WO2021014305A1 - Diagnostic device and its uses - Google Patents
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- WO2021014305A1 WO2021014305A1 PCT/IB2020/056735 IB2020056735W WO2021014305A1 WO 2021014305 A1 WO2021014305 A1 WO 2021014305A1 IB 2020056735 W IB2020056735 W IB 2020056735W WO 2021014305 A1 WO2021014305 A1 WO 2021014305A1
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Definitions
- This invention concerns an integrated device to perform different laboratory procedures, in particular of molecular and/or cellular biology.
- an integrated device is configurated for clinical practice and research applications, such as those generally intended primarily for:
- Genetics is the future of medicine: the so-called “tailored-medicine” or “targeted medicine” is mainly based on the identification of genetic variants, constitutional or acquired, of clinical relevance for patients.
- the presence or absence of a specific variant may confirm a diagnostic suspect, establish the individual risk of developing a given condition over time, define a prognosis and/or identify the more appropriate and effective treatment for the patient.
- molecular genetics tests based on the identification of the genome of specific pathogens, allow the rapid diagnosis of infection in conditions of suspected epidemics.
- ICSI in vitro fertilisation
- IVF in vitro fertilisation
- these methodologies of medically assisted fertilization are carried out in highly specialized laboratories.
- assisted fertilization is now also used in the veterinary/zootechnical field in which, especially in the case of valuable animals, cross-breedings are performed in laboratory.
- research laboratories are generally well-structured and with personnel adequately trained, there are some applications requiring repetitive operations, sometimes with experimental times not compatible with regular working hours.
- the automation of laboratory procedures allows to reduce the burden on staff, standardize procedures and results, and achieve reproducible scientific data at lower times and costs.
- Spartan rx (Spartan Bioscience Inc, Ottawa, Canada) is only marketed for analysis of the CYP2C19 gene; moreover it can only be used for cells obtained from buccal swab from which the DNA is not extracted and purified but subjected to direct amplification after cell lysis. This device is also not fully satisfactory as:
- Q-POC Quantum dx, Newcastle, UK
- the International Applications WO2007149791 and W02007100986 refer to automated systems for the purification of substances from biological samples, in particular RNA.
- the application WO2014144548 refers to a system and a method for rapid analysis, quantification and identification of nucleic acids or proteins.
- the application W0200403950 refers to a microfluidic system for the purification and/or analysis of a nucleic acid in a sample.
- the application W0200403950 refers to a device for simultaneously extracting and fractionating DNA from a lysate or a whole sample.
- the application US2011229897 refers to a device for extracting and analyzing DNA.
- Aim of the invention is to provide a composite device, conceived as a portable and automated laboratory, which allows to overcome the problems of traditional solutions.
- Another aim of the invention is to provide a device that allows to perform in vitro experimental procedures on cell cultures.
- Another aim of the invention is to provide with a composite and compact, portable, automatic device that produces reliable and reproducible results.
- Another aim of the invention is to provide with a device that provides easily interpretable results.
- Another aim of the invention is to provide with a device that provides molecular tests with high standardization.
- Another aim of the invention is to provide with a device that allows the analysis of information deriving from laboratory procedures also by non-expert operators.
- Another aim of the invention is to provide with a device that can be used for applications of genetics and molecular biology.
- Another aim of the invention is to provide with a device that can be used for applications of reproductive medicine.
- Another aim of the invention is to provide with a device that can be used for research applications.
- Another aim of the invention is to provide with a device that allows to quickly perform tests of molecular genetics.
- Another aim of the invention is to provide with a device that can be used in microgravity conditions. Another aim of the invention is to provide with a device that is easy to implement and at low costs. Another aim of the invention is to provide with a device that is alternative and/ or improved compared to the known devices.
- an apparatus for handling fluids (2) configured to allow the transfer of fluidic substances, comprising the sample to be analysed/treated and/or at least one reagent, between corresponding containers (4) and at least one microfluidic reactor (10), said microfluidic reactor (10) comprising at least one chamber (30) in which molecular and/or cellular biology reactions occur between said sample to be analysed and at least a reagent,
- heating and/or refrigerating unit (16) which is configured to control the thermal conditions in at least one chamber (30) of said reactor (10),
- an optical measuring apparatus (22) which is configured to perform optical measurements on the fluidic substances that enter and/or pass through said reactor (10),
- the Device (1) comprises means ( 18) for immobilizing the fluid substances, when they enter and pass through the reactor (10), said means comprising:
- an electromagnet (20) configured to generate a variable magnetic field
- At least one syringe (4) is of the precision type and is configured to pipette volumes up to 0.01 ml
- optical measuring apparatus (22) comprises:
- an excitation light source preferably at least one LED lamp and/or at least one laser source
- - includes inside a plurality of chambers (30) of circular or polygonal shape with beveled corners, said chambers (30) being fluidically connected, by means of cylindrical connecting ducts (32), and in that said inlet/outlet doors (34) are defined in correspondence with the edges of said reactor (10) and each of said doors (34) is connected by a connecting tube (6) of the manipulation apparatus (2) to a corresponding container (4) in which the sample to be analysed/treated and/or a reagent is present.
- cylindrical connecting ducts (32) are equipped with antireflux valves, both between them and to inlet/outlet doors (34) to/from said reactor (10).
- said reactor ( 10) comprises a plurality of fertilization chambers (601) fluidly connected in series through at least one connecting duct (32) and in that:
- said reactor (10) comprises at least one culture chamber (701) to which are connected:
- said reactor ( 10) comprises at least two analytical chambers (801, 802, 803) fluidically independent from each other, to each analytical chamber (801, 802, 803) being connected:
- Another object of the invention is the use of the device as above defined to:
- a) perform analysis of gene variants, for example aimed to the diagnosis/prognosis or epidemiology, or quantification/qualification of gene transcripts, where said analysis include the extraction phases from one or more isolated human or animal biological samples, purification and amplification of nucleic acid and where said phases are performed inside the microfluidic reactor (10); or
- Another object of the invention is the use of the device as above defined to perform in vitro fertilization procedures.
- Figure 1 show in schematic view the device according to the invention
- Figure 12 shows in schematic perspective view the reactor according to the invention in a fourth embodiment, in solid
- the fluid handling apparatus 2 is advantageously derived from the device described in patent US 8,669,096, the content of which is intended here fully incorporated as a reference. Compared to the device described in US 8,669,096, in a preferred form of the present invention, the extraction and purification chambers containing nucleic acid binding membranes have been eliminated.
- syringes 4 can be arranged parallel to each other in order to reduce the size of the device 1.
- the heating and/or cooling unit 16 allows constant and controlled temperature incubation but also repeated temperature cycles suitable for DNA amplification by polymerase chain reaction (PCR).
- the heating unit 16 can be made up of a block of aluminium which temperature variation can be controlled by a Peltier system or other known temperature control systems that allow a ramping rate of at least rc/sec.
- the heating and/or cooling unit 16 can be manually controlled or, preferably, is controlled by a control and operating unit (for example a microprocessor), which for example can be the same unit 14 that controls the manipulation apparatus 2.
- Device 1 also preferably includes tools 18 for specifically immobilizing analytes of interest, in particular present in the sample, when they enter and pass through the reactor 10. Immobilization equipment 18 shall be properly used to prevent loss of analytes during the washing phase.
- the signal recorded by detector 26 can be converted to an analytical data, qualitative or quantitative. Properly the conversion, as well as the control of the excitation light sources 24 may be carried out manually, or preferably controlled by a control and operating unit. For example, it can be the same unit 14 that controls the handling apparatus 2 and/or the heating and/or cooling unit 16 and/or the electromagnet 20.
- reactor 10 is made of optical and bio-compatible plastic material.
- reactor 10 is small in size, for example about 56.5 x 35 x 2.5-4 mm.
- Rooms 30 may be circular or polygonal in shape, preferably with rounded corners, to facilitate fluid dynamics and avoid the stagnation of reagents.
- chambers 30 may have a rhomboidal shape with rounded corners.
- entry/exit doors 34 for connecting pipes 32.
- a first embodiment may be used for: -the identification of variants with diagnostic/prognostic/epidemiological significance;
- nucleic acids DNA or RNA
- biological samples such as blood, plasma, tissues
- RT-PCR and the amplification of specific DNA/cDNA regions in a fully automatized mode occur inside the reactor 10.
- channels 512, 513, 514, 515 for entering reagents into chamber 301 are advantageously equipped with anti-reflux valves and are located on the opposite ends of duct 32, which connect the extraction chamber 301 to the first reaction chamber 501.
- the reactor 10 has the form of a parallelepiped with, for example, external sizes of about 56.5 mm (width), 35 mm (depth) and 4 mm (height), and shall consist of at least one (preferably three) fertilization chambers 601, possibly connected by a series of ducts 32.
- Each room 601 has a central portion 602 which is appropriately depressed compared to the remaining part of chamber 601.
- the portion 602 is cylindrical (preferably with 1.6 mm of height of and 9 mm of diameter, containing a volume of approximately 125 mI), and is the part where the gametes are laid.
- Each fertilization chamber 601 is equipped with an additional duct 630 for gamete injection.
- each additional duct 630 is connected to a corresponding portion 602.
- this additional duct 630 is closed and sealed before the lodging of reactor 10 in device 1.
- a fourth channel 603 (which is connected to a first fertilization chamber 601) for the input of the culture medium and a fifth efflux channel 604 which is connected to another fertilization chamber 601.
- the reactor 10 is a parallelepiped of external sizes of about 56.5 mm (width), 35 mm (depth) and 3.5 mm (height). According to this embodiment, it includes one or more culture chambers 701 not communicating with the others. The number of culture chambers 701 varies according to the number of experimental conditions which are simultaneously analysed.
- Figures 10 and 11 show the embodiment with three culture chambers 701.
- the culture chambers 701 have preferably form of prism with rhomboidal base and rounded sides and this in order to facilitate fluid dynamics and avoid liquid stagnation near the corners.
- culture chambers 701 have a base of about 25x15 mm and a height of about 2 mm, containing a volume of about 750 pi.
- - Syringe n. 2 lysis buffer, connected through channel 513 to the extraction chamber 301,
- the electromagnet 20 is activated and the sample is incubated for 5 minutes at room temperature.
- Syringe No 11 IX reaction mix (Taqman ® Fast Virus 1-Step Master Mix, Thermofisher Scientific, P/N: 444432); 400 nm of BCR-ABLela2-F and BCR-ABL-R primers; 100 nm of the BCR-ABLela2-P probe; FI20 by volume. Connected to channel 518 of second reaction chamber 502,
- Syringe No 2 empty, connected through channel 604 to the reactor. 2) The oocytes are manually inserted (one per chamber) into the fertilisation chambers 601 through the 630 ducts, directly connected to the 602 cylindrical depression.
- Phases 2) and 3) should be monitored by inverted light microscope observation.
- the fertilisation chambers are analysed under the inverted light microscope and those in which fertilisation and embryo growth have occured are identified.
