EP3850344A1

EP3850344A1 - Sensor for detection of biomolecules in a biological fluid via chemiluminescence reaction

Info

Publication number: EP3850344A1
Application number: EP19779596.6A
Authority: EP
Inventors: Cecilia PEDERZOLLI; Cristina POTRICH; Lorenzo LUNELLI; Maurizio Boscardin; Lucio Pancheri; Laura PASQUARDINI
Original assignee: Fondazione Bruno Kessler
Current assignee: Fondazione Bruno Kessler
Priority date: 2018-09-12
Filing date: 2019-09-05
Publication date: 2021-07-21
Also published as: WO2020053714A1

Abstract

Described herein is a sensor (1) for detection of biomolecules in a biological fluid via chemiluminescence reaction, said sensor comprising: - a microfluidic layer (2) defining a reaction volume (3), said reaction volume being in fluid communication with an inlet microchannel (4) for inlet of at least one fluid into said reaction volume and with an outlet microchannel (5) for discharge of at least one fluid from said reaction volume; - an optoelectronic layer (6) comprising at least one photodetector (7) for detection of electromagnetic radiation generated, in use, by a chemiluminescence reaction in said reaction volume (3); - a separation layer (8) set between said microfluidic layer (2) and said optoelectronic layer (6), said separation layer being at least partially transparent to electromagnetic radiation in a given range of wavelengths; and - a microstructured surface (9) facing into said reaction volume (3), said microstructured surface comprising a plurality of micro-pillars (12) preferably having an approximately cylindrical shape, said micro¬ pillars having a longitudinal dimension comprised between 50 pm and 400 pm and a transverse dimension comprised between 5 pm and 20 pm.

Description

"Sensor for detection of biomolecules in a biological fluid via chemiluminescence reaction"

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TEXT OF THE DESCRIPTION

Field of the invention

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.

Prior art

Sensors of the above-indicated type are known in the art, for example from the paper by Laura Pasquardini et al . : "SPAD aptasensor for detection of circulating protein biomarkers", BIOSENSORS AND BIOELECTRONICS, vol . 68, June 2015, pp . 500-507.

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:

- depositing on the surface a self-assembled monolayer (SAM) of silanes using a wet deposition or dry deposition technique, thus providing a plurality of reactive sites, such as thiol groups, on the surface; - immobilizing the molecular probes (for example, DNA aptamers) on the silane layer by having the molecular probes react with at least a part of the reactive sites provided on the surface; and

- passivating the functionalized surface by saturating the reactive sites that have remained free, i.e., not bound to a molecular probe, with other molecules not having the functionality of molecular probe in regard to the analyte sought.

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.

Likewise, 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.

In known sensors, 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.

In known sensors, 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) 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.

In the case of a direct assay, 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 . In the case of 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) is then introduced, which gives rise to the chemiluminescence reaction and to development of the optical signal.

Once the step of detection of the optical signal (or incubation) is through, the reaction chamber is typically emptied of the fluids introduced therein through an outlet microchannel .

In view of a re-use of the sensor for analysis of a subsequent sample, 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 .

Known sensors of the type described above present, however, a certain number of disadvantages.

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.

For instance, from an analysis of the prior art regarding solid state sensors for single photon detection (as those available, for example, from companies such as Hamamatsu, SensL, etc.), it emerges that it is possible to provide devices with a dark count rate of the order of 50 kHz/mm². This means that, in conditions of total dark, the aforesaid optical sensors detect a certain number of false positives, i.e., they generate a certain number of electrical pulses that do not correspond to any photon received.

For the optical sensors mentioned above, which are characterized by a dark count rate of the order of 50 kHz/mm², the aforesaid number of false positives is equal to approximately 50000 pulses per second per square millimetre of area of the sensor. The simple solution of increasing the area of the SPAD in order to increase the intensity of the signal detected and reduce the lower detection limit of the sensor would lead to an increase of the noise due to the dark count rate, which is superimposed on the useful signal, this possibly resulting in a saturation of the reading circuit of the SPAD.

In addition, an increase of the area of the SPAD would also result in an increase in the capacitance of the sensor itself, with consequent increase in the time necessary for recharging and reduction in the maximum detectable signal.

Therefore, in sensors of a known type there is a consolidated use of 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.

Known sensors are hence re-usable exclusively provided that the reaction chamber is subjected to thorough flushing between one assay and the next. This practice proves inconvenient in terms of time and cannot in any case eliminate completely the risk of contamination between one analysis and the next.

