WO2020053714A1 - Capteur pour la détection de biomolécules dans un fluide biologique par réaction de chimioluminescence - Google Patents

Capteur pour la détection de biomolécules dans un fluide biologique par réaction de chimioluminescence Download PDF

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Publication number
WO2020053714A1
WO2020053714A1 PCT/IB2019/057489 IB2019057489W WO2020053714A1 WO 2020053714 A1 WO2020053714 A1 WO 2020053714A1 IB 2019057489 W IB2019057489 W IB 2019057489W WO 2020053714 A1 WO2020053714 A1 WO 2020053714A1
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Prior art keywords
micro
pillars
layer
reaction volume
sensor according
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PCT/IB2019/057489
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English (en)
Inventor
Cecilia PEDERZOLLI
Cristina POTRICH
Lorenzo LUNELLI
Maurizio Boscardin
Lucio Pancheri
Laura PASQUARDINI
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Fondazione Bruno Kessler
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Priority to EP19779596.6A priority Critical patent/EP3850344A1/fr
Publication of WO2020053714A1 publication Critical patent/WO2020053714A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/028—Modular arrangements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0819—Microarrays; Biochips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0877—Flow chambers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0887—Laminated structure
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N2021/0346—Capillary cells; Microcells

Definitions

  • the present invention relates to sensors for quantitative detection of biomolecules (for example, markers of a certain pathological condition) in biological fluids, in particular to sensors comprising a reaction chamber in which a chemiluminescence reaction takes place between biomolecules (analytes) and molecular recognition elements immobilized on a functionalized surface of said reaction chamber, and an optical sensor facing said reaction chamber for detecting electromagnetic radiation generated, in use, by a chemiluminescence reaction.
  • biomolecules for example, markers of a certain pathological condition
  • the aforesaid sensors of a known type are typically characterized by the presence of a reaction chamber having an inner surface functionalized with molecular probes (or molecular recognition elements, MREs ) .
  • Functionalization of an inner surface of such a reaction chamber may be carried out, for example, according to a known procedure comprising:
  • SAM self-assembled monolayer
  • the molecular probes used may be selected, for example, from among DNA or RNA aptamers, entire antibodies or fragments (Fab), and peptides. Selection of a particular species of molecular probe is made as a function of the particular analyte that is to be detected in the biological fluid examined.
  • the molecules used for passivating the reactive sites on the surface not bound to a molecular probe may be chosen, for example, from among short molecules with double functionality, proteins, peptides, and polyethylene glycols of various lengths and with various functionalities. Also this choice may be dictated by the particular analyte that is to be detected in the biological fluid examined.
  • the reaction chamber having a functionalized inner surface usually has a wall that is at least partially transparent to electromagnetic radiation in a given range of wavelengths, this range of wavelengths comprising the wavelengths corresponding to those of the radiation emitted, in use, by a chemiluminescence reaction that takes place in the reaction chamber.
  • an optical sensor typically faces the reaction chamber through the above transparent wall in such a way as to detect electromagnetic radiation produced by a chemiluminescence reaction that takes place inside the reaction chamber, and transduce the optical signal into an electrical signal indicating the intensity of electromagnetic radiation emitted.
  • Such an optical sensor may, for example, be a single-photon detector or single-photon avalanche diode (SPAD) , the structure of which is known to the person skilled in the art.
  • An optical sensor of a SPAD type is typically able to supply a current pulse containing a number of electrons of the order of 10 s for each photon detected. This current pulse corresponds to an electrical signal that can be detected, for example, using an electronic circuit comprising a comparator and a counter, as illustrated in the aforementioned paper by Laura Pasquardini, et al .
  • the above sensors for quantitative detection of biomolecules are typically used by introducing a sample to be analysed, i.e., a biological fluid in which a given biomolecule (the analyte) is to be sought, into the reaction chamber through an inlet microchannel .
