Field of the invention

The present invention relates to an SPR image unit for biochemical assays, more specifically to a portable unit conceived for use in cooperation with an electronic mobile apparatus, in particular a smartphone.

Background art

In recent years several devices and apparatuses for biochemical assays have been developed. Such assays are aimed at identifying and quantifying contaminants in food and water, as well as pathogens in blood and other sera. Therefore, the purpose of such apparatuses is to find a certain analyte in a certain specimen. The increasing demand for reliability of results and speed to obtain them has triggered an important development of the art in this field. Among the different paths followed by the operators in the field, the one relating to assays performed by microscopy (that is, by means of images) taking advantage of the phenomenon of surface plasmon resonance (more often referred to as "SPR") has proven to be of particular interest.

An apparatus for biochemical assays of known type comprises a reflecting surface having first and second opposite sides. Molecular detection probes are applied at the first side. The apparatus further comprises an optical system. In detail, such an optical system comprises

- a lens applied to the reflecting surface at the second side;

- a laser irradiating, by means of such lens, the reflecting surface at a certain wavelength and at different incidence angles, and

- a camera for capturing images in response to laser radiation.

An electric motor is connected to the laser, so as to move it to irradiate a photon beam according to the above-mentioned incidence angles.

The probes are molecules capable of capturing a specific analyte, are secured to the reflecting surface generating a covalent bond with the same. The analyte is captured by the corresponding probe by chemical affinity, for example a probe obtained by a molecule of a certain antibody will capture a certain antigen (analyte for that specific measurement carried out). Chemical affinity is an aspect that typically leads to the creation of an intermolecular bond between probe and analyte.

The photon beam irradiated in particular conditions of wavelength and incidence angle pairs with the free electrons present on the reflecting surface, generating a plasmon wave on the same. The analyte captured by the respective probe produces a corresponding variation in the local refraction index, i.e., of the region in which it is captured. Such a variation, in turn, produces a perturbation of the plasmon wave. Therefore, the combination of photon radiation (under precise conditions of wavelength and refraction angle) with the analyte capturing corresponds to an absolute minimum of sensitivity induced by energy absorption of the plasmon wave; in such a condition a variation of the photon beam reflected by the surface is produced, quantifiable as the variation in intensity of the light reflected on the camera sensor. Such a variation is thus a direct measurement of the presence of that analyte in the solution of interest.

The reflecting surface is a fully reflecting plate of material (typically gold), in different conditions with respect to those occurring at the absolute minimum of sensitivity it reflects 100% of light irradiated thereon.

Therefore, the reflectivity function may be considered as a function of a parameter accounting for the wavelength and incidence angle per captured analyte. Such a function has a minimum for a certain wavelength and incidence angle value, while outside of these precise parameters it has a reflectivity of about 100% of the irradiated value. The value of this minimum further allows determining the concentration of the analyte in the solution of interest. In order to obtain an accurate measurement on the presence of the analyte and its concentration it is therefore fundamental to irradiate the surface in the minimum conditions: that is, produce a photon beam with exact wavelength and incidence angle. By setting a determined wavelength the incidence angle can be varied so as to obtain a minimum reflection. The electric motor must have such a resolution to move the laser of extremely close subsequent angles and needs to be very accurate: the minimum region of this function is located in a very narrow range and finding the absolute minimum requires setting the laser at an accurately determined incidence angle. The specimen to analyze is contained in a fluidic module applied to the reflecting surface at the side on which the molecular probes are provided. Such a module brings the specimen into contact with the reflecting surface so that any analytes contained therein may pair with the molecular probes and be detected. The same Applicant actively participates in research in this field; in particular, she has developed a nanostructured device conceived for replacing the reflecting surfaces of known apparatuses, as described in the European patent application with publication number "EP2546635" entitled "SPR sensor device with nanostructure". Such a device has an active surface with which the specimen to be analyzed comes into contact, on which a plurality of molecular probes are arranged to find corresponding analytes in the assay steps. Therefore, each probe on the active surface defines a corresponding assay region where a specific analyte is identified. The different assay regions are "isolated" according to a special production process so that the captured image of a determined assay region is not affected by interferences due to adjacent regions. The thus designed surface therefore allows amplifying sensitivity in conditions of irradiation of the photon beam and variation of the refraction index following immobilization of an analyte, thereby very significant and reliable measurements on the presence of analytes may be obtained; in this case, the sensitivity minimum corresponds to a plurality of values located at a relatively broad minimum region: so the measurement is resistant against external disturbances and more reliable. Furthermore, the number of analytes detectable by a single device can be drastically increased, in fact the construction process allows obtaining assay areas within which the plasmon waves having very low sizes are confined, therefore on a small-sized surface it is possible to analyze a very large plurality of analytes.

