Patent Application
20220154260
The present invention relates to a method of immobilising an oligonucleotide on a polypropylene surface. The invention has utility in biomolecular assays, and in particular in hybridisation techniques where it can be used to attach single-stranded deoxyribonucleic acid (ssDNA) probes to the surface of polypropylene components such as microfluidic cassettes and plugs.
Microfluidic cassettes, or microfluidic flow cells, have a microfluidic structure in which one or more fluid channels are formed. The fluid channel typically comprises a flowable carrier medium, with which reagents or probes, housed in microchannels or chambers, or immobilised on the surface, are brought into contact for the purposes of analysis or testing. In most microfluidic systems, it is necessary to optimise flow of the carrier through the channels to ensure efficient throughput. This can be manipulated by careful selection of the material making up the microfluidic flow cell or components thereof.
While traditionally composed of silicon, and then glass, the use of polymeric materials in microfluidic applications has increased in recent years. For many applications, polycarbonate is now the preferred material for microfluidic applications involving biomaterials. The use of polycarbonate in microfluidic applications strikes a balance between flowability, and the ability to immobilise reagents on its surface.
Polypropylene is an inert, non-polar material which exhibits excellent flow characteristics. In addition, the low background fluorescence of polypropylene makes it well suited for techniques involving fluorescence detection. However, the inherent inert nature of polypropylene makes immobilisation on its surface difficult, thereby limiting its use in assays where attachment of reagents such as biomolecules, is often required. Hybridisation assays, for instance, typically require the flow of a medium over an immobilised single-stranded oligonucleotide, such as single-stranded DNA (ssDNA). Immobilisation of the ssDNA must be sufficiently stable to ensure that the strands of DNA remain attached to the surface during the performance of the assay, and must allow selective and sensitive detection in a reproducible manner. In addition, the immobilisation technique itself should be efficient and not require vast quantities or excesses of expensive materials, such as labelled biomolecules. To date, such immobilisation has proven challenging.
“Oligonucleotide” means a molecule which comprises a chain of nucleotides. Oligonucleotides are commonly in the form of DNA or ribonucleic acid (RNA). The term “oligonucleotide” includes polynucleotides of genomic DNA or RNA, complementary DNA (cDNA), and polynucleotides of semisynthetic, or synthetic origin. Standard nucleotide bases may also be substituted with nucleotide isoform analogs. In this context the term “oligonucleotide” is somewhat interchangeable with “polynucleotide” although generally refers to nucleotide chains of shorter length.
It is an object of the present invention to obviate or mitigate one or more of these problems, and in aspects of the invention, to provide an improved method of immobilising an oligonucleotide onto a polypropylene surface.
The present invention relates to a method of immobilizing an oligonucleotide on a polypropylene surface, the method comprising oxidising at least a portion of the polypropylene surface; and bringing an amine-terminated oligonucleotide into contact with the oxidised polypropylene surface in the presence of a carbodiimide coupling agent to immobilise the oligonucleotide to the oxidised polypropylene surface via the formation of amide bonds.
The invention also relates to a microfluidic cassette or portion thereof, e.g. a plug insertable to form part of a microfluidic channel, having an oligonucleotide immobilised thereon by the method of the invention.
Various further features and aspects of the invention are defined in the claims.
Embodiments of the present invention will now be described by way of example only with reference to the accompanying drawings where like parts are provided with corresponding reference numerals and in which:
The present invention relates to a method of immobilizing an oligonucleotide on a polypropylene surface, the method comprising oxidising at least a portion of the polypropylene surface; and bringing an amine-terminated oligonucleotide into contact with the oxidised polypropylene surface in the presence of a carbodiimide coupling agent to immobilise the oligonucleotide to the oxidised polypropylene surface via the formation of amide bonds.