  • Incubation i.e., the step in which the sample remains in the reaction chamber for conduct of the analysis
  • Incubation may be static or dynamic and may last from a minimum of a few minutes up to a maximum of a few hours, according to the type of assay and the amount of analyte present in the sample.
  • the assay may be of a direct or competitive type.
  • a further molecular recognition element (MRE) in solution is used, which may have, directly bound thereto, an enzyme that causes development of the chemiluminescence reaction, or else form part of a system that comprises a chain of molecular-recognition elements that further amplifies the optical signal generated by the chemiluminescence reaction .
  • MRE molecular recognition element
  • a competitive assay instead, into the reaction chamber analyte is introduced bound to a chemiluminescence enzyme, which saturates the binding sites left available.
  • a development solution for example, a mixture of hydrogen peroxide and luminol
  • a development solution for example, a mixture of hydrogen peroxide and luminol
  • the reaction chamber is typically emptied of the fluids introduced therein through an outlet microchannel .
  • the reaction chamber of the sensor may be subjected to a flushing step, carried out by introducing into the reaction chamber an appropriate buffer solution through the inlet microchannel. The buffer solution is then removed through the outlet microchannel .
  • a first disadvantage derives from the use of a basically planar functionalized surface, which enables adhesion of a limited amount of molecular probes.
  • a small amount of molecular probes on the functionalized surface results in a weak chemiluminescence reaction, which generates an optical signal that is of low intensity and hence difficult to detect. Consequently, deriving therefrom is a sensor characterized by a lower detection limit that does not enable detection of analytes in small concentrations within the sample.
  • a second disadvantage of known sensors derives from the use of an optical sensor of a SPAD type, which is characterized by a dark count rate that is proportional to the area of the optical sensor itself. This choice prevents the area of the optical sensor from being increased beyond a certain threshold, which would otherwise introduce an excessive noise that would saturate the reading circuit of the sensor itself.
  • the aforesaid number of false positives is equal to approximately 50000 pulses per second per square millimetre of area of the sensor.
  • an optical sensor of a SPAD type having a limited area, typically having a photosensitive area much smaller than the area of the functionalized surface.
  • Such an optical sensor is unable to detect entirely the electromagnetic radiation generated by a chemiluminescence reaction over the entire functionalized surface, this resulting in a deterioration of the resolution of the sensor.
  • a third disadvantage of known sensors is represented by the impossibility of re-using only some of the components of the sensor, for example only the optical sensor (or, more in general, only the optoelectronic components), as a result of the manufacturing process, which involves permanent assembly of all the components of the sensor together.
  • the object of the present invention is to solve the technical problems mentioned previously in relation to known sensors.
  • an object of the invention is to provide a sensor for detecting biomolecules in a biological fluid via a chemiluminescence reaction with improved lower detection limit as compared to known solutions, enabling detection of biomolecules in particularly low concentrations.
  • a further object of the invention is to provide a sensor for detecting biomolecules that is provided with an optical sensor (or, more in general, optoelectronic components) that can be re-used in association with different reaction chambers, thus eliminating the need to carry out flushing of the reaction chamber and removing any possibility of contamination between different samples, without increasing excessively the cost of the single analysis.
  • the object of the invention is achieved by a sensor for detection of biomolecules in a biological fluid via a chemiluminescence reaction, said sensor comprising:
  • reaction volume being in fluid communication with an inlet microchannel for inlet of at least one fluid into said reaction volume and with an outlet microchannel for discharge of at least one fluid from said reaction volume;
  • an optoelectronic layer comprising at least one photodetector for detecting electromagnetic radiation generated, in use, by a chemiluminescence reaction in said reaction volume;
  • the senor according to the invention is characterized in that said microstructured surface is functionalized with molecular-recognition elements immobilized at least partially on the surface of said micro-pillars.