Object of the invention

The object of the present invention is to solve the technical problems mentioned previously in relation to known sensors.

In particular, 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.

Summary of the invention

The object of the present invention is achieved by a sensor having the characteristics set forth in one or more of the ensuing claims, which constitute an integral part of the technical teaching provided herein in relation to the invention.

In particular, 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:

- a microfluidic layer defining a reaction volume, said 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;

- a separation layer arranged between said microfluidic layer and said optoelectronic layer, said separation layer being at least partially transparent to electromagnetic radiation in a given range of wavelengths; and

- a microstructured surface facing into said reaction volume, said microstructured surface comprising a plurality of micro-pillars preferably having an approximately cylindrical shape, said micro pillars having a longitudinal dimension comprised between 50 pm and 400 pm and a transverse dimension comprised between 5 pm and 20 pm. According to a further preferred characteristic, the sensor 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.

Brief description of the drawings

The invention will now be described with reference to the annexed drawings, which are provided purely by way of non-limiting example and in which:

- Figures 1 and 2 are exploded perspective views of two embodiments of the invention;

- Figure 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;

- Figure 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 ;

- Figure 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;

- Figure 6 shows a portion of the surface illustrated in Figure 5a at an enlarged scale; and

- 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.

Detailed description of the invention

With reference to Figure 1, 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:

- a microfluidic layer 2 defining a reaction volume 3, the 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;

- a 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; and

- a 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 mΐ and 10 mΐ . 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 mm² and 1 mm², preferably in the region of 0.2 mm². 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. In a preferred embodiment, 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.

Therefore, in one embodiment, 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. Alternatively, 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) .

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.

Advantageously, 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.

In the embodiment illustrated in Figure 1, 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.

In particular, in the embodiment illustrated in Figure 1, 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. In the condition in which the sensor 1 is assembled, 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.

In the embodiment illustrated in Figure 1, the possibility of reversible assembly of the layers 2, 8 and 6 hence makes it possible to provide sensors that can be used according to various modalities:

- according to a first modality, all the layers 2, 8, and 6 (the microfluidic layer 2, the optoelectronic layer 8, and the separation layer 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;

- according to a second modality, also 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 ;

- according to a third modality, only the optoelectronic layer 6 is re-used, whereas the microfluidic layer 2 and the separation layer 8 are replaced, as likewise the plate-like element 11; this solution does not require any flushing with buffer solutions, in so far as the optoelectronic layer 6 is not contaminated by the sample introduced into the reaction volume 3.

Figure 2 represents, once again in exploded view, a sensor 1 for detection of biomolecules according to a second embodiment of the invention.

In all the figures annexed herein, parts or elements that are similar are designated by the same references/numbers and the corresponding description will not be repeated for reasons of brevity.

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.

In the second embodiment described herein, 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. In this case, 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.

Consequently, whereas in the first embodiment 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, in the second embodiment 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.

Also in this second embodiment, assembly of the layers 2, 8, and 6 may be reversible, and the following modalities of use of the sensor 1 may be envisaged:

- according to a first modality, 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; and

- according to a second modality, only the optoelectronic layer 6 is re-used, whereas the microfluidic layer 2 and the separation layer 8 are replaced at each analysis; this solution does not require any flushing with buffer solutions.

Both of the embodiments described herein are characterized in that the 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.

The above microstructures (and, in various preferred embodiments, also the above nano-structures) 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. There is thus an improvement in performance of the sensor 1 with respect to the typical performance of known sensors: for example, it may be noted a decrease in the lower limit of detection of the sensor and a simultaneous increase in the sensitivity of the sensor itself .

The 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. In this particular example, the micro-pillar 12 has an approximately cylindrical shape, with a top surface 13 and a lateral surface 14.

According to the manufacturing techniques used for providing the micro-pillars 12, 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. For instance, 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. The 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. 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.

For instance, 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.

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.

For instance, whilst the 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. In the context of the present description, by "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. For instance, in the case of cylindrical micro-pillars, the 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.

Moreover, 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.

Such surface roughness, obtained by means of nanomachining techniques further described in the sequel of the present description, favours adhesion of the molecular probes.

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. The term "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. For instance, 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.

For instance, 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.

An alternative solution, exemplified in Figure 4b, envisages, instead, that the micro-pillars 12 are arranged according to an array having a triangular elementary cell, identified, for example, by the micro pillars 12i, 122, 123.

It is evident that the possibilities of orderly arrangement of micro-pillars on the microstructured surface 9 are multiple, and that the scope of protection is in no way restricted to the possibilities illustrated herein purely by way of example.