- Example 5 Determination of SMN protein levels compared to Glyceraldehyde -3-Phosphate-
- - Syringe No 5 anti-SMN (e.g., Abeam ab232784, polyclonal antibody produced in rabbit) and anti-GAPDH (e.g., Sigma-Aldrich SAB2500450, polyclonal antibody produced in goat), diluted 1/1000 in Dulbecco's modified Phosphate Buffer (PBS)/BSA 1%, anti-rabbit conjugated with FITC and anti-goat conjugated with Alexa-fluor, diluted 1/1000; the syringe No 5 is connected via channels 813, 817 and 821 to the 801, 802, 803 reactor chambers in the fourth form of implementation,
- PBS Dulbecco's modified Phosphate Buffer
- APOE-C.364-F 5'-GGGCGCGGACATGGA-3' (SEQ I D NO: 1)
- APOE-C.517-T 5'-FAM-TGCAGAAGTGCCTGGC-MGB-3'(SEQ ID NO:8)
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Abstract
The present invention refers to an integrated and automated device (1) for carrying out molecular and/or cellular biology laboratory procedures, characterized in that it comprises: - an apparatus for handling fluids (2) configured to allow the transfer of fluidic substances, comprising the sample to be analysed/treated and/or at least one reagent, between corresponding containers (4) and at least one microfluidic reactor (10), said microfluidic reactor (10) comprising at least one chamber (30) in which molecular and/or cellular biology reactions occur between said sample to be analysed and at least a reagent, - a heating and/or refrigerating unit (16) which is configured to control the thermal conditions in at least one chamber (30) of said reactor (10), - a pressure applicable/lockable lid (21) of an optically indifferent material, configured to superiorly cover the reactor (10), which prevents the deformation of said reactor as a result of temperature variations and allows the detection of the fluorescent signal, without phenomena of light refraction or diffraction, - an optical measuring apparatus (22) which is configured to perform optical measurements on the fluidic substances that enter and/or pass through said reactor (10), - a command and control unit (14) configured to control and/or coordinate the fluid handling apparatus (2), the heating and/or refrigerating unit (16), and/or said optical measuring apparatus (22).
Description
DIAGNOSTIC DEVICE AND ITS USES
FIELD OF THE INVENTION
This invention concerns an integrated device to perform different laboratory procedures, in particular of molecular and/or cellular biology.
In particular, according to the present invention, an integrated device is configurated for clinical practice and research applications, such as those generally intended primarily for:
Health professionals in first level hospitals/laboratories (outpatient clinics, hospitals) located in developed or developing regions;
Health professionals in level second or third level hospital/laboratories who need to perform a limited number of tests with quick responses;
Personnel involved in medium and long-term space missions or missions in remote or difficult to reach areas on Earth.
These applications can be substantially divided into two groups, those in molecular and cellular biology, for nucleic acids, protein or cellular cultures analysis, and those in reproductive medicine.
BACKGROUND OF THE INVENTION AND STATE OF ART
Genetics is the future of medicine: the so-called "tailored-medicine" or "targeted medicine" is mainly based on the identification of genetic variants, constitutional or acquired, of clinical relevance for patients. The presence or absence of a specific variant may confirm a diagnostic suspect, establish the individual risk of developing a given condition over time, define a prognosis and/or identify the more appropriate and effective treatment for the patient. Moreover, molecular genetics tests based on the identification of the genome of specific pathogens, allow the rapid diagnosis of infection in conditions of suspected epidemics.
In the present scenario, access to molecular genetic tests is limited by the relatively high costs/sample, the long turnaround time, the need of highly qualified personnel in highly specialized laboratories, and result interpretation issues. For these reasons, in several Countries laboratory diagnostic facilities have a pyramidal structure: the vast majority of molecular test are conveyed to and performed in second or third level laboratories, usually large hospitals or university centers. However this system strongly penalizes patients of the first level centers, especially those located in Developing Countries or, more in general, in remote areas.
Further needs, arising in the medium term, although initially limited to a few users, will be related to space journeys of medium or long duration, in preparation for human missions to the Moon or Mars, now scheduled by all international Space Agencies. Although comparable to that of remote areas of the Earth,
this scenario is further complicated by microgravity, tight room, lack of medical and laboratory staff, and emerging health problems, still unknown nowadays. Differently from the current scenario on the Ground, where one of the major morbidity sources (i.e. the number of cases of disease recorded during a given period in relation to the total number of people examined) are environmental pathogens, It can be expected that during space travel, prolonged exposure to cosmic radiation will be among the major causes of disease for astronauts, resulting in increased risk of neoplastic diseases.
Currently, the routine clinical application of genetic tests is limited and prevented by some drawbacks:
Molecular genetics laboratories currently tend to the extreme automation which, to be cost- effective, requires huge number of samples, multiple manual interventions of qualified personnel, in addition to the presence of numerous devices, each dedicated to a part of the experimental process, with relatively high costs;
The results of genetic testing are difficult to interpret: in particular, although the current diagnostic methods allow to identify a very large number of gene variants, these often have no impact on the clinical practice, mainly because of the lack of data in the scientific literature. To date, for example, although there are several mutations identified in cancer, only a few are recognized by the EMA or the FDA as 'druggable';
The medium-long turnaround-time of genetic tests often limits their routine application in patients' clinical management protocols. For example, in some hematological diseases therapeutic choices may be oriented by the detection of minimal signs of disease, which can only be diagnosed by molecular tests. There are also clinical scenarios where the molecular diagnosis is even an urgency/emergency: it is the case of the identification of some pathogens, in which the prompt isolation of the patient and/or the immediate initiation of therapy may be crucial.
Regarding the reproductive medicine application, it should be noted that couple infertility is a common problem in both developed and developing countries, with an overall prevalence of about 10%, even if it is due to different causes. In developed countries, the increase in the average reproductive age and lifestyles make male causes of infertility relatively more frequent. In developing countries, where the average reproductive age is lower, the female causes of infertility are more common, especially the obstructive ones, such as tubal tuberculosis or sexually transmitted infections (Stevenson et al., 2016; Benagiano et al., 2006). In the first scenario, the main method for medically assisted fertilization is the ICSI (Intra Cytoplasmic Sperm Injection), which execution requires well-equipped laboratories and highly specialized personnel. In the second scenario, ICSI can be replaced by in vitro fertilisation (IVF), for which it is sufficient to put male and female gametes in contact, with a suitable culture medium and under sterile conditions. To date, these methodologies of medically assisted fertilization are carried out in highly specialized laboratories. Moreover, assisted fertilization is now also used in the veterinary/zootechnical field in which, especially in the case of valuable animals, cross-breedings are performed in laboratory.
In the field of research, although research laboratories are generally well-structured and with personnel adequately trained, there are some applications requiring repetitive operations, sometimes with experimental times not compatible with regular working hours. In these cases, the automation of laboratory procedures allows to reduce the burden on staff, standardize procedures and results, and achieve reproducible scientific data at lower times and costs.
In this context, the "Point Of Care" (POC) devices are a new category of available instruments; these are diagnostic laboratory instruments for first level centers, which have a high degree of operational simplicity and do not require special skills from the operator. POCs for routine biochemical tests (such as blood sugar, cholesterolemia, etc.) are now quite common. Genetic tests have much higher levels of complexity, both in terms of experimental procedures, with consequent automation difficulties, and of operator's manual skills. The category of devices used for this type of application could be called Genetic-Point Of Care" (G-POC). To date, only very few G-POCs are commercially available and are intended for a limited number of applications. All G-POCs share the use of DNA amplification by PCR and the signal detection by fluorescent oligonucleotide probes/primers. This approach is the basis of almost the whole molecular genetics, regardless of the portability and/or the degree of automation of the device.
Among the devices on the market there is, for example, Revogene (GenePoc, Quebec, Canada), an instrument intended solely for the identification of two bacteria (C. difficile and S. agalactiae) by application of real time PCR. However, this device is not fully satisfactory as:
- allows the extraction of genomic DNA only from bacterial cells (and not from samples of eukaryotic cells/tissues),
- does not allow the identification of single nucleotide variants,
- cannot be used for applications other than DNA amplification in PCR.
Cepheid Xpert (Cepheid, California, USA) has been on the market with different configurations (based on the number of samples) for several years; the only applications are DNA and RNA extraction and DNA amplification.
Spartan rx (Spartan Bioscience Inc, Ottawa, Canada) is only marketed for analysis of the CYP2C19 gene; moreover it can only be used for cells obtained from buccal swab from which the DNA is not extracted and purified but subjected to direct amplification after cell lysis. This device is also not fully satisfactory as:
It is not usable for diverse tissues, namely whole blood, due to the common presence of DNA polymerase inhibitors
The lack of purification of the extracted DNA reduces its amplification efficiency, the standardization of the results and prevents its use for quantitative approaches,
It is not suitable for applications other than DNA amplification.
Q-POC (Quantum dx, Newcastle, UK) has been in development for several years and is not yet on the market. It is based on a microfluidic semiconductor system. However, even this device is not fully satisfactory as the use of lyophilized reagents and the fluid circulation system in micro-cartridges limit the application of the instrument only to the analysis of nucleic acids; it is also little versatile because it can perform only few genetic tests, developed by the producers.
Gene Radar (Nanobiosym, Massachusetts, USA) is a microfluidic-based device that allows to perform capillary electrophoresis on consumable devices. However, even this device is not fully satisfactory as it is not designed for the various applications of cell biology and research that can be obtained with the present invention.
There are also several patent applications for genetic analysis devices. For example, the International Applications WO2007149791 and W02007100986 refer to automated systems for the purification of substances from biological samples, in particular RNA. The application WO2014144548 refers to a system and a method for rapid analysis, quantification and identification of nucleic acids or proteins. The application W0200403950 refers to a microfluidic system for the purification and/or analysis of a nucleic acid in a sample. The application W0200403950 refers to a device for simultaneously extracting and fractionating DNA from a lysate or a whole sample. The application US2011229897 refers to a device for extracting and analyzing DNA.
None of the above documents describes a device that allows to obtain the various applications of cellular biology and research obtainable with the present invention.
DESCRIPTION OF THE INVENTION
Aim of the invention is to provide a composite device, conceived as a portable and automated laboratory, which allows to overcome the problems of traditional solutions.
Another aim of the invention is to provide a device where all the chemical/biochemical reactions of the experimental process occurs.
Another aim of the invention is to provide a device that allows to perform biology and molecular genetics experiments/analyses, based on analysis of both nuclear acids and proteins.
Another aim of the invention is to provide a device that allows to perform in vitro experimental procedures on cell cultures.
Another aim of the invention is to provide a device that allows to perform human or animal embryo cultures, for medically assisted reproduction methods such as IVF.
Another aim of the invention is to provide small Health centers, also located in deprived areas, with a device that meets the growing demand of autonomy in performing genetic tests with diagnostic, prognostic or therapeutic significance in the context of tailored medicine applications.
Another aim of the invention is to provide small Health centers, also located in deprived areas, with a device that meets the growing demand of autonomy in the identification of pathogen genome on biological samples of patients.
Another aim of the invention is to provide with a device for zoological applications for studies of genetic epidemiology in insects.
Another aim of the invention is to provide with a composite and compact, portable, automatic device that produces reliable and reproducible results.
Another aim of the invention is to provide with a device easily manageable even by unskilled personnel.
Another aim of the invention is to provide with a device that provides easily interpretable results.
Another aim of the invention is to provide with a device that provides molecular tests with high standardization.
Another aim of the invention is to provide with a device able to carry out assisted reproduction technology, without the need of traditional infrastructures, both in terms of human resources and equipment.
Another aim of the invention is to provide with a device that is suitable for use in laboratories not particularly structured or in areas that are geographically distant from the most specialized centers.
Another aim of the invention is to provide with a device that allows the analysis of information deriving from laboratory procedures also by non-expert operators.
Another aim of the invention is to provide with a device that can be used for applications of genetics and molecular biology.
Another aim of the invention is to provide with a device that can be used for applications of reproductive medicine.