The images captured (using both the reflecting surface and the nanostructured device) in response to the radiation of the photon beam are (for both assay techniques) processed by dedicated software with which the presence of an analyte, its amount in percentage terms as compared to the specimen in which it has been detected and other similar information are determined.

The apparatuses for biochemical assays described hereinabove have an important disadvantage, in that both the electric motor and the injection system of the specimens are significantly bulky. This causes the device to have such a size excluding an easy handling by the operator, which therefore has to go to an equipped laboratory to perform the assays.

Consider a group of people infected by a particular epidemic or a contaminated watercourse; taking a specimen of material to be assayed, taking it to a laboratory equipped with suitable equipment of the type described may be ineffective, both because the specimen may mutate its organic features during transport, thus altering the assays; and because the time needed to perform the assays for recognizing any contaminating substances may be too long and thus make any countermeasures ineffective. Known solutions are particularly disadvantageous in this regards; in fact, they require: the presence of an electric mains to supply power to the various units, ideal working conditions (the working environment needs to be protected against any external and potentially contaminating atmospheric agents), dedicated spaces for installing an apparatus. In essence, such solutions are not portable, therefore it is not possible to operate on site when real-time assays are required.

Summary of the invention

The Applicant has realized that the known solutions partially described hereinabove do not allow to provide a portable device for biochemical assays which may be used anywhere it is necessary to perform assays. In particular places and upon occurrence of some events, making biochemical assays in real time is required to provide the results as soon as possible and undertake appropriate countermeasures. Consider for example an epidemic or a contamination of waters; taking a specimen to be assayed and taking it to a laboratory provided with suitable equipment may be ineffective both because the specimen may mutate during transport, thus altering the assay, and because the time needed may be long, thus causing any countermeasures to be late and/or ineffective. Furthermore, in many different (for example, pharmaceutical, food, environmental protection) fields, health care regulations require frequent controls; therefore, it would be very useful for the operators of these fields to be able to perform specimen-based biochemical assays in a quick and affordable manner and on site.

For this purpose, the known solutions are particularly disadvantageous; in fact they require: the presence of an electric mains to supply power to the equipment, protected working conditions (for example against atmospheric agents and contaminants), dedicated spaces for installing the equipment.

The inventive idea at the basis of the present invention is to make a portable and passive SPR unit for biochemical assays to be used in cooperation with an electronic mobile device, in particular a smartphone; thereby, the processing and storing, lighting, image capturing and power supply means of the latter may be used to perform the biochemical assays.

In general, an SPR unit for biochemical assays according to the present invention is intended for use in cooperation with an electronic mobile device, in particular a smartphone, provided with lighting means and image capturing means; such a unit comprises:

- an insertion element adapted to receive a specimen to be assayed from outside the unit;

- a nanostructured detector device comprising an active surface adapted to receive said specimen to be assayed;

- an optical assembly adapted to cooperate with the lighting means and with the capturing means so as to receive lighting from said lighting means, light the device, in particular the active surface thereof, and propagate at least one image towards the capturing means in response to the lighting;

- a microfluidic assembly operatively connected to the insertion element and to the device, and adapted to take said specimen to the active surface.

In general, in the present invention nanostructured detector device means an L- SPR device having the sensitive area configured in the shape of a regular arrangement of nanorecesses within which receptors are applied.

Further advantageous technical features of the present invention are expressed in the dependent claims.

Brief description of the drawings

The invention will now be described with reference to the accompanying drawings, which show some non-limiting embodiments thereof, in which:

- figure 1 shows a perspective view of a unit according to the present invention; figure 2 shows a microfluidic assembly of a first embodiment of a unit according to the present invention;

figure 3 shows a microfluidic assembly of a second embodiment of a unit according to the present invention;

figure 4 shows a perspective view of a typical usage method of a unit according to the present invention;

figure 5 shows a diagrammatic view of some components of a unit according to the present invention;

figure 6 shows a perspective view of the components in figure 5 with parts removed for clarity;

figures 7 to 9 respectively show three different embodiments of some components of a unit according to the present invention;

figures 10 to 12 respectively diagrammatically show three subsequent steps of some operations performed during an assay process by means of a unit according to the present invention;

figure 13 shows a first embodiment of the result obtained by the assay of a specimen according to the teachings of the present invention;

figure 14 shows a second embodiment of the result obtained by the assay of a specimen according to the teachings of the present invention; figure 15 shows a first embodiment of a unit according to the present invention;

figure 16 shows a second embodiment of a unit according to the present invention;

figure 17 shows a perspective view of a unit according to the present invention in the step of securing to a support frame to make such a unit integral with an electronic mobile device;

figure 18 shows a perspective view of a further embodiment of a unit according to the present invention;

figure 9 shows a top cutaway view of the unit in figure 18, according to the present invention, integral with an electronic mobile device;

figure 20 shows a diagrammatic view of some components of a unit according to the present invention, relating in particular to a unit for Raman assay;

Detailed description of the invention

Both this description and these drawings are intended for illustration purposes only and therefore non-limiting; therefore, the present invention may be implemented according to other and different embodiments; moreover, it should be noted that such figures are diagrammatic and simplified.