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 .

In the case described previously, where the transverse dimension of the micro-pillars 12 is comprised between 5 pm and 20 pm, 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.

In various preferred embodiments, 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 .

In Figure 5a, which is a view at an enlarged scale of a portion of the lateral surface 14 of a micro pillar 12 in a respective top portion 15, nano structures are visible in the form of 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.

The shape and dimensions of such 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.

Purely by way of example, in Figure 5b 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. It will moreover be noted that the lateral surfaces 14 of the micro-pillars 12, with the exclusion of the respective top parts 15 characterized by the nano-structures 16, 17, 18 described previously, have mean roughness values typically comprised between 2 nm and 5 nm.

In various embodiments, 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. For instance, 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.

Experimentally, some preliminary tests conducted with fluorescent biomolecules (in order to enable identification and quantification thereof by optical microscopy) highlight the fact that a surface characterized by the presence of micro-pillars 12 - with lateral surface possibly having at least one top portion 15 that has nano-structures 16, 17, 18 - is able to bind fluorescent biomolecules effectively after prior surface functionalization. It has been experimentally found that the presence of micro-pillars 12 arranged in regular fashion on the microstructured surface 9 enables an increase by approximately four times of the intensity of the optical signal emitted with respect to the signal emitted by a functionalized surface without micro pillars (planar surface) .

Moreover, it has been experimentally found that the capacity of functionalizing the surface - and hence the capacity of getting biomolecules to adhere to the microstructured surface - is markedly dependent upon the nature of the surface deposition possibly provided on the surface of the micro-pillars 12. According to the type of material deposited, there has been noted an increase in the intensity of the optical signal even thirty times higher than in the case of a non- functionalized surface.

For instance, 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. In variant embodiments, deposition of one or more different materials on the surface of the micro-pillars 12 can favour adhesion of different molecular probes.

By examining, by means of fluorescence microscopy, a cross section of a microstructured surface 9 having a plurality of micro-pillars 12, it has also been noted how adhesion of the biomolecules is especially increased (up to 60 times) at the top portion 15 of the lateral surface 14 of the micro-pillars where the nano structures 16, 17, 18 are present.

In this regard, Figure 7a shows a cross-sectional view of the micro-pillars 12 in clear field, and Figure 7b shows the same cross-sectional view of the micro- pillars 12 with fluorescence signal. There may be noted a particular increase in the optical fluorescence signal at a top portion 15 of the lateral surface 14 of the micro-pillars 12.

As discussed previously, a further technical problem of known sensors is linked to the use of optical sensors of a SPAD type, the area of which must necessarily be limited in order to limit the thermal noise introduced in the output signal by the optical sensor itself.

Consequently, in one or more embodiments of the present invention, it is advantageous to use an optical sensor 7 of a different type, such as a silicon photomultiplier (SiPM) . Such a silicon photomultiplier 7 is made up of a set of SPAD devices connected in parallel, possibly arranged in a matrix.

In a device of a SiPM type, 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.

In one or more embodiments, the optical sensor 7 may be an integrated circuit containing arrays of SPADs with a corresponding reading circuit. In this type of circuits, also referred to as digital SiPMs, 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 adoption of a SiPM device having a photosensitive area equal to the sum of the photosensitive areas of a number of SPAD devices connected in parallel instead of a single SPAD device of similar area enables improvement of the signal collected as a function of the area of the optical sensor (photodetector) .

Assuming that the detection limit of the optical sensor 7 is dictated by the Poisson noise, the signal is represented by the number of photons detected N_Ph, whereas the noise is represented by the standard deviation of the dark count ratio (DCR) plus the number of photons detected N_Ph. The signal-to-noise ratio (SNR) of the optical sensor 7 is hence represented by

Both N_ph and DCR are proportional to the photosensitive area A of the optical sensor 7. Thus,

N_ph = a - A

DCR = b A

where 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

a

SNR = VI

a +b

Passing from an area Ai to an area A₂ the signal- to-noise ratio SNR is thus scaled according to a factor A₂/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.

Consequently, in one or more embodiments, the area of the SiPM photodetector 7 sensitive to electromagnetic radiation is comparable with the area in plan view of the microstructured surface 9. In particular, the area sensitive to electromagnetic radiation of the photodetector 7 is preferably greater than 1 mm².

It will be noted that the use of 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 .

Of course, the details of construction and the embodiments may vary widely with respect to what has been described and illustrated herein, without thereby departing from the present invention, as defined by the annexed claims.