Another aim of the invention is to provide with a device that can be used for research applications.
Another aim of the invention is to provide with a device that allows to quickly perform tests of molecular genetics.
Another aim of the invention is to provide with a device that can be used in microgravity conditions. Another aim of the invention is to provide with a device that is easy to implement and at low costs.
Another aim of the invention is to provide with a device that is alternative and/ or improved compared to the known devices.
All these aims, individually taken or in any combination, in addition to others aims resulting from the following description, are reached through a device with the characteristics indicated in claim 1, according to the invention. 1.
It is an object of the present invention and integrated and automated device (1) for carrying out molecular and/or cellular biology laboratory procedures, characterized in that it comprises:
- an apparatus for handling fluids (2) configured to allow the transfer of fluidic substances, comprising the sample to be analysed/treated and/or at least one reagent, between corresponding containers (4) and at least one microfluidic reactor (10), said microfluidic reactor (10) comprising at least one chamber (30) in which molecular and/or cellular biology reactions occur between said sample to be analysed and at least a reagent,
- a heating and/or refrigerating unit (16) which is configured to control the thermal conditions in at least one chamber (30) of said reactor (10),
- a pressure applicable/lockable lid (21) of an optically indifferent material, configured to superiorly cover the reactor (10), which prevents the deformation of said reactor as a result of temperature variations and allows the detection of the fluorescent signal, without phenomena of light refraction or diffraction,
- an optical measuring apparatus (22) which is configured to perform optical measurements on the fluidic substances that enter and/or pass through said reactor (10),
- a command and control unit (14) configured to control and/or coordinate the fluid handling apparatus (2), the heating and/or refrigerating unit (16), and/or said optical measuring apparatus (22).
Preferably the Device (1) comprises means ( 18) for immobilizing the fluid substances, when they enter and pass through the reactor (10), said means comprising:
- an electromagnet (20) configured to generate a variable magnetic field,
- a plurality of paramagnetic beads configured to specifically bind with some substances present inside the reactor (10).
Preferably said containers (4) comprise at least two syringes (4) and in that:
- said syringes (4) are disposed in parallel to each other, e
- at least one syringe (4) is of the precision type and is configured to pipette volumes up to 0.01 ml,
- said syringes (4) comprise a plunger (8) which is actuated in an automated and controlled manner and
- the plunger (8) of the syringes (4) is actuated by a telescopic piston (12) which is part of the manipulation apparatus (2).
Preferably said optical measuring apparatus (22) comprises:
- an excitation light source (24), preferably at least one LED lamp and/or at least one laser source,
- a detector (26) placed above said reactor (10),
- configured filters to isolate the emission wavelengths of the single fluorochromes present inside said reactor (10).
Preferably said reactor (10):
- has a substantially laminar conformation,
- is of a single-use type,
- is of optical and bio-compatible plastic material
- includes inside a plurality of chambers (30) of circular or polygonal shape with beveled corners, said chambers (30) being fluidically connected, by means of cylindrical connecting ducts (32), and in that said inlet/outlet doors (34) are defined in correspondence with the edges of said reactor (10) and each of said doors (34) is connected by a connecting tube (6) of the manipulation apparatus (2) to a corresponding container (4) in which the sample to be analysed/treated and/or a reagent is present.
Preferably the cylindrical connecting ducts (32) are equipped with antireflux valves, both between them and to inlet/outlet doors (34) to/from said reactor (10).
Preferably said reactor (10) comprises:
- an extraction chamber (301) for m ixing said fluidic substances, said extraction chamber (301) being fluidically connected to:
• at least one first bidirectional channel (511, 516) for the entry/exit into/from said extraction chamber (301) of the sample to be treated/analyzed,
• at least one second channel (512,513,514,515) for introducing the reactants into said extraction chamber (301),
- at least one reaction chamber (501, 502, 503) which is in fluid connection with said extraction chamber (301) and/or with any other reaction chambers (501, 502, 503), to each reaction chamber being fluidically connected at least one third channel (517, 518, 519) for the input of further biological sample and/or reagents within the corresponding reaction chamber (501, 502, 503).
Preferably said reactor (10) comprises at least one fertilization chamber (601) configured to contain the embryos to be cultivated in a depressed portion (602) with respect to the rest of the chamber
(601), to said fertilization chamber (601) being fluidically connected :
- at least one further conduit (630) for the deposit of gametes,
- at least one channel (603) for the input of the culture medium,
- at least one efflux channel (604) of the culture medium.
Preferably said reactor ( 10) comprises a plurality of fertilization chambers (601) fluidly connected in series through at least one connecting duct (32) and in that:
- said at least one channel (603) for the introduction of the culture medium is connected to the fertilization chamber (601) further upstream,
- said efflux channel (604) is connected to the fertilization chamber (601) further downstream. Preferably said reactor (10) comprises at least one culture chamber (701) to which are connected:
- at least one access channel (703) for cell deposition,
- at least one inflow channel (704) of the culture medium, and
- at least one efflux channel (714) of the culture medium.
Preferably said reactor ( 10) comprises at least two analytical chambers (801, 802, 803) fluidically independent from each other, to each analytical chamber (801, 802, 803) being connected:
- at least one corresponding channel (804, 805, 806) for loading the biological sample into said chamber,
- a plurality of channels (811-822) for the introduction of culture media and/or reagents.
- at least one corresponding channel (823, 824, 825) for the reagents outflow.
Another object of the invention is the use of the device as above defined to:
a) perform analysis of gene variants, for example aimed to the diagnosis/prognosis or epidemiology, or quantification/qualification of gene transcripts, where said analysis include the extraction phases from one or more isolated human or animal biological samples, purification and amplification of nucleic acid and where said phases are performed inside the microfluidic reactor (10); or
b) identify the presence of the genome of pathogen agents in human or animal biological samples, wherein said analysis include the extraction phases from one or more isolated biological samples, purification and amplification of nucleic acids and wherein said phases are performed inside the microfluidic reactor (10).
Another object of the invention is the use of the device as above defined to perform in vitro fertilization procedures.
Another object of the invention is the use of the device as above defined to cultivate adherent cells in controlled conditions and/or to measure over time specific biological/biochemical parameters of the cells cultivated in the microfluidic reactor (10) and/or to evaluate over time the effect of pharmacological treatments in adherent cells cultivated in vitro in the microfluidic reactor ( 10). Another object of the invention is the use of the device as above defined to purify and quantify proteins extracted from biological samples entered into the microfluidic reactor (10) or from cells cultivated in vitro in the microfluidic reactor (10).
In the present device or microfluidic reactor, means and/or chambers for capillary electrophoresis are preferably not present. Therefore, preferably the molecular and/or cellular biology reactions do not comprise capillary electrophoresis.
The present device or microfluidic reactor preferably comprises gene specific primers and/or probes, preferably fluorescent.
The present device or microfluidic reactor preferably comprises anti-reflux valves.
FIGURE DESCRIPTION
The present invention is further defined below in some of its preferred embodiments, described below as examples and not limiting purposes, with reference to the attached drawings tables, where:
Figure 1 show in schematic view the device according to the invention,
Figure 2 shows in perspective view the device according to the invention without the pipes connecting the syringes to the reactor,
Figure 3 shows the device of Fig. 2 in exploded perspective view,
Figure 4 shows in top view the Fig. 2 device,
Figure 5 shows in schematic perspective view the reactor of the device according to the invention in a first embodiment, in solid,
Figure 6 shows the schematic side view of the reactor in Fig. 5,
Figure 7 shows in perspective schematic perspective a horizontal section of the reactor of the device according to the invention in a second embodiment
Figure 8 shows in perspective side view the realization of the reactor of Fig. 7, in solid,
Figure 9 shows in perspective schematic view the construction of the reactor of Fig. 7, in solid,
Figure 10 shows schematic perspective view of the reactor according to the invention in a third embodiment, in solid,
Figure 11 shows in schematic perspective view the construction of the reactor in Fig. 10,
Figure 12 shows in schematic perspective view the reactor according to the invention in a fourth embodiment, in solid,
Figure 13 shows the schematic side view of the reactor in Fig. 12,
Figure 14 shows a schematic representation of the molecular strategy used for the determination of the APOE genotype. The reactor used is that in fig. 5. DNA is extracted in the extraction chamber 301 and subsequently amplified and analyzed in reaction chambers 501 and 503. The expected result is
schematized according to the observed genotype. The colour of the obtained fluorescence varies according to the presence of the specific alleles.
Figure 15 shows a schematic representation of the molecular strategy used for the determination of the presence of BCR-ABL fusion transcripts. The reactor used is the one in fig. 5. The total RNA is extracted from peripheral blood samples in the extraction chamber 301 of the reactor and subsequently amplified and analyzed in the three reaction chambers 501, 502 and 503. The expected result is schematized according to the chimeric gene identified. The colour and intensity of the obtained fluorescence may vary according to the rearrangement in the sample and its relative quantity, respectively.
Figure 16 shows a schematic representation of the molecular strategy used for the determination of SMN protein levels in FleLa cells treated with salbutamol. The reactor used is the one in Fig. 12. A) After total protein extraction, SMN and GAPDFI are immobilized by binding to a specific antibody, bound to paramagnetic beads. B) Protein detection occur through specific polyclonal antibodies, produced in different species, which are recognized by fluorescent antibodies directed against the Fc fragment of immunoglobulins. C) The amount of protein is determined on the basis of the fluorescence emitted by the two secondary antibodies (represented with grey tones), measured by the device. The variation of SMN protein levels is determined in relation to those of the control protein (GAPDFI).
DETAILED DESCRIPTION OF THE INVENTION
As from figures, device 1 includes a handling apparatus 2 configured to transfer fluids between corresponding containers 4 - preferably defined by a plurality of syringes 4 - and a reactor 10.
In particular, fluid-transferred substances may include the testing sample and/or air and/or appropriate reagents, either individually or in a suitable mixture. In addition, fluid-transferred substances may be in the liquid or gaseous state, or in a mixture thereof, as appropriate.
The fluid handling apparatus 2 is advantageously derived from the device described in patent US 8,669,096, the content of which is intended here fully incorporated as a reference. Compared to the device described in US 8,669,096, in a preferred form of the present invention, the extraction and purification chambers containing nucleic acid binding membranes have been eliminated.
The preferred features of this device, which differentiate it from that described in US patent 8,669,096, are shown below. In particular, apparatus 2 includes a plurality of containers 4, and preferably of syringes 4, even of different capacity and placed in suitable slots 5 mounted on a common basement 11.
Properly, apparatus 2 also includes a number of connecting tubes 6 configured to collect the fluids from the end of each syringe 4 and to transfer them to a reactor 10. Each tube 6 is properly welded at a first end at the outlet of a corresponding syringe 4 and, at the other end, is welded to reactor 10, so that the transfer of fluids takes place without leakage.
Properly, each syringe 4 and the corresponding slot 5 can be numbered progressively. In addition, each syringe 4 can be conveniently equipped with a QR-code for the recognition of the content and of the specific kit, which is located in the device 1, as well as to avoid incorrect placement of syringes 4 by the user.
Properly, syringes 4 can be arranged parallel to each other in order to reduce the size of the device 1.
Conveniently, one or more of the syringes 4 can be of precision type, which is configured to adjust volumes up to 0.01 ml, in order to allow the addition of precise volumes of reagents.
Conveniently, the piston 8 of the syringes 4 is implemented by a telescopic piston 12 part of the manipulation apparatus 2 and, properly, connected to a control and operating unit 14, which allows automated command and control.
