Patent Application
20210033518
The present invention discloses beads comprising at least one structure based on DNA origami and fluorophores for use in microfluidics, such as flow cytometry.
The commercial calibrators on the market today that are used to quantify numbers of fluorophores per particle(cell) from the flow cytometric fluorescence intensity are associated with several drawbacks: (a) The current calibrator beads are made of plastic and thus exhibit a high auto-fluorescence corresponding to about 200-2.000 fluorophores per bead. (b) The degree of fluorophore-labelling of commercial beads is associated with a large variation, (c) These calibrations are based on thousands of fluorophores per bead (including high auto-fluorescence) and thus the extrapolation into the lower range (50-2.000 fluorophores is associated with very high uncertainty).
DNA-origami-based standards for microscopy have been described in US 20140057805 A1.
The present invention provides beads comprising a structure based on DNA-origami, wherein said structure comprises a predetermined number of fluorophores for microfluidic applications, such as flow cytometry. The bead has several advantages over the prior art. One advantage is that the bead exhibits no or very little auto-fluorescence. Moreover, the number of fluorophores attached to the structure based on DNA-origami is known and can be precisely controlled so that the bead can be labelled with a distinct number of fluorophores down to as few as one fluorophore. Thus, calibration with the methods of the present disclosure allows quantification of antigens present in a sample at a very low level.
One advantage of the present invention lies in that the origami structure allows fluorescence quantification in microfluidics, where the signal is obtained differently compared to fluorescence microscopy. In microscopy, the object is stationary and thus the fluorophores attached to the object have more excitation exposure and emission time to generate the signal, while in microfluidics, such as flow cytometry, the beads are moving rapidly past the detector, which substantially limits the exposure and detection time. The calibration of flow cytometry systems may require beads with specific number of fluorophores, up to 1500-3000 fluorophores.
One aspect of the present disclosure relates to a method of calibrating a microfluidics system comprising:
Calibration of a microfluidics systems, such as flow cytometry setups, using calibration beads as defined herein allows precise biochemical analysis of small biological particles e.g. extracellular vesicles, viruses and bacteria. Thus, the provided methods for calibrating microfluidics systems can advantageously be used in academic, industrial as well as for clinical analyses, including diagnostic methods based on microfluidics, such as flow cytometry.
Another aspect of the present disclosure relates to a method for determining the level of an antigen in a sample by a microfluidics system comprising
Another aspect of the present disclosure relates to a method for determining the presence or state of a haematological disease in an individual by a microfluidics system, such as a flow cytometry system, the method comprising
Another aspect of the present disclosure relates to a kit for calibration of microfluidic instruments comprising a bead having a structure based on DNA origami wherein said structure comprises a predetermined number of fluorophores.
Definitions
The term “structure based on a DNA origami” as used herein refers to a DNA origami formed from a scaffold DNA strand and shorter DNA segments, also called staple strands, which form a predetermined structure of the scaffold DNA strand. The scaffold DNA strand is usually a long single-stranded DNA molecule. The length of the scaffold DNA strand depends on the system used for its production. Scaffold DNA strands of approximately 7.000 nucleotides may be produced using for example M13mp18 phage. Scaffold DNA strands of approximately 50.000 nucleotides may be produced using for example a λ/M13 hybrid virus as described in Marchi et al. (2014). The structure based on a DNA strand can comprise further components such as dyes, plasmonic structures, biological molecules such as proteins, enzymes, nanoparticles, and small molecules such as biotin. Alternatively, the structure based on a DNA origami can also be constructed solely from short DNA segments, as recently described by Ong et al. (2017). Constructing a structure based on a DNA origami solely from short DNA segments is also possible for large structures, such as structures in the gigadalton range.
The term “DNA” as used herein, is understood to mean not only strands of deoxyribonucleic acid, but also analogous structures, such as strands of ribonucleic acids (RNA), peptide nucleic acid (PNA), as well as hybrid structures.
