Patent Application
20090305291
The present invention relates to a method and a microdevice for identification and/or quantification, particularly in real time, of an analyte, particularly a biomolecule present in a biological sample, and is particularly applicable to the field of diagnostic analysis (genome and/or proteome analysis) and to the production of biochips.
Over the last ten years, there has been a considerable proliferation of biochips, and numerous terms have been used to denote them (for example “gene-chip”, “gene-array”, “DNA micro-array”, “protein chip”, “lab-on-a-chip”). Essentially, these chips, whether developed in simple formats or integrated into more complex devices or architectures, are planar structures of various materials (commonly glass or plastics), on which (bio)molecules such as DNA, protein and cells capable of selectively recognizing target molecules are immobilized (by chemical modification of the surface or by in situ synthesis) [Fan, 2006].
Biochip technology has revolutionized molecular biology, and is widely used for studying gene and protein expression (genomic and/or proteomic expression) in various fields such as experimental diagnosis and clinical medicine, the discovery of new medicines (biomarker discovery) and pharmacogenomics.
Different types of chip have been developed for a very wide range of applications such as enzyme assays [Hadd, 1997], immunochemical assays [Wang, 2001], analysis for recognition of polymorphism in gene variants [Dunn, 2000], and nucleic acid sequencing [Scherer, 1999], together with chips for carrying out DNA ligation and amplification reactions on microvolumetric scales (the ligase chain reaction [Cheng, 1996] and the polymerase chain reaction (PCR) [Kopp, 1998; Daniel, 1998]).
In particular, because of the high specificity of the hybridization reaction between oligonucleotide sequences (selective pairing of A-T, G-C), DNA chips have been developed more rapidly than protein chips. Although the market for the latter is enormously valuable to the scientific world, its development has been slower than that predicted initially for this technology, because of the complexity of the interactions underlying the biorecognition mechanism between protein molecular species [Bodovitz, 2005].
In general, in a chip the event of recognition between the (bio)molecule and the target species must be translated into a detectable and measurable signal. The physical transducers for converting the signal can be of different types, based on different operating mechanisms, including optical, electrical and electrochemical transduction, or devices sensitive to variations of mass, current or frequency [Wang, 2002; Murphy, 2006].
Optical sensors therefore play a central role in the large field of biosensors. Optical detection is essentially based on the measurement of the fluorescence which develops at the biorecognition site following the event of binding between the interacting species (between complementary oligonucleotides, for example). Typically, in the most common biochip formats, one of the two biomolecular species is conjugated with a fluorescent marker.
The first optical transducers used fairly simple optical detection systems: typically, the fluorescence signal was derived from a fluorophore conjugated with the target biomolecule, and the signal was detected at the end of the hybridization (biorecognition) reaction. These approaches can be used for the purpose of purely qualitative detection, concerning the presence or absence of the target biomolecule in the solution being examined (the “on/off” approach), but cannot be used to obtain real-time (quantitative) data on the analyte interaction events.
A number of innovative optical detection systems were subsequently developed to provide greater sensitivity and flexibility in the detection system. For example, the use of optical fibres for signal transduction has become widespread, new detection strategies (such as FRET, molecular beacons, TIRF, SPR, phosphorescent probes and beads) have been developed, and new fluorescent markers (for example, PicoGreen) have been designed.
These strategies have yielded considerable improvements in respect of the signal detection limits, but they do not provide a means of quantitative determination of the biomolecules concerned, especially in planar formats, such as microarrays, in which specific probes for biorecognition have to be immobilized on a solid substrate.
CN 1 358 867 describes a “gene chip” device which uses the FRET (Fluorescence Resonance Energy Transfer) phenomenon for the detection of nucleic acids, where the FRET phenomenon is caused by the resonant energy exchange between a donor fluorophore and an acceptor fluorophore, one of which is bound to the probe species while the other is bound to the target species.
WO2006/066977 describes the use of FRET resonance for protein detection, in which a marked protein, comprising an energy donor marker and at least one acceptor group which can accept energy from the donor marker by Forster energy transfer, is exposed to incident electromagnetic energy in order to excite the donor group and measure the fluorescence emission of the donor.
One object of the present invention is to provide a method and a new optical transduction device which use the FRET phenomenon, and which enable the target molecular species to be measured quantitatively in real time.
