IT202100015821A1 - Device for detecting nano particles in a fluid. - Google Patents

Description

Device for detecting nano particles in a fluid.

The present invention relates to a device for detecting nanometric-sized particles in a fluid.

Field of invention

Pi? in detail, the invention relates to a device of the aforementioned type, designed and manufactured in particular to detect the presence of viruses, such as SARS-Cov-2, within a biological fluid, but which can be used for any detection of pathogenic material and/or DNA or RNA nucleotide sequences within a biological fluid, such as saliva, exhaled condensate, urine or blood.

In the following, the description will be? aimed at a device for detecting viral particles in a biological fluid, but ? it is clear how the same should not be considered limited to this specific use.

Known technique

How?? well known, devices for detecting ionic currents inside a biological fluid can be used to detect the presence of particular elements inside this biological fluid, such as for example DNA sequences.

These detection devices usually include two containers equipped with cavities? able to accommodate fluids, separated by a membrane in which? obtained at least one detection hole, used to detect the transit of particles present in the fluid contained in the two cavities.

The set of the two containers and the separation membrane can? be arranged in superimposed or side by side layers, where the membrane with the detection hole ? the intermediate layer, which ? disposed between, and coupled to a lower layer in which ? is there a cavity? or channel suitable for hosting fluids, and an upper layer where? Is there a second cavity?

The cavities? they usually house two electrodes, positioned so as to be in direct contact with the liquids hosted by the cavities.

The electrodes support an electric field and measure the ionic currents through the sensing hole, and their possible variations due to the passage of particles contained in the biological fluid through the sensing hole itself.

However, the devices for detecting ionic currents of the prior art are not capable of precisely identifying the presence of viruses, and in particular of SARS-Cov-2, in biological fluids.

Currently, the presence of SARS-Cov-2 can be accurately detected within a biological fluid through the polymerase chain reaction technique, called PCR, which amplifies and finally quantifies the viral mRNA after breaking the virus shell.

This process has very high costs and requires reagents which may not be present on the market in large quantities. adequate, especially in times of great demand, requires highly specialized personnel, and provides the result only after several hours, allowing a reduced number of tests per day.

Other rapid antigen tests used for the identification of SARS-Cov-2 are based on the chromatic change of a compound when in contact with the virus.

Do rapid antigen tests of this type have sensitivity? variables, but usually much lower than the sensitivity? of PCR, compared to reduced processing times to provide a result, usually equal to a few minutes.

In particular, the sensitivity of these rapid antigen tests, i.e. the minimum number of detectable virus particles, ? usually greater than or equal to 2.08E6, and the specificity? ? variable from 88.24% to 94.85%.

In light of the above, The object of the present invention is to provide a device for measuring ionic currents which is capable of detecting the presence of particles on the nanometric scale, in particular viral particles of the SARS-Cov-2 virus, within a biological fluid in a rapid manner , without the help of specialized personnel and directly in the field, without having to resort to specialized laboratories.

In particular, the purpose of the present invention ? that of providing a highly sensitive, specific, rapid and reliable detection device.

Another purpose of the invention? that of providing a virus detection device which is economical, both in terms of production costs and operating costs, for example due to the consumables used to perform the examination.

Finally, the purpose of the present invention ? to provide a detection device capable of detecting different types of viruses or biomarkers.

Object of the invention

These and other results are obtained according to the invention.

The object of the present invention is therefore a device for detecting nanoparticles in a fluid, comprising a lower layer, in which obtained a channel, an upper layer, in which? obtained a through channel and an intermediate layer, on which? obtained at least one detection hole, for the passage of said biological fluid, in which said intermediate layer ? disposed between and coupled to said lower layer and said upper layer, and in which said sensing hole is arranged in correspondence with said through channel, so as to identify with it and with a portion of said channel a cavity for containing said biological fluid, said sensing device comprising at least a first and a second electrode for sensing an ionic current signal generated by the passage of said fluid in said sensing hole, wherein said at least a first and a second electrode are disposed on said intermediate layer.

Further according to the invention, said intermediate layer ? coupled to said lower layer and/or to said upper layer by glue.