The term “shorter DNA segments” as used herein refers to the nucleotide molecules which are also referred to as “staple strands” or “staple DNA strands” and which have a sequence complementary to a sequence of the scaffold DNA strand or another shorter DNA segment. Furthermore, said shorter DNA segments can be used to fold the long DNA strand into the predetermined structure. Alternatively, the shorter DNA segments can be those which hybridize with the DNA scaffold strand of the DNA origami in predetermined regions. The shorter DNA segments may comprise a sequence that does not hybridize to the DNA scaffold strand, called “sticky end” herein, which can be used for decorating the structure with fluorophores. The shorter DNA segments can also be used to bind/stick individual DNA origami structures together into large DNA origami oligomers in a controllable manner.
The term “calibration” is understood here to mean quantifying a measured variable on the basis of one or more reference samples or determining the properties of an apparatus, such as the sensitivity and resolution. For example, the methods disclosed herein may be used for calibration of a flow cytometer.
Structure Based on DNA Origami
In one aspect the present disclosure relates to a method of calibrating a microfluidics system, such as a flow cytometer, comprising providing at least one bead having a structure based on DNA origami, wherein said structure comprises a predetermined number of fluorophores.
The structure based on DNA origami usually comprises a single stranded DNA molecule, also called DNA scaffold strand, which is folded into a predetermined structure with the help of a plurality of staple strands. Each staple strand hybridizes to specific parts of sequence on the single stranded DNA molecule thereby determining how the single stranded DNA molecule folds.
Depending on the length of the DNA scaffold strand, a certain number of staple DNA strands will be required for folding the scaffold strand into the predesigned structure.
Thus, in one specific embodiment the structure comprises a predetermined number of fluorophores, a single stranded DNA molecule and staple DNA strands, wherein the number of staple DNA strands (Ns) is as in the formula below:
Ns=Lss/n,
wherein Lss is the length of the single stranded DNA molecule, and
wherein n is an integer number comprised between 26 and 34.
In another specific embodiment, the structure comprises a predetermined number of fluorophores, a single stranded DNA molecule and staple DNA strands, wherein the number of staple DNA strands (Ns) is as in the formula below:
Ns=Lss/n,
wherein Lss is the length of the single stranded DNA molecule, and
wherein n is an integer number comprised between 28 and 32.
However, the present invention is not limited to embodiments meeting the above equation. Indeed, it is possible to produce many different DNA origami structures, which do not follow this equation.
The length of the staple DNA strands may differ from one staple DNA to the other. The minimum length is a length that allows the staple DNA strand to be stable at room temperature.
In one embodiment, the length of the staple DNA strands is in the range between 12 and 60 nucleotides, preferably between 15 and 50 nucleotides.
Thus, in one embodiment the present disclosure relates to a method of calibrating a flow cytometer comprising providing at least one bead having a structure based on DNA origami, wherein said structure comprises a predetermined number of fluorophores, a single stranded DNA molecule and staple DNA strands.
The length of the single stranded DNA molecule is important for the size of the bead as well as for the number of fluorophores that the bead can accommodate.
Thus, the single stranded DNA molecule or DNA scaffold strand may have a length in a range between 1.500-nucleotides and 70.000-nucleotides, such as between 2.000-nucleotides and 68.000-nucleotides, such as between 5.000-nucleotides and 65.000-nucleotides, for example between 10.000-nucleotides and 60.000-nucleotides, such as between 15.000-nucleotides and 55.000-nucleotides, preferably between 20.000-nucleotides and 55.000-nucleotides, such as between 50.000 and 52.000-nucleotides. Thus, the single stranded DNA molecule or DNA scaffold strand may have a length of 51.466-nucleotides.