It is useful to state beforehand that the present invention takes advantage of the technology known as "MEMS" [Micro Electro-Mechanical Systems] and the technology known as "NEMS" [Nano Electro-Mechanical Systems] which is an extension thereof, and advantageously uses the solutions thereof.

With reference to figure 1 , a unit 1 for biochemical assays by means of SPR images according to the present invention is shown. Such a unit 1 is intended for cooperating with an electronic mobile device 100, in particular a smartphone, not shown in figure 1 , but visible, for example, in figures 3, 11 , 12 and 16. Device 100, of known type from the prior art, is equipped with camera 180 for images and videos, in turn comprising lighting means 181 and image capturing means 182. From the following description the mode in which unit 1 and device 100 cooperate to perform SPR assays will become apparent.

Unit 1 according to the present invention comprises:

- an insertion element 19 adapted to receive a specimen to be assayed from outside unit 1 ;

- a nanostructured detector device 10 comprising an active surface 10a adapted to receive said specimen to be assayed;

- an optical assembly 16 adapted to cooperate with the lighting means 181 and with the capturing means 182 so as to receive lighting from the lighting means, light device 10, in particular the active surface 10a thereof, and propagate at least one image towards the capturing means 182 in response to the lighting;

- a microfluidic assembly 2 operatively connected to the insertion element 19 and to the device 10, and adapted to take said specimen to the active surface 10a; It should be noted that on the active surface 10a of the nanostructured device 10 there are specific receptors for the analytes present in the specimen, in particular molecular probes which bound by affinity to the analytes of the specimen, which are thus immobilized. As will be better appreciated in the following description, the lighting means 181 light the active surface 10a with a light beam; in response to such lighting there occurs a variation in the light reflection of the active surface 10a based on the presence of a certain analyte. Such a variation is captured by the capturing means 182, thus having a direct measurement on the presence of an analyte in the specimen.

The insertion element 19, in the practice, defines an interface by which a specimen to be assayed is inserted from the outside of unit 1. The thus inserted specimen is contained by the microfluidic assembly 12, operatively connected to the insertion element 19, to be transported to the active surface 10a of the nanostructured device 0 and assayed.

With reference to figure 2, a diagrammatic representation of a first embodiment of the microfluidic assembly 12 of a unit 1 according to the present invention is shown. The assembly comprises a plurality of capillary (actually small pumps or "micropumps") pumps 22 to contain and "pump" said specimen, connected to the insertion element 19 and to a corresponding plurality of transport lines 11 to transport said specimen to the active surface 10a. In particular, these are pumps of the passive type, i.e., they do not require external power to work, which take advantage of the phenomenon of capillarity to generate a liquid flow, i.e., "to pump"; as diagrammatically shown in the figure, first there is a triangular grating of short capillary conduits which ends at the entrance of each line 1 ; by suitably sizing the triangular grating, flow speed and flow rate in the line may be determined; it is also possible to provide (but this makes the solution slightly more complicated) a microvalve, for example, between the outlet of the grating and the inlet of the line.

As mentioned, the method underlying the SPR assay includes detecting the presence of an analyte based on the assay of the change in the reflection index by means of images of the specimen captured at the active surface 10a. It is therefore preferred to provide for a common reference to be used to assess the variation in the different reflection indexes of the images of the specimen captured. Such a common reference is obtained by making a measurement of a reference liquid. The embodiment of the microfluidic assembly 12 includes a plurality of micropumps 22 connected to a corresponding plurality of lines 11. One of these lines is dedicated to the reference measurement, therefore it is pre-loaded with the reference liquid and during the assay time (that is, the time during which images of the specimen are captured on the active surface 10a) contains and transports reference liquid only. The other micropumps 22 are operatively connected with the insertion element 19 and with the corresponding plurality of lines 11 , to transport the specimen (inserted taking advantage of the element 19) to the active surface 10a. According to this embodiment, the specimen is manually inserted in unit 1 by an operator, driving it into the element 19 by means of a suitable dispenser tool, such as a pipette. The amounts of the specimen to be introduced are very limited, in the order of microliters. The micropumps 22, by taking advantage of the phenomenon of capillarity, allow transporting through the lines 11 the specimen introduced on the active surface 10a of the nanostructured device 10. The lines 11 extend from the micropumps 22 to the device 10 getting closer to each other (however remaining separate) at the active surface 10a. The active surface 10a is provided with a corresponding plurality of receptors, each adapted to capture a certain analyte, and determine the presence thereof by assaying the captured images. The number of micropumps 22, lines 11 and receptors on the active surface 10a thus determine the number of analytes detectable in a specimen. It is therefore possible, by means of the microfluidic assembly 12 according to the present embodiment, to analyze analytes present in a specimen in parallel during the testing period; the degree of parallelism is given by the number of lines 11 at the active surface 10a and could be for example and typically two, three, ... seven, eight.