  • FIG. 3 comprises a portion a) that represents, for various embodiments, a microstructure in the form of micro-pillars, and a portion b) that represents a plurality of micro-pillars arranged in an orderly way to form a microstructured surface;
  • FIG. 4 comprises a portion a) and a portion b) , which are two schematic top plan views of possible orderly micro-pillar arrangements in various embodiments ;
  • FIG. 5 comprises a portion a) that represents a portion of a lateral surface of a micro-pillar in various embodiments, and a portion b) that is a schematic view of a portion of a lateral surface of a micro-pillar in various embodiments;
  • FIG. 6 shows a portion of the surface illustrated in Figure 5a at an enlarged scale
  • Figure 7 comprises a portion a) and a portion b) , which are side views of the plurality of micro pillars of Figure 3b in the absence and in the presence, respectively, of a fluorescence reaction.
  • the reference number 1 designates as a whole a sensor for detection of biomolecules according to a first embodiment of the invention, here represented in exploded view.
  • the sensor 1 comprises:
  • reaction volume 3 being in fluid communication with an inlet microchannel 4 for inlet of at least one fluid into the reaction volume and with an outlet microchannel 5 for discharge of at least one fluid from the reaction volume;
  • an optoelectronic layer 6 comprising at least one photodetector 7 for detecting electromagnetic radiation generated, in use, by a chemiluminescence reaction in the reaction volume 3;
  • separation layer 8 arranged between the microfluidic layer 2 and the optoelectronic layer 6, the separation layer 8 being at least partially transparent to electromagnetic radiation in a given range of wavelengths;
  • microstructured surface 9 housed in the reaction volume 3 on a portion of at least one surface of said reaction volume, preferably on a surface of said reaction volume that is opposite to the photodetector 7.
  • the microfluidic layer 2 may be made, for example, of a material such as silicon, silicon oxide, glass, polymeric material, and/or similar and/or equivalent materials.
  • the reaction volume 3 and the microchannels 4, 5 may be obtained in the microfluidic layer 2 using micromachining techniques known to the person skilled in the art .
  • the reaction volume 3 typically has dimensions in the region of a few microlitres, preferably between 0.1 m ⁇ and 10 m ⁇ . Such small dimensions of the reaction volume 3 enable saving of reagents and likewise reduction of the dimensions required for the sample to be analysed.
  • the microchannels 4, 5 typically have an internal cross-sectional area comprised between 0.01 mm 2 and 1 mm 2 , preferably in the region of 0.2 mm 2 .
  • These microchannels may be coupled to a microfluidic system external to the sensor 1 (not represented in Figure 1), implemented, for example, via miniaturized motor- syringes and/or pumps, for regulating inlet and outlet of the fluids and enabling precise control of the amount of fluids introduced and/or discharged, and possibly control of flushing of the reaction volume 3.
  • the optoelectronic layer 6 comprises a photodetector 7 facing the reaction chamber 3.
  • the optoelectronic layer 6 also comprises a printed-circuit board (PCB) that houses the electronic circuitry for control and/or reading of the photodetector 7.
  • PCB printed-circuit board
  • the photodetector 7 is mounted in a respective package 7b fixed to the optoelectronic layer 6, the package being provided with terminals for electrical connection to the electronic circuitry for control and/or reading of the photodetector 7.
  • the photodetector 7 may be soldered directly on the PCB, in the case where this is housed on the optoelectronic layer 6.
  • the separation layer 8 is made of polymeric material, for example an elastomeric material, such as polydimethylsiloxane (PDMS) .
  • PDMS polydimethylsiloxane
  • a first function of the separation layer 8 is to electrically insulate the optoelectronic layer 6 from the reaction volume 3, which in use contains one or more fluids, in order to guarantee proper operation of the photodetector 7 and of the electronic circuitry associated thereto.
  • a second function of the separation layer 8 is to prevent contamination of the optoelectronic layer 6 with the biological fluids introduced into the reaction volume 3.
  • the sensor 1 is assembled by coupling together the layers 2, 8, and 6 by means of a pressure system.
  • This pressure system may be, for example, a snap-fit system, or else a gluing system, or else again a system in which the separation layer 8 is made of silicone material and simultaneously performs the function of seal of the reaction volume 3 and of mechanical coupling of the optoelectronic layer 6 and microfluidic layer 2.