The single stranded DNA molecule may have a length in a range between 5.000-nucleotides and 70.000-nucleotides, for example between 7.000-nucleotides and 70.000-nucleotides, such as between 10.000-nucleotides and 70.000-nucleotides, preferably between 13.000-nucleotides and 70.000-nucleotides, such as between 15.000-nucleotides and 70.000-nucleotides, for example between 18.000-nucleotides and 70.000-nucleotides, such as between 20.000-nucleotides and 70.000-nucleotides, preferably between 23.000-nucleotides and 70.000-nucleotides, such as between 25.000-nucleotides and 70.000-nucleotides, for example between 28.000-nucleotides and 70.000-nucleotides, such as between 30.000-nucleotides and 70.000-nucleotides, preferably between 33.000-nucleotides and 70.000-nucleotides, such as between 35.000-nucleotides and 70.000-nucleotides, for example between 38.000-nucleotides and 70.000-nucleotides, such as between 40.000-nucleotides and 70.000-nucleotides, preferably between 43.000-nucleotides and 70.000-nucleotides, such as between 45.000-nucleotides and 70.000-nucleotides, for example between 48.000-nucleotides and 70.000-nucleotides, such as between 50.000-nucleotides and 70.000-nucleotides, preferably between 53.000-nucleotides and 70.000-nucleotides, such as between 55.000-nucleotides and 70.000-nucleotides, for example between 58.000-nucleotides and 70.000-nucleotides, such as between 60.000-nucleotides and 70.000-nucleotides, preferably between 63.000-nucleotides and 70.000-nucleotides, such as between 65.000-nucleotides and 70.000-nucleotides, for example between 5.000-nucleotides and 65.000-nucleotides, such as between 5.000-nucleotides and 60.000-nucleotides, preferably between 5.000-nucleotides and 55.000-nucleotides, such as between 5.000-nucleotides and 50.000-nucleotides, for example between 5.000-nucleotides and 45.000-nucleotides, such as between 5.000-nucleotides and 40.000-nucleotides, preferably between 5.000-nucleotides and 35.000-nucleotides, such as between 5.000-nucleotides and 30.000-nucleotides, for example between 5.000-nucleotides and 25.000-nucleotides, such as between 5.000-nucleotides and 20.000-nucleotides, preferably between 5.000-nucleotides and 15.000-nucleotides, such as between 5.000-nucleotides and 10.000-nucleotides, for example between 5.000-nucleotides and 7.000-nucleotides.
The single stranded DNA molecule may have a length of at least 5.000-nucleotides, for example at least 7.000-nucleotides, preferably at least 10.000-nucleotides, such as at least 15.000-nucleotides, for example at least 20.000-nucleotides, preferably at least 25.000-nucleotides, such as at least 30.000-nucleotides, for example at least 35.000-nucleotides, preferably at least 40.000-nucleotides, such as at least 45.000-nucleotides, for example at least 50.000-nucleotides.
In one embodiment the single stranded DNA molecule may be produced using a phage and has a length in a range between 5.000-nucleotides and 10.000 nucleotides.
In one embodiment the single stranded DNA molecule may be produced using a hybrid virus and has a length in a range between 5.000-nucleotides and 55.000 nucleotides.
It may be beneficial to produce long single stranded DNA molecules, such as single stranded DNA molecules having a length of at least 30.000-nucleotides as these long molecules can carry a higher number of fluorophores compared to single stranded DNA molecules having a length in the range of between 5.000 and 10.000-nucleotides, such as at the most 30.000-nucleotides.
For example, a bead comprising a single stranded DNA molecule of 7.000 to 12.000-nucleotides may accommodate 1 to 200 fluorophores; a bead comprising a single stranded DNA molecule of 40.000 to 50.000-nucleotides may accommodate 1 to 1500 fluorophores.
Another way to increase the number of fluorophores comprised in a bead is the production of dimeric, such as trimeric structures through hydrophobic interactions and hydrogen bond interactions.
Thus, in one embodiment the bead comprises a dimeric structure based on DNA origami. In another embodiment, the bead comprises a trimeric structure based on DNA origami. However, it is also possible to produce larger oligomer structures such as tetrameric, pentameric, hexameric, heptameric and/or larger multiple oligomeric structures.
The structure based on DNA origami may have any shape and it may be flat or curved. A certain geometry may be preferred over other geometries. For example a geometry that hides the fluorophores inside the structure to prevent undesired oligomerization of structures through possible hydrophobic interactions between the fluorophores may be preferred. Thus, in one embodiment the structure based on DNA origami may be a sphere, an hexagon, a barrel, a tube or a combination of these structures.
In one embodiment the structure based on DNA origami may be a sphere.
In one embodiment the structure based on DNA origami may be a barrel or a tube.
In one embodiment the structure based on DNA origami may be an hexagon.
In one embodiment, the structure based on DNA origami may be a combination of the proposed geometrical structures.
