Patent Application
20160108471
The present invention regards a kit for the optical detection of a micro-RNA of interest in a sample extracted from a body fluid.
The present invention also regards a method for detecting such micro-RNA of interest.
As is known, there is increasing interest in the possibility to make an early prediction of pathologies with dire prognoses by means of examinations that are well-directed, precise, possibly little-invasive as well as inexpensive.
For such purpose, micro-RNA are considered very promising molecules in the diagnostic field, even if they are present in body fluids with low concentration. These are small, non-codifying RNA molecules (about 21-23 nucleotides), which mediate the post-transcriptional regulation of the gene expression, acting as master switches on the human genome. There have been numerous studies which attest how such molecules exert a key role on genes specifically involved in some pathologies (e.g. cancer, cardiovascular diseases, liver diseases, neurological diseases etc.), and in processes such as differentiation, programmed cellular death and tumor transformation.
It is therefore clearly important to provide sensitive and reliable techniques for the detection of such molecules, extracted from a sample of a body fluid.
Most of the methods for detecting a micro-RNA of interest extracted from a sample of body fluid, e.g. blood (serum and/or plasma), urine or saliva, are based on the hybridization principle, in which each molecule of micro-RNA of interest (also called “target” in jargon), present in a sample C under examination, interacts (“hybridizes” in technical jargon) with a respective complementary oligonucleotide synthetic probe with single strand (also called “probe” in jargon). In the hybridization, the chain of nucleotide bases, which constitute an oligonucleotide probe, is linked in a specific manner to the complementary sequence of nucleotides present in a strand of target micro-RNA, thus giving rise to a molecule with double strand. The hybridization event indicates the presence of a specific micro-RNA contained in the sample C under examination.
Once such hybridization has occurred, the detection methods of the prior art provide for the actual measurement of the quantity of the target micro-RNA thus hybridized, contained in the sample C under examination, by means of the generation of a measurable signal obtained, e.g. electrochemically (by means of redox reactions) or optically due to fluorescence or bioluminescence or by means of autoradiography, generatable by a molecular marker (called “tag”, “label” or “reporter” in jargon) that can be conjugated to the hybridized micro-RNA of the sample or to the oligonucleotide probe or as an intercalating agent inserted in the double strand or in any case associable with the hybridization event between the two complementary strands.
As is known, both the electrochemical approach and the optical fluorescence or bioluminescence approach can be conducted in solid state or in solution, depending on whether the oligonucleotide probe specific for the recognition of the complementary micro-RNA has been previously constrained to the surface on which the hybridization occurs (e.g. comprising micro-particles or nanoparticles or a flat surface) or is found in solution (suspended in a liquid). For such purpose, various devices and methods were developed over the years for measuring micro-RNA, including for example the microarray or deep sequencing, or other biochemical methods such as Northern blotting, quantitative reverse-transcriptase-PCR (qRT-PCR) or in situ hybridization (ISH).
The devices and the methods mentioned briefly above suffer from several drawbacks.
One of these is poor sensitivity. Indeed, given the low concentrations of micro-RNA in the body fluids, they are unsuitable for obtaining a reliable direct reading of the quantity of a micro-RNA of interest present in the sample under examination and therefore require a step of amplification of the micro-RNA of interest in the sample C to be analyzed by means of enzymatic reaction (PCR). Such preliminary step, in addition to being arduous and costly, lengthens the analysis times of the sample itself. The average times of amplification by means of PCR of the micro-RNA of interest in the sample C to be analyzed can even reach 2 hours. Overall, therefore, the execution of such methods typically requires a time greater than 2 hours.
Therefore, the main object of the present invention is to provide a kit for the optical detection of a micro-RNA of interest in a sample extracted from a body fluid which is very sensitive, in the sense that it allows detecting very low concentrations of micro-RNA and that it requires a minimum quantity of sample to be analyzed.
Still another object of the present invention is to provide a method for the optical detection of a micro-RNA of interest extracted from a body fluid which is quicker than the conventional methods.
A further object of the present invention is to provide a method for the optical detection of a micro-RNA of interest extracted from a body fluid which is precise and reliable.
Not least object of the present invention is to provide a method for the optical detection of a micro-RNA of interest extracted from a body fluid which is practical to implement.