With reference to figure 3, a diagrammatic view of a second embodiment of the microfluidic assembly 12 of a unit 1 according to the present invention is shown, which is adapted to cooperate with a mobile device 100 provided with vibrating means (not illustrated in the figure), i.e., those means which cause the mobile device 100 to vibrate, for example as a notification signal for incoming calls.

The assembly 12 according to this embodiment of unit 1 comprises:

- a pumping element 13 adapted to generate a thrust force and connected to a first containment reservoir 23, such a reservoir 23 being adapted to contain a reference liquid 230. The thrust produced by the pumping element 13 is transmitted to the reference fluid 230.

- activator means 14 adapted to be operatively connected to the vibrating means, and adapted to actuate the pumping element 13 in response to vibrations produced from the vibrating means.

Again with reference to figure 3, it may be noted that the microfluidic assembly 12 of unit 1 comprises:

- a first reservoir 23 operatively connected to the pumping element 13 and adapted to contain a reference liquid 230;

- a second reservoir 24, in fluid connection with the first reservoir 23 and with the insertion element 19; such a second reservoir 24 being adapted to receive from the insertion element 19 at least a part of the specimen 240 and to contain it;

- at least one fluid transport line 25 in fluid connection with the second reservoir 24 for taking the reference liquid and the specimen to the active surface 0a.

According to the latter embodiment, the reference liquid and the specimen are transported to the active surface sequentially: in a first step a measurement of the reference liquid is performed, in a subsequent second step the specimen is transported to the active surface 10a and analyzed. To this end, the line 25 is preloaded with reference liquid, so that the first measurement performed is that of reference. The reference liquid 230, contained in the first reservoir 23 is used as a means for transmitting the thrust exerted by the element 13 on the specimen 240. Thereby, this latter is taken to the active surface 10a running along the line 25. It should be noted that the channels which fluidly connect the first reservoir 23, the second reservoir 24 and the line 25 are microfluidic, i.e., they have such sections which produce transport flow rates in the order of nanoliters.

Thereafter, also the reference liquid 230 reaches the surface 10a to thus perform a further detection of the common reference. Downstream of device 0 (that is, opposite the arrangement of the element 13 and the first reservoir 23), a discharge reservoir 26 is fluidly connected, where portions of the specimen and portions of the reference liquid are deposited after the detections.

The active surface 10a of this embodiment comprises a plurality of detecting cells for a certain analyte arranged as a matrix on the same surface 10a, each of these detecting cells comprises at least one probe for the specific analyte of the detecting cell. This structure of the surface 10a allows multiplying the assay, it is in fact possible to assay a plurality of analytes in one detecting cycle. The mode by which the specimen is assayed exploiting the microfluidic assembly illustrated in figure 3 will become clear from the following description, in particular with reference to figures 9 to 11. For the purposes of the present invention, the term "specimen in detecting conditions" means a specimen (or a portion thereof) on the active surface 10a so that the analytes present bind to the molecular probes and are therefore immobilized by the receptors.

With reference to figure 4, a typical usage configuration of a unit 1 according to the present invention is shown. As mentioned, unit 1 is designed to cooperate with an electronic mobile device 100, in particular a smartphone. In essence , the camera 180, the processing and storing, lighting, image capturing and electrical supply means may be used. In order to maintain unit 1 in a fixed and predetermined position, so as to cooperate with the electronic device 100, a support frame 50 is provided. The operation of unit 1 in cooperation with the electronic device 100 will become apparent from the following description.

With reference to figure 6, an optical assembly 16 of a unit 1 according to the present invention is shown. Such an optical assembly 16 is conceived to receive the light beam 120 emitted by the lighting means 181 and focus a corresponding second light beam 130, emitted by device 10 (in particular by the active surface 10a) in response to the first beam 120 received, on the capturing means 182. An image of the specimen in detecting conditions is therefore obtained. To this end, on the storing means of the mobile device 100 a dedicated program is loaded, run by the respective processing means, to manage the lighting means 181 and capture the corresponding images by means of the capturing means 182. The images captured may be stored on the storing means of the mobile device 00, to be then processed (by the same mobile device 100, or by another processing unit, to which they are sent) for determining the presence and concentration of one or more analytes.