Conveniently, both syringes 4 and tubes 6 can be made of materials resistant to heat and chemical compounds such as alcohols, ethers, acids and bases commonly used in the biological and/or chemical field; in addition, these materials are preferably non-toxic in order to avoid reactions in the inserted biological samples and, for example, can be propylene medical in the formulation Purell HP548N®.
Device 1 also includes a heating and/or cooling unit 16 configured to control the reactor temperature 10.
Properly, the heating and/or cooling unit 16 can be complementary to the shape of reactor 10, in order to host it at least partially, to obtain an adequate heat exchange. In particular, the heating and/or cooling unit 16 can provide a hollowed slot, substantially complementary to that of reactor 10.
The heating and/or cooling unit 16 allows constant and controlled temperature incubation but also repeated temperature cycles suitable for DNA amplification by polymerase chain reaction (PCR). Properly, the heating unit 16 can be made up of a block of aluminium which temperature variation can be controlled by a Peltier system or other known temperature control systems that allow a ramping rate of at least rc/sec. Properly, the heating and/or cooling unit 16 can be manually controlled or, preferably, is controlled by a control and operating unit (for example a microprocessor), which for example can be the same unit 14 that controls the manipulation apparatus 2.
To avoid deformation of the reactor as a result of heating, the device 1 is equipped with a lid 21 configured to cover the reactor 10 from above. In particular, the lid 21 can be applied/placed by pressure and is made of transparent material, for example plexiglas. Suitably, the material of the lid 21 shows characteristics of optical indifference, to prevent refractive phenomena or light diffraction during the fluorescence detection.
Preferably, the device 1 includes a cover 21 that can be applied/placed by pressure, made of an optically neutral material, configured to cover the reactor at the top 10, which prevents the reactor from deflecting
as a result of temperature changes and allows the fluorescent signal to be detected, without refraction or light diffraction phenomena.
Device 1 also preferably includes tools 18 for specifically immobilizing analytes of interest, in particular present in the sample, when they enter and pass through the reactor 10. Immobilization equipment 18 shall be properly used to prevent loss of analytes during the washing phase.
Advantageously, the tools for immobilization 18 include an electromagnet 20 and a plurality of paramagnetic beads, configured for binding specifically analytes of interest and/or reagents. Unlike a permanent magnet, electromagnet 20 generates a magnetic field by activation only when required. The orientation of the magnetic field can, for example, be oscillating in order to allow the movement of the paramagnetic beads and thus mix the fluid substances present in reactor 10. This fluctuation is obtained by alternating the electric field that generates the magnetic field. Properly, the electromagnet 20 can be controlled manually or, preferably, by a control and operating unit, which for example can be the same unit 14 that controls the handling apparatus 2 and/or the heating and/or cooling unit 16.
Properly, reactor 10, with heating and/or cooling unit 16 and immobilization equipment 18, are located in a structure 13 mounted on a basement 11.
Device 1 also includes an optical measuring apparatus 22, configured to carry out optical measurements on fluids present inside the reactor 10. In particular, the optical measuring apparatus 22 is configured to perform fluorescence measurements.
The optical measuring apparatus 22 includes:
- an excitation light source 24, preferably composed of one or more LED lamps and/or one or more laser sources,
- a detector 26, preferably a diode or alternatively a CCD chamber, and
- filters configured to isolate the emission wavelengths of individual fluorochromes, used in experimental procedures.
Properly, detector 26 can be located above reactor 10 so that the excitation and the measurement of the emitted light take place in a geometry that involves the reflection of the light radiation. Detector 26 measures and records the light produced by fluid substances - in particular of the sample - contained inside the reactor 10, for example by fluorescence, during different experimental procedures. Properly, the fluorescence emission at an appropriate wavelength can be used to monitor over time a reaction of interest.
The signal recorded by detector 26 can be converted to an analytical data, qualitative or quantitative. Properly the conversion, as well as the control of the excitation light sources 24 may be carried out manually, or preferably controlled by a control and operating unit. For example, it can be the same unit
14 that controls the handling apparatus 2 and/or the heating and/or cooling unit 16 and/or the electromagnet 20.
Device 1 also includes the control and operating unit 14 configured to control and coordinate the various components 2, 16, 20, 22 of the device. Properly, unit 14 can include and/or be associated with a memory unit so that the detected data can be saved and/or elaborated.
Properly, Unit 14 can include a programmed processor to control and coordinate the various components and, advantageously, to process the detected data. More in details, in the control and operating unit 14 a software unit that allows the control of the different components 2, 16, 20, 22 is properly loaded. For example, the software module is able to define the sequence and timing of activation/deactivation of the various components 2, 16, 20, and 22 of the device 1 and, properly, the accurate sequential injection of the appropriate volumes of the different reagents contained in the different syringes 4. The control and operating unit 14 is also configured to collect and convert raw data into analytical data, easy to decode even for unskilled operators.
In addition, the control and operating unit 14 may include communication equipment to send the collected and/or processed data to another device. The communication equipment may include broadcast facilities such as wireless, even of short-range, or cable.
Preferably, the control and operating unit 14 includes and/or is associated with a user interface of simple and intuitive use even for non-specialist personnel. Preferably, this user interface consists of a touch screen display or a keyboard associated display or other input facilities.
As mentioned above, device 1 includes a microfluidic reactor 10 which constitutes the enclosure within which the different chemical/biochemical reactions take place.
Advantageously, reactor 10 is disposable. Preferably, the reactor 10 is made of plastic material, such as medical polypropylene. Properly, reactor 10 can be made of the same material as syringes 4 and/or tubes 6.
Advantageously, reactor 10 is made of optical and bio-compatible plastic material.
Properly, reactor 10 is small in size, for example about 56.5 x 35 x 2.5-4 mm.
Reactor 10 comprises of one or more chambers 30 connected by pipes 32, preferably cylindrical. In particular, these pipes 32 define the flow and outflow of fluids to/from chambers 30. In particular, pipes 32 are used by the fluid samples under analysis/treatment, which may be added with appropriate reagents, and/or by reaction rejects and/or gases (in particular air) and/or other substances.
Properly, reactor 10 consists of a parallelepipeds body 29 (block), laminar in shape (length and width greater than the thickness), in which are obtained cavities, defining the chambers 30, and the hollow sections that connect these cavities and define these connecting pipes 32.
Rooms 30 may be circular or polygonal in shape, preferably with rounded corners, to facilitate fluid dynamics and avoid the stagnation of reagents. For example, chambers 30 may have a rhomboidal shape with rounded corners.
Suitably, at the perimeter walls of body 29, are provided entry/exit doors 34 for connecting pipes 32.
In addition, at connecting ducts 32 anti-reflux valves are provided for both to avoid reagent backflow and to avoid contamination of stock solutions.
Properly, reactor 10 may have different forms of implementation depending on the reaction to be analyzed.
The device as above defined can be used: to carry out analyses of gene variants, for example for the purpose of diagnosis/prognosis or epidemiology,
- to quantify/qualify gene transcripts, where those analyses include extraction from one or more human or animal isolated biological samples, purification and amplification of nucleic acids and where these phases are carried out inside the microfluidic reactor 10;
- to identify the presence of the genome of pathogens in human or animal biological samples, where such analyses include extraction from one or more isolated biological samples, purification and amplification of nucleic acids and where these phases are carried out within the microfluidic reactor 10; or to carry out in vitro fertilisation procedures; or to cultivate adherent cells under controlled conditions and/or to measure specific biological/biochemical parameters of cells grown in the microfluidic reactor 10 over time and/or to evaluate the effect of pharmacological treatments in adhering cells cultured in vitro in the microfluidic reactor 10 over time; or to purify and quantify proteins extracted from biological samples entered into the microfluidic reactor 10 or from cells cultured in vitro in the microfluidic reactor 10.
First form of implementation
A first embodiment (see Fig. 5, 6) may be used for:
-the identification of variants with diagnostic/prognostic/epidemiological significance;
-the qualification and/or quantification of gene transcripts;
-the identification of pathogen genomes, in human or animal biological samples.
Properly, the extraction of nucleic acids (DNA or RNA) from different biological samples (such as blood, plasma, tissues), and possibly the RT-PCR and the amplification of specific DNA/cDNA regions in a fully automatized mode, occur inside the reactor 10.
In this first embodiment, the reactor 10 has the shape of a parallelepiped with rounded vertices and external sizes of about 56.5 mm (width), 35 mm (depth) and 3.5 mm (height).
In the context of the present invention the extraction chamber is configured for mixing said fluidic substances in order to carry out nucleic acid extraction.
In this particular case, the reactor 10 consists of an extraction chamber 301 and a variable number of reaction chambers 501, 502, 503 joined by connecting ducts 32 equipped with anti-reflux valves. The number of reaction chambers 501, 502 and 503 can properly vary according to the number of analyses to be performed at the same time. The model with three reaction chambers 501, 502 and 503 is shown in Figures 5 and 6.
Regardless of the number of reaction chambers 501, 502 and 503, the extraction chamber 301 is advantageously shaped as a prism with a rhomboidal base and rounded corners, to facilitate fluid dynamics and avoid stagnation of liquids in proximity of the corners.
Preferably, the extraction chamber 301 has a base of approximately 28x18 mm of size and approximately 2 mm of height, in order to contain approximately 1000 pi of fluid volume. Each reaction chamber 501, 502 and 503 is preferably cylindrical with a base of about 4 mm of diameter and a height of about 2 mm, thus defining a volume of about 25 mI.
Extraction chamber 301 includes:
- Two bidirectional channels 511, 516 for the fluid(s) injection/ejection into/from reactor 10; in particular, the two channels 511 and 516 are located at two opposite ends of the rhomboid which defines the extraction chamber 301;
- Four channels 512, 513, 514, 515 for entering reagents into chamber 301; these channels are advantageously equipped with anti-reflux valves and are located on the opposite ends of duct 32, which connect the extraction chamber 301 to the first reaction chamber 501.
The number of channels 512, 513, 514 and 515 can be appropriately defined according to the number of reagents to be introduced into extraction chamber 301.
A series of corresponding ducts 32 connects the extraction chamber 301 to the first reaction chamber 501, the latter to the second 502 reaction chamber, and the latter to the third 503 reaction chamber. In this way, liquids and reagents placed in the extraction chamber 301 sequentially pass through the first reaction chamber 501, the second reaction chamber 502 and the third reaction chamber 503.
Each reaction chamber 501, 502 and 503 has a corresponding third channel -respectively, 517, 518 and 519 - for the entry of further biological sample and/or reagents into the corresponding chamber.
Advantageously, each third channel is equipped with an anti-reflux valve.
In addition, from the third reaction chamber 503 originates an efflux channel 510 for the waste material, which is also preferably equipped with an anti-reflux valve.
Preferably, the above-mentioned channels have a diameter of about 0,8 mm.
Second form of implementation
A second embodiment (see Fig. 7, 8, 9) of reactor 10 can be used for in vitro fertilization procedures which do not involve intracytoplasmic injection of sperm (ICSI). In this form of realization reactor 10 allows the entry of gametes and the growth of zygote up to the stage of 8-16 cells in a sterile environment, with controlled temperature and CO2 tension.
In this form of implementation, the reactor 10 has the form of a parallelepiped with, for example, external sizes of about 56.5 mm (width), 35 mm (depth) and 4 mm (height), and shall consist of at least one (preferably three) fertilization chambers 601, possibly connected by a series of ducts 32.
The model with three fertilization chambers 601 is shown in figures 7, 8 and 9, but it is understood that there could also be one single chamber.