Still according to the invention, said detection hole has a conical shape.

Always according to the invention, said detection hole has a cylindrical, funnel or hourglass shape.

Preferably according to the invention, said sensing hole has a section width of 1 micrometre.

Further according to the invention, said intermediate layer ? a polyimide membrane.

Still according to the invention, said membrane has a thickness comprised between 5 micrometres and 50 micrometres, preferably 12.5 micrometres.

Still according to the invention, said first and second electrodes are made of silver chloride.

Preferably according to the invention, said detection hole ? made using a laser source.

Further according to the invention, said cavity? has a volume between 10 microlitres and 100 microlitres, preferably 60 microlitres.

Still according to the invention, said upper layer ? of plastic material, such as Polymethylmethacrylate - PMMA or Polycarbonate ? pc.

List of figures

This invention will come now described for illustrative but not limiting purposes, according to its preferred embodiments, with particular reference to the figures of the attached drawings, in which: figure 1 shows an exploded perspective view of a device for detecting viral particles in a biological fluid , object of the present invention, comprising an upper layer, an intermediate layer and a lower layer;

figure 2 shows a perspective view of the sensing device of figure 1 assembled in all its parts;

figure 3 shows a top view of the detecting device of figure 1, in which the intermediate layer ? visible in transparency;

figure 4 shows a side view in section of the detection device of figure 1;

figure 5 shows a bottom view of the detection device of figure 1, in which the intermediate layer ? visible in transparency;

figure 6 shows a bottom view of the intermediate layer of the detection device of figure 1;

figure 7 shows a top view of the intermediate layer of the detection device of figure 1;

figure 8 shows the graph of a typical trace of ion current passing through a detection hole of the device at the potential of 400 mV, following the filling of the cavities? and the canal with a phosphate buffered saline solution;

figure 9a shows the graph of a typical current trace in which the impedance variations due to the passage of two analytes having different dimensions through the detection hole are observed;

Figure 9b shows a close-up of two single translocation events representative of the two single analytes; And

figure 10 shows a dispersion graph which shows the relationship between the amplitude of the impedance variations and the area of the graph subtended by the single variations detected in the current trace shown in figure 9a; the dashed crosses show the mean and standard deviation of each population, where ?n? indicates the number of events detected for each variation population.

In the various figures, similar parts will be indicated with the same reference numbers.

Description of the embodiments

The detection device according to the invention essentially comprises a multilayer structure in which at least one detection hole and the electrodes for detecting viral particles in a biological fluid are arranged on the same layer, in particular on an intermediate layer.

With the reference number 1 ? indicated a device 1 for detecting viral particles in a biological fluid according to the present invention.

With reference to figures 1-5, in particular, the detection device 1 comprises a lower layer 10, an upper layer 11 and an intermediate layer 12, arranged between the lower layer 10 and the upper layer 11.

On said lower layer 10 ? obtained a channel 100, able to collect the biological fluid.

Said channel 100 has a substantially "U" shape.

Said lower layer 10 ? preferably made of plastic material, such as for example Polymethylmethacrylate - PMMA or Polycarbonate - PC, or materials capable of containing a biological fluid without altering its characteristics.

On said upper layer 11 ? obtained a through channel 110, shaped to receive a quantity? of biological fluid to be analyzed and ? capable of conveying said biological fluid to the subsequent layers.

By way of example, the through channel 110 can? be a substantially cylindrical channel having a diameter and depth? such as to have a volume for the biological fluid of between 10 microlitres and 100 microlitres, preferably equal to 60 microlitres. In fact, this volume ensures that the viruses present in a sample of biological fluid, such as saliva, can be identified with a good degree of accuracy, as it guarantees that a sufficient number of particles are present. In alternative embodiments, the shape and/or volume of the through channel 110 may however? be different, being able to depend on the biological fluid analyzed and/or on the particle to be detected.

On said upper layer 11 there are also made through holes 111, through which ? It is possible to fill said channel 100 with an electrically conductive solution.