With reference to the figure 5, an embodiment of the optical assembly 16 is shown. It is worth mentioning that the lighting means and the capturing means are typically arranged at two different places and spaced from one another by the outer enclosure of a smartphone. To this end, the optical assembly 16 is substantially divided into two focusing portions 29 and 30; the first portion 29 for cooperating with the lighting means 181 , the second portion 30 for cooperating with the capturing means 182. In particular, the first portion 29 comprises a first collimating lens 18, a first beam splitter 19 and a second collimating lens 20; such three elements are aligned along a first rectilinear axis 50 coinciding with the symmetry axis of beam 120 produced by the lighting means 181. The second portion 30 comprises a second beam splitter 21 and a third lens; these two elements are aligned along a second rectilinear axis 60 coinciding with the symmetry axis of the reflected beam 130, directed towards the capturing means 182 and produced in response to the lighting of the surface 0a with beam 120. The first axis 50 along which the optical elements of the first portion 29 (perpendicular to the active surface 10a of device 10) are aligned is parallel and spaced apart with respect to the second axis 60 along which the optical elements of the second portion 30 are aligned.

Between the first portion 29 and the second portion 30 there is a passive optical filter 27, arranged so as to be aligned to the first beam splitter 19 and to the second beam splitter 21 along a third axis 70, substantially perpendicular to the first and second axis 50 and 60.

Preferably, the first portion comprising the optical elements arranged in the following sequence, assessed by the portion of unit 1 , in use, closest to device 100:

- the first lens 18;

- the first beam splitter 19;

- the second collimating lens 20;

therefore, beam 120 irradiates the active surface 10a of device 10 crossing, in the above-mentioned sequence, the optical elements (the representation of beam 120 in figure 5 is merely indicative).

In response to the lighting of the surface 10a with beam 120, a reflected beam 130 is produced (which transports an image, namely an image sequence, if such a beam is considered within a time interval): such a reflected beam needs to be captured by the camera 182, is in fact an indication of the analytes immobilized on the active surface 10a.

Beam 130 therefore crosses the second collimating lens 20 and the first beam splitter 19, in this order. The optical configuration of the first beam splitter 19 allows modifying the focusing axis of beam 30, which is thus focused along the third axis 70. Therefore, beam 130 crosses the filter 27 and the second beam splitter 21 , and similar to the previous crossing, is focused along the second axis 60, substantially perpendicular to the third axis 70. Beam 130 then crosses the third lens 28 to propagate to the capturing means 182.

In this example of embodiment, the lighting means 181 and the capturing means 182 with which the mobile device 100, which is a smartphone, is equipped are not expressly designed for performing SPR image assays according to the present invention. In fact, the beam 120 produced has a spectrum which is suitable for photography applications, therefore not optimized to energize the active surface 10a; the presence of the filter 27 allows selecting the wavelengths of interest of beam 130, i.e., those relevant and affected by less noise created by the presence of wavelengths not suitable for use according to the present invention: For example, the filter 27 may filter beam 130 at about 750 nm, such a value being interpreted in a non-peremptory, but variable manner depending on the actual application.

However, the lighting spectrum of the beam 120 produced by the lighting means 181 may be entirely unsuitable for certain applications. In fact, the lighting peak of such sources, in the case for example of smartphones, is typically focused at about 550 nm, with tails up to 750 nm. Some applications may require a photon beam centered at about 1000 nm (or in certain cases even higher). To this end, the first lens 18 may be suitably configured to translate the wavelength of the first beam 120 about the optical wavelength of interest. In order to achieve this effect, preferably, lens 18 may be doped with substances capable of absorbing the light produced by the means 181 and emit it at a (different) desired wavelength. Such substances may comprise fluorescent molecules, elements belonging to the rare earth group, "quantum dots". In particular, lens 18 may be doped with erbium (element belonging to the lanthanide group) to achieve this effect. Beam 120 propagating through a so designed lens is brought to the desired wavelength. With reference to figures 7 to 9, three different embodiments of activator means 14 of the pumping element 13 are shown.

The embodiments described hereinafter implement the general technical teaching which provides the pumping element 13 movable between a first position (indicated in figures 7, 8 and 9 with reference A) and a second position (indicated in the same figures with reference B) in which the movement from the first position to the second position generates a thrust force for moving at least part of the reference liquid and at least part of the specimen. The pumping element 13 is movable between the first and the second position by the effect of the activator means 14.

All three embodiments are designed to take advantage of the vibrations produced by the vibrating means of the mobile device 100 to actuate the activator means 4 and start pumping the specimen and the reference element. To this end, a coupling module to device 100 is provided, which is adapted to transmit the vibrations produced by the related vibrating unit to the unit 1 according to the present invention. According to an exemplary embodiment, the coupling module is obtained in part of the same support frame 150, thus besides making unit 1 integral with device 100 also the vibrations produced by the latter are transmitted to the activator means 14.