In one embodiment, the structure based on DNA origami comprises a positioning domain ,a fluorescent domain and/or triggering domain
A positioning domain is a domain where a fluorophore is placed on the structure. It can be one of the staple strands that are fully complementary to the scaffold strand or it can have a single stranded overhang sequence that is not complementary to scaffold strand. The overhang is used to attach a fluorophore labelled oligo that is complementary to the overhang.
The fluorescent domain is the domain where the fluorophores are attached. Thus, the fluorescent domain comprises the predetermined number of fluorophores. In addition, each bead may comprise more than one different type of fluorophore, for example, each bead may comprise two or more different fluorophores or fluorescent domains, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 different fluorophores or fluorescent domains. In a preferred embodiment, the beads comprises 1-4 different fluorophores or fluorescent domain.
In another embodiment, the DNA based origami structure comprises a triggering domain.
The triggering domain is a domain where a different type of fluorophore or nanoparticle can be attached. The nanoparticle e.g. gold or silver nanoparticle can efficiently spread light. Thereby, these fluorophores or nanoparticles in the triggering domain can be used to trigger detection of a calibration bead.
An advantage of using beads comprising a structure based on DNA origami is that a precise and known number of fluorophores can be attached on the structure, and therefore accurate calibration of the measuring instrument, in this case of the flow cytometer, is possible.
In one embodiment the fluorescent domain comprises sticky ends and the fluorophores are conjugated to a DNA fragment complementary to said sticky ends, so that the fluorophores are attached to said sticky ends via said complementary DNA fragment.
A bead of the present disclosure comprises a predetermined number of fluorophores. Based on the length and the design of the structure based on DNA origami, the bead may comprise a certain maximum number of fluorophores.
In one embodiment the structure based on DNA origami comprises between 1 and 50 fluorophores, such as between 1 and 80 fluorophores, such as between 1 and 100 fluorophores, such as between 1 and 150 fluorophores, such as between 1 and 200 fluorophores, such as between 1 and 300 fluorophores, such as between 1 and 400 fluorophores, such as between 1 and 500 fluorophores, such as between 1 and 600 fluorophores, such as between 1 and 700 fluorophores, such as between 1 and 800 fluorophores, such as between 1 and 900 fluorophores, preferably at the most 1000 fluorophores, such as at the most 1200 fluorophores, such as at the most 1500 fluorophores, such as at the most 1800 fluorophores, such as at the most 2000 fluorophores, such as at the most 2200 fluorophores, such as at the most 2500 fluorophores, such as at the most 2700 fluorophores, such as at the most 3000 fluorophores.
In one embodiment the bead comprises a dimeric or trimeric structure based on DNA origami, and each monomer comprises between 1 and 1800 fluorophores.
In one embodiment the bead comprises a monomeric structure based on DNA origami and said structure comprises between 1 and 900 fluorophores, such as between 1 and 1500 fluorophores, preferably between 1 and 1800 fluorophores, such as between 1 and 2700 fluorophores, preferably at the most 3000 fluorophores.
In one embodiment the bead comprises a structure based on DNA origami, wherein said structure comprises a single stranded DNA molecule of at least 30.000-nucleotides and comprising between 1 and 1000 fluorophores.
In one embodiment the bead comprises a structure based on DNA origami, wherein said structure comprises a single stranded DNA molecule of at least 50.000-nucleotides and comprising between 1 and 1800 fluorophores.
Any fluorophore suitable for flow cytometry can be used. However, the preferred fluorophores are selected from small organic molecules.
In one embodiment, the small organic molecules are smaller than 5 nm.
In one embodiment, the fluorophore is selected from a group consisting of Quasar® 566 nm (Cy 3), Quasar® 670 nm (Cy 5), and fluorescein isothiocyanate (FITC).
In one embodiment the structure based on DNA origami comprises 120 fluorophores, for example 120 Cy5 fluorescence molecules.
In one embodiment the structure based on DNA origami comprises 242 fluorophores, for example 242 Cy5 fluorescence molecules.