A specific object of the invention relates to the possibility of quantitatively analysing the DNA amplification process (Polymerase Chain Reaction, PCR), since this technology, in its different versions (reverse transcriptional, RT-PCR; multiplex PCR; real-time PCR [Speers, 2006; Wrong, 2005]), combined with new methods of automated synthesis of nucleic acids, provides an important research tool for diagnosis and has been widely used in genotyping and the phenotype expression of pathogenic or viral antigens [Cockerill, 2003; Domiati-Saad, 2006].
The operating principle of real-time PCR is typically based on the measurement of a fluorescent marker, whose signal increases proportionally to the quantity of DNA amplified in each reaction cycle. As a general rule, use is made of probes suitably conjugated with fluorescent molecules having different operating mechanisms (SYBR Green, TaqMan, Molecular Beacons, Scorpions, Sunrise) [Wrong, 2005].
Various examples of real-time PCR on chips are currently available. The miniaturization of this technology yields considerable advantages such as the higher speed of the thermal cycles, a significant reduction in the consumption of reagents and biological samples, the portability of the microdevice and the intrinsic possibility of use for diagnostic applications at the place of treatment [Lee, 2003].
In view of the aforesaid objects, the invention proposes a method, a device and equipment as defined in the following claims.
In one embodiment of the invention, the probe molecules are fixed to a support coated with a film comprising a polymer matrix, in which the fluorescent nanocrystals are dispersed, or a polymer matrix comprising or formed from one or more photoluminescent polymers, in which the said nanocrystals or the said photoluminescent polymer can induce a FRET phenomenon with the fluorophore; the coating film is then irradiated with a radiation having a wavelength such that it selectively excites the nanocrystals or the photoluminescent polymer, but not the fluorophore bound to the target analyte.
Alternatively, the probe molecule can be fixed to a polymer coating film comprising an electroluminescent polymer; in this case, the electroluminescent polymer is excited electrically by applying a potential difference to the polymer film.
The fluorescence signal induced by FRET is then detected in the spectral emission region of only the fluorophore which is bound to the target analyte.
Other advantages and features of the method and device according to the invention will become clear from the following description which is given with reference to the appended drawings, in which:
The optical transduction device proposed by the invention for the real-time quantitative determination of biomolecules, adaptable to genomic and proteomic analysis, comprises a reaction microchamber 1, intended to receive a solution containing the biological sample including the analyte or analytes A to be detected; the reaction microchamber comprises a wall 2, formed by a solid substrate coated, or at least partially coated, with a thin film 3 of polymer material (
The fluorescent nanocrystals NCs are uniformly dispersed in this film, and specific probes S for the analytes to be determined are immobilized on its surface.
The analytes A present in the biological sample to be detected and quantified are marked directly (by synthesis in the microchamber for example), or indirectly, with one or more suitably selected fluorophores. The specific interaction between an analyte biomolecule present in the sample and the probes immobilized on the optically active wall of the microchamber is detected by a FRET process between the NCs (which act as donors) and the fluorophores bound to the biomolecule (which act as acceptors).
The optical system is shown in
In the first case, the specific optical parameters used in the microdevice are shown in the drawing, as a non-limiting example.
The system exploits the specific photophysical characteristics of FRET processes: the efficiency of the donor-acceptor interaction is strongly dependent on the relative distance (d) between the two species (being typically a function of d−6), and consequently there can be resonant transfers of excitation energy from the NCs to the target fluorophores only for distances which are typically less than 10 nanometres.
In the optical detection system integrated into the microdevice, these photophysical characteristics are such that an efficient process of energy transfer from the NCs to the fluorophores bound to the target molecules can be established only at the point where these biomolecules have interacted specifically with the corresponding probe (except for a negligible background which is constant in time and which is due to the biomolecules which are statistically in solution in the region within the first 10 nm from the surface of the polymer film.
By exploiting the specific spectral properties of the nanocrystals (essentially the very broad absorption spectrum; see for example the spectrum of
The essence of this approach is that none of the free biomolecules in solution are excited, since they cannot directly absorb the exciting radiation, and therefore they make no contribution to the fluorescence signal which is to be detected. Thus this feature enables the biomolecular interactions with the specific probes to be monitored selectively in real time and in a quantitative way, and also makes it possible to analyse and quantify the biomolecules which are of interest for genomics and/or proteomics.
The nanocrystals used in the field of the invention are preferably colloidal semiconductor nanocrystals, preferably of the core-shell type; for example, CdSe/ZnS core-shell nanocrystals can be used. Examples of other materials which are frequently used to form the core are ZnSe, CdS, and CdTe. Clearly, a wide range of other materials can also be used.