Figure 7 shows the first embodiment of the activator means 14, comprising:

- said coupling module;

- a battery adapted to store electric energy;

- a first converter mechanically connected to the coupling module and to the battery, and adapted to convert the mechanical energy of the vibrations produced by the vibrating means into electric energy which is stored into the battery,

- a second converter electrically connected to the battery and mechanically connected to the pumping element 13, to thus move the pumping element from the first position A to the second position B.

For example, both the first and the second converters may be piezoelectric crystals, but used in a slightly different manner. In the case of the first converter, vibration may cause a repeated mechanical deformation of the crystal and a subsequent alternating potential difference at its terminals (to be rectified). In the case of the second converter, the continuous potential difference, available thanks to the battery, is applied to the terminals of the piezoelectric crystal. In response to the application of such a continuous potential difference, the piezoelectric crystal undergoes a mechanical deformation, which is exploited to move the pumping element 13.

Figure 8 shows the second embodiment of the activator means 14, comprising:

- said coupling module;

- a micro-mechanical converter connected to the coupling module and to the pumping element 13, and configured for converting the vibrations received, that is, an alternating rectilinear motion, into movement, i.e., a translatory motion, of the pumping element 13 from the first position A to the second position B.

Figure 9 shows the third embodiment of the activator means 14, comprising:

- said coupling module;

- a first compartment containing a reaction liquid and a second compartment, divided from the first compartment by a separation membrane at a first end and delimited by the pumping element 13 at a second end opposite to the first one, containing an expandable polymer.

The coupling module is connected to the first compartment and transmits thereto the vibrations of the mobile device 100. The vibrations induced by the coupling module allow the liquid contained in the first compartment to tear the membrane and come into contact with the expandable polymer. The result of the chemical reaction following the contact between the reaction liquid and the expandable polymer causes the latter to increase its volume. The polymer acts on the element 13, a volume increase thereof moves the latter from the first position A to the second position B.

With reference to figures 10 to 12, three subsequent steps of the specimen pumping and of the reference liquid to perform assays by means of a unit according to the present invention are shown.

Figure 10 shows the first step: the line 25 extending to the active surface 10a of device 10 is pre-loaded with some reference liquid 230. According to this embodiment, the first measurement will therefore be that of reference. Figure 11 shows a step following that in figure 10; the passage from the previous to the present step takes place in cooperation with the mobile device 100. As mentioned, on the mobile device 100 a program is preloaded and run to perform assays by means of a unit according to the present invention. In particular, such a program may provide an interface (with which the same device 100 can interact) to activate the vibrating means and therefore actuate the activator means, and to activate the lighting means 181 and the capturing means 182 in a synchronized manner with the assay time of the specimen. Therefore, according to one embodiment, an operator needs to introduce the specimen to be assayed through the insertion element 19 and by means of the program loaded on device 100 to activate the assay procedure. The vibration produced by the device 100 is transmitted to unit 1 , as already described, and the pumping element 13 exerts its thrust force on the reference liquid 230 contained in the first reservoir 23. Such an action produces an equal thrust on the specimen 240 contained in the second reservoir 24 to which the first one is fluidly connected. In turn, the specimen 240 exerts a corresponding thrust on the reference liquid contained in the line 25 at the active surface 10a. The reference liquid 230 previously contained, therein is pushed inside the discharge reservoir 26. The specimen 240, at the active surface 10a, is thus subjected to assays, according to the already described modes.

Figure 12 shows the step following that illustrated in figure 1 . In such a step, a further reference measurement is performed by the assay of the reference liquid 230. It should be noted that such a step is performed if carrying out a further reference measurement is required, otherwise (that is, if the previous reference measurement, figure 0, is sufficient to determine a measurement of the analytes) such a step may be skipped. As described in figure 11 , the thrust produced by the element 13 on the liquid 230 allows taking the latter to the active surface 10a and performing assays. The specimen 240 (in the previous step at the active surface 10a) is taken, by the thrust produced by the liquid 230 in the discharge reservoir 26.

Figures 13 and 14 show two possible images obtained capturing beam 130 according to two different embodiments of unit 1 and show how such images are a direct measurement of the analytes contained in the respective specimens assayed.

With reference to figure 13, the result obtained by capturing beam 130 obtained following the energization of the active surface 10a by means of the lighting with the light beam 120 is shown. As mentioned, capturing beam 130 produces images capable of providing a direct measurement of the presence of certain analytes in the specimen. Figure 13 shows a possible image 300 produced by assaying a specimen with a unit 1 integrating a device 0 of the type indicated in figure 3. In fact the image of a plurality of detecting cells 301 is shown: the color of the image of each cell indicates whether the corresponding analyte with which it is associated has been detected. The number of detecting cells may vary on a case- to-case basis, for example, from 400 to 40000; the lower limit derives from detecting cells having a side of 200 microns and a visual field having a side of 4 mm; the lower limit derives from detecting cells having a side of 20 microns and a visual field having a side of 4 mm. For example, cell 302 (black) indicates that in the specimen the presence of a corresponding analyte has not been detected, while the cell 303 (clear) indicates that the corresponding analyte has been detected. It should be noted that the clear color when detecting an analyte is thus indicated for simplicity, indeed the color of a cell which has detected the presence of an analyte is shown by means of a grey scale where the intensity of the grey allows deriving the concentration of that analyte in the specimen.