Suitable fluorophores are known to the skilled person and include Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 647, ATTO 488 and ATTO 532 and 5/6 carboxy-tetramethyl rhodamine (TMR), 6-carboxyfluorescein (6-FAM), Alexa Fluor® 350, DY-415, ATTO 425, ATTO 465, Bodipy® FL, fluorescein isothiocyanate, Oregon Green® 488, Oregon Green® 514, Rhodamine Green™, 5′-Tetrachloro-Fluorescein, ATTO 520, 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluoresceine, Yakima Yellow™ dyes, Bodipy® 530/550, hexachloro-fluorescein, Alexa Fluor® 555, DY-549, Bodipy® TMR-X, cyanine phosphoramidites (cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.5), ATTO 550, Rhodamine Red™, ATTO 565, Carboxy-X-Rhodamine, Texas Red (Sulforhodamine 101 acid chloride), LightCycler® Red 610, ATTO 594, DY-480-XL, DY-610, ATTO 610, LightCycler® Red 640, Bodipy 630/650, ATTO 633, Bodipy 650/665, ATTO 647N, DY-649, LightCycler® Red 670, ATTO 680, LightCycler® Red 705, DY-682, ATTO 700, ATTO 740, DY-782, IRD 700 and IRD 800, CAL Fluor® Gold 540 nm, CAL Fluor® Gold 522 nm, CAL Fluor® Gold 544 nm , CAL Fluor® Orange 560 nm, CAL Fluor® Orange 538 nm, CAL Fluor® Orange 559 nm, CAL Fluor® Red 590 nm, CAL Fluor® Red 569 nm, CAL Fluor® Red 591 nm, CAL Fluor® Red 610 nm, CAL Fluor® Red 590 nm, CAL Fluor® Red 610 nm, CAL Fluor® Red 635 nm, Quasar® 570 nm, Quasar® 548 nm, Quasar® 566 nm (Cy 3), Quasar® 670 nm, Quasar® 647 nm, Quasar® 670 nm (Cy 5), Quasar® 705 nm, Quasar® 690 nm, Quasar® 705 nm (Cy 5.5), Pulsar® 650 Dyes, SuperRox® Dyes.
In one embodiment the structure based on DNA origami comprises a DNA scaffold of a length between 50.000 and 52.000-nucleotides, for example of a length of 51.466-nucleotides; a number of staple strands between 1000 and 2000, for example between 1500 and 1600, preferably 1550; and between 50 and 1800 fluorophores.
In one embodiment the structure based on DNA origami comprises a DNA scaffold of a length between 50.000 and 52.000-nucleotides, for example of a length of 51.466-nucleotides; a number of staple strands between 1000 and 2000, for example between 1500 and 1600, preferably 1550; and between 50 and 900 fluorophores.
In one embodiment the structure based on DNA origami comprises a DNA scaffold of a length between 50.000 and 52.000-nucleotides, for example of a length of 51.466-nucleotides; a number of staple strands between 1000 and 2000, for example between 1500 and 1600, preferably 1550; and between 50 and 1800 fluorophores, for example 80 fluorophores, for example 160 fluorophores, such as 900 fluorophores, such as 1800 fluorophores.
In one embodiment the structure based on DNA origami comprises a DNA scaffold of a length between 50.000 and 52.000-nucleotides, for example of a length of 51.466-nucleotides; a number of staple strands between 1000 and 2000, for example between 1500 and 1600, preferably 1550; and 80 fluorophores, for example 80 Cy5 fluorescence molecules.
In one embodiment the structure based on DNA origami comprises a DNA scaffold of a length between 50.000 and 52.000-nucleotides, for example of a length of 51.466-nucleotides; a number of staple strands between 1000 and 2000, for example between 1500 and 1600, preferably 1550; and 160 fluorophores, for example 160 Cy5 fluorescence molecules.
Method of Calibrating a Microfluidics System
Microfluidic systems involves setups with very small volumes of fluids, and microfluidic devices utilises the principles of microfluidics and can thereby exploit the physical and chemical properties of liquids and gases at the microscale. Many operations can be executed at the same time thanks to their compact size and the shortened experiment time while they still offer an excellent data quality.
The current invention relates to methods for using DNA origami in calibration of a microfluidics system.
So, one aspect the present disclosure relates to a method of calibrating a microfluidic device comprising:
Another aspect of the present disclosure relates to a method for determining the level of an antigen in a sample by a microfluidic technology comprising
In a preferred embodiment, the microfluidic system is a flow cytometer system, where structures based on DNA origami, wherein said structures comprise a predetermined number of fluorophores, are used for calibration; cf. herein below. However, any specific embodiment described herein below in respect of flow cytometer systems can also be applied to microfluidic systems in general.