  • the aforesaid assembly of the layers 2, 8, and 6 may be reversible, thus enabling separation of the layers 2, 8, and 6 after use of the sensor 1, for example in order to re-use the optoelectronic layer 6 and/or the microfluidic layer 2.
  • the microstructured surface 9 is provided on a plate-like element 11, and in particular on a surface of the plate-like element 11 that faces into the reaction volume 3, and that as such constitutes an inner surface of the reaction volume 3 itself.
  • the above plate-like element 11 is typically made of a material similar to the material that constitutes the microfluidic layer 2, namely, as described previously, silicon, silicon oxide, glass, polymeric material, and/or similar and/or equivalent materials.
  • the surface of the plate-like element 11 that bears the microstructured surface faces the photodetector 7 in such a way as to facilitate the detection thereby of the electromagnetic radiation emitted by a chemiluminescence reaction that takes place on the microstructured surface 9. This significantly increases the number of photons that reach the photodetector 7 after emission from the microstructured surface 9.
  • the separation layer 8 has two reliefs 10, projecting from the surface of the separation layer 8 towards the inside of the reaction volume 3.
  • the reliefs 10 are configured for exerting a pressure on the microstructured surface 9, and in particular on the plate-like element 11, pushing the latter against an inner wall of the reaction volume 3, and thus blocking it in the desired position.
  • all the layers 2, 8, and 6 are re-usable, provided the reaction volume 3 is flushed with buffer solutions, and only the plate-like element 11 comprising the functionalized surface 9 is replaced at each new analysis;
  • the separation layer 8 is replaced at each new analysis, but re-use of the microfluidic layer 2 in any case requires flushing of the reaction volume 3 with buffer solutions ;
  • Figure 2 represents, once again in exploded view, a sensor 1 for detection of biomolecules according to a second embodiment of the invention.
  • the embodiment illustrated in Figure 2 differs from the one illustrated in Figure 1 in that the microstructured surface 9 is not provided as element distinct and separate from the layers 2, 6, and 8.
  • the optoelectronic layer 6 and the microfluidic layer 2 are substantially the same as those of the first embodiment described with reference to Figure 1.
  • the separation layer 8 is not provided with reliefs 10 and houses the microstructured surface 9 in an approximately central region thereof, and in any case in a region arranged at the reaction volume 3.
  • the molecular probes can be immobilized on a surface of the plate-like element 11 held in position within the reaction volume 3 via the reliefs 10
  • the microstructured surface 9 is obtained directly on a portion of the surface of the separation layer 8 that faces into the reaction volume 3.
  • the above second embodiment is advantageous in so far as it reduces the complexity of assembly of the sensor 1, eliminating the need to position and hold the plate-like element 11 in position within the reaction volume itself. Moreover, in the second embodiment, the distance between the microstructured surface 9 - where the chemiluminescence reaction takes place - and the photodetector 7 is reduced, with an overall increase in the sensitivity of the sensor 1.
  • assembly of the layers 2, 8, and 6 may be reversible, and the following modalities of use of the sensor 1 may be envisaged:
  • the layers 2 and 6 are re-usable, provided the reaction volume 3 is flushed with buffer solutions, and only the separation layer 8 comprising the functionalized surface 9 is replaced at each analysis;
  • microstructured surface 9 comprises a plurality of microstructures, which are in turn possibly characterized by the presence of surface nano-structures, obtained via the manufacturing processes described in detail in what follows.
  • microstructures make it possible to obtain an enlargement of the surface available for immobilization of the molecular probes as compared to known sensors.
  • the possibility of increasing the number of molecular probes that can be immobilized (and are immobilized) on the surface 9 by virtue of its microstructure determines, in various embodiments, a greater intensity of the optical signal produced, given the same concentration of analyte in the biological fluid examined, in so far as it is possible to capture a greater amount of analyte via the molecular probes.