According to the present invention, at least a portion of the polypropylene surface is oxidised. The polypropylene surface may be, for example, an internal or external surface of a cassette, slide, plate or any component of a microfluidic assay. As would be understood by one skilled in the art, in this embodiment, immobilisation of the oligonucleotide onto a portion of the surface only would be required, in order to allow the reagent to flow over the polypropylene surface and bind or react selectively with the immobilised oligonucleotide, such as in a hybridisation assay.
In an embodiment, the oxidising step is carried out using plasma activation, flame treatment, chemical oxidation or UV-ozone.
The plasma activation may be arc discharge, corona discharge, dielectric barrier discharge or piezoelectric direct discharge.
In an embodiment, the oxidising step is carried out using a low-pressure oxygen plasma system.
The parameters for performing the low-pressure oxygen plasma would be readily ascertained by one skilled in the art; however typically the process is carried out for from 1 minute to 15 minutes, with 70% to 100% generator power and from 2 sccm to 20 sccm, or from 2 sccm to 10 sccm oxygen, typically 5 sccm.
Typically, the low-pressure oxygen plasma activation generates multiple oxygen-containing groups such as hydroxyl, aldehyde and carboxylic acid groups, on the polypropylene surface. The carboxylic acid groups can react with the amine-terminated oligonucleotide in the presence of the carbodiimide coupling agent to form amide bonds. Advantageously, when oxygen plasma is used for functionalisation of the polypropylene surface, typically only oxygen-containing functional groups are generated, thereby minimising undesirable side-reactions which can result from the generation of other functional groups, such as nitrogen-containing groups which can result from ammonia plasma functionalisation. Oxygen plasma is also readily available and inexpensive, making it particular suitable for use in the method of the present invention.
Alternatively, the oxidising step can be carried out using chemical oxidation. Suitable chemical oxidation systems include chromium oxide, sodium dichromate, sulphuric acid, fuming nitric acid, potassium permanganate solution, ammonium peroxydisulphate solution, acid piranha solution, base piranha solution, methanol/hydrochloric acid; and combinations thereof.
In an embodiment, chemical oxidation can be performed using sodium dichromate and sulphuric acid.
Oxidation using acid etching leads to a high concentration of acid groups on the oxidised surface, making it particularly useful for the process of the present invention in which the carbodiimide coupling agent reacts with the acid groups, to form the active ester.
According to the invention, the amine-terminated oligonucleotide is brought into contact with the oxidised propylene surface. The amine-terminated oligonucleotide may be a labelled or unlabelled oligonucleotide.
In an embodiment, the oligonucleotide is single-stranded DNA (ssDNA).
In an embodiment, the ssDNA is a labelled DNA probe. Optionally, the labelled DNA probe is a fluorescent-labelled DNA probe.
For instance, the immobilised oligonucleotide can be amine-terminated ssDNA which can be incorporated in a hybridisation assay as a probe for target DNA. The ssDNA can be labelled, such as, for example, with a fluorescent cyanine dye, or a ruthenium dye. Polypropylene has low background fluorescence, and therefore the immobilisation of fluorescent labelled biomolecules to a polypropylene surface is particularly advantageous.
The terminal amine group may be a 5′ amine group or a 3′ amine group. In an embodiment, the terminal amine group is a 5′ amine group. Amine-terminated oligonucleotides can be purchased commercially or can be prepared using known methods.
According to the invention, the amine-terminated oligonucleotide is brought into contact with the oxidised polypropylene surface in the presence of a carbodiimide coupling agent to immobilise the oligonucleotide to the oxidised polypropylene surface via the formation of amide bonds.
A carbodiimide coupling agent is a coupling agent comprising an RN═C═NR functional group. In a coupling reaction, the carbodiimide functional group activates a carboxylic acid group to form an activated carboxylic ester intermediate, which reacts with an amine group to yield an amide and urea. A method of immobilising an oligonucleotide on a polypropylene surface according to the method of the present invention is illustrated schematically in
Carbodiimide coupling agents are known in the art and include, for example, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC); dicyclohexylcarbodiimide (DCC); diisopropylcarbodiimide (DIC); phenyl isopropyl carbodiimide (PIC); phenyl ethyl carbodiimide (PEC); N-tert-butyl-N′-methylcarbodiimide (BMC), N-tert-butyl-N′-ethylcarbodiimide (BEC), and bis[[4-(2,2-dimethyl-1,3-dioxolyl)]methyl]-carbodiimide (BDDC).