Said upper layer 11, similarly to said lower layer 10, is preferably made of plastic material, such as for example PMMA or PC, or materials suitable for containing biological fluids without altering their characteristics.

Said intermediate layer 12 ? arranged between the lower layer 10 and the upper layer 11.

On said intermediate layer 12 ? at least one detection hole 120 is obtained in fluid connection with the through channel 110 and a portion of the channel 100, so as to form a cavity 13 for the biological fluid.

A first electrode 16 and a second electrode 17 are arranged on said intermediate layer 12, capable of being in contact respectively with the fluids contained in said through channel 110 and channel 100.

Said first electrode 16 has a first electrical connection 14 and said second electrode 17 has a second electrical connection 15, for connecting said first 16 and second 17 to an external measuring device.

Said middle layer ? coupled to said lower layer 10 and upper layer 11 preferably by means of an adhesive.

However, not all adhesives can be used due to the extreme sensitivity of the glue. of the measuring instrument, as these could alter the conductivity? of the device 1, for example having a conductivity? greater than that allowed for the particular application, this cos? to alter the measurement.

Furthermore, the adhesive must be resistant to the liquid solutions used in the preparatory phases, such as isopropanol, sodium hypochlorite, 1M sodium chloride, and the like.

AND? it is preferable that the glue has an impedance higher than 10E12 Ohm.

Furthermore, the intermediate layer 12 can be constituted by a membrane, preferably having a thickness between 5 and 50 micrometers, more? preferably equal to 12.5 micrometres.

This thickness, together with the electric potential applied by said first 16 and second 17 electrodes, determines the transit time of the particle through the membrane.

Greater ? the thickness, greater ? the transit time at parity? of applied electric potential.

However, major ? the thickness, the greater are also the difficulties? related to the creation of a hole of suitable dimensions for the detection channel 120, as better illustrated below.

The identified thickness therefore maximizes the detectable transit time, ensuring that the detection hole 120 is made of the desired dimensions.

Lower thicknesses cause a very fast particle transit time, therefore undetectable.

In particular, this membrane can be made of polyimide, or polyimide, which improves the accuracy of the detections by means of said first 16 and second 17 electrodes, as better illustrated below.

As ? known in scientific literature, in particular the polyimide does not interact with the virus, presenting, at physiological PH, few surface charges capable of attracting other electrically charged particles, at the same time guaranteeing a high electrical insulation and excellent wettability qualities.

Said first 16 and second 17 electrodes support an electric field which favors the passage of nanoparticles to be detected through the detection hole 120 itself and detect the ionic currents associated with the biological fluid.

Said first 16 and second 17 electrodes are made of AgCl.

Alternatively, said first 16 and second 17 electrodes are covered with a layer of silver chloride AgCl.

In particular, said first 16 and second 17 electrodes can be made of copper on which a layer of silver, also called chemical silver, is deposited. Subsequently ? It is possible to create a layer of silver chloride on this layer of chemical silver.

The AgCl electrodes allow the establishment of an electric current thanks to the exchange of Cl ions which takes place at the AgCl interface with the KCl liquid solution with which they are in contact.

In particular, the cavity 13 and channel 100 are configured to be filled, in use, with an electrically conductive liquid solution, such as, for example, potassium chloride KCl or phosphate buffered saline PBS.

Reagents capable of binding to the particles to be detected can also be dissolved within this liquid solution, in such a way as to increase the specificity of the particles. of the detecting device 1, as better illustrated below.

The biological fluid to be analyzed is added to said through channel 110, so as to be diluted in the liquid solution.

In this way, only the particles to be detected are pushed through said detection hole 120 under the action of the electric field generated by said first 16 and second 17 electrodes.

The passage of these particles, and the consequent partial obstruction of the detection hole 120, determines the variation of the impedance associated with the detection hole 120, allowing to identify the particles present in the biological fluid, as shown in figures 9a and 9b.

In particular therefore, the detection device 1 ? configured in such a way as to receive the biological fluid in the cavity? 13, what? that it is diluted in the liquid solution.

If this biological fluid contains the particles to be detected, these can be bound to the reagents so as to form mega-aggregates which will pass through the detection hole 120, significantly modifying their impedance as shown in figures 9a and 9b.