For example, the cameras with which smartphones are usually equipped (and therefore adapted to be uses for the purposes according to the present invention) have color depths of at least 8 bits. In the case of a grey scale representation, a cell with one among at least 256 grey tones may be shown, each indicative of the degree of concentration of that analyte in the specimen. Therefore, each cell 301 indicates the presence of an analyte and shows its concentration in the specimen. With reference to figure 14, the result obtained by capturing beam 30, as in figure 13, using a unit 1 with device 10 of the type indicated in figure 2 is shown. In this case, the analytes are detected identifying the sensitive areas 301 of the active surface 10a. In the embodiment in the figure, 7 sensitive areas are shown, each aimed at identifying a specific analyte. The color of each sensitive area detected while capturing the image indicates whether the related analyte is present or not. As in the case of figure 13, the color of each sensitive area may be shown by means of a grey scale, to have a direct measurement on the presence of an analyte and the concentration thereof in the specimen.

An aspect according to the present invention therefore provides the program loaded on the mobile device 100 to acquire the images 300 and based on such images to calculate the presence and concentration of analytes in the specimen. With reference to figure 15, an embodiment of a unit 1 according to the present invention is shown, substantially having the shape of a plate and in which the insertion element defined on the outer surface of the enclosure comprises a micro- tub 21 connected to the second reservoir 24: the specimen inserted in the micro- tub 21 flows into the reservoir 24 to be later transported to the active surface 10a and assayed (according to the different modes described herein).

With reference to figure 16, an embodiment of a unit 1 according to the present invention is shown, in which the insertion element comprises a collection interface 22 in turn comprising a plurality of needles 22a, 22b, 22c each connected to the second reservoir 24. Such a configuration is particularly advantageous if the specimen 240 to be collected is an organic fluid (for example, mammalian blood): by means of the application of said needles to the individual (or animal) being examined it is possible to collect the specimen while taking it to the second reservoir 24 to be then taken to the active surface 10a and assayed.

With reference to figure 17, an embodiment of a unit 1 according to the present invention is shown, provided with a containment enclosure 90 for the detecting device 10, the microfluidic assembly 12 and the optical assembly 16. Preferably, the insertion element 19 is obtained flushed with the outer surface of the enclosure 90 (providing the plurality of needles which protrudes from said surface in the related embodiment in which these are provided). As previously said, according to a preferred embodiment, such an enclosure is substantially shaped as a plate, and may have, for example, a thickness of 2-8 mm and surface of 2000-6000 mm2, therefore compatible so that, in use, it can be coupled to the mobile device 100. Therefore, it may be easily coupled to the support frame 150. To this end, frame 150 firmly receives the mobile device 100 and the unit 1 , the frame is further fastened to device 100 by means of fastening means 151 , while it firmly receives unit 1 by means of the coupling surface 152, in use substantially parallel to and spaced apart from the surface of device 100 where the camera 180 is provided. In use, unit 1 is applied to the surface 152 of frame 150; to ensure a correct coupling on this surface 152 there is provided at least one abutment opening 153 obtained in order to align, in use, the optical assembly 16 of unit 1 to the camera 180 of the mobile device 100. The dimensions of frame 150 are designed considering the features of the optical assembly 16 and the capturing means 180. In particular, the dimensions of the fastening means 151 define the distance between the optical assembly 16 and the capturing means 180, while the dimensions of the opening 153 allow the passage of the reflected light beam reflected by the capturing means. Such dimensions are therefore suitably designed depending on the type of device 100 used and the features of the optical assembly 16 of unit 1.

It should be noted that by means of a unit 1 according to the present invention the multiplation degree of the analyte detection as a function of the features of the mobile device 100 which will be used. In fact, unit 1 may be designed having as a constraint the performance of the camera of the mobile device (in particular, its resolution and visual field); as a function of such a constraint the multiplation degree of the analytes may be determined (for example, designing a device whose active surface 10a allows simultaneously assays many hundreds or even many thousands of analytes in a specimen).

With reference to figure 18, a further embodiment of a unit 1 according to the present invention is shown. Such a unit 1 provides the optical assembly 16 and the microfluidic assembly 12 made in two different bodies, thereafter coupled. According to a preferred embodiment, the optical assembly 16 is made by discrete optical components joined together to form the same optical assembly. In the practice, the optical assembly comprises a plurality of passive optical elements, of the type already described with reference to figure 5, mutually arranged in a suitable manner and coupled to base 80 to focus the beam 120 produced by the light source 181 on the device 10 (in particular, on the active surface 10a) and later focus the reflected beam 120 on the camera 182.