In these fertilization chambers 601, embryos fertilization and culture takes place, prior to be implanted in the uterus. The number of fertilization chambers 601 varies according to the number of embryos being cultured simultaneously (one embryo per chamber). Regardless of their number, each fertilization chamber 601 may have the shape of a prism with rhomboidal base and rounded sides, to facilitate fluid dynamics and avoid stagnation of liquids in the proximity of the corners. For example, fertilization chambers have a base size of about 28x18 mm and a height of about 1 mm, with a volume of about 500 pi.
Each room 601 has a central portion 602 which is appropriately depressed compared to the remaining part of chamber 601. The portion 602 is cylindrical (preferably with 1.6 mm of height of and 9 mm of diameter, containing a volume of approximately 125 mI), and is the part where the gametes are laid.
Each fertilization chamber 601 is equipped with an additional duct 630 for gamete injection. In particular, each additional duct 630 is connected to a corresponding portion 602. In addition, this additional duct 630 is closed and sealed before the lodging of reactor 10 in device 1.
In the case of an embodiment which includes multiple fertilization chambers 601 connected in series, is properly provided a fourth channel 603 (which is connected to a first fertilization chamber 601) for the input of the culture medium and a fifth efflux channel 604 which is connected to another fertilization chamber 601.
In addition, the fertilization chambers 601 are suitably in fluid connection through ducts 32. Preferably, the connecting ducts 32 are sloping from top to bottom according to the flow direction and this in order to reduce the risk of air bubbles entrapment.
In the embodiment with a single fertilization chamber 601 (not represented), the fourth inlet channel 603 and the fifth outlet channel 604 are both connected to the same fertilisation chamber 601.
Third form of implementation
In a third embodiment (see Fig. 10, 11), reactor 10 can be configured for
- the performance of experimental methods involving the cultivation of adhering cells, under controlled conditions of temperature, C02 tension and culture medium and/or
- the longitudinal repeated measurement of specific biological/biochemical parameters of cultured cells and/or
- the longitudinale evaluation of the effect of pharmacological treatments on in vitro cultured adhering cells.
The reactor 10 is a parallelepiped of external sizes of about 56.5 mm (width), 35 mm (depth) and 3.5 mm (height). According to this embodiment, it includes one or more culture chambers 701 not communicating with the others. The number of culture chambers 701 varies according to the number of experimental conditions which are simultaneously analysed.
Figures 10 and 11 show the embodiment with three culture chambers 701. The culture chambers 701 have preferably form of prism with rhomboidal base and rounded sides and this in order to facilitate fluid dynamics and avoid liquid stagnation near the corners. Preferably, culture chambers 701 have a base of about 25x15 mm and a height of about 2 mm, containing a volume of about 750 pi.
Each culture chamber 701 is equipped with a access channel 703 for cell injection. Each culture chamber 701 is connected with two channels, respectively a sixth channel 704 of inflow of the culture medium and a seventh channel 714 of efflux of the culture medium.
Properly, the channels 703, 704 and 714 can be equipped with anti-reflux valves. All channels 703-714 preferably have a diameter of 0.8 mm.
Advantageously, for reactor 10 models comprising more than 3 culture chambers 701, the size of the chambers can be reduced, for example, to 25x8 mm base and 2 mm height, with a volume of about 400 pi.
Fourth form of implementation In a fourth embodiment (see Fig. 12 and 13) reactor 10 can be configured to:
- perform analyses that provide purification and quantification of proteins of interest, either extracted from biological samples injected into the reactor or from cells grown inside the reactor itself, and subjected to various experimental conditions and/or
- longitudinal measurements of specific biological/biochemical parameters of cells grown inside reactor 10 and/or
- evaluate the longitudinal effect of pharmacological treatments on adhesion cells grown in vitro inside the reactor 10.
In this fourth embodiment, the reactor 10 has the shape of a parallelepiped with external sizes of 56.5 mm (width), 35 mm (depth), 2.5 mm (height), containing one or more analytical chambers (801, 802 and 803 respectively), not in communication with each other. The number of analytical chambers may properly vary depending on the number of experimental conditions being analysed at the same time. Figures 12 and 13 show the embodiment with three analytical chambers 801, 802, 803.
The analytical chambers 801, 802, 803 have a prismatic shape with rhomboidal base and rounded sides, to facilitate fluid dynamics and avoid stagnation of liquids in the proximity of the corners. Their sizes can be for example a base of about 25x15 mm and a height of about 1 mm, containing a volume of approximately 375 pi.
Each analytical chamber 801, 802 and 803 is connected to:
- a corresponding channel 804, 805, 806, for example for the injection of biological samples;
- a corresponding channel 823, 824 and 825 for the efflux of reagents;
- multiple channels 811-822 for the injection of culture media/reagents; for example, four channels could be provided for each 801, 802, 803 analytical chamber.
Properly, the 811-822 channels are equipped with anti-reflux valves. All 804-825 channels are preferably 0.8 mm in diameter.
For example, in reactor models 10 which include more than three analytical chambers, the dimensions of the latter can be advantageously reduced to 25x8 mm at the base and 1 mm of height, with a volume of about 200 mI.
EXAMPLES OF APPLICATION
Some detailed protocols of possible applications of device 1 according to this invention are described below as examples. Reagents, kits, probes, sequences, culture media and antibodies cited as examples, may be replaced by other elements known to the expert and/or equivalents.
Example 1: Molecular test for the presence/absence of the e4 allelic variant of the APOE gene, on DNA extracted from peripheral blood samples
This application requires the reactor in the first embodiment and includes a phase of cell lysis, a phase of DNA extraction-purification and a phase of target sequence amplification.
In Humans, the APOE gene is present in three main allelic variants, encoding proteins differing in only two aminoacid residues. The combination of the different alleles changes the individual's risk of developing Alzheimer's disease, the most common form of dementia in the general population. The three most common alleles in the general population are referred to as e2, e3 and e4. The e4 allele significantly increases the risk of Alzheimer's disease, 4-10 folds that of the general population. These three alleles are determined by two SN Ps, a T-C transition (rs429358, chrl9:44908684, Grch38.pl2) which has an allelic frequency of about 0.15, and a C-T transition (rs7412, chrl9:44908 Grch38.pl2) with an allelic frequency of about 0.05. The different combinations of the two SnNP determine the three alleles.
The implemented protocol allows, starting from samples of peripheral blood or other tissue, to perform DNA extraction and amplification and to determine the genotype at the locus APOE, in a fully automatized mode. The experimental strategy is outlined in figure 14.
For DNA extraction and purification the MagMAX™ DNA Multi-sample kit (Applied Biosystems/Thermo Fisher) is used according to protocol 4425070, modified to adapt to the volumes and materials specific for the device 1.
In particular, the following operations shall be carried out:
1) Device 1 is initialized and equipped with 12 syringes 4:
- Syringe n. 1: Protein Kinase (PK) buffer/enzyme mix, connected through channel 512 to the extraction chamber 301 of the reactor 10,
- Syringe n. 2: lysis buffer, connected through channel 513 to the extraction chamber 301,
- Syringe n. 3: paramagnetic beads, connected through channel 513 to the extraction chamber 301,
- Syringe n. 4: 100% isopropanol, connected through channel 514 to extraction chamber 301,
- Syringe n. 5: wash buffer 1 with isopropanol, connected through channel 514 to the extraction chamber 301,
- Syringe n. 6: Wash buffer 2 with ethanol, connected through channel 514 to the extraction chamber 301
- Syringe n. 7: elution buffer, connected through channel 515 to the extraction chamber 301,
- Syringes n 8 and n. 9: empty, connected through channels 511 and 516 to the extraction chamber 301,
- Syringe n. 10: Taqman Mastermix II IX (Thermofisher PN 4440040); 900 nm of primers APOE- C.364-F and APOE-C.417-R; 200 nm of the APOE-C.381-T and APOE-C.381-C probes; bi-distilled nuclease-free water. Connected through channel 517 to the first reaction chamber 501,
- Syringe n. 11: Taqman Mastermix II IX (Thermofisher PN 4440040); 900 nm of primers APOE- C.500-F and APOE-C.550-R; 200 nm of the APOE-C.519-C and APOE-C.517-T probes; bi-distilled nuclease-free water. Connected through channel 518 to the second reaction chamber 502,
- Syringe N. 12: Empty, connected to channel 510 of third chamber 503.
Reagents other than those indicated may be properly used, if equivalent. Other oligonucleotides than Taqman MGB-probes or even oligonucleotidic probes with modified nucleotides (such as LNA or similar) could be used. In addition, this application is to be considered purely as an example, since the same experimental approach could be used for the identification of any constitutional or acquired genomic variant.
2) As the reactor 10 is still outside the device 1, the operator inserts 100 pi of whole blood (with anticoagulant) into the 301 extraction chamber of reactor 10 using a needle syringe through channel 511.
3) The reactor containing the whole blood is inserted into the corresponding lodgement 17 in device 1, and the extraction and analysis protocol is initiated.
4) At this point the DNA extraction phase is started. The initial step provides the injection of 100 mI of solution in the extraction chamber 301, through the syringe n. 1 connected through channel 512. The syringe n. 8 connected via channel 511 aspirates an equal volume. Protein digestion occurs in this phase.
5) Solution is homogenised by pipetting mixing between syringes 8 and 9 connected via channels 511and 516.
6) The sample is then incubated for 20 minutes at 65 °C, heated by the heating/refrigerant unit 16.
7) The next step consists of a cell lysis phase, obtained by injection of 300 mI of lysis buffer into the 301 extraction chamber, through the syringe n. 2 connected through channel 513. The syringe n. 8 connected through channel 511 aspirates an equal volume.
8) The solution is homogenized by pipetting mixing between the syringes n. 8 and n. 9, connected through channels 511 and 516.
9) 20 pi solution containing paramagnetic beads is pipetted through the syringe n. 3 connected through channel 513. The sample is homogenised by mixing through syringes 8 and 9 connected through channels 511 and 516. This passage allows DNA binding to the paramagnetic beads.
10) 400 mI of 100% isopropanol is injected (syringe n. 4, channel 514), to facilitate the precipitation of nucleic acids and their binding to paramagnetic beads. The syringe n. 8 connected through channel 511 aspirates an equal volume.
11) The solution is homogenised by pipetting mixing between the syringes n. 8 and n. 9 connected through channels 511 and 516.
12) The electromagnet 20 is activated and the sample is incubated for 5 minutes at room temperature.
13) With the electromagnet 20 still on, the supernatant is slowly aspirated through the syringe n. 8 connected through channel 511. The air intake valve is open. 14) The washing phase: 150 mI of Washl solution (containing isopropanol) (syringe n. 5, channel 514) is injected, with the electromagnet 20 on. The syringe n. 9, connected through channel 516, aspirates an equal volume.
15) The sample is incubated for one minute at room temperature.
16) The supernatant is slowly aspirated through the syringe n. 9 connected through channel 516. 17) Steps 14, 15 and 16 are repeated.
18) Then 150 mI of Wash2 solution is injected (syringe n. 6, channel 514). The syringe n. 9 connected via channel 516 aspirates an equal volume.
19) The sample is incubated for one minute at room temperature.
20) The supernatant is slowly aspirated through the syringe n. 9 connected through channel 516. 21) Steps 18, 19 and 20 are repeated.
22) The residual ethanol is evaporated due to air intake through the syringe n. 8 connected via channel 511 and opening of the air-admitting valve.