The analysis of the aforementioned impedance variations also makes it possible to obtain a quantitative measure of the particles analysed, which can be extrapolated as a fraction of the quantity? of reagent introduced into the sample, as shown in figure 10.

In this way it will be Is it possible to recognize and quantify with high specificity? and sensitivity? the presence of these particles through the detection of the ionic currents which pass through the detection hole 120 by means of said first 16 and second 17 electrodes, arranged on the intermediate layer 12, as better illustrated below.

In particular, the presence of said first 16 and second 17 electrodes on the intermediate layer 12 allows a fine adjustment of the electric field, which can be adapted to the different needs? of detection.

As regards the detection hole 120, this has a section of much smaller dimensions than the dimensions of the through channel 110.

The dimensions of the detection hole 120 must be comparable with the dimensions of the particles to be detected. The optimal relationship between the size of the detection hole and the size of the particles to be detected? less than or equal to 1/10.

In the case of a detection device 1 for detecting the presence of a virus, in particular SARS-Cov-2, inside a biological fluid such as saliva, the detection hole preferably has a section with a dimension or width of section, equal to about 1 micrometer. In fact, as known, SARS-Cov-2 has a size between 50nm and 200nm, usually being equal to about 100nm.

This section size ? been expressly studied to guarantee a variation of the impedance measured on said first 16 and second 17 electrodes, when the virus passes through the detection hole 120, both if in free form, but even more so? when bound to reagents in solutions in the form of macro-aggregates.

In the case of a very pure biological fluid, such as a condensed exhaled fluid, what is it? a fluid coming directly from the lungs free of other particles or corpuscles in addition to the possible viral particles, the detection device 1 will be? capable of detecting the presence of viruses only by identifying the presence of particles passing through said detection hole 120.

However, the use of reagents allows to increase the specificity? of the test, allowing, for example, to distinguish whether the biological fluid analyzed contains the simple influenza virus or SARS-Cov-2.

In fact, thanks to the use of these specific reagents able to aggregate exclusively to the target element, for example the SARS-Cov-2 virus, the size of the macro-aggregates virus-reagents that form inside the cavity 13 pu? get to 300 or 400nm. Other viruses similar in size to those of SARS-Cov-2 will not, on the other hand, be bound to the reagents and, therefore, will produce a smaller impedance variation as they pass through the detection hole 120. What? allows to obtain a high specificity test?.

The detection hole 120 can? moreover, present different shapes according to the particles to be identified.

In particular, the detection hole 120 can have a conical shape, having a greater section width at the upper layer 11.

As is known, this shape is particularly suitable for detecting spherical or similar particles, for example spheroids, a category to which viral particles belong.

The conical shape, as known, in fact has the advantage of generating a linear relationship between the amplitude of the variation of the impedances and the size of the particles of spherical morphology, making it more? it is easy to estimate the relative sizes of analytes having this shape.

In alternate embodiments, the sensing hole 120 may also present a shape other than conical, for example having a cylindrical, funnel or hourglass shape.

The detection hole 120 can? be made for example by drilling with a laser source. This technology ensures, in fact, the formation of holes of micrometric size with high precision, high reproducibility? and with a very low drilling time (a few ms per hole). Furthermore ? It is possible to integrate laser drilling systems on automatic production lines, to increase production volumes by reducing times and costs compared to other processes.

The detection device 1 described above operates as follows.

The cavity? 13 and channel 100 are initially filled with conductive ionic liquid solution, for example 1M KCl.

An electric potential? applied between said first 16 and second 17 electrode.

The correct filling of the cavity? 13 and of the channel 100 is then detected by verifying that the current passing through the sensing hole 120 is equal to the nominal design current, for example being equal to approximately 150nA with approximately 400mV of applied potential as illustrated in figure 8.

Subsequently, the biological fluid to be analyzed is inserted into the cavity 13 through the through channel 110 of the upper layer 11.

The application of an electric field by means of said first 16 and second 17 electrodes triggers the passage of the electrically charged particles, if present in the biological liquid, through the hole 120.