In an embodiment of the invention, the carbodiimide coupling agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, but also referred to in the art as EDAC or EDCI).
The step of bringing the amine-terminated oligonucleotide into contact with the oxidised polypropylene surface in the presence of a carbodiimide coupling agent is carried out under conditions sufficient to immobilise the amine-terminated oligonucleotide via the formation of amide bonds with oxygen-containing functional groups on the activated polypropylene surface.
Typically, the contacting step is performed in the presence of a buffer.
Suitable buffers would be known to one skilled in the art and include, for example, 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic add (MOPS), saline-sodium citrate (SSC), phosphate buffers, 2,2-Bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol (BIS-TRIS), piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), 3-morpholinopropanesulfonic acid (MOPSO), 1,3-Bis[tris(hydroxymethyl)methylamino]propane (BIS-TRIS propane), N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, N,N-Bis(2-hydroxyethyl)taurine (BES) and 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS).
In an embodiment, the buffer is 2-(N-morpholino)ethanesulfonic acid (MES).
In an embodiment, the contacting step is performed by printing the amine-terminated oligonucleotide onto the oxidised polypropylene surface. The printed surface may be allowed to incubate for a period of from less than 1 hour to 14 days, e.g. from 5 minutes to 14 days, from 5 minutes to 10 days. Preferably the incubation is allowed to take place for from 5 minutes to 24 hours. Incubation may take place under ambient temperature and humidity conditions. If a fluorescent probe is being used, the printed surfaces may be kept in darkness during any incubation period.
In an embodiment of the invention, the polypropylene surface is an internal surface of a microfluidic channel in a microfluidic cassette for use in a hybridisation array or a surface of a plug that forms part of a microfluidic channel. The ssDNA-immobilised PP surface may be used in a hybridisation assay.
It will be understood that the immobilisation of an oligonucleotide onto the internal surface can occur during the fabrication of a microfluidic cassette.
In an embodiment, the method further comprises the step of blocking the oligonucleotide-immobilised surface with a blocking agent.
According to this embodiment of the invention, non-specific binding of other biomolecules or other components of the assay to the activated polypropylene surface during performance of the assay can be minimised by blocking unoccupied binding sites with a blocking reagent. Any suitable blocking reagent, i.e. one which can reduce or eliminate non-specific binding, which does not participate in the reactions that form part of the assay, and which does not otherwise damage components of the assay, can be used.
Blocking agents which could be used include alkali and alkaline earth metal borohydrides such as sodium borohydride and lithium borohydride; aluminium borohydride, zinc borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, and acidic solutions of Zn2+ or Fe2+.
In an embodiment, the blocking agent is sodium borohydride.
In an embodiment, the method further comprises one or more washing steps. The washing steps may be performed prior to the blocking step. A detergent, such as sodium dodecyl sulfate (SDS) may be used in the washing step.
The following examples are intended to be illustrative only and are not intended to limit the scope of the invention.
Unlabelled DNA probes modified on the 5′ end with an amine termination, a C12 linker and 2 hexaethylene glycol spacer modifications were purchased from Integrated DNA Technologies (Illinois, USA).
Cy5 and Cy3 labelled DNA probes, modified on the 5′ end with an amine termination and a C6 linker were purchased from Integrated DNA Technologies (Illinois, USA).