  • a greater intensity of the optical signal produced given the same concentration of analyte in the biological fluid examined, in so far as it is possible to capture a greater amount of analyte via the molecular probes.
  • microstructures provided on the surface 9 for increasing the surface involved in the chemiluminescence reaction comprise a plurality of micro-pillars of the type represented in Figure 3a. It should be noted, on the other hand, that such microstructures (as well as the nanostructures possibly provided thereon) enable an increase of the surface involved in the chemiluminescence reaction, keeping the overall dimensions in plan view of the microstructured surface 9 unvaried, to full advantage of compactness of the sensor 1.
  • Figure 3a illustrates a micro-pillar 12 on the microstructured surface 9.
  • the micro-pillar 12 has an approximately cylindrical shape, with a top surface 13 and a lateral surface 14.
  • the area of the cross section of the micro-pillars 12 may remain approximately constant throughout the longitudinal direction of the micro-pillar 12, or else present slight variations, without thereby affecting functionality of the sensor.
  • the micro pillar illustrated in Figure 3a has a cross section the area of which decreases slightly in the proximity of the base of the micro-pillar itself.
  • Other embodiments may comprise micro-pillars having an approximately constant cross section, or also a cross section the area of which increases slightly in the proximity of the base of the micro-pillar.
  • micro-pillar structures of the type illustrated in Figure 3a for definition of a microstructured surface 9 may be obtained using micromachining techniques (for example, so-called bulk micromachining techniques) of the type used for the fabrication of electronic and/or MEMS (Micro-Electro- Mechanical Systems) devices.
  • micromachining techniques for example, so-called bulk micromachining techniques
  • Such technologies may include, for example, processes based upon chemical etching obtained by dipping the substrate in solutions of potassium hydroxide (KOH) or tetramethylammonium hydroxide (TMAH) , or else physical technologies based upon plasma etching, in combination with photolithographic techniques (whether optical of electron-beam lithography - EBL) for the definition of the structures.
  • a process of selective removal such as DRIE (Deep Reactive Ion Etching) enables provision of micro-pillar structures with different geometries and of different heights.
  • DRIE Deep Reactive Ion Etching
  • a microstructured surface comprising pillars 12 may be obtained both on silicon substrates (or substrates made of similar materials) for application in embodiments like the ones illustrated in Figure 1, and on polymeric substrates, for example PDMS substrates, for application in embodiments like the ones illustrated in Figure 2, varying accordingly the micromachining techniques used.
  • micro-pillar structures may be obtained directly on silicon substrates (or substrates made of similar materials) using micromachining techniques
  • similar micro-pillar structures may be obtained indirectly on polymeric substrates (for example, made of PDMS) using moulding techniques .
  • the above moulding techniques envisage fabrication of complementary structures (i.e., micro-cavities instead of micro-pillars) via micromachining techniques on silicon substrates (or substrates made of similar materials), and using silicon moulds thus obtained to form micro-pillar structures on polymeric substrates.
  • the micro-pillars 12 may have an approximately cylindrical shape, with a longitudinal dimension comprised between 50 pm and 400 pm and a transverse dimension comprised between 5 pm and 20 pm.
  • longitudinal direction with reference to the micro-pillars 12 is meant a direction of main development parallel to the axis of the micro-pillar structure 12, and by “transverse direction” is meant a direction of secondary development, transverse to the axis of the micro-pillar structure 12.
  • longitudinal direction is to be understood as being parallel to the axis of the cylinder, whereas the transverse direction is to be understood orthogonal to the axis of the cylinder.
  • combinations of micromachining techniques via wet etching and dry etching enable control of roughness both on the top surface 13 and on the lateral surface 14 of the micro-pillars 12.
  • a plurality of micro-pillars 12 may be provided on the microstructured surface 9, to obtain substantially an array of pillars as illustrated in Figure 3b.
  • array is intended to indicate an orderly and regular arrangement of micro-pillars, where the distance between one given micro-pillar and the next (i.e., the "pitch" of the array) is approximately constant.