Method of Calibrating a Flow Cytometer
In one aspect the present disclosure relates to a method of calibrating a flow cytometer comprising:
In one embodiment the method of calibrating a flow cytometer comprises providing a series of beads each comprising a different predetermined number of fluorophores. For example at least two beads may be provided, such as at least three beads, preferably at least four beads, even more preferably at least five beads may be provided, each bead comprising a different predetermined number of fluorophores.
For example, a series of 2 beads comprising 50 and 100 fluorophores, respectively, may be provided; preferably a series of 3 beads comprising 50, 100, 200 fluorophores, respectively, may be provided; preferably a series of 4 beads comprising 50, 100, 200 and 500 fluorophores, respectively, may be provided; preferably a series of 5 beads comprising 50, 100, 200, 500 and 1000 fluorophores, respectively, may be provided; for example a series of 6 beads comprising 50, 100, 200, 500, 1000 and 1500 fluorophores, respectively, may be provided.
Thus, the fluorescence of each bead will be measured according to step b. of the method above.
Although it is possible to calibrate a flow cytometer with the methods of the present disclosure by using only two beads having a structure based on DNA origami as disclosed herein, it is preferable to use more than two beads. In fact, the precision of the calibration will increase with the number of beads used.
When the method of calibrating a flow cytometer comprises providing a series of beads each comprising a different predetermined number of fluorophores, it may be beneficial that at least one bead comprises a number of fluorophores as close as possible to the sensitivity of the method.
For example, a first bead may comprise a number of fluorophores between 1 and 50; a second bead may comprise a number of fluorophores between 51 and 100; a third bead may comprise a number of fluorophores between 101 and 150; a fourth bead may comprise a number of fluorophores between 151 and 300; a fifth bead may comprise a number of fluorophores between 301 and 500. If a series of beads comprising a number of fluorophores as in this example are used, the data for a higher number of fluorophores may easily be extrapolated.
For example, a first bead may comprise a number of fluorophores between 1 and 50; a second bead may comprise a number of fluorophores between 51 and 100; a third bead may comprise a number of fluorophores between 101 and 300; a fourth bead may comprise a number of fluorophores between 301 and 750; a fifth bead may comprise a number of fluorophores between 751 and 1500; a sixth bead may comprise a number of fluorophores between 1501 and 2700.
In one embodiment the method of calibrating a flow cytometer comprises measuring the fluorescence of the at least two beads provided, such as of the at least three beads provided, preferably of the at least four beads provided, even more preferably of the at least five beads provided.
In one embodiment the method of calibrating a flow cytometer comprises calibrating the flow cytometer on the basis of the fluorescence measurements of the at least two beads provided, such as of the at least three beads provided, preferably of the at least four beads provided, even more preferably of the at least five beads provided.
The calibration of a flow cytometer according to the methods disclosed herein allows precise determination of the sensitivity of the instrument and generation of a calibration curve. The calibration curve allow us to correlate fluorescence intensity with the number of fluorescent molecules (fluorophores) on the entity of interest for a given fluorophore.
One advantage of the calibration method of the present disclosure is that, thanks to the use of beads comprising a structure based on DNA origami and a predetermined number of fluorophores, fluorescence can be determined with precision and a linear correlation between fluorescence and number of fluorophores is determined, allowing the establishment of a zero-point. The zero-point is a reference value, which functions as a measurement of the background fluorescence e.g. the auto-fluorescence of the utilized buffer.
Thanks to this zero-point, measurements performed with different flow cytometer systems can be compared. The zero-point, together with the calibration curve, also allows precise extrapolation of values based on the fluorescence signal.