An external measuring device, via said first 14 and second 15 electrical connection, measures the intensity? of the ionic currents of the biological fluid which passes through the detection hole 120.

When a particle present in the biological fluid having dimensions that are not negligible with respect to the dimensions of the detection hole 120 passes through the detection hole 120 itself, is a variation in the intensity detected? of electric current, as shown in figures 9a and 9b, which can? be uniquely associated with that particle.

As described above, the presence of said first 16 and second 17 electrodes directly on the intermediate layer 12 advantageously allows to distinguish a greater number of particles with respect to detectors of the known art, also allowing to detect the presence of viruses.

Furthermore, the use of reagents capable of binding to the particle to be detected, so as to form macro-aggregates passing through the detection hole 120 improves the sensitivity? and the specificity? of the detection device 1 in detecting a particular virus compared to others.

Finally, the coupling of the intermediate layer 12 by gluing it to the other layers 10, 11, the realization of the intermediate layer as a polyimide membrane of micrometre thickness, the choice of a detection hole 120 with shape and dimensions of the aforementioned type, and/or the ? The use of silver or silver chloride electrodes are all characteristics that can improve the sensitivity? of this detection device 1, making it particularly effective for the detection of SARS-Cov-2 in saliva samples or other biological fluids.

Advantages

The detection device 1 described allows reliable measurements to be obtained for verifying the presence of particles in a biological fluid.

In particular, the detection device allows to detect the presence of viruses, and specifically SARS-Cov-2 in biological fluids in a rapid, safe and economic way, with a sensitivity? up to 1000 times greater than other rapid tests based on colorimetric reactions.

The present invention ? been described by way of illustration, but not limitation, according to your preferred embodiments, but ? it should be understood that variations and/or modifications may be made by experts in the art without thereby departing from the relative scope of protection, as defined by the attached claims.

Claims (11)

1. Detection device (1) of nanoparticles in a fluid, comprising: a lower layer (10), in which ? obtained a channel (100); an upper layer (11), in which ? obtained a through channel (110); and an intermediate layer (12), on which ? obtained at least one detection hole (120), for the passage of said biological fluid, wherein said intermediate layer (12) ? disposed between and coupled to said lower layer (10) and said upper layer (11), ed wherein said detection hole (120) ? arranged in correspondence with said through channel (110), so as to identify with it and with a portion of said channel (100) a cavity (13) for containing said biological fluid; said detection device (1) being characterized in that it comprises at least a first (16) and a second (17) electrode for detecting an ionic current signal generated by the passage of said fluid in said detection hole (120), wherein said at least one first (16) and one second (17) electrode are disposed on said intermediate layer (12). 2. Detection device (1) according to the preceding claim, characterized in that said intermediate layer (12) is coupled to said lower layer (10) and/or to said upper layer (11) by means of glue. 3. Detection device (1) according to any one of the preceding claims, characterized in that said detection hole (120) has a conical shape. 4. Detection device (1) according to any one of claims 1 or 2, characterized in that said detection hole (120) has a cylindrical, funnel or hourglass shape. 5. Detection device (1) according to any one of the preceding claims, characterized in that said detection hole (120) has a section width of 1 micrometre. 6. Detection device (1) according to any one of the preceding claims, characterized in that said intermediate layer (12) is a polyimide membrane. 7. Detection device (1) according to the preceding claim, characterized in that said membrane has a thickness comprised between 5 micrometres and 50 micrometres, preferably 12.5 micrometres. 8. Detection device (1) according to any one of the preceding claims, characterized in that said first (16) and second (17) electrodes are made of silver chloride. 9. Detection device (1) according to any one of the preceding claims, characterized in that said detection hole ? made using a laser source. 10. Detection device (1) according to any one of the preceding claims, characterized in that said cavity? (110) has a volume of between 10 microliters and 100 microliters, preferably 60 microliters. 11. Detection device (1) according to any one of the preceding claims, characterized in that said upper layer (11) is of plastic material, such as Polymethylmethacrylate - PMMA or Polycarbonate ? pc.