  • Figure 3b represents an array of pillars having a triangular elementary cell.
  • Figure 4 is a schematic illustration of some possible orderly arrangements of micro-pillar 12 on the microstructured surface 9.
  • Figure 4a is a top plan view of an array of micro-pillars having a rectangular elementary cell, characterized by two pitches A and A' in respective directions (approximately) orthogonal to one another. It is of course possible to provide an array with square elementary cells, wherein the dimensions of the pitches A and A' are the same.
  • micro-pillars 12 are arranged according to an array having a triangular elementary cell, identified, for example, by the micro pillars 12i, 122, 123.
  • the values of the pitches A and A' - and in general, the distances between successive micro-pillars in the matrix arrangement - are typically comprised between 2 and 5 times the respective transverse dimension D of the micro-pillar, so as to result in particularly hydrophilic spatial micro-pillar configurations .
  • the pitches A and A' can hence assume values roughly comprised between 10 pm and 100 pm.
  • the micro-pillars 12 enable increase in the surface available for immobilization of molecular probes, exposing their respective lateral surfaces 14 for this purpose.
  • the lateral surface 14 of each micro-pillar 12 is in turn further characterized by nano-structures that have the purpose of increasing further their surface and controlling the roughness thereof.
  • Nano-structuring of the lateral surface 14 of the micro-pillars may involve the surface 14 in its entirety, or else may be limited to a top portion 15 of the surface 14. For instance, such top portion 15 of the surface 14 extends for at least 10 pm, and possibly up to a maximum of 50 pm, from the top of the respective micro-pillar.
  • a first form of nano-structures obtained in a top portion 15 of the surface 14 is illustrated in Figure 5a .
  • Nano-grooves 16 that extend in a longitudinal direction with respect to the axis of the micro-pillar.
  • the nano-grooves 16 typically have a width, measured in a transverse direction on the surface of the micro-pillar, comprised between 100 nm and 600 nm, and a depth comprised between 100 nm and 600 nm.
  • Figure 5a illustrates also a second form of nano structures obtained in a top portion 15 of the lateral surface 14 of a micro-pillar 12.
  • These nano-structures have the form of nano-grooves 17 that extend at least in part in a transverse direction over the lateral surface 14 of the micro-pillar.
  • the nano-grooves 17 typically have a width, measured in a longitudinal direction on the surface of the micro-pillar, comprised between 200 nm and 600 nm, and a depth comprised between 30 nm and 150 nm.
  • nano-grooves 17 may be modulated, for example, by varying the parameters of a step of micromachining of the microstructured surface 9 that comprises a DRIE process .
  • Figure 5b is a schematic view of the same surface represented in Figure 5a, for clarity of representation. It will be noted that, as visible in Figure 5b, the nano-structures 16 and 17 may be simultaneously present on the lateral surface 14, giving rise to a surface 14 that has a nano-structure repeated both in a longitudinal direction and in a direction transverse with respect to the lateral surface of the micro-pillar 12.
  • the nano structures 16 and 17 are characterized by values of width and depth that are practically constant. Notwithstanding this, the width and depth of the nano structures 16 and 17 may vary, in different regions of the lateral surface of one and the same micro-pillar, in the respective ranges of variability mentioned above .
  • the top portion 15 of the surface 14 of the micro pillars 12 may be characterized by a third type of nano-structures, in particular by globular nano structures 18, as illustrated in Figure 6, which shows a further enlargement of Figure 5a.
  • the globular nano structures 18 typically have an approximately ellipsoidal shape, characterized by a major dimension L comprised between 20 nm and 150 nm and a minor dimension 1 comprised between 15 nm and 100 nm.
  • L major dimension
  • minor dimension 1 comprised between 15 nm and 100 nm.
  • the surface of the micro pillars 12, possibly nano-structured as described previously, is coated alternatively by a layer of insulating material, preferably silicon oxide, or silicon nitride, or aluminium oxide, or by a layer of metal material.