However, the invention is not to be interpreted as being limited to a specific choice of fluorescent nanocrystals.
In one embodiment of the invention, the thin film of polymer material can comprise two or more types of fluorescent nanocrystals, having different spectral absorption characteristics.
The fluorophores bound to the biomolecules can be common commercial organic fluorophores or any other type of novel fluorophore, including NCs, oligothiophenes, GFPs (Green Fluorescent Proteins), beads, and hybrid systems), provided that they have suitable characteristics to act as efficient acceptor species in FRET processes using NCs dispersed in the polymer matrix (essentially, they must have a good spectral overlap with the NCs used).
The emission of the fluorophores bound to the target molecules can range over a wide spectral interval (from blue to infrared), according to the specific biomolecular application.
The analytes included in the biological sample to be analysed can be marked with different fluorophores which emit in distinct spectral regions; thus the possibility of using different NC-fluorophore pairs as donor-acceptor pairs enables analyses to be conducted in parallel on different analytes (multiplexing).
For this purpose, it is simply necessary to monitor the emission of the different fluorophores simultaneously, acquiring the different signals in different spectral windows.
Clearly, the method according to the invention is not limited to the detection and quantification of specific analytes; the analytes to be determined can, for example, comprise molecules of DNA, proteins and ligands which can be marked with one or more fluorophores.
These analytes (target compounds) can be present in biological or non-biological samples, such as clinical samples extracted from blood, urine, feces, saliva, pus, serum, tissue, fermentation solutions or culture media. The analytes (target compounds) can preferably be isolated, purified, split, copied and/or amplified, if necessary by using methods known in the prior art.
Similarly, the specific probes immobilized on the surface of the thin polymer film can comprise specific sequences of ssDNA, antibodies, receptors, aptamers, etc.
The optically active film integrated in the microdevice according to the invention is preferably made from a solution of NCs and a polymer, preferably an elastomer, which is deposited on the solid substrate, preferably by spin-coating or by other deposition techniques which can ensure a uniform distribution of NCs in the polymer matrix; typically, the thickness of the polymer film varies from a few nanometres to several tens of microns, for example from 1 nm to 50 μm; preferred thicknesses are of the order of 10 to 100 nm.
The polymer material used must not be optically active (in the absence of NCs, it must have zero or very low intrinsic fluorescence), must be transparent in the spectral range from the near UV to the visible and to the infrared, and must have chemical and physical characteristics enabling NCs to be dispersed uniformly without significantly perturbing the specific optical properties of the film, and chemical characteristics of the surface which allow chemical functionalization with molecules capable of biorecognition.
In one embodiment of the invention, it is possible to use a polymer material with electronic resist properties (such as PMMA) which enables the optically active surface of the device to be patterned by lithographic methods.
A possible alternative to the use of an optical film formed by an inert polymer in which colloidal nanocrystals are dispersed is the use of an optical film composed of, or comprising, other active materials, such as photoluminescent or electroluminescent polymers or phosphorescent compounds, which may optionally be incorporated in polymer matrices, on which the sequences of probe DNA, or more generally (bio)molecules with the function of specific recognition of target analytes, are fixed in a similar way to that described above.
The operating mechanism (for molecular recognition of analytes by solid state FRET processes) is entirely identical to what has been described for the case in which colloidal nanocrystals are used. In particular, in the case of photoluminescent polymers, the device operates in the same way as that specified for the hybrid PMMA-NCs system, used in the following example of embodiment: the exciting radiation is selected so as to excite only the photoluminescent polymer and not the fluorophores conjugated with the target biomolecules; the latter are excited by the polymer by means of FRET processes only as a result of the biorecognition event. In the case of electroluminescent polymers, however, there is no need for an external excitation source; the polymer is excited (causing the excitation of the fluorophores conjugated with the target biomolecules via FRET, as a result of biorecognition) by an electrical method (electroluminescence), by applying a suitable potential difference to the polymer film (according to the conventional geometries and configurations of the LED devices). In the latter case, evidently, the device has the further advantage of not requiring an external excitation source, but in all other respects it operates in an identical way to the system described in relation to the use of nanocrystals.
The following examples of luminescent substances can be cited:
The substrate which acts as the support for the polymer matrix does not have to meet specific requirements, except that it must be physically and chemically compatible with the polymer material used; for example, silicon, silica, glass, quartz or plastics materials can be used.
The reaction chamber can be made from various materials, such as polymer or glass materials, which may or may not be suitably functionalized and passivated, provided that they are compatible with the integrated polymer matrix and with the solution containing the sample and the reagents, which is usually an aqueous solution.