Method for Determining the Level of an Antigen
The calibration of a flow cytometer setup performed using calibration beads as described herein will allow precise biochemical analysis of small biological particles e.g. extracellular vesicles, viruses and bacteria. Such analyses can be applied in academic, industrial as well as for clinical settings
Extracellular vesicles includes exosomes and microvesicles and are secreted by almost all cells. Extracellular vesicles are generally difficult to characterize at single particle level because of their small size. Exosomes are the smallest entities with a size of 30-150 nm. These are formed by inward budding of the endosomal membrane during maturation of large multivesicular endosomes. Fusion of multivesicular endosomes with the plasma membrane results in the release of exosomes into the extracellular space. Microvesicles are larger with a size of 50-500 nm. These are released into the extracellular space by outward budding directly from the plasma membrane. Extracellular vesicle cargo is very heterogeneous and can include nucleic acids, proteins and lipids. These extracellular vesicles play an important role mediating intercellular communication. The surface proteins embedded in these exosomes can reveal their subcellular origin, producer and recipient cell type. Characterization of these vesicles at single particle level will help to understand the intercellular communication code but also allow diagnosing e.g. tumor development and progression.
One aspect of the present disclosure relates to a method for determining the level of an antigen in a sample by a microfluidic system, such as flow cytometry comprising
In one embodiment, the method for determining the level of an antigen in a sample by flow cytometry as disclosed herein can detect as few as one single antigen in the sample provided.
In one embodiment, the sample comprises at least one antigen, such as at least 10 antigens, for example at least 25 antigens, preferably at least 50 antigens, wherein said antigen or said antigens are attached to one or more cells and/or vesicles and/or virus-like particles and/or viral particles and/or exosomes.
Method for Diagnosis of an Haematological Disease
One aspect of the present disclosure relates to a method for determining the presence or state of a haematological disease in an individual by a microfluidic system, such as flow cytometry, the method comprising
In one embodiment, the fluid is selected from a group consisting of blood, bone marrow, spinal fluid, bronchoalveolar lavage and serous effusions.
In one embodiment, the individual is human.
In one embodiment the method for diagnosis of a haematological disease by flow cytometry further comprises a step of comparing said level and/or concentration of said at least one antigen on the blood cell with a cut-off value,
wherein said cut-off value is determined from the concentration range of said at least one antigen in healthy individuals, such as individuals who do not present with a haematological disease, and
wherein a level and/or concentration that is lower or greater than the cut-off value indicates the presence or absence of said haematological disease.
The fluid sample can be a blood sample, and the blood sample can be full blood, but more preferably plasma, serum, cell-free or a sample comprising at least one blood cell or blood cell derived microvesicle.
For example the methods disclosed herein can be used for diagnosis of leukemia or lymphoma by performing an immunophenotyping of a fluid sample, such as by determining the level of certain markers expressed on cells of any of the above mentioned fluid samples.
For example the following leukemias and lymphomas may be diagnosed using the methods disclosed herein: acute myelogenous leukemia (or acute myeloid leukemia), acute lymphoblastic leukemia, chronic lymphocytic or myelocytic leukemias, B-cell and T-cell non-Hodgkin lymphomas, erythroleukemia (RBC leukemia), megaloblastic leukemia (platelets), and multiple myeloma.
In one embodiment, the level of any one of the following antigens may be determined: ZAP-70, HLA-DR, TdT, CD34, CD38, CD117, CD19, CD20, CD22, CD79a, immunoglobulin heavy (gamma, alpha, mu or delta) and light chains (kappa or lambda), CD10 (pre-B cell), CD2, CD3, CD5, CD7, CD4, CD8, MPO (myeloperoxidase), CD11, CD13, CD15, CD16b, CD33, CD66, CD11 b, CD16, CD56, CD31, CD36, CD41, CD42, CD61, CD235a, CD14, CD33, CD64, CD68, CD11c, and CD103. The skilled person will know which antigens are expressed by the different types of blood cells and which levels are expected in an individual affected by a leukemia or lymphoma; see for example Morice et al. 2004; Jaffe 1987; Hanson et al. 1999; Langerak et al. 2001.
Kit for Calibration of Flow Cytometry Instruments
One aspect of the present disclosure relates to a kit for calibration of microfluidic instruments, such as flow cytometry instruments comprising at least one bead having a structure based on DNA origami wherein said structure comprises a predetermined number of fluorophores.
In one embodiment of the present disclosure, the at least one bead is as disclosed herein in the section “Structure based on DNA origami”.
In one embodiment of the present disclosure, the kit comprises at least two beads, for example at least three beads, such as at least 4 beads, preferably at least 5 beads, wherein each bead comprises a different predetermined number of fluorophores, as described in the section “Structure based on DNA origami”.
