in turn to carry
out specific measurements or observations of one or several
parameters and/or activities inside the medium where they are
found. More specifically, said chemical functionalization may
comprise several molecules of natural or synthetic origin, with
chemical and/or biological activity, which include, but are not
restricted to, simple organic compounds, polymers, peptides,
proteins, nucleotides and nucleic acids. The molecules can be
deposited on the upper face of the microparticles preferably using
techniques from the field of micrometric- and nanometric-scale
molecule printing, such as microcontact printing, dip-pen
nanolithography or polymer-pen lithography.
[0043] As explained previously, it may be possible to release the
microparticles from the array produced by means of the method
described. In this particular embodiment of the invention, after
stage d) for functionalization of the surface of the
microparticles, the described method further comprises: [0044] e)
proceeding to break, in a controlled manner, the feet that support
the microparticles by applying directed mechanical loads, in order
to separate them from the substrate (individualize them). These
loads may be applied by means of a variety of techniques, such as
rasping the foot; applying an adhesive substance on the already
functionalized surface of the microparticles and subsequently
pulling it off, then dissolving the adhesive in media that do not
affect the molecular functionalization of the microparticle;
cryofracture, etc.
[0045] In this way, by applying directed mechanical stress, the
feet can be broken in a controlled manner, for example with a clean
cut, in order to release the microparticles from the substrate of
the array without breaking or damaging them, preserving intact the
functionalization that was previously applied to them, since it is
a completely physical method that is not chemically aggressive.
[0046] Preferably, the mechanical rupture of the feet to
individualize the microparticles that they sustain may be carried
out by means of a directed cut, applying a controlled lateral force
strong enough to break the foot. Said cut may be done with a
micro-tool appropriately designed for this purpose, comprising a
sharpened flat-tipped spatula having micrometric dimensions. In
another preferred embodiment, the mechanical rupture may be carried
out by means of cryofracture, freezing the entire structure of the
array (the substrate with functionalized microparticles and their
respective feet), which comprises: wetting the substrate with a
solution such as a phosphate-buffered saline solution (PBS) with a
content of 0.05% of Tween 20 solution (PBS-T); submerging the
entire structure in liquid nitrogen until freezing the solution;
re-wetting in the same way and re-freezing with liquid nitrogen;
then finally applying a force or movement of leverage with a
gripper or similar element until breaking the foot. The frozen
solution that contains the microparticles is left to melt at
ambient temperature in order to release them. In another preferred
embodiment, an adhesive substance may be deposited on top of the
chemically functionalized microparticles, for instance a layer of a
polymer matrix such as Fluoromount.RTM., in liquid phase so that it
can enter even underneath the microparticles, which partially
hardens after polymerizing. This substance is an aqueous-based
biological mounting medium that is commonly used to cover tissues
containing fluorescent markers, for subsequent inspection in
optical microscopes such as confocal microscopes, and fluorescence
microscopes, including scanning and transmission electron
microscopes (SEM and TEM). At this point, said layer of the polymer
matrix can be manually separated from the substrate, carrying along
with it the microparticles and breaking the feet as it separates
them, after which this hardened layer can be dissolved in a medium
which does not affect the chemical functionalization, for example
in an aqueous medium, in order to eliminate it from the surface of
the micro particles.
[0047] Likewise, in a more preferred version of the foregoing
embodiment, the method further comprises: [0048] f) gathering the
functionalized and separated (or individualized) microparticles in
a suspension medium, which may be any medium that does not affect
the chemical functionalizations.
[0049] Thus, the microparticles, once they have been separated from
the substrate by mechanical means, can be kept for storage in a
suspension in an aqueous medium that may be an acid, neutral or
base, it matters not which, as required by the type of
functionalization carried out.
[0050] Through the fabrication method it is possible to produce an
array of planar microparticles with surface molecular multiplexing.
Likewise, in the particular embodiment in which the feet of the
structure are mechanically broken, these same functionalized
microparticles are obtained but individualized. In an more
preferred embodiment, a suspension of these microparticles is
achieved, according to the aforementioned. Any of those products,
array, microparticle(s) and microparticle suspension may be used to
analyze, by way of example, "chemical parameters", which are all
the measurable chemical magnitudes, such as the pH or the redox
(oxidation/reduction) potential. What is more, it may be used to
simultaneously measure several "biological parameters", thus
referring to any magnitude that proves the presence of specific
biological compounds, or the action thereof in the medium in which
the microparticles are found. Said parameters may be ion
concentration in solution, the activity of a specific enzyme, the
presence of proteins and/or ligands, even the study of DNA, among
others. These parameters in a sample medium may be measured by
means of the signal emitted by one or more microparticles of the
array, by one or more released and individualized microparticles or
by one or more individualized microparticles and in suspension that
is added to the sample medium. The sample medium in which the array
may be used, the individualized microparticle or microparticles or
the suspension thereof may be used as a sensor, actuator or the
like, may be any chemical or biological medium, for example, an in
vitro cell sample. In fact, a single cell may be a suitable sample
medium in which to measure specific parameters due to the
functionalization of the microparticles, which means that, in this
embodiment, a microparticle may be separated from the array to
insert it into the cell.