The chamber can be produced in various ways, for example by etching, embossing, moulding or any other suitable manufacturing method. A suitable system of heaters and/or coolers can be integrated into the reaction chamber, to ensure that a controlled temperature of the solution in the chamber can be maintained, or that specific thermal cycles can be executed according to the application concerned.
A preferred material for the microchamber at the present time is polydimethylsiloxane (PDMS), which is currently one of the most widely used plastics materials for the production of chips for stand-alone or integrated systems. PDMS has a number of advantages: it is biologically inert, non-toxic, gas-permeable, and inexpensive, and adheres easily to other materials such as glass, polystyrene and PMMA, thus making it possible to construct hybrid systems composed of different materials.
However, the high hydrophobicity and low chemical reactivity of the groups exposed on the surface considerably limit its use in many applications [Xiang, 2005]. To improve the hydrophily of the surface, PDMS can be subjected to surface modification treatments using physical and chemical methods.
For this purpose, PDMS can be treated with oxygen plasma in experimental conditions in order to produce a minor modification of the surface (hydrolysis of the exposed methoxyl groups) rather than true etching. The action of the oxygen plasma is effective, resulting in very low typical contact angles of about 12.5°±6.25°measured after treatment.
In order to improve the stability of this treatment over time and prevent the treated PDMS surfaces from returning in a short time to a hydrophobic state (for example, for periods in excess of 24 hours), surface passivation procedures can be carried out. A preferred passivation procedure is a treatment with an acid solution of diamine-PEG which enables good wettability characteristics to be obtained for periods of more than two weeks.
The surface of the optically active thin film can be functionalized by known conventional methods for biochip preparation. In general, it is possible to use reactions of hydrolysis or aminolysis of the exposed free methoxyl groups and activation of the resulting groups with reactive chemical groups (mono-linkers and/or bi-linkers) for the (bio)molecules, such as oligonucleotides, proteins and peptides, with which the surface is to be functionalized.
One preferred method is to fix biological molecules such as proteins, peptides or nucleic acid sequences by a chemical bond with amine groups present on the optically active surface, bound to activating mono-linkers such as glutaraldehyde.
The insertion of amine groups (which in turn bind the activated groups on the surface) at the terminal ends of the nucleic acid chain is easily carried out by synthesis using animated nucleotides.
It is to be understood that the probe molecules can be bound to the optically active surface in a matrix arrangement, according to known methods of biochip production.
A prototype microreactor for real-time optical detection for genome and/or proteome analysis was constructed, using PDMS as the material for the microchamber. In order to improve the hydrophily characteristics of the surface, the PDMS was initially treated with oxygen plasma, as mentioned above, to cause a minor modification of the surface with hydrolysis of the exposed methoxyl groups.
Surface passivation procedures were also carried out, using acid solutions of diamine PEG, to increase the stability of the treatment time. This procedure was successful, and the device showed good wettability characteristics for more than two weeks.
The PDMS microreactor was constructed by pouring a solution of PDMS and polymerizing agent, in suitable concentration ratios, in circular metal moulds whose dimensions impart a constant shape and thickness to the microreactor. Following the polymerization of the solution, carried out in a furnace at 140° C. for 30 minutes, the PDMS microchamber was removed from the metallic mould and cooled.
The resulting microchambers had dimensions suitable for volumes from 50 μL to 5 μL; a typical prototype microchamber is shown in
The possibility of producing hybrid systems, composed of different polymer materials, was also investigated, and a PDMS microreactor was constructed with a base composed of a layer of PMMA functionalized according to the procedures described below. Various PDMS sheets were also produced for the purpose of sealing the whole system and thus preventing the evaporation of solvents, since it is very important to control this phenomenon in PCR processes.
For the construction of the prototype, PMMA was selected as the polymer matrix for dispersing the nanocrystals, since it conforms to the chemical and physical characteristics described above. Various substrates (glass, silica and PDMS) were initially used as the support for the polymer matrix, and the characteristics of chemical and physical compatibility with a PMMA solution were investigated for each of these, following deposition by spin-coating. Samples of PMMA with and without colloidal semiconductor nanocrystals (standard samples and optically active samples) were prepared, and the resulting film was characterized using a profile gauge, atomic force microscopy (AFM) and confocal microscopy.