According to a first aspect of the present invention, a kit is provided for the detection of a micro-RNA of interest in at least one sample extracted from a body fluid, comprising:
According to a further aspect of the present invention, a method is supplied for the detection of a micro-RNA of interest in a sample extracted from a body fluid, comprising the following operative steps:
providing a kit according to the first aspect of the present invention;
arranging such sample to be examined in such at least one container means;
arranging, in such at least one container means, at least one oligonucleotide probe, the oligonucleotide probe being designed to establish a specific bond with a respective complementary sequence of nucleotides present in a strand of such micro-RNA of interest;
providing, in such at least one container means, such at least one molecular marker associable with such micro-RNA of interest in such sample;
inserting such at least one container means in such housing seat of such device;
activating such at least one optical excitation group;
activating such at least one detection group;
detecting, by means of such at least one detection group thus activated, such emission light radiation coming from such at least one container means and correlated with the quantity of such micro-RNA of interest in such sample and
applying at least one electric output signal correlated with such quantity in such sample of such micro-RNA of interest.
Further aspects and advantages of the present invention will be clearer from the following detailed description of a currently preferred embodiment thereof, illustrated as a merely non-limiting example in the accompanying drawings, in which:
In the accompanying drawings, equivalent or similar parts or components have been marked with the same reference numerals.
With reference now to
In the housing casing 2a of the device 2 of the kit according to the present invention, an optical excitation group 5 is mounted, designed to deliver an excitation light radiation λ towards the housing seat 2b and hence towards the container 3 that, in use, is insertable in such seat.
More particularly, the optical excitation group 5 comprises a light source 5a, e.g. of LED type, suitable for delivering an excitation light radiation λ. Such light radiation λ has a wavelength in the excitation range of a molecular marker that is optically excitable and associable in a known manner with the molecules of micro-RNA of interest in the sample C under examination. The LED light source will for example be selected with emission peak around 470 nm.
As will be noted, one such type of light source 5a, being fairly inexpensive, allows maintaining limited production costs as well as limited power consumption of the kit according to the present invention. In any case, other types of light sources 5a can be used, such as light sources of laser type or one or more white light sources, e.g. a halogen lamp.
The optical excitation group 5 of the kit according to the present invention also comprises collimator means 5b of such excitation light radiation λ, for example comprising a converging optical lens set to collimate the light radiation λ towards the housing seat 2b of the casing 2a of the device 2. Such collimator means 5b for example comprise an optical lens with focal length equal to about 10 mm placed at a distance equal to the focal length itself from the LED light source 5a.
Advantageously, the optical excitation group 5 according to the present invention also comprises an optical filtering means 5c between the collimator means 5b and the housing seat 2b. The optical filtering means 5c are set to limit the spectrum of the light radiation component λ, emitted by the source 5a, which reaches the housing seat 2b. The light radiation λ1 which crosses through the optical filtering means 5c has a spectrum such to obtain, in use, the optical excitation of the marker associable with the sample C under examination. The optical filtering means 5c also performs the function of reducing to a minimum the light radiation λ that is not useful for the optical excitation of the molecular marker and which is also inevitably propagated to the other components of the device, as will be better stated below.
Optionally, the optical excitation group according to the present invention comprises one or more means 5d for screening the light radiation λ, λ1 emitted by the light source 5a. The screening means 5d, also called “optical slits” in technical jargon, are provided arranged between the optical filtering means 5c and the housing seat 2b of the device 2. They make a physical barrier for a part of the light radiation λ and/or λ1 laterally delimiting an emission zone within which such radiation λ, λ1 is free to be diffused towards the housing seat 2b. The light radiation λ, λ1 thus incident at the housing seat 2b and, in use, on the container 3, has parallel beams with greater or lesser amplitude depending on the width of the emission zone delimited by the screening means 5d.
The kit for the optical detection of a micro-RNA of interest in a sample C according to the present invention also comprises a group 6 for detecting an emission light radiation λ2 that is generated, in use, by the molecular marker associable with the optically excitable micro-RNA molecules of interest. Such detection group 6 is provided housable in the device 2 at the housing seat 2b. The emission light radiation λ2 generated by the molecular marker is emitted by the sample C under examination in response to the emission, by the light source 5a, of the excitation light radiation λ1. The emission light radiation λ2 signals that the hybridization of the micro-RNA of interest with a respective oligonucleotide probe has occurred. The detection group 6 is designed to detect the light radiation λ2 and to supply one or more electric output signals SO (signal output) correlated with the quantity, in the sample C, of the micro-RNA of interest.
The kit according to the present invention furthermore comprises a processing unit 7, preferably mounted in the device 2, designed to process in a known manner the electric signal or signals SO supplied by the detection group 6 and, to in turn, to output an index or measurement of such quantity of micro-RNA of interest in the examined sample C.
Advantageously, the detection group 6 comprises at least one sensor means of silicon photomultiplier type 6a (or SiPM), which, as is known, is extremely sensitive to low-intensity light signals. The sensor means SiPM 6a is suitable for detecting the photons emitted by the molecular marker associable with the micro-RNA of the sample C, once it has been optically excited. In output, the SiPM 6a supplies an electric signal SO. A typical SiPM sensor usable in the kit according to the present invention has a peak of sensitivity comprised between 380-480 nm.