A DNA ink for deposition was prepared as follows:
A 0.1 M MES (w/0.9% NaCl, pH 4.6) buffer solution was prepared as follows: 1.066 g of MES monohydrate was weighed into a 50 ml falcon tube. 45 ml of ultrapure water was added from a measuring cylinder. 0.450 g of NaCl was weighed in a weighing boat and added to the MES solution, which was shaken until complete dissolution occurred. The pH of the solution was measured, and adjusted to pH 4.6 using a 0.1 M or 1 M NaOH solution prepared from NaOH pellets (Sigma-Aldrich, product #221465). The remaining volume of ultrapure water needed to make up to 50 ml was added via pipette. The final pH was measured to confirm it fell within the range of pH 4.55 and pH 4.65. The solution was filtered through a 0.22 μm filter into a clean falcon tube. The buffer was stored in a fridge before use (up to 1 week maximum).
A 20 mM EDC solution in 0.1 M MES buffer was prepared as follows: 3.83 mg of EDC was dissolved in 1 mL of the 0.1 M MES buffer prepared in example 1.2.1 above, in a glass sample vial. The solution was used within 10 hours of its preparation.
When the solution was required, 160 μL of 20 mM EDC in 0.1 M MES buffer (section 1.2.2.1) was pipetted into an Eppendorf tube. 2 μL of glycerol was pipetted into the solution, followed by 18 μL of ultrapure water which was pipetted into the solution.
A 20 μl 100 μM (unlabelled) DNA probe solution was prepared as follows:
18 μl of the 17.78 mM EDC in 0.089 M MES buffer with 1.11% glycerol solution prepared in section 1.2.2.2 was pipetted into an Eppendorf tube. 2 μl of 1 mM unlabelled DNA probe was pipetted into the solution.
A 20 μl 50 μM (Cy5 or Cy3 labelled) DNA probe solution was prepared as follows:
18 μl of the 17.78 mM EDC in 0.089 M MES buffer with 1.11% glycerol solution prepared in section 1.2.2.2 was pipetted into an Eppendorf tube. 1 μl of ultrapure water was pipetted into the solution and then 1 μl of 1 mM Cy5 or Cy3-labelled DNA probe was pipetted into the solution.
Polypropylene (PP) slides (thinXXS, Zweibrücken, Germany) and polypropylene array plugs (thinXXS, Zweibrücken, Germany) were rinsed with isopropyl alcohol (IPA) from a squeezy bottle at a minimum of 1 ml IPA per cm2 of PP surface, and then dried using a stream of nitrogen until all visible traces of IPA had disappeared. The slides were then inserted into a glass holder and baked in the oven at 70° C., atmospheric pressure, for 15 minutes.
Although the PP surface was pre-cleaned in this specific example, the cleaning step is optional, and subsequent testing has confirmed that it is not an essential step.
The clean polypropylene samples (slides and array plugs) from example 1.3.1 were placed into a plasma asher (Diener™ Pico, Serial number 116299) with the PP surface to which immobilisation is required facing upwards. The plasma asher generator power was set to 90%, the O2 flow rate to 5 sccm, and the PP surface was activated for 15 minutes. Samples were then removed from the asher and placed in the printer chamber (Scienion SX, Berlin, Germany) until needed. All activated samples were used within 10 hours of activation.
10 minutes after plasma activation, the DNA ink solutions prepared in example 1.2 above were deposited onto the activated PP surfaces using a Scienion sciFLEXARRAYER SX printer (Scienion SX, Berlin, Germany). The humidity was set to 65% (cold mist), and the inks were printed with 2 drops per spot with a drop volume of between 200 pl and 220 pl. Immediately after deposition, the polypropylene slides and plugs were placed into a clean dish or tray, wrapped in foil, and left to incubate at ambient temperature and humidity for between 6 and 8 days.
A blocking solution was prepared by adding 15 ml 10× phosphate-buffered saline (PBS), 135 ml ultrapure water and 50 ml ethanol to a 500 ml Duran bottle. 0.5 g of sodium borohydride (Sigma, product code 213462) was added to the solution. The solution was stirred for 15 minutes.
Five reservoirs were prepared with 1) 0.2% SDS, 2) ultrapure water; 3) the blocking solution prepared above; 4) 0.2% SDS and 5) ultrapure water. