ITTO20100865A1 - PURIFICATION AND AMPLIFICATION OF NUCLEIC ACIDS IN A MICROFLUIDIC DEVICE INCLUDING POLIDIMETHYLSILOSSAN SURFACES - Google Patents

Description

DESCRIPTION of the industrial invention entitled: "Purification and amplification of nucleic acids in a microfluidic device comprising polydimethylsiloxane surfaces"

DESCRIPTION

The present invention refers to the sector of microfluidic devices, of the type currently referred to as LAB-ON-A-CHIP, suitable for carrying out a biological assay on a sample comprising a nucleic acid (DNA) extraction / purification step from the sample and a nucleic acid amplification / detection stage by polymerase chain reaction (PCR and RT-PCR).

Recently, microfluidic devices have been proposed and described which allow the three operations of nucleic acid extraction, amplification and detection to be carried out in a single integrated structure.

Chen et al. in Biomed. Microdevices (2010) 12: 705-719, describes a device of the type mentioned above, in which the operations of lysis, isolation of the nucleic acid, enzymatic amplification and detection are carried out in a polycarbonate structure. In this microdevice, the DNA extraction step is carried out with chaotropic salts on silicon membranes and subsequent elution, and the PCR reaction takes place with dynamic passivation with the use of BSA (bovine albumin); although the microfluidic device allows to carry out the biological assay in its entirety, the reactions of the single stages must be carried out by moving the sample to physically different areas of the device.

WO2006 / 085948 discloses an integrated microfluidic device including a nucleic acid extraction / purification region and an amplification / detection region, performed by RT-PCR. This device presents, in the DNA extraction region, microfabricated columns with silica coating and the DNA purification is carried out using a lysis buffer, containing chaotropic salts, able to open the cells and to bind the DNA to the microfabricated columns. After a washing step for the removal of unwanted material, the DNA is released from the columns and used for the RT-PCR reaction in an amplification region with passivated surfaces by BSA.

EP 2 160243 A1 describes a microfluidic device suitable for carrying out cell lysis and DNA extraction; PDMS (polydimethylsiloxane) is indicated among the various materials that can be used for the structure of the microdevice; however, the microdevice has a modular type structure with different regions and parts, specifically designed to use and execute part of the contemplated protocols; the DNA amplification operation is performed by transferring the DNA to an amplification unit.

Microfluidic devices of PDMS are also known predisposed for gene amplification and / or detection; passivating agents are always used in these microdevices to modify the PDMS surfaces and obtain the amplification of the gene material.

Frey et al. in Biomed. Microdevices (2007) 9: 711-718 describes a miniaturized amplification system in PDMS, in which a dye specific for double-stranded DNA is inserted into the PCR mix; the walls of the PDMS device are coated with poly "L-lysine" conjugated with poly ethylene glycol (PLL-g-PEG), to make the surface hydrophilic and increase the PCR yield.

The present invention is based on the discovery of the fact that polydimethylsiloxane surfaces, by virtue of their chemical and morphological characteristics, have the spontaneous ability to adsorb nucleic acids. In view of this discovery, the invention thus provides a simplified procedure for carrying out biological assays comprising the extraction / purification of nucleic acids from a biological sample, and also the amplification of the nucleic acid, with the use of a single microfluidic device.

Thus an object of the invention is a process for carrying out a diagnostic / biological test having the characteristics as defined in the following claims.

Following the invention, it has been found that the adsorption of nucleic acids on PDMS surfaces is spontaneous and does not require any type of treatment or surface modification to be promoted. Consequently, the DNA extraction / purification operation can be performed by placing the biological sample, previously used or optionally in a lysis solution, in direct contact with PDMS surfaces, where such surfaces are not functionalized or modified prior to contact with the biological sample.

Within the scope of the invention, the use of PDMS surfaces is thus contemplated which are:

two-dimensional layers (where this term means very thin layers) used as a covering of other materials, in the form of flat surfaces or with small curvatures (for example internal coatings of microfluidic devices) or circular or spherical, with high curvatures (for example coating of materials in the form of beads or capillaries);

surfaces of massive parts in flat or small curvature PDMS (e.g. microfluidic devices or parts of them) or circular or spherical shaped parts with high curvatures (e.g. beads or capillaries).

Therefore, in a first embodiment, these PDMS surfaces can be part of microfluidic devices manufactured with different materials, such as silicon, silicon-glass, glass, composite metals, oxides, polymers, thermoplastic polymers, thermosetting polymers, elastomers, thermoplastic elastomers .

In these cases, the PDMS can:

cover the internal surfaces of the device obtained according to the technology required for the class of materials used for manufacturing;

it can also constitute auxiliary structures inserted in the primary fluidic structure for the sole purpose of increasing the specific internal surface of PDMS in order to spontaneously adsorb DNA.

The maximum benefit that the system, object of the present invention, is able to bring in terms of cost reduction and analysis times, is obtained by building microfluidic systems using only PDMS. The device made entirely of polydimethylsiloxane, by means of an open mold casting process, allows a significant reduction in cost, compared to both traditional materials such as glass and silicon, and other polymeric materials such as resins and thermoplastic materials.

In a preferred embodiment, the device is formed by a main part in which the different functionalities are defined (chip), and by a flat base formed by a thin membrane that allows the closure of the fluidic elements defined in the main body through a process bonding by means of a thin layer of the same material (PDMS) cross-linked in place. Furthermore, the processes of cell lysis of the blood sample, DNA purification / extraction, PCR and finally detection, are performed in the same structure without the need for distinct areas for the different operations. Being able to use whole blood (but not only) as a biological sample, the proposed system is suitable for the diagnosis of genetic diseases, as well as for the detection of pathogens.

This, together with the constructive simplification and the reduction of costs, characteristics of the proposal, makes it suitable for the development of "point-of-care" systems. DNA purification takes place by exploiting the simple chemical-morphological interaction with the material of the device, and the PCR reaction is carried out directly on the DNA adsorbed on the walls of the device, without the need for special treatments with passivating agents in order to be carried out effectively.

The invention consists in the innovative use of a miniaturized system for the detection of pathologies starting from reduced quantities of biological fluids (about 7 μΐ of blood).

The main component of the device is a chip made of PDMS coupled to a base (membrane) in the same material, possibly obtained by spreading a thin veil (for example by spinning) on a rigid, smooth and more thermally conductive surface than the polymer (for example silicon). The fabrication exploits the simple and inexpensive casting technology (open mold casting).

Following crosslinking and extraction, the two parts (base and chip) are glued by means of a thin veil of the same polymer, interposed between the base and the chip, and then made crosslinked.

This chip must perform the function of containing the reaction volume, without interfering in biological reactions and guaranteeing airtightness in all phases of the reactions.

The DNA extraction process can be performed using approximately 5-10 μΐ of biological material, in less than thirty minutes. The extraction system does not require surface treatments of the device, but exploits the natural adsorption of the biological material on the walls of the device itself.

Conventional PCR systems require the intervention of specialized personnel for their use and are conducted using polypropylene reaction tubes with volumes of 25 μΐ or greater which are characterized by low conductivity and high thermal inertia. The use of the silicon base allows to shorten the time for the PCR reaction thanks to the better thermal conductivity of the material.

The fluorescence detection takes place through the use of marked probes, and can be integrated directly into the system, by carrying out an on-line monitoring of the fluorescent signal and / or a measurement at the end of the amplification process directly on the device.

The system is designed to allow diagnosis in a fast and economical way; this is applied in mass screening, in on-site analyzes, for example in favor of hospital patients to identify nosocomial infections directly at the patient's bed.

In the attached drawings, provided by way of non-limiting example:

fig. 1 is an image of a demonstration device, made entirely of PDMS;

fig. 2A is a vertical sectional view of the device of fig. 1; And

fig. 2B is a schematic top view. The microfluidic device (or chip) consists of two parts:

1. a main body 2, in which the functionalities of the chip are defined; And

2. a base 4 which serves to close the device, ensuring the confinement of the fluids in the working volume.

Both parts are manufactured in PDMS through an open mold casting process of the polymer previously mixed with the crosslinking agent. PDMS is an elastomeric polymer, available as a two-component product, which is prepared by mixing the polymer with a cross-linking agent.

After degassing, the mixture is poured into a mold with a geometry that is dual to that of the object to be obtained, it is allowed to degas and the material in the mold is heated to a temperature compatible with that of the material that forms the mold itself (usually 75 ° C for 45 minutes) or the reaction is carried out at room temperature for at least 24 hours.

The composition of the final PDMS, defined as the weight ratio of polymer with respect to the crosslinking agent, can be chosen on the basis of final characteristics of use, such as for example stiffness. For the devices manufactured by way of example for the present invention, it has been chosen to use, purely for demonstration purposes, a composition equal to 10 parts by weight of polymer and 1 of cross-linking agent (10: 1).

The mixing is done manually. The polymer thus obtained is poured into the molds and subjected to heating in order to promote the crosslinking reaction. The material is then taken out of the mold and can be used.

The devices prepared for this demonstration are characterized by the presence of a single reaction chamber 6 with an approximately elliptical shape, connected to two holes 8, 10 (inlet and outlet), arranged along the major axis of the structure (see image proposed in fig. 1). The total volume available in this demonstration device is 15 μΐ.

The mold used for this demonstration was manufactured with high precision mechanical processing using a numerically controlled milling cutter. The working volume, using this technology, and keeping the shape of the chamber unchanged, can go down to 10 μΐ. A further reduction can be obtained by using buffer oils compatible with the solutions, in order to occupy the channels with those oils (minimum volume 5 μΐ).

A further reduction in volume can be achieved by manufacturing the mold through the use of micro-technologies and by changing the shape of the working area from chamber to microfluidic channel. In the literature there is evidence of minimum volumes that can be manufactured in devices made entirely of PDMS via soft-lithography, of the order of 1.75 μΐ [A. Ranjit Prakash et al. Sensors and Actuators B 113 (2006) 398-409].

The base of the device can be manufactured as:

self-standing membrane, through a mold casting process having a dual structure of the one of interest;

film deposited and thinned (with tools such as, for example, spatulas) or by spinning on a rigid support surface, which conducts heat more efficiently than PDMS (metals, composites, silicon, silicon-glass, glass).

The two parts (body and base) are joined by non-cross-linked PDMS layers positioned between the body and the base and then left cross-linked (but other bonding modes can be chosen).

No device treatments are required for the DNA extraction and amplification process. The device can be fed with a biological material consisting of previously purified nucleic acids, or a biological material including nucleic acids in a solvent or in a lysis solution; preferably, this material does not comprise solvents or chaotropic salts. The biological material is incubated on the device and, thanks to the chemical and morphological structure of the PDMS surface, is absorbed on it. The washes, carried out to eliminate components that could interfere with the subsequent amplification reaction, leave the biological material available on the walls of the device.

The device allows to obtain DNA sufficiently purified from cellular debris, hemoglobin, proteins, lipids and other material deriving from the lysis of blood cells without the need for surface treatments, and can be used without further purification processes in subsequent analyzes.

This occurs with the use of a minimum quantity of biological starting material, reduced quantities of reagents and above all a reduced duration of the extraction process (30 minutes) compared to the approximately 2-3 hours required for an extraction under standard conditions that uses volumes of the order of at least tens of μΐ.

The amplification reaction is carried out directly on the biological material adsorbed on the surface of the device, which is sufficiently exposed and available for the reaction, without the need to detach it from the walls. Furthermore, the amplification reaction takes place without the need to use passivating agents such as BSA (bovine serum albumin) or PVP (polyvinylpyrrolidone), commonly used on this type of material.

By means of an appropriate fluorescent detection protocol it is also possible to use the microchip for the specific determination of the increase in amplified DNA. Two specific probes may be present in the PCR reaction mix, each bound to a different fluorophore, or there may be a single specific probe or fluorescent indicator of amplification. This system allows, through the analysis of the fluorescence detected at the end of the PCR reaction, to define the identity of the investigated nucleotide.

This therefore lends itself to be used in any type of mutational screening (it is therefore used in genetic diagnostics relating to hereditary genetic diseases such as cystic fibrosis, hemochromatosis, deafness, Mediterranean anemia, chondroplasia, etc.), as well as in genotyping analyzes (study of single nucleotide polymorphisms or SNPs) related to susceptibility studies to diseases with complex traits (obesity, cardiovascular disease, osteoporosis, etc.) and, potentially, opens up the possibility of carrying out gene expression studies on a good number of samples, monitoring the fluorescence growth during the PCR reaction, in real time.

Details of implementation examples

Example 1: Purification of nucleic acids

The purification system and the related protocol described in this example were developed on microchips with a volume of about 15 μΐ, initially using purified human genomic DNA. The extraction efficiency of the devices was verified with quantities of purified genomic DNA up to 100 ng.

Given the positive outcome of the extraction process and considering that in the quantity of blood to be used there are about 200 ng of DNA, the tests were carried out using whole blood, developing an in-chip lysis protocol.

7 μΐ of biological material (whole blood in EDTA and thawed) was combined with 7 μΐ of lysis solution containing no chaotropic salts or solvents and 0.7 μΐ of proteinase K and inserted into the chip. The chip was subsequently housed in the appropriate thermal cycler and the following thermal protocol was applied: raising to 56 ° C, staying at this temperature for 10 minutes.

At the end of the thermal cycle, the micro-chip is removed from the thermal cycler and the lysed biological material is left to adsorb for 20 minutes at room temperature on the walls of the device.

At the end of the incubation, the device is washed by inserting 15 μΐ of ultrapure water for at least 5 times consecutively, finally removing the water remaining in contact. At the end of the washing process, the DNA that remains adsorbed on the walls of the device is available for the subsequent PCR amplification process without the need to detach it from the walls.

Example 2: PCR

The DNA used as template comes from the purification process described in example 1 and is used directly in the PCR mixture, without the need to detach it from the walls of the device, giving rise to products that are clearly visible at the end of the amplification. The PCR reaction is then carried out in the thermal cycler, with the following protocol, described below by way of example.

A standard PCR reagent mix contains 2.5 mM MgCl2, 0.2 mM dNTPs, 0.5 μΜ of each C282Y forward and reverse primer, 1.5U of Roche HotStartTaQ polymerase in 15 μΐ. The protocol used consists of the following cycles: 1 cycle at 95 ° C for 5 minutes, 35 cycles at 95 ° C for 30 seconds, 62 ° C for 30 seconds and 72 ° C for 30 seconds, 1 cycle at 72 ° C for 7 minutes. The amplified gene region in this example is related to a point mutation causing hemochromatosis.

At the end of the reaction, the solution obtained is used in this example in an electrophoretic cell to check the purity of the DNA thus obtained (2% agarose gel containing ethidium bromide). Alternatively, using a special protocol described in the next point, it is detected by monitoring a fluorescence signal.

Example 3: Fluorescence detection Currently, the functionality of the fluorescence detection system has been demonstrated using the same PCR chips used for electrophoretic detection, which use a reaction volume of approximately 15 μΐ.

A fluorescence microscope with the following characteristics was used as a system for excitation and detection of the fluorescent signal:

2 lOx objective (acquired area 1000x800 pm), 120W mercury lamp, 480/40 nm bandpass excitation filter, 527/30 nm bandpass emission filter, integration time about 0.6 sec.

The fluorescence values were measured on

2

of an area of about 400,000 µm, which corresponds to a small portion of the microchip used and therefore to a reaction volume of less than 500µm. TaqMan® type fluorescent probes were used, complementary to an internal region of the gene sequence to be amplified whose presence induces a fluorescent signal following the repression of a fluorescence quenching.

The result is therefore a specific revelation of the amp to be monitored, even in Real-Time. The protocol used and described below is an example of those successfully tested in experiments that integrate PCR and detection.

A standard mix of PCR reagents contains 2.5 mM MgCl2, 0.2 mM dNTPs, stock solution containing primer for and rev and the concentrated TaqMan IX probes (TaqMan® SNP Genotyping Assay), 1 Unit of Roche HotStartTaQ polymerase . The protocol used is composed of the following cycles: 1 cycle at 95 ° C for 10 minutes, 40 cycles at 92 ° C for 15 seconds, 60 ° C for 1 minute.

In our case, the measurements were performed both by comparing the fluorescence signal of the same chip before and after the PCR and by comparing the signal of the chips containing the mixture described above ("positive") with that of the control chip ("negative": for example without template, or alternatively complete with all reagents, but not subjected to PCR thermal cycles). Typical values detected range from approximately 1,000 (in negative controls) to approximately 10,000 arbitrary units for positive samples, with an increase in the signal of approximately 10 times and therefore easily detectable.

MAIN AREAS OF APPLICATION

The proposed system, integrating the functions of: 1) cell lysis and DNA purification;

2) amplification by PCR; And

3) online fluorescence detection system,

it can be used in the diagnosis of pathologies caused by mutations. Specific pairs of primers located across the zone of the investigated gene due to the presence / absence of the mutation, a system of pairs of fluorescent probes designed to define the identity of the nucleotide in the position under examination, can be used in the miniaturized PCR reaction; the analysis of the fluorescence signal present after the gene amplification step will provide the identity of the nucleotide in question and therefore the presence or absence of the mutation.

The system lends itself well to the parallelization of the process; in this sense, by means of multiple amplification reactions, multiple individuals can be characterized for the same mutation or, vice versa, more mutations per subject can be tested at the same time.

The applications of the system in question are numerous and concern all hereditary pathologies caused by mutations of the genetic code (cystic fibrosis, hemochromatosis, deafness, Mediterranean anemia, chondroplasia, Huntington's Còrèa, etc.); the cost-effectiveness of the analysis, the simplicity with which the blood sample can be recovered from the patient, the speed of execution make the system usable in a hospital setting.

Furthermore, the possibility of identifying the nucleotide identity in a specific point of the genetic code makes the system useful for the genotyping of a certain number of individuals and therefore makes it possible to extrapolate information relating to the so-called pathologies with complex traits, i.e. those pathologies for which it is not valid. the axiom "a mutation = a pathology", but which rather originate from a sum of causes including lifestyle, environmental factors and not least genetic causes; specific patterns of polymorphisms could be predisposing for some of these pathologies (obesity, cardiovascular diseases, osteoporosis, tumors and others).

More generally, therefore, the device object of this patent would find use in the field of Predictive Tests (with regard to complex and common diseases) which identify situations of susceptibility or resistance to the diseases in question; this type of determination does not confer any certainty regarding the onset of the disease, rather it gives indications about an increased risk of developing it, in conjunction with triggering environmental factors.

Furthermore, it is understood that any application and field that envisages the use of a PCR reaction will be able to benefit from the system in question, in an economical and fast way. All areas that draw information from genotyping (molecular medicine, pharmacogenomics, nutrigenomics, personalized medicine) will be able to make use of the device covered by this patent, with the individual patient as the preferred user; in fact, the device is not designed for determinations on an unlimited number of patients, but, vice versa, to give advantage to personalized diagnosis.

Claims (9)

CLAIMS 1. Process for carrying out a diagnostic assay, comprising at least one step of extraction and / or purification of nucleic acid from a biological sample and a step of amplification and optionally detection of the nucleic acid, said steps being performed in a single device microfluidic positive, comprising at least one microfluidic chamber or channel having at least one surface in direct contact with the biological sample, capable of adsorbing the nucleic acid, characterized in that said at least one surface is a polydimethylsiloxane surface placed in direct contact with the biological sample, said surface being devoid of coatings and not being functionalized or modified prior to contact with the biological sample. 2. Process according to claim 1, characterized in that the nucleic acid amplification step is carried out on the nucleic acid adsorbed on said at least one adsorption surface without causing its desorption and in the absence of a passivation treatment of said at least one adsorption surface. 3. Process according to claims 1 or 2, characterized in that the extraction / purification stage and the nucleic acid amplification stage are carried out in the same chamber or microfluidic channel, without requiring the displacement of the nucleic acid from the adsorption surface to another surface of said chamber or channel. 4. Process according to any one of claims 1 to 3, characterized in that said nucleic acid extraction / purification step in said microfluidic device is carried out by injecting into said device a biological sample in a non-lysis solution. containing chaotropic salts or solvents and subjecting the injected sample to a heat treatment suitable for determining the cell lysis of the biological sample. 5. Process according to any one of claims 1 to 4, characterized in that said microfluidic device is a device integrally formed by a first and a second substrate, jointly defining a microfluidic channel, integrally formed by polydimethylsiloxane. 6. Process according to any one of claims 1 to 4, characterized in that said microfluidic device is formed by a first and a second substrate jointly defining a microfluidic channel where said first and second substrate are formed by a material selected from silicon, silicon-glass, metallic, ceramic or polymeric materials, having surfaces intended for contact with the biological sample, coated with polydimethylsiloxane. 7. Process according to claim 6, characterized in that said microfluidic device includes in said microfluidic chamber or channel auxiliary structures entirely constituted of polydimethylsiloxane or with surfaces coated with polydimethylsiloxane. 8. Process according to any one of the preceding claims, characterized in that said microfluidic device comprises a single chamber or microfluidic channel. 9. Use of unmodified polydimethylsiloxane (PDMS) as material for the adsorption of nucleic acids in microfluidic devices prepared for extraction / purification and / or amplification of nucleic acids assays.