[0051] It must be noted here that if the array, the individualized
microparticles or the microparticle suspension are used as
actuators, these may also serve in the more preferred embodiment
for substance vehiculation, such as for example, drugs or specific
reagents. As such, in some of the examples of the use of the array
or the microparticles thereof once individualized through the
methods described above, it must be noted that they may be used in
the field of pharmaceuticals and biomedicine as drug transport
systems or drug delivery systems.
EXAMPLES
Example 1
[0052] Producing an array of planar microparticles, each one
functionalized with three different proteins and produced through
the method proposed in the present invention, and release of
functionalized microparticles to produce a suspension.
[0053] The aim of this example is to demonstrate the possibility of
manufacturing an array of planar microparticles, with dimensions of
3 .mu.m.times.3 .mu.m.times.1 .mu.m functionalized with three types
of different molecules. In this particular embodiment, the method
for placing the molecules on the planar surface is based on the
polymer-pen lithography technique. Three different proteins have
been printed using this technique.
A--Producing the Microparticles.
[0054] To produce microparticles, a monocrystalline silicon sheet
with crystallographic orientation (100) with a diameter of 100 mm
and thickness of 525 .mu.m was taken. A thermal silicon oxide was
thermally grown on it at 1100.degree. C. This grown material was
used for the subsequent structuration or molding of microparticles.
Then, as set forth in the paragraphs of the Detailed Description,
the photolithographic process is carried out, that is, the
definition of the structures of the microparticles. To do so, 1.2
.mu.m of positive photoresin (HiPR 6512) was deposited on the
sheet. Using a glass grid as a mask on which the geometry of the
microparticles was defined in chromium, the resin was irradiated
with monochromatic light (wavelength 435 nm). For the specific
embodiment of this Example of the Invention, square geometric
shapes were arranged on the plane, which were 3 .mu.m long and
separated from one another by 3 .mu.m. After irradiating the
photoresin for 5 to 8.5 s, it was partially removed in a developer
solution ODP 462 so that resin only remained in areas of the
silicon oxide layer that subsequently defined the microparticles.
Then, the remaining resin was annealed at 200.degree. C. for 30 min
in order to increase the strength thereof against subsequent
etching. The following process consisted of carrying out vertical
etching on the entire surface, in order to engrave the silicon
oxide layer in the area that was not protected by the resin. To do
so, a dry reactive ion etching equipment was used, using a mixture
of C.sub.2H.sub.6 and CHF.sub.3. This etching ended when the
silicon sheet was reached. After this step of the process, the
microparticles were already well defined but still joined to the
silicon sheet. In the following stage of the manufacturing process,
an isotropic etching of the silicon sheet was carried out, using
the silicon oxide structures as a mask, along with the remaining
resin layer, in a deep reactive ion etching (DRIE) process. To do
so, SF.sub.6 and C.sub.4F.sub.8 gases were used. This process
laterally etched 1.3 .mu.m, from all sides, the silicon located
below the silicon oxide microparticles for the formation of the
feet the held the microparticles joined to the silicon sheet during
the chemical functionalization process. Lastly, the photoresin used
as a mask was removed until the microparticle surface was clean of
organic compounds, leaving the microparticles ready for their
molecular functionalization and subsequent rupture to gather and
suspend them.
B--Functionalization of the Surface of the Microparticles
[0055] As an example of functionalization of the surface of the
microparticles described above, the technique referred to as
polymer-pen lithography was applied. This technique (Fengwei Huo,
Zijian Zheng, Gengfeng Zheng, Louise R. Giam, Hua Zhang and Chad A.
Mirkin, Science (2008) 321, 1658-1660) combines the possibility of
printing or assembling molecular monolayers on a large surface,
characteristic of the microcontact printing technique, with the
accuracy of individualized printing using the dip-pen
nanolithography technique.
[0056] This technique previously required the manufacture of a mold
or stamp made of soft polymeric material to transfer the molecules
to the surface of the sample. In this exemplary embodiment,
polydimethylsiloxane (PDMS), an organic polymer-based silicon in
liquid state, was used, the components of which (a curing agent and
the base elastomer) are mixed in a ratio of 10:1 by weight and are
cured at a temperature between 60.degree. C. and 100.degree. C. for
a period of time that may vary between 45 min and 120 min,
depending on the hardness desired. To manufacture the mold for the
PDMS stamp, another silicon sheet was used where a 1 .mu.m layer of
silicon oxide is thermally grown at 1100.degree. C. A
photo-lithography process, such as the one described above was
used, but with an inverted mask compared to the one used to define
the microparticles (where before there was resin, now there is not,
and vice versa). Similarly to the previous embodiment, the silicon
oxide layer was engraved through the existing mask and the
remaining resin was subsequently removed. Once in this state, an
anisotropic KOH etching was carried out, with which inverted
pyramids were defined in the area where there was no silicon oxide.