Optionally, between the container 3 and the sensor means 6a, the detection group 6 of the kit according to the present invention provides for an optical filtering means 6b, designed to filter the light radiation λ1 which inevitably reaches the sensors means 6a, even if in limited quantity.
The optical filtering means 6b is for example mounted or deposited on the surface itself of the sensor means 6a. With one such configuration, the optical path followed by the emission light radiation λ2 is quite limited, with consequent increase of the sensitivity of the kit with respect to the sensitivity of conventional techniques.
In addition, it is quite clear that one such configuration with limited optical path for the radiations λ2 allows reducing the background noise coming from the surrounding environment and hence detecting the emission light radiation λ2 in a more efficient manner. As will be observed, with one such configuration the size of the device 2 of the kit according to the present invention can also be quite limited, which renders the device easy to transport by an operator.
The kit according to the present invention also comprises administration means (not illustrated in the drawings) of any suitable type suitable for administrating, in the container means 3 of the sample C to be analyzed, the molecular marker and, if the hybridization is provided in liquid phase, oligonucleotide probes 4 complementary to the micro-RNA to be detected.
In the particular case in which the hybridization, between the micro-RNA of interest and the oligonucleotide probe(s) 4, is provided to occur in solid phase, the probes 4 will already be provided in the container means 3, e.g. bonded or constrained in a known manner to the internal surface of the container 3 or to the surface of micro-or nanoparticles present in the container 3 and not illustrated in the figure.
In this case, the kit according to the present invention comprises, in addition to the administration means of the sample C, administration means (also not illustrated in the drawings) of any suitable type that are designed to insert, in the container 3, at least one molecular marker associable with the plurality of oligonucleotide probes 4, complementary to the molecules of the micro-RNA of interest, and optically excitable.
According to the first embodiment of the present invention, illustrated in
In this first embodiment, the container means 3, permeable to the light radiations λ, λ1, λ2, is directly supported by the optical filtering means 6b. The container 3 is for example made of quartz, COC, PC, PET; in use, the sample C to be analyzed extracted from the body fluid is inserted inside the container, where both the hybridization with the plurality of oligonucleotide probes 4 and the association with the molecular marker take place. In the case of hybridization in solid phase, the probe(s) 4 will already be present in the container 3. In the case of hybridization in solution, the probes can be added in a known manner together with the sample C and with the molecular marker by means of suitable administration means.
With this configuration, the optical path of the emission light radiation λ2 is reduced to the minimum and therefore the sensitivity of the kit is increased. The sensor means SiPM 6a can have reduced size in this case, e.g. on the order of 1×1 mm or 2×2 mm.
As an alternative, the detection group 6 can be provided anchored in the device 2, directed towards the housing seat, and the container means 3 can be provided integrated in a disposable cartridge 8 insertable/disconnectable in/from the housing seat 2b.
According to a second embodiment of the kit 1 of the present invention (see
This configuration is advantageous since it allows drastically reducing the background noise caused by the excitation light radiation A emitted by the light source 5a. Such light radiation, as stated above, is never fully eliminated even if filtered by the optical filtering means 6b of the detection group 6, and given that it is detected (even minimally) by the sensor means SiPM 6a, it actually represents the lower limit of detection of the light radiation λ2 emitted by the sample C under examination.
According to a first variant of the second embodiment of the present invention, the container means 3 is housable in the housing seat 2b along the axis x-x and in such seat it laterally receives the excitation light radiation λ1.
In this case (
The sensor means SiPM 6a can have limited size, e.g. on the order of 1×1 mm or 2×2 mm.
According to a second variant of such second embodiment, see
This second variant of the second embodiment allows an improved lighting of the sample C by the light source 5a, at the same time ensuring a reduction of the background noise caused by the same. The detection group 6 of the kit according to the present invention comprises, in this second embodiment, an optical sensor means SiPM 6a with large size, e.g. 4×4 mm.
According to a further variant of the second embodiment of the present invention, the detection group 6 comprises a plurality of sensor means SiPM 6a, illustrated with a dashed line in
Advantageously, the kit according to this further variant of the present invention provides for two sensor means 6a arranged on opposite sides with respect to the housing seat of the device 2b. With one such configuration, in addition to obtaining a double signal SO emitted by the detection group 6 (one emitted by each sensor means), the disturbance caused by the excitation light radiation λ1 is limited.
The man skilled in the art can easily understand that other configurations of the sensor means are possible. Thus, for example, the detection group 6 can also comprise a plurality of sensor means SiPM 6a arranged, in a plan orthogonal to the axis x-x, angularly offset from each other around the housing seat 2b to form a ring of sensors. In this case, the size of the sensor means SiPM can have various dimensions, e.g. ranging from 1×1 mm to 4×4 mm, depending on the number of sensors mounted in the device.