  • Insulating materials can be deposited on the surface of the micro-pillars, for example, using PECVD, LPCVD, or ALD techniques, whereas metal materials can be deposited via evaporation or sputtering techniques.
  • the above coating materials provided on the surface of the micro-pillars 12 make it possible to alter the chemical properties of the microstructured surface 9.
  • one or more of these materials may introduce a certain (positive or negative) surface electrical charge, or else may provide chemical groups that can facilitate a process of bio-functionalization of the microstructured surface 9, for example favouring formation of bonds with the molecular probes or MREs.
  • deposition of a thin layer of silicon nitride on the surface of the micro-pillars 12 can favour surface functionalization and subsequent adhesion of nucleic acids (for example, microRNA) as molecular probes.
  • deposition of one or more different materials on the surface of the micro-pillars 12 can favour adhesion of different molecular probes.
  • Figure 7a shows a cross-sectional view of the micro-pillars 12 in clear field
  • Figure 7b shows the same cross-sectional view of the micro- pillars 12 with fluorescence signal.
  • optical fluorescence signal at a top portion 15 of the lateral surface 14 of the micro-pillars 12.
  • an optical sensor 7 of a different type such as a silicon photomultiplier (SiPM) .
  • SiPM silicon photomultiplier
  • Such a silicon photomultiplier 7 is made up of a set of SPAD devices connected in parallel, possibly arranged in a matrix.
  • each SPAD device connected in parallel is an independent sensor (or "cell") , and the current that flows at the terminals of the SiPM optical sensor is equal to the sum of the current pulses of the individual cells.
  • Current reading of a SiPM using an integrator or transimpedance circuit enables a signal to be obtained that is limited by the statistics of arrival of the photons, i.e., by their Poisson distribution, with an intrinsic amplification of the signal of the order of 10 s .
  • the complexity of the electronic reading circuits is in this case reduced as compared to the case where the optical sensor 7 is implemented by means of a simple photodiode.
  • the optical sensor 7 may be an integrated circuit containing arrays of SPADs with a corresponding reading circuit.
  • the signal can be supplied directly as digital count of the number of photons detected, but their efficiency is lower than that of analog SiPMs.
  • the signal-to-noise ratio (SNR) of the optical sensor 7 is hence represented by
  • N ph and DCR are proportional to the photosensitive area A of the optical sensor 7.
  • N ph a - A
  • A is the photosensitive area of the optical sensor 7 and and b are parameters that depend upon the specific implementation of the system, whence
  • the signal- to-noise ratio SNR is thus scaled according to a factor A 2 /A I . From the optoelectronic standpoint, it is hence reasonable to consider an increase of the surface of the optical sensor 7 up to dimensions comparable with the area of the reaction site (i.e., with the area of the microstructured surface 9) in order to obtain an improvement of the sensitivity.
  • the area of the SiPM photodetector 7 sensitive to electromagnetic radiation is comparable with the area in plan view of the microstructured surface 9.
  • the area sensitive to electromagnetic radiation of the photodetector 7 is preferably greater than 1 mm 2 .
  • an optical sensor of an SiPM type instead of a sensor of a SPAD type of comparable area is further advantageous in so far as it enables a wide signal dynamics; i.e., it enables detection of optical signals that may be either very weak or very intense, since the signal measured is produced as the sum of the currents delivered by independent cells in parallel. It is possible to use known techniques of current-to-voltage conversion of the signal that enable the wide dynamic range to be maintained .