23) 150 mI elution buffer are injected and incubated for 5 minutes at room temperature (syringe n. 7, channel 515). The syringe n. 9 connected through channel 516 aspirates an equal volume. In this way, the DNA is eluted from the paramagnetic beads. Electromagnet 20 is still on.
24) The eluate is aspirated into the three reaction chambers through channel 510 until the 3 reaction chambers 501, 502 and 503 are filled. The air-admitting valve is open.
25) Through channel 517, 15 pi of the reaction mix is injected into the 501 reaction chamber from the syringe n. 10. Syringe n. 12 connected through channel 510 aspirates an equal volume. 26) Through channel 519, 15 mI reaction mix are injected by syringe n. 11 into the chamber reaction
503. Syringe n. 12 connected through channel 510 aspirates an equal volume.
27) DNA amplification is initiated in real-time mode. After the initial denaturation of genomic DNA (Temperature:95 °C; Time:10'), 40 cycles are carried out under the following conditions: i. Temperature : 95 °C, Time : 15" ii. Temperature : 60 °C, Time : 60"
28) Fluorescence, produced by the degradation of theTaqman-MGB probe, is measured and recorded by the optical measuring apparatus 22 during each amplification cycle. Based on the specific genotype of the individuals under analysis, the presence of different fluorochromes in the first 501 and in the third reaction chamber 503 is detected. In particular, each allele provides the following results:
The different combinations of fluorophores will enable to establish the patient's genotype at the APOE locus. Example 2: Molecular genetic test for the presence/absence of the BCR/ABL fusion gene on cDNA from peripheral blood samples
This application is carried out using the Processor in the first form of implementation, and is based on the extraction of total RNA from peripheral blood samples of patients with chronic myeloid leukaemia (CML) or acute lymphoblastic leukaemia (ALL).
This test is commonly used in clinical practice to monitor over time the evolution of the patient's disease and of the response to treatment. The classic cytogenetic marker of CM L (but also of some types of ALL) is the formation of the Philadelphia chromosome (Ph), originating from a balanced translocation between a chromosome 9 and a chromosome 22. At the molecular level, this cytogenetic rearrangement determines the formation of a chimeric fusion protein between the BCR gene (located on chromosome 22) and the tyrosine-kinase domain of ABL oncogene (located on chromosome 9). The breaking points observed in most patients with CM L determine the formation of a chimeric protein (p210), encoded by the el3a2 m RNA (Fig. 15) with breaking points in the intron 13 of BCR and in the intron 1 of ABL. An alternative product, encoded by the el4a2m RNA, is obtained when the breakpoint on chromosome 22 occurs between exons 14 and 15 of BCR. 95% of patients with CM L have one of these two chimeric transcripts; in 5% of cases, due to an alternative splicing phenomenon, both isoforms can be observed. In addition, 50% of patients with ALL Ph+ have a chimeric gene between exon 1 of BCR and exon 2 of ABL (ela2 mRNA); the same rearrangement can be observed in 45% of CML patients.
A molecular test has been developed to identify the three most frequent chimeric genes, with a sensitivity of 95% in both CM L and ALL Ph+. The experimental strategy is schematized in figure 15. The wild-type BCR transcript is amplified as positive control of the amplification. For the extraction of the total RNA, the kit MagMAX™-96 Blood RNA Isolation Kit (Thermofisher scientific, P/N AM 1837) was used, modified with respect to the manufacturer's instructions and adapted to Device 1. The same kit can be used to extract RNA from tissue samples other than whole blood or cell cultures. Appropriately, another kit could be used for RNA extraction. In addition, this application is to be considered purely as an example, since the same experimental approach could be used for the identification of any other fusion gene.
The phases of the RNA/cDNA extraction, retro-transcription and amplification protocol are as follows:1 ) Device 1 is initialized and equipped with thirteen syringes:
- Syringe No 1: Lysis/Binding Solution, containing isopropanol, connected to channel 512 of reactor
10,
- Syringe No 2: Paramagnetic beads, connected to channel 513 of reactor 10,
- Syringe n, 3: Wash Buffer 1 with isopropanol, connected to channel 514 of reactor 10,
- Syringe No 4: Wash Buffer 2 with ethanol, connected to channel 514 of reactor 10,
- Syringe No 5: DNAse solution, connected to channel 514 of reactor 10,
- Syringe No 6: Elution Buffer, connected to channel 515 of reactor 10,
- Syringes 7 and 8: empty, connected to channels 511 and 516 of reactor 10,
Syringe No 10: reaction mix IX (Taqman® Fast Virus 1-Step Master Mix, Thermofisher Scientific, P/N :
444432); 400 nm of BCR- exl4F and BCR-exl5R primers; 100 nm of the BCR-P probe; H20 by volume. Connected to channel 517 of first reaction chamber 501,
Syringe No 11: IX reaction mix (Taqman® Fast Virus 1-Step Master Mix, Thermofisher Scientific, P/N: 444432); 400 nm of BCR-ABLela2-F and BCR-ABL-R primers; 100 nm of the BCR-ABLela2-P probe; FI20 by volume. Connected to channel 518 of second reaction chamber 502,
Syringe No 12: reaction mix IX (Taqman® Fast Virus 1-Step Master Mix, Thermofisher Scientific, P/N: 444432); 400 nm of BCR-ABLel3a2-F, BCR-ABLel4a2-F and BCR-ABL-R primers; 100 nm of BCR- ABLel3a2-P and BCR-ABLel4a2-P probes; FI20 by volume. Connected to channel 519 of third reaction chamber 503,
Syringe No 13: empty, connected to channel 510.
2) The operator inserts in the extraction chamber of the molecular reactor 50mI of whole fresh peripheral blood with anticoagulant (EDTA, heparin, citrate or other anticoagulants; the choice of the anticoagulant is indifferent to the yield of the extraction protocol). For this purpose a needle syringe is used and the sample is inserted into the reactor through the load channel 511. The reactor containing the sample is placed in the appropriate housing of device 1 which was previously fitted with the 4 syringes containing the reagents.
3) 130 mI of solution from the syringe No 1 are injected through channel 512. An equal volume of air is aspirated through the syringe No 7, connected to the channel 511. The solution is mixed by pipetting through syringes 7 and 8, connected to channels 511 and 516.
4) At this point 20 mI of solution from the syringe No 2 are injected through channel 513. The sample is mixed for 5 minutes at room temperature, pipetting through the syringes No 7 and No 8 connected to channels 511 and 516. This phase allows the complete lysis of the sample and the binding of the RNA to the paramagnetic beads.
5) The electromagnet 20 is activated and the sample is incubated for 5 minutes at room temperature. The supernatant is aspirated through the syringe No 7 connected to channel 511. The air intake channel is open.
6) 150 mI from the syringe No 3 are injected into the extraction chamber, through channel 514. An equal volume is aspirated through the syringe No 7, connected to channel 511. The sample is incubated for one minute at room temperature. In these steps the electromagnet 20 remains active. This washing step is repeated once.
7) 150 mI of solution are injected through channel 514 (syringe No 4). An equal volume is aspirated through the syringe No 7, connected to the channel 511. The sample is incubated for one minute at room temperature. In these steps the electromagnet 20 remains active.
8) The solution inside the reactor is aspirated through the syringe No 7, connected to channel
511.
9) 50 mI of solution are injected through channel 514, (syringe No 5) and incubated 5 minutes at room temperature. An equal volume is aspirated through the syringe No 7, connected to channel 511. The electromagnet 20 is not active.
10) 130 mI of solution are injected from the syringe No 1 through channel 512. An equal volume is aspirated through the syringe No 7, connected to channel 511. The solution is incubated for 3 minutes at room temperature. Electromagnet 20 is active.
1 1 ) The reagents inside the reactor are aspirated through the syringe No 7 connected to channel 511.
12) 150 mI of solution are injected through channel 514 (syringe No 4). An equal volume is aspirated through the syringe No 7, connected to the channel 511. The sample is incubated for one minute at room temperature. In these steps electromagnet 20 remains active. The supernatant is aspirated through the syringe No 8, connected to channel 516. The washing step is repeated.
13) The residual ethanol is evaporated by air-aspiration through the syringe No 8, connected to channel 516. The air-admitting valve remains open.
14) 150 mI are injected from syringe No 6 through channel 515, and incubated 5 minutes at room temperature. In this phase, RNA is eluted from paramagnetic beads. Electromagnet 20 remains active.
15) Total RNA is now conveyed into the 501, 502, 503 reaction chambers, by aspiration through the syringe No 13 connected to channel 510 and to the channels connecting the chambers.
16) The control gene (BCR) is amplified in reaction chamber 501. 12.5 mI are injected from syringe No 10 through channel 517. An equal volume is aspirated from syringe No 13, through channel 510.
17) The BCR-ABL ela2 fusion gene is amplified in reaction chamber 503. 12.5 mI are injected from the syringe No 11 through channel 518. An equal volume is aspirated from the syringe No 13, through channel 510.
18) The BCR-ABL el3a2 and el4a2 fusion genes are amplified in reaction chamber III. 12.5 mI are injected by the syringe No 12 through channel 519. An equal volume is aspirated from the syringe No 13, through channel 510.
19) The following temperature conditions are used for RT-PCR and DNA amplification:
a. T=50°C time 5' b. T=95° C time 20"
40 cycles:
c. T=95°C time 3" d. T=60° C time 30"
20) During the amplification cycles, the optical measuring apparatus 22 measures the fluorescence emitted during the extension phase at 60°C of the amplification reaction.
21) Based on the amount of fluorescence emitted, the device management software determines the presence/absence of the fusion gene and its relative amount, compared to the initial volume of blood introduced into the reactor.
Example 3: Human/animal assisted fertilisation through IVF
This protocol makes use of one of the processors in the second form of implementation (figg. 7,8,9), which number of fertilization chambers 601 is variable depending on the clinical indication to IVF and on the number of female gametes available. In the following example, reactor 10 is used in second form of implementation with three chambers of fertilization 601, usable when involving ovarian stimulation cycles. In the case of a single oocyte, the operational protocol is invariable; in this case, a version with one fertilisation chamber 601 may also be used.
Fertilization and embryo culture (one per chamber) up to the implantation in utero, take place inside the chambers described in figure 7-9. Reactor 10 used in this procedure requires the use of an inverted light microscope as an ancillary monitoring device. In order to guarantee the sterility of the experimental procedures, the entire process is carried out in surgical operating room environment both in the human and zootechnical fields.
The female gametes are picked up according to the standard operating procedures validated and currently in use in clinical practice, both in the human and zootechnical fields. In order to increase the yield of assisted fertilisation, it is preferable to use freshand non-crystalled gametes (Ozcavukcu et al., 2008). Once male gametes are collected, the mobile and vital fraction is selected by migration-sedimentation process, as described by Kiratli et al. (2018). The maintenance of a finely regulated fluid handling speed, during oocyte fertilisation as during the initial embryo growth, is considered a critical factor to determine an increasedyield of the entire process on platforms that use microfluidics, compared to static systems (Swain JE et al., 2013).
The operational protocol includes the following phases:
1) Device 1 is initialized and equipped with two syringes: a) Syringe No 1: containing Quinn's culture medium Advantage™ Fertilization ( HTF) Medium, 5% C02 balanced, connected through channel 603 to the reactor; if appropriate, another culture medium could be used.
b) Syringe No 2: empty, connected through channel 604 to the reactor.
2) The oocytes are manually inserted (one per chamber) into the fertilisation chambers 601 through the 630 ducts, directly connected to the 602 cylindrical depression.