The processing group 7 of the kit according to the present invention comprises known components suitable for receiving and processing the signal(s) SO emitted by the sensor means 6a of the detection group 6. For example, it comprises signal amplifier means, set for amplifying the signal(s) SO emitted by the SiPM 6a. Such signal amplifier means comprise, for example, low-noise operational amplifiers. The processing group 7 then comprises, for example, integrator means and analog-digital converters, designed to convert the analog current signal SO into a corresponding electric voltage level which is actually an index corresponding to the quantity of micro-RNA of interest in the analyzed sample. Optionally, the index to output from the processing unit 7 can be correlated with the concentration of the micro-RNA of interest in the examined sample C.
The method for detecting a micro-RNA of interest in a sample C extracted from a body fluid is very practical in execution and reliable.
Initially, such method provides for arranging a kit as described above and arranging a sample C to be examined in the container 3 of the kit.
If the hybridization of the micro-RNA of interest in the sample C under examination occurs in solution, the method provides for administering, together with the sample C, one or more oligonucleotide probes that are complementary to the micro-RNA of interest in order to allow the formation of double-strand hybridized molecules, as well as molecule markers associable with the hybridization event.
If instead the hybridization occurs in solid phase, the probe(s) 4 will already be arranged in the container 3 and it will suffice to wait a certain time period (a few minutes) in order to allow the hybridization to take place; in a subsequent step, an optically excitable molecular marker will be added to the sample C thus hybridized, by means of the above-described administration means, and such marker will be bonded to the hybridized micro-RNA molecules of interest.
The detection method according to the present invention then provides for a step for activating the optical excitation group 5, during which the sample C is optically excited, and then a step for detecting the emission light radiation λ2 emitted from the molecular marker bonded to the hybridized micro-RNA molecules of the sample C and correlated with the quantity of such micro-RNA of interest in the sample itself.
In this detection step, the sensor means SiPM 6a of the detection group 6 detect the emission light radiation λ2 and each means emit, in response to such radiation, one or more electric signals SO.
The above-described method also comprises, between the step of administering the molecular marker and the step of activating the optical excitation group 5, a step for the manual insertion of the container 3 in the housing seat 2b of the device 2, integrated or not integrated in the disposable cartridge 8 depending on the case.
According to a variant of the above-described method, the step for administering the sample C can occur in an automatic manner.
Preliminary tests have demonstrated that the use of the hybridization method (which uses probes constituted by PNA oligomers and nucleobases joined with fluorophore) sold by DestiNA Genomics and the subject matter of the international application WO 2009/037473 allows increasing the selectivity of the kit according to the present invention, hence making it even more precise and reliable in the executing of the invention.
The limited times for executing the method (several minutes for the hybridization and several seconds for the optical excitation of the sample and the analysis of the corresponding signal(s) SO), given that no preliminary amplification step is necessary for the sample C to be analyzed, as well as the limited size of all the components of the kit, make it practical in use and reliable.
The above-described Kit and method are susceptible to numerous modifications and variants within the protection scope defined by the following claims.
Thus, for example, the kit according to the second embodiment of the present invention can comprise the container 3, at least one sensor means SiPM 6a and, if desired, an optical filtering means 6b, integrated in a disposable cartridge 8 insertable/disconnectable in/from the housing seat 2b of the device 2.
Moreover, if the container 3 of the kit according to the present invention is directly supported by, or integrated with a respective detection group 6, it can be provided lacking the wall thereof in contact with such detection group 6, such that the sample C of body fluid to be analyzed together with the oligonucleotide probes 4 and with the molecular marker will be directly in contact with the sensor means SiPM 6a or, if provided, with the optical filtering means 6b.
Furthermore, the method for detecting a micro-RNA of interest described above can be made even more precise and reliable by means of a sequential control of the turning on/turning off or activation-deactivation of the various components of the kit.
For example, it can thus be provided to activate the optical excitation group 5, so as to induce the emission by the sample C under examination of the respective emission light radiation λ2, and to deactivate such optical excitation group 5 before activating the detector group(s) 6 of the device 2.
In this manner, the light radiation λ, λ1 emitted by the optical excitation group 5 towards the container 3 will be nearly fully eliminated, and therefore the light radiation emitted by the group 5 and effectively detected by the detection group(s) 6, will comprise only the emission light radiation λ2 correlated with the quantity of micro-RNA of interest contained therein.
| Number | Date | Country | Kind |
|---|---|---|---|
| VR2013A000132 | May 2013 | IT | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2014/060793 | 4/17/2014 | WO | 00 |