Assembly of the Calibration Beads
Calibration beads are self-assembled into designed structure a circular single stranded scaffold strand (8064 bp) and 211 shorter single-stranded staple DNA strands. The scaffold strand is bought from Tilibit nanosystems and staple strands are produced chemically by Integrated DNA technologies. Staples strands bind to scaffold strand parts of the scaffold strand making it fold into a predesigned structure as described in (Rothemund 2006).
In the present case, the designed bead is assembled into a hexagonally shaped barrel (
Some of the staple strands are labelled with fluorophores (
Assembly of the Calibration Beads
Calibration beads are self-assembled into designed structure from a circular single-stranded DNA scaffold strand (51.466 bp) and 1550 shorter single-stranded staple DNA strands. The scaffold strand is produced biologically as described in (Marchi, Saaem et al. 2014) while staple strands are produced chemically. Staple strands bind to complementary parts of the scaffold strand making it fold into a predesigned structure as described in (Rothemund 2006).
In the present case, the designed bead is assembled into two hexagonally shaped structures forming an 8 number (
Alternative way of labelling structures: some of the staple strands contain additional DNA sequences without complementarity to the scaffold strand, so called sticky ends which serve as a docking sequence for fluorophore labelled DNA strands. Hence, fluorophore labelled strands have a complementary sequence to sticky ends and are labelled with a fluorophore (
Fluorophores Attachment
Fluorophore labelled strands are produced by chemically attaching a fluorophore to a DNA strand and subsequently purifying the conjugate using high pressure liquid chromatography. The number of strands in the structure containing sticky ends determines the number of annealed fluorophore strands which equals the number of fluorophores on a bead and can range from 1 to 4000. Labelled fluorophore strands dock onto complementary sticky ends that point into the structure (
Assembly of the Structure.
Design and assembly of DNA origami calibration beads. CadNano software (http://cadnano.org/) was used for generation of staple strand sequences AutoCAD Maya (http://www.autodesk.com/) was used for evaluation of the 3D shape and designing shape complementary domain for dimerization into fully assembled bead. The self-assembly of DNA origami structures was performed by mixing Single-stranded Scaffold DNA (p8064, tilibit nanosystems) with 10× excess of staple strands in 100 μL TAE/Mg2+ buffer (40 mM Trisacetate, 1 mM EDTA, pH=8.3, 12 mM Mg2+). Annealing of staple strands was done using a following ramp: 65° C. for 5 min, followed by a temperature rapid decrease to 50° C. and slow cooling for 200min for each 1° C. from 50-43° C. then rapid cooling to 20° C. In structures assembled with fluorophores, fluorophore strands were conjugated with a fluorophore in 5′end. Assembled structures were purified from excess of staple strands gel extraction. Purified structures were characterized by transmission electron microscopy and used directly for flow cytometry analysis.
Results
A Cytoflex (Beckman Coulter) cytometry system was used to test calibration beads assembled according to the description of example 1 above and labelled with none, 120 and 242 Cy5 fluorescence molecules. Cytoflex system showed a significant difference between the obtained fluorescence signal coming from unlabelled, labelled with 120 Cy5 fluorescence molecules and labelled with 242 Cy5 fluorescence molecules, respectively. Remarkably, the unlabeled calibration beads do not have any autofluorescence and the obtained fluorescence signal is equal to the signal of buffer. This allows to define a “0” point as a real point without any need for down extrapolation of the dataset. This allows the making of a calibration curve for flow cytometry systems that enables a precise quantification of flow cytometry output signals of low abundant antigens.
Bigger beads compared to the one of Example 1 can be assembled either by using a longer scaffold strand as described in Example 2 or by organizing the beads in dimers or trimers through hydrophobic interactions. This allows each bead to accommodate a larger number of fluorophores, such as up to 1500, 1800, 2000, 2500, 2700 fluorophores, and so a more diverse pool of beads to choose from for the calibration.
The quality of the synthesized beads is evaluated by cryo-TEM and agarose gel.
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Tables
| Number | Date | Country | Kind |
|---|---|---|---|
| 18154868.6 | Feb 2018 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2019/052640 | 2/4/2019 | WO | 00 |