These pyramids enabled the subsequent production of the polymer
points. Due to the use of the same mask, but inverted, it is
possible to produce a polymer point for each microparticle.
Therefore, in this specific example a matrix of square-based
inverted pyramids, of 3 .mu.m by 3 .mu.m, separated by 3 .mu.m,
with a depth of 2.12 .mu.m. Once the mold has been obtained, a
surface treatment was carried out with fluorosilane
trichloro-1,1,2,2-tetrahydroperfluorooctylsilane at 97% to prevent
the polymer from adhering to the mold. In this state, liquid PDMS
was deposited on this mold and after the curing thereof, the PDMS
stamp was removed.
[0057] This stamp was used to transfer the absorbed molecules to
the point of the pyramids on the surfaces of the microparticles. In
order to put the molecules on the PDMS mold, the so-called inks are
used. These inks are solutions that may contain any type of
substance that one wishes to print; from organic molecules, such as
for example fluorescent or fluorophore markers, as well as
biomolecules such as single-strand DNA, proteins, etc. depending on
the subsequent application thereof. In this exemplary embodiment,
three types of different inks were used: i) wheat germ agglutinin
(WGA) lectin conjugated with the Streptavidin Texas Red.RTM.
fluorescent marker (SAV-TR) in red; ii) bovine serum albumin (BSA)
protein conjugated with the Neutravidin OregonGreene fluorescent
marker (NAV-OG) in green; iii) Goat Anti-Rabbit antibody IgG
conjugated with the AMCA (7-Amino-4-methyl-3-coumarinylacetic acid)
in blue, respectively. As a process control and to visualize the
results obtained, a fluorescence microscope was used.
C--Mechanical Release of the Microparticles Previously
Functionalized by Means of Controlled Mechanical Fracture.
[0058] To release the printed microparticles of the silicon sheet,
a drop of Fluoromount.RTM. mounting medium is deposited on the
sheet, forming a layer that homogeneously covered the
microparticles of the sheet. The medium was left to polymerize at
room temperature for 1 hours, creating a solid layer that covered
the microparticles. This layer is mechanically separated from the
sheet, taking the microparticles that had been broken at the feet
with it. This method prevents the deterioration of the molecules
previously printed since the medium was chemically inert.
[0059] The polymerized layer that is separated from the sheet with
the separated microparticles was able to be stored in this state,
for the subsequent use thereof in suspension. In order to obtain
the microparticles in suspension, the separated layer was dissolved
in an aqueous medium, such as for example, de-mineralized water or
buffer solutions.
Example 2
[0060] Molecular recognition of proteins: demonstration of the use
of the suspension of microparticles with molecular multiplexing
prepared in Example 1 as sensor and/or actuator.
[0061] In order to demonstrate that the functionalized molecules on
the surface of the microparticles continue to be active (they
maintain their integrity and functionality and therefore, are able
to react with different elements of the medium) after being
immobilized and once the microparticles have been released from the
array substrate by means of controlled rupture of the feet, an
antibody binding assay was carried out. For this assay, Goat
anti-WGA IgG was chosen as the primary antibody and anti-Goat IgG
(H+L) conjugated with the fluorescent marker AMCA
(7-Amino-4-methyl-3-coumarinylacetic acid) in blue was chosen as
the secondary antibody. These antibodies were orderly incorporated
(in first place the primary antibody and then the secondary
antibody) in an aqueous medium, following the standard methods of
these assays, wherein the suspension of microparticles had
previously been incorporated, giving rise to the recognition of
proteins by the primary antibodies and the resulting bonding of
both molecules (primary and secondary antibodies) to said
proteins.
[0062] As a result, the expected changes were noted in the
fluorescence emissions of the proteins previously printed on the
microparticles due to the correct combination of emissions of the
fluorescent markers present in both the proteins and the
antibodies; perfectly visible changes using a conventional
fluorescence microscope and that show that the molecular
recognition centers of the proteins continue to function. Said
changes were the following: [0063] a) the WGA lectin conjugated
with SAV-TR that initially emitted a red fluorescence signal,
changed to magenta, [0064] b) the BSA protein conjugated with
NAV-OG that initially emitted a green fluorescence signal, changed
to cyan, and [0065] c) the Goat Anti-Rabbit IgG antibody conjugated
with AMCA that initially emitted a blue fluorescence signal,
continued to emit in said color.
[0066] As a control of the functionality of the multiplexing
system, said immunoassay was successfully carried out with the
manufactured array, that is, between steps B and C of Example 1 of
the embodiment (after the manufacture and functionalization of the
microparticles and before their release from the substrate), in
order to demonstrate that the molecule multiplexing system by means
of the polymer-pen lithography technique followed by the mechanical
release system of the microparticles did not affect the correct
activity of the molecules.
* * * * *