3) 100 pi of the viable fraction of the sperm, suitably selected, are taken by an insulin sterile disposable syringe; 30 mI are inserted in each fertilization room through the ducts 630, near the deposition point of the oocytes. Phases 2) and 3) should be monitored by inverted light microscope observation.
4) Ducts 630 are sealed with methyl-methacrylate.
5) Reactor 10 is inserted into device 1, maintaining sterility conditions.
6) The medium is injected by the syringe No 1, at a rate of 1 ml/min, to avoid displacement of the gametes, until saturation of the entire fluidic. At the same time, syringe No 2 aspirates an equal volume.
7) The assembled reactor is incubated at 37°C for 48 hours. The hermetic closure of the system and the previous medium balancing ensure the correct maintenance of the oxygen tension and the pH of the medium.
8) At the end of the 48-hour incubation period, the fertilisation chambers are analysed under the inverted light microscope and those in which fertilisation and embryo growth have occured are identified.
9) The embryos, at the stage of 4-8 cells, are taken with an intrauterine insemination cannula with flexible distal end (Ainsenblue type or similar), and implanted in the uterus, according to the validated gynecological or veterinary protocols.
Example 4: Evaluation of the effect of treatment with ouabain on intracellular concentration of Na+, in SH- SY5Y human neuroblastoma cell cultures
In this example a fluorescent tracker specific to the ion Na+ (Piacentini et al., 2015) is used to measure the effect of treatment with ouabain on SH-SY5Y human neuroblastoma cells. Ouabain is a compound that inhibits the activity of the Na/K ATPase pump (Marck et al., 2018).
Following treatment with ouabain, cell cultures are treated with gramicidine (Doebler et al., 1999) as a normalizer of the intracellular concentration of the Na+ ion. Gramicidine is a compound that allows to short-circuit the sodium/potassium pump and to reach intracellular sodium concentrations of about 150 mMol, as in the extracellular environment.
The same protocol, suitably modified, can be used for any other compound that modifies intracellular concentrations of ions and/or for any analyte for which a fluorescent vital tracker is available; some examples are H+, Ca++, specific markers of mitochondrial membrane potential, necrosis or apoptosis activation markers, etc. In addition, any other type of human or non-human cell culture, primary or stabilized, may be used, if it grows in adhesion.
Reactor 10 is used for this protocol in the third form ( Figg. 10, 11); up to 3 samples or treatments
with alternative molecules can be analysed with this type of reactor. In this case, the number and content of the syringes 4 installed may be adjusted accordingly and the other culture chambers 701 shall be used. In this example only one culture chamber is used, the median one. A cell culture facility with a cell incubator and a laminar flow hood is required for cell maintenance and preparation.
The operational protocol shall comprise the following phases:
1) Device 1 is initialized and equipped with four syringes 4:
- Syringe No 1: containing DMEM High glucose, Ham's F12 medium, phenol red free, with 5% fetal bovine serum, balanced at 5% C02. Syringe No 1 is connected via channel 714 to reactor 10 in the third form of implementation; appropriately, different culture media could be used,
- Syringe No 2: containing the same culture medium as indicated above, with an addition of 100 pm ouabain, 5pm Corona green (Invitrogen) and 300ng/ml Pluronic f-127 (Sigma- Aldrich). Syringe No 2 is connected via channel 714 to reactor 10 in its third form of implementation,
- Syringe No 3: containing the same culture medium as above, with an addition of lOpg/ml gramicidine, 5pm Corona green (Invitrogen) and 300ng/ml of Pluronic f-127 (Sigma- Aldrich). Any other fluorescent sodium marker may also be used. Syringe No 3 is connected through channel 714 to reactor 10 in the third form of implementation,
- Syringe No 4: empty, is connected through channel 704 to reactor 10 in the third form of implementation,
2) The day before the experiment, SH-SY5Y cells are detached from the culture flask by trypsinization, according to standard protocols. After counting, cells are resuspended in DMEM High glucose w/o phenol red, Ham's F12 medium, 5% foetal bovine serum at a concentration of 600 cells/pl; 100 pi of cell suspension is loaded into the culture chamber 701 of reactor 10 through channel 703, which is subsequently sealed with methyl methacrylate.
3) Reactor 10 is loaded into device 1.
) 650 mI of basal medium are injected through the syringe No 1 at a rate of 1 ml/minute; at the same time, an equal volume is aspirated through the syringe No 4.
) The cells are incubated at 37°C for 16 hours.
) During the last two hours of incubation, fluorescence emitted by plastic and cell culture (autofluorescence) is measured by reading at 450 nm every 30 minutes at 6 different positions of the reactor surface. This measurement constitutes the baseline.
) 750 mI of medium are injected through the syringe No 2 the culture chamber 701, at a rate of 1 ml/minute. At the same time, an equal volume is aspirated through the syringe No 4.
) Cell culture is maintained at 37°C. ) From time 0, the fluorescence emitted by the Corona green probe is measured every 15 minutes for 6 hours at the same positions of the reactor surface, used for the baseline. 0) At the end of the incubation, 750 mI of medium is injected into the culture chamber through the syringe No 3; at the same time, an equal volume is aspirated through the syringe No 4.
1 ) After 15 minutes of incubation, fluorescence levels emitted by the Corona green probe are measured at the same locations as the culture previously used.
2)The concentration of Na+ measured at each time is extrapolated from the fluorescence measured after gramicidine.
3) The concentration curve of Na+ which constitutes the final output of the analysis is constructed.
Example 5: Determination of SMN protein levels compared to Glyceraldehyde -3-Phosphate-
Dehydrogenase levels in Hela cell cultures treated with salbutamol for 12 and 24 hours.
In this example a hybrid method between ELISA and Western blot is described, for the quantification of SMN protein levels compared to those of Glyceraldehyde-3-Phosphate- Dehydrogenase (GAPDH) in HeLa cells treated with salbutamol (Angelozzi et al., 2008).
The same protocol, suitably modified, may be used to determine the levels of any protein of interest for which specific antibodies are available. In addition, although human HeLa cells have been used in this example, any other type of human or non-human cell culture, either primary or stabilized, may be used if growing adherent. The use of the different reagents and culture media indicated in the example is not mandatory and can therefore be amended accordingly.
For this operational protocol the reactor 10 is used in the fourth form of implementation, with three analytical chambers 801, 802, 803. A cell culture facility with a cell incubator and laminar flow hood is required for cell culture and preparation.
The operational protocol includes the following phases:
1) Device 1 is initialized and equipped with 9 syringes:
- Syringe No 1: containing the appropriate culture medium, depending on the cell line being tested, balanced at 5% C02. In the case of HeLa cells, DM EM High glucose with Pen strep/l-glutamine may be used. The syringe No 1 is connected via channels 811, 815 and 819 to the 801, 802 and 803 analytical chambers of the reactor 10 in the fourth form of implementation,
- Syringe No 2: containing the same culture medium as above, supplemented with 0.05 pm of salbutamol (Angelozzi et al., 2008). The syringe No 2 is connected via channels 815 and 819 to the 802 and 803 reactor chambers in the fourth form of implementation,
- Syringe No 3: containing RIPA buffer (25mm Tris, pH 7.8; 150 mm Nad; 0.5% sodium deoxycholate; 1% NP-40; protease inhibitors cocktail; 10 mg/ml DNAse I), connected via channels 812, 816 and 820 to the 801, 802, 803 analytical chambers of reactor 10 in the fourth form of implementation,
- Syringe No 4: containing anti-SMN (e.g., 2B1 monoclonal antibody, Sigma-Aldrich, mouse product) and GAPDH (e.g., ab8245 Abeam, mouse monoclonal antibody), diluted 1/1000 in RIPA buffer/1% Bovine Serum Albumin (BSA), containing 1% v/v of Absolute Mag™ Anti-
Igg Mouse (Fc) Magnetic Particles (Creative Diagnostics, WHM-S070), connected via
channels 812, 816 and 820 to the 801, 802, 803 analytical chambers of reactor 10 in the fourth form of implementation. If appropriate, any other antibody directed against the proteins of interest may be used. Any other protein may be dosed in suitable samples, using specific antibodies,
- Syringe No 5: anti-SMN (e.g., Abeam ab232784, polyclonal antibody produced in rabbit) and anti-GAPDH (e.g., Sigma-Aldrich SAB2500450, polyclonal antibody produced in goat), diluted 1/1000 in Dulbecco's modified Phosphate Buffer (PBS)/BSA 1%, anti-rabbit conjugated with FITC and anti-goat conjugated with Alexa-fluor, diluted 1/1000; the syringe No 5 is connected via channels 813, 817 and 821 to the 801, 802, 803 reactor chambers in the fourth form of implementation,
- Syringe No 6: containing PBS lx/Tween 20 0.1%, connected via channels 814, 818 and 822 to the 801, 802, 803 analytical chambers of the reactor 10 in fourth form of implementation,
- Syringes 7-9: empty, each connected via channels 823, 824 and 825, to the 801, 802, 803 analytical chambers of reactor 10 in the fourth form of implementation,
2) The day before the experiment, the HeLa cells are detached from the culture flask by trypsinization, according to standard protocols. After counting, cells are resuspended at a concentration of 100,000 cells/ml in DMEM High glucose with 15% of fetal bovine serum and loaded into the reactor through channels 804, 805, 806 in number of 50000 for each analytical chamber 801, 802, 803 (see Figure 5).
3) The reactor is placed into device 1.
4) 500 pi of medium are injected through the syringe No 1 at a rate of 1 ml/minute into each 801, 802, 803 analytical chamber. Syringes 7-9 aspirate an equivalent volume.
5) Cells are incubated at 37°C for 16 hours.
6) After the indicated time, 500 mI of medium are injected from the syringe No 1 through channels 821 and 825 in the incubation chambers 801, 802, 803. Empty syringes 7 and 8 aspirate an equivalent volume, through channels 825 and 824.
7) Subsequently, 500 mI of medium containing salbutamol are injected into the incubation chamber 803, via channel 819 (syringe No 2). The syringe No 9 aspirates an equal volume through channel 823.
8) Cell culture is maintained at 37°C.
9) After 12 hours of incubation, 500 mI of medium with salbutamol are injected into the incubation chamber 802 through channel 815 (syringe No 2). Syringe No 8 aspirates an equivalent volume of medium through channel 824.
10) After a further 12 hours, the medium contained in the three chambers of incubation is aspirated through syringes 7-9.
1 1 ) In the three incubation chambers, 250 mI of RIPA buffer are injected through channels 812, 816 and 820 (syringe No 3) and incubated for 30 min at 4°C.
12) 250 mI of antibodies conjugated with paramagnetic beads are injected into the incubation chambers through channel 812, 816 and 820 (syringe No 4), and incubated at 37°C for 30 seconds. At this stage, the electromagnet 20 is active.
13) After the incubation time, the RIPA buffer is aspirated through channels 823, 824, 825.
14) 375 mI of 0.1% PBS/Tween are injected into each analytical chamber 801, 802, 803 from the syringe No 6 through channels 811, 818, 822; the reactor is incubated at 20°C for 10 minutes.
15) The washing step 14) is repeated
16) The PBS-Tween is aspirated and 500 mI of secondary antibodies are injected into the three analytical chambers from the syringe No 5 through channels 813, 817 and 821, and incubated for 30 minutes at 37°C.
17) After the incubation time, two washes are carried out, as indicated in step 14)
18) The fluorescence emitted by the two secondary antibodies is measured and recorded by the device for 5 minutes.