The microfluidic assembly 12 and the device 10 are instead integrated into a base 80, later made integral with the optical assembly 16. With reference to figure 19, unit 1 can be viewed in the embodiment with discrete components (that is, optical assembly 6 obtained separately with respect to base 80 integrating microfluidic assembly and detecting device 10) applied to a device 100 by means of a special frame 150. Preferably, frame 150 is connected to the mobile device 100 by means of two fastening portions 151 and maintains base 80 in a fixed and predetermined position, joining thereto at two opposite points thereof. The frame 150 of this embodiment is designed so that, in the fixed and predetermined position of use, the optical assembly 16 aligns with respect to the camera 180 so as to correctly cooperate with the latter.

It is clear from the above description that the SPR unit for biochemical assays according to the present invention may be defined as "passive" since it does not require an internal energy (electric) source. The term "passive" is particularly applicable in the embodiments of the present invention comprising a passive (capillary) pump because, in this case, the unit does not require any energy to operate.

Typically, the SPR unit for biochemical assays according to the present invention will be made in the form of a plate, i.e. a plate having a 2-8 mm thickness and a 2000-6000 mm2 surface.

The SPR unit for biochemical assays according to the present invention may be used together with a smartphone; for some applications and/or in certain fields, however, a specific electronic mobile device may also be designed and created which is dedicated to the use in combination with such a unit. In the case of a dedicated device, the optical assembly of the unit may be very simple; the optical assembly may comprise for example two beam splitters only if the lighting means and capturing means of the device were on the same side of the device enclosure; the optical assembly may comprise for example one beam splitter only if the lighting means and capturing means of the device were on two perpendicular sides of the device enclosure. Among other things, by means of appropriate lighting means and capturing means, the optical assembly may consist of a space where only the lighting beam and the resulting beam propagate.

With reference to figure 20, an alternative embodiment of a unit according to the present invention is shown. As previously said, the first focusing portion 29 is adapted to cooperate with the lighting means 181 of the smartphone. In particular, the first focusing portion 29 comprises in sequence:

- a first lens 18;

- a first beam splitter 19;

- a second lens 20.

Preferably and as described above, lens 18 may be suitably doped so that incid 120 has an incidence on the active surface 10a at a predetermined wavelength. In particular, the wavelength is selected so as to energize the Raman vibrating modes in the specimen being assayed. To this end, the lens 18 is suitably designed to produce a light beam centered at a predetermined wavelength and provided with a rather narrow band, typically 5 nm.

The second focusing portion 30 is adapted to cooperate with the capturing means 182 of the smartphone. In particular, the second focusing portion 30 comprises in sequence:

- a passive optical filter 27;

- a mirror 21 b;

- a diffraction grating 21 a;

- a third lens 28.

The specimen being assayed is suitably irradiated by the optical beam 120 so as to energize the Raman vibrating modes (or the vibrating mode) and emits a corresponding optical beam 130. The passive optical filter 27 is configured to select only the wavelengths indicative of the Raman spectrum from the optical beam 130. The filtered optical beam 130 has an incidence on the mirror 21 b, thus translating the direction of its optical path by 90°. It then passes through the diffraction grating 21 a and the third lens 28. The effect of the diffraction grating is to transform the optical beam 130, indicative in this condition of the Raman vibrating mode of the specimen being assayed, in an image which may be captured by the capturing means 182 indicative of the Raman spectrum of the specimen being assayed.

The diffraction grating 21 a may be made according to various modes. In other embodiments it may be made as a lens and/or prism and/or mirror system aimed at obtaining the same effect, i.e. to transform the optical beam 130 in an image which may be captured by the capturing means 182 indicative of the Raman spectrum of the specimen being assayed.

Therefore, a unit designed according to this embodiment may - very conveniently - be used as a unit for obtaining the Raman spectrography of a specimen to be assayed. The nanostructured active surface 10a advantageously allows amplifying (also by a factor of 100,000) the signal indicative of the Raman spectrum of the specimen being assayed. In particular, the nanocavities of an active surface 10a allow collecting parts of the specimen to be assayed and to optimize the amplification of the signal indicative of the Raman spectrum.

It should be noted that the active surface 10a may be suitably functionalized to selectively receive substances to be assayed, locally concentrating and attracting substances to be assayed on parts of the surface. This effect is obtained by means of an active surface 10a made with porous or ultraporous, hydrophobic or hydrophilic materials, etc.

According to an alternative embodiment, however, the active surface 10a may be a simple carrier for the specimen being assayed, therefore free from specific receptors for the analytes present in the specimen.

It is also contemplated that the SPR image unit for biochemical assays according to the present invention may be also implemented as a disposable article for predetermined assays in certain environments.