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Abstract

L'invention concerne un capteur (1) pour la détection de biomolécules dans un fluide biologique par réaction de chimioluminescence, ledit capteur comprenant : - une couche microfluidique (2) définissant un volume de réaction (3), ledit volume de réaction étant en communication fluidique avec un microcanal d'entrée (4) pour l'entrée d'au moins un fluide dans ledit volume de réaction et avec un microcanal de sortie (5) pour la décharge d'au moins un fluide à partir dudit volume de réaction ; - une couche optoélectronique (6) comprenant au moins un photodétecteur (7) pour la détection d'un rayonnement électromagnétique généré, lors de l'utilisation, par une réaction de chimioluminescence dans ledit volume de réaction (3) ; - une couche de séparation (8) disposée entre ladite couche microfluidique (2) et ladite couche optoélectronique (6), ladite couche de séparation étant au moins partiellement transparente au rayonnement électromagnétique dans une plage donnée de longueurs d'onde ; et - une surface microstructurée (9) faisant face au dit volume de réaction (3), ladite surface microstructurée comprenant une pluralité de micro-piliers (12) ayant de préférence une forme approximativement cylindrique, lesdits micro-piliers ayant une dimension longitudinale comprise entre 50 pm et 400 pm et une dimension transversale comprise entre 5 pm et 20 pm.
PCT/IB2019/057489 2018-09-12 2019-09-05 Capteur pour la détection de biomolécules dans un fluide biologique par réaction de chimioluminescence WO2020053714A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP19779596.6A EP3850344A1 (fr) 2018-09-12 2019-09-05 Capteur pour la détection de biomolécules dans un fluide biologique par réaction de chimioluminescence

Applications Claiming Priority (2)

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IT102018000008541 2018-09-12
IT201800008541 2018-09-12

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WO2020053714A1 true WO2020053714A1 (fr) 2020-03-19

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Citations (5)

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US20020037249A1 (en) * 2000-09-21 2002-03-28 Kaoru Konakahara Method of producing oxide whiskers, oxide whiskers, and photoelectric conversion apparatus
WO2005085806A1 (fr) * 2004-03-05 2005-09-15 Canon Kabushiki Kaisha Puce de reconnaissance pour substance cible, et procédé et dispositif de détection pour ladite puce
WO2014184673A2 (fr) * 2013-04-17 2014-11-20 King Abdullah University Of Science And Technology Nouvelle microstructure d'échafaudage 3d
WO2017040966A1 (fr) * 2015-09-02 2017-03-09 SeLux Diagnostics, Inc. Systèmes et procédés de détection multiplexée de biomarqueurs
US20180221876A1 (en) * 2015-10-01 2018-08-09 The Regents Of The University Of Michigan Assay plate and uses thereof

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US20020037249A1 (en) * 2000-09-21 2002-03-28 Kaoru Konakahara Method of producing oxide whiskers, oxide whiskers, and photoelectric conversion apparatus
JP2002167300A (ja) * 2000-09-21 2002-06-11 Canon Inc 酸化物針状結晶の製造方法、酸化物針状結晶および光電変換装置
WO2005085806A1 (fr) * 2004-03-05 2005-09-15 Canon Kabushiki Kaisha Puce de reconnaissance pour substance cible, et procédé et dispositif de détection pour ladite puce
WO2014184673A2 (fr) * 2013-04-17 2014-11-20 King Abdullah University Of Science And Technology Nouvelle microstructure d'échafaudage 3d
WO2017040966A1 (fr) * 2015-09-02 2017-03-09 SeLux Diagnostics, Inc. Systèmes et procédés de détection multiplexée de biomarqueurs
US20180221876A1 (en) * 2015-10-01 2018-08-09 The Regents Of The University Of Michigan Assay plate and uses thereof

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LAURA PASQUARDINI ET AL: "SPAD aptasensor for the detection of circulating protein biomarkers", BIOSENSORS AND BIOELECTRONICS, vol. 68, 1 June 2015 (2015-06-01), AMSTERDAM, NL, pages 500 - 507, XP055312961, ISSN: 0956-5663, DOI: 10.1016/j.bios.2015.01.042 *
M. BASZCZYK ET AL: "A readout circuit dedicated for the detection of chemiluminescence using a silicon photomultiplier", JOURNAL OF INSTRUMENTATION, vol. 13, no. 05, 8 May 2018 (2018-05-08), pages P05010 - P05010, XP055569629, DOI: 10.1088/1748-0221/13/05/P05010 *

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