Claims

1. A sensor (1) for detection of biomolecules in a biological fluid via chemiluminescence reaction, the sensor comprising:

- a microfluidic layer (2) defining a reaction volume (3), said reaction volume being in fluid communication with an inlet microchannel (4) for inlet of at least one fluid into said reaction volume and with an outlet microchannel (5) for discharge of at least one fluid from said reaction volume;

- an optoelectronic layer (6) comprising at least one photodetector (7) for detection of electromagnetic radiation generated, in use, by a chemiluminescence reaction in said reaction volume (3);

- a separation layer (8) arranged between said microfluidic layer (2) and said optoelectronic layer

(6), said separation layer being at least partially transparent to electromagnetic radiation in a given range of wavelengths; and

- a microstructured surface (9) facing into said reaction volume (3), said microstructured surface comprising a plurality of micro-pillars (12) preferably having an approximately cylindrical shape, said micro pillars having a longitudinal dimension comprised between 50 pm and 400 pm and a transverse dimension comprised between 5 pm and 20 pm.

2 . The sensor according to claim 1, wherein said microstructured surface (9) is functionalized with molecular recognition elements immobilized on the surface of said micro-pillars (12) .

3 . The sensor according to claim 1 or claim 2, wherein said microstructured surface (9) corresponds to at least one portion of at least one surface of said reaction volume (3) .

4. The sensor according to any one of the preceding claims, wherein said microstructured surface (9) corresponds to at least one portion of the surface of said separation layer (8) that faces said reaction volume (3) .

5. The sensor according to any one of the preceding claims, wherein said micro-pillars (12) are arranged on said microstructured surface (9) in a regular fashion according to a matrix arrangement, and wherein the distance (A, A' ) between said micro-pillars is comprised between 2 and 5 times the respective transverse dimension (D) .

6. The sensor according to any one of the preceding claims, wherein a lateral surface (14) of said micro-pillars (12) is characterised, in a respective top portion (15) extending for at least

10 pm from the top of said micro-pillars, by first nano-structures in the form of nano-grooves (16) extending in a longitudinal direction of said micro pillars (12), said nano-grooves (16) having a width comprised between 100 nm and 600 nm and a depth comprised between 100 nm and 600 nm.

7. The sensor according to any one of the preceding claims, wherein a lateral surface (14) of said micro-pillars (12) is characterised, in a respective top portion (15) extending for at least

10 pm from the top of said micro-pillars, by second nano-structures in the form of nano-grooves (17) extending at least in part in a transverse direction over the lateral surface of said micro-pillars (12), said nano-grooves (17) having a width comprised between 200 nm and 600 nm and a depth comprised between 30 nm and 150 nm.

8. The sensor according to any one of the preceding claims, wherein a lateral surface (14) of said micro-pillars (12) is characterised, in a respective top portion (15) extending for at least 10 pm from the top of said micro-pillars, by third nano-structures, said third nano-structures being globular nano-structures (18) having a major dimension (L) comprised between 20 nm and 150 nm and a minor dimension (1) comprised between 15 nm and 100 nm.

9. The sensor according to any one of the preceding claims, wherein said at least one photodetector (7) comprises a silicon photomultiplier, SiPM, said silicon photomultiplier comprising a set of single photon avalanche diodes, SPADs, electrically coupled in parallel.

10. The sensor according to claim 9, wherein the area sensitive to electromagnetic radiation of said at least one photodetector (7) is comparable with the area of said microstructured surface (9), the area sensitive to electromagnetic radiation of said at least one photodetector (7) being preferably larger than 1 mm².

11. The sensor according to any one of the preceding claims, wherein said optoelectronic layer (6) comprises sensing circuitry and/or control circuitry of said at least one photodetector (7) .

12. The sensor according to any one of the preceding claims, wherein the surface of said micro pillars (12) is coated, alternatively, with:

- a layer of insulating material, preferably silicon oxide, or silicon nitride, or aluminium oxide; or

- a layer of metal material.

13. The sensor according to any one of the preceding claims, wherein said microfluidic layer (2), said separation layer (8), and said optoelectronic layer (6) are configured to be coupled together by applying pressure, preferably with a system of a reversible type.

14 . The sensor according to claim 13, wherein said reversible coupling system comprises one of a snap-fit system or a glue-based system.

15 . The sensor according to claim 13 or claim 14, wherein said optoelectronic layer (6) is removable and can be reassembled for subsequent use.