The characterizations which were carried out showed that the resulting polymer film had excellent uniformity. Additionally, the analysis of the NC emission spectra showed that the nanocrystals were dispersed uniformly in the film and that they had spectral characteristics identical to those measured in newly synthesized standard NC solutions.
In particular, in this specific working example, the thickness of the PMMA layer in which the NCs were dispersed was optimized to about 40-50 mm, since the fluorescence signals measured in these conditions were sufficiently intense for optimal detection.
As mentioned above, it is to be understood that films with different thicknesses can be used, and therefore the choice of the thickness of the optically active film is not particularly critical. In the case under consideration, CdSe/ZnS core-shell NCs were used, emitting at 530 nm; polymer films with the same optical and uniformity characteristics were also produced with NCs of different dimensions which emitted in a range from the blue to the near infrared.
NCs were dispersed in a solution of PMMA-950K (typically with the following final chlorobenzene concentrations: CNCs=7×10−6 M and CPMMA=1.9×105 M). The resulting dispersed solution was deposited on the substrate by spin-coating (typical parameters: 3000 revolutions per 40 seconds) and the polymerization of the PMMA was then induced by heating on a plate at 180° C. for two minutes.
In the example described here of a microdevice for quantitative determination of oligonucleotide sequences (real-time PCR), the PMMA film, made optically active by NCs dispersed in them, was functionalized by aminolysis with a single layer of primary amine groups. These groups were conjugated with oligonucleotides with terminal amine functionality by means of a linker such as glutaraldehyde.
ssDNA molecules immobilized on the PMMA form the complementary target DNA biorecognition elements which represent the product of the amplification chain reaction (PRC) conducted in the reaction chamber located above the PMMA. All the experimental conditions, for both the aminolysis reaction and the reactions of activation with glutaraldehyde and hybridization with ssDNA, were suitably optimized.
The density of the resulting primary amine groups was evaluated by reaction with the reagent fluorescein-5-isothiocyanate (FITC). The concentration of exposed amine groups was estimated to be 2-3 picomoles/cm2 (in accordance with the published data [Patel, 2006]).
After the activation of the PMMA layer, which was animated with glutaraldehyde, the density of bound molecules per cm2 of surface was also quantified with ssDNA, using ssDNA marked with Cy3 and measuring the fluorescence: a concentration of 0.3-0.5 picomole/cm2 was found for the probes.
A similar reaction can be used not only for DNA but also for the functionalization of the surface of PMMA or other polymer materials with molecules of different kinds and for different applications.
In order to evaluate the operation of the resulting microreactor for real-time PCR applications, a plasmid of interest (pMPSV-RM1) was initially amplified with PCR at different reaction volumes (25, 12, 6 e 3 μL). In this case, the microreactor was treated in advance by various passivation procedures (using BSA solutions, for example) and the detection was carried out at the end point of the reaction with agarose gel electrophoresis. The results show that PCR takes place in the microreactor even at minimum reaction volumes (
In
The real-time detection of the hybridization event between the two species (immobilized ssDNA and target DNA) was then conducted.
The target DNA was marked with commercial organic fluorophore (Cy3) which has a large spectral overlap with the NCs used and enables the optical detection of the interaction to be conducted beyond 600 nm (the region in which selected NCs do not emit).
The optical detection was then carried out by measuring the fluorescence signal of the Cy3 marker present in the amplified target DNA (as shown in the general diagram in
The Cy3 fluorescence signal is produced by exciting the microreactor at a low wavelength, specifically λ≈400 nm, which selectively and efficiently excites the NCs but not the fluorophores conjugated with the free biomolecules in solution, and then transferring energy (FRET) from the NCs present in the PMMA to the fluorophore conjugated with the target DNA (which has interacted with the probe DNA).
In this case, the detected fluorescence signal increases as a function of the increase in the number of DNA amplification cycles (PCR cycles), as can be observed in the signal measured in real time for the amplification of the plasmid pMPSV-RM1 (
Briefly, the method and device according to the invention have the following innovative and advantageous features:
Furthermore, since a material with electronic resist properties (such as PMMA) is used as the polymer material for making the optically active film of the device, the film can be patterned by lithographic methods (such as e-beam lithography) with resolutions down to a few nanometres.
Thus this approach makes it possible to obtain, with high precision and very high resolution, matrices of pixels (each pixel being a different optical transduction element) for quantitative analyses of biomolecules in parallel.
| Number | Date | Country | Kind |
|---|---|---|---|
| TO2006A000883 | Dec 2006 | IT | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2007/055112 | 12/14/2007 | WO | 00 | 6/11/2009 |