19)The variation in SM N protein levels in response to treatment is determined in relation to GAPDH levels and normalised to the untreated cell culture present in analytical chamber 801.
DESCRIPTION OF THE SEQUENCES ANALYSED IN THE EXAMPLES
APOE-C.364-F: 5'-GGGCGCGGACATGGA-3' (SEQ I D NO: 1)
APOE-C.417-R: 5'-CCTCGCCGCGGTACTG-3' (SEQ I D NO:2)
APOE-C.381-T: 5'-VIC-GACGTGTGCGGCCG-MGB-3' (SEQ I D NO:3)
APOE-C.381-C: 5'-FAM-GACGTGCGCGGCC-MGB-3'(SEQ I D NO:4)
APOE-C.500-F: 5'-CCGCGATGCCGATGAC-3'(SEQ ID NO:5)
APOE-C.550-R: 5'-GCCCCGGCCTGGTACA-3'(SEQ I D NO:6)
APOE-C.519-C: 5'-VIC-CAGAAGCGCCTGGCA-MGB-3'(SEQ ID NO:7)
APOE-C.517-T: 5'-FAM-TGCAGAAGTGCCTGGC-MGB-3'(SEQ ID NO:8)
BCR-exl4F: 5'- TTCTGAATGTCATCGTCCACTCA -3'(SEQ ID NO:9)
BCR-exl5R: 5'-CCAGGGTGCAGTACAGATTTGA-3'(SEQ ID NO:10)
BCR-P: 5'-FAM-CCACTGGATTTAAGCAGAG-MGB-3'(SEQ ID NO:ll)
BCR-ABLel3a2-F: 5'-AGCATTCCGCTGACCATCA-3'(SEQ ID NO:12)
BCR-ABLel3a2-P: 5'-VIC-ATAAGGAAGAAGCCCTTCAG-MGB-3'(SEQ I D NO: 13)
BCR-ABLela2-F: 5'-CGAGGGCGCCTTCCAT-3'(SEQ ID NO:14)
BCR-ABLela2-P: 5'-VIC-AGACGCAGAAGCC-MGB-3'(SEQ ID NO:15)
BCR-Ablel4a2-F: 5'-CAGCCACTGGATTTAAGCAGAGT-3'(SEQ ID NO:16)
BCR-Ablel4a2-P: 5'-FAM-CAAAAGCCCTTCAGCG-MGB-3'(SEQ ID NO:17)
BCR-ABL-R: 5'-GAGGCTCAAAGTCAGATGCTACTG-3'(SEQ ID N0:18)
REFERENCES
- Angelozzi C, Borgo F, Tiziano FD, Martella A, Neri G, Brahe C. Salbutamol increases SM N mRNA and protein levels in spinal muscular atrophy cells J. Med. Genet. 2008 Jan;45(l):29-31
- Benagiano G, Bastianelli C, Farris M. [Infertility: a global perspective] Minerva Ginecol. 2006 Dec;58(6):445-57
- Doebler JA. Gramicidin toxicity in NG108-15 cells: protective effects of acetamidine and guanidine Cell. Biol. Toxicol. 1999; 15(5):279-89
Kiratli S, Yuncu M, Kose K, Ozkavukcu S. A comparative evaluation of migration sedimentation method for sperm preparation Sysf. Biol. Reprod. Med. (2018) 64:122-129
- Marck PV, Pierre SV. Na/K-ATPase Signaling and Cardiac Pre/Postconditioning with Cardiotonic Steroids. Int J Mol Sci. 2018 Aug 9;19(8).
- Ozkavukcu S, Erdemli E, Isik A, Oztuna D and Karahuseyinoglu S. Effects of
cryopreservation on sperm parameters and ultrastructural morphology of human spermatozoa J. Assist. Reprod. Genet. (2008) 25(8):403-411
- Piacentini R, Li Puma DD, Ripoli C, Marcocci M E, De Chiara G, Garaci E, Palamara AT, Grassi C. Flerpes Simplex Virus type-1 infection induces synaptic dysfunction in cultured cortical neurons via GSK-3 activation and intraneuronal amyloid-b protein accumulation Sci. Rep. 2015 Oct 21;5:15444
- Stevenson EL, Hershberger PE, Bergh PA. Evidence-Based Care for Couples With Infertility J. Obstet. Gynecol. Neonatal. Nurs. 2016 Jan-Feb;45(l): 100-10
- Swain J E, Lai D, Takayama S and Smith GD. Thinking big by thinking small: application of microfluidic technology to improve ART Lab Chip (2013) 13: 1213
Claims
1. Integrated and automated device ( 1) for carrying out molecular and/or cellular biology laboratory procedures, characterized in that it comprises:
- an apparatus for handling fluids (2) configured to allow the transfer of fluidic substances, comprising the sample to be analysed/treated and/or at least one reagent, between corresponding containers (4) and at least one microfluidic reactor (10), said microfluidic reactor (10) comprising at least one chamber (30) in which molecular and/or cellular biology reactions occur between said sample to be analysed and at least a reagent,
- a heating and/or refrigerating unit (16) which is configured to control the thermal conditions in at least one chamber (30) of said reactor (10),
- a pressure applicable/lockable lid (21) of an optically indifferent material, configured to superiorly cover the reactor (10), which prevents the deformation of said reactor as a result of temperature variations and allows the detection of the fluorescent signal, without phenomena of light refraction or diffraction,
- an optical measuring apparatus (22) which is configured to perform optical measurements on the fluidic substances that enter and/or pass through said reactor (10),
- a command and control unit (14) configured to control and/or coordinate the fluid handling apparatus (2), the heating and/or refrigerating unit (16), and/or said optical measuring apparatus (22).
2. Device (1) according to claim 1, characterized in that it comprises means (18) for immobilizing the fluid substances, when they enter and pass through the reactor (10), said means comprising:
- an electromagnet (20) configured to generate a variable magnetic field,
- a plurality of paramagnetic beads configured to specifically bind with some substances present inside the reactor (10).
3. Device (1) according to one or more of the previous claims, characterized in that said containers (4) comprise at least two syringes (4) and in that:
- said syringes (4) are disposed in parallel to each other, and
- at least one syringe (4) is of the precision type and is configured to pipette volumes up to 0.01 ml,
- said syringes (4) comprise a plunger (8) which is actuated in an automated and controlled manner and
- the plunger (8) of the syringes (4) is actuated by a telescopic piston (12) which is part of the manipulation apparatus (2).
4. Device (1) according to one or more of the previous claims, characterized in that said optical measuring apparatus (22) comprises:
- an excitation light source (24), preferably at least one LED lamp and/or at least one laser source,
- a detector (26) placed above said reactor (10),
- configured filters to isolate the emission wavelengths of the single fluorochromes present inside said reactor (10).
5. Device (1) according to one or more of the previous claims, characterized in that said reactor
( 10) :
- has a substantially laminar conformation,
- is of a single-use type,
- is of optical and bio-compatible plastic material
- includes inside a plurality of chambers (30) of circular or polygonal shape with beveled corners, said chambers (30) being fluidically connected, by means of cylindrical connecting ducts (32), and in that said inlet/outlet doors (34) are defined in correspondence with the edges of said reactor (10) and each of said doors (34) is connected by a connecting tube (6) of the manipulation apparatus (2) to a corresponding container (4) in which the sample to be analysed/treated and/or a reagent is present.
6. The device according to claim 5 wherein the cylindrical connecting ducts (32) are equipped with antireflux valves, both between them and to inlet/outlet doors (34) to/from said reactor (10).
7. Device (1) according to one or more of the preceding claims, characterized in that said reactor (10) comprises:
- an extraction chamber (301) for mixing said fluidic substances, said extraction cham ber (301) being fluidically connected to:
• at least one first bidirectional channel (511, 516) for the entry/exit into/from said extraction chamber (301) of the sample to be treated/analyzed,
• at least one second channel (512,513,514,515) for introducing the reactants into said extraction chamber (301),
- at least one reaction chamber (501, 502, 503) which is in fluid connection with said extraction chamber (301) and/or with any other reaction chambers (501, 502, 503), to each reaction chamber being fluidically connected at least one third channel (517, 518, 519) for the input of further biological sample and/or reagents within the corresponding reaction chamber (501, 502, 503).
8. Device (1) according to one or more of claims 1-6 characterized in that said reactor ( 10) comprises at least one fertilization chamber (601) configured to contain the embryos to be
cultivated in a depressed portion (602) with respect to the rest of the chamber (601), to said fertilization chamber (601) being fluidically connected:
- at least one further conduit (630) for the deposit of gametes,
- at least one channel (603) for the input of the culture medium,
- at least one efflux channel (604) of the culture medium.
9. Device (1) according to claim 8, characterized in that said reactor (10) comprises a plurality of fertilization chambers (601) fluidly connected in series through at least one connecting duct (32) and in that:
- said at least one channel (603) for the introduction of the culture medium is connected to the fertilization chamber (601) further upstream,
- said efflux channel (604) is connected to the fertilization chamber (601) further downstream.
10. Device (1) according to one or more of claims 1-6 characterized in that said reactor ( 10) comprises at least one culture chamber (701) to which are connected :
- at least one access channel (703) for cell deposition,
- at least one inflow channel (704) of the culture medium, and
- at least one efflux channel (714) of the culture medium.
11. Device (1) according to one or more of claims 1-6 characterized in that said reactor ( 10) comprises at least two analytical chambers (801, 802, 803) fluidically independent from each other, to each analytical chamber (801, 802, 803) being connected:
- at least one corresponding channel (804, 805, 806) for loading the biological sample into said chamber,
- a plurality of channels (811-822) for the introduction of culture media and/or reagents.
- at least one corresponding channel (823, 824, 825) for the reagents outflow.
12. Use of the device according to one of claims 1-7 to:
a) perform analysis of gene variants, for example aimed to the diagnosis/prognosis or epidemiology, or quantification/qualification of gene transcripts, where said analysis include the extraction phases from one or more isolated human or animal biological samples, purification and amplification of nucleic acid and where said phases are performed inside the microfluidic reactor (10); or
b) identify the presence of the genome of pathogen agents in human or animal biological samples, wherein said analysis include the extraction phases from one or more isolated biological samples, purification and amplification of nucleic acids and wherein said phases are performed inside the microfluidic reactor (10).
13. Use of the device according to one of claims 1-6 and 8-9 to perform in vitro fertilization procedures.
14. Use of the device according to one of claims 1-6 and 10 to cultivate adherent cells in controlled conditions and/or to measure over time specific biological/biochemical parameters of the cells cultivated in the microfluidic reactor ( 10) and/or to evaluate over time the effect of pharmacological treatments in adherent cells cultivated in vitro in the microfluidic reactor ( 10).
15. Use of the device according to one of claims 1-6, 11 and 14 to purify and quantify proteins extracted from biological samples entered into the microfluidic reactor (10) or from cells cultivated in vitro in the microfluidic reactor (10).
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|---|---|---|---|
| IT102019000012345A IT201900012345A1 (en) | 2019-07-19 | 2019-07-19 | DIAGNOSTIC DEVICE AND ITS USES |
| IT102019000012345 | 2019-07-19 |
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| WO2021014305A1 true WO2021014305A1 (en) | 2021-01-28 |
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|---|---|---|---|
| PCT/IB2020/056735 WO2021014305A1 (en) | 2019-07-19 | 2020-07-17 | Diagnostic device and its uses |
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| IT (1) | IT201900012345A1 (en) |
| WO (1) | WO2021014305A1 (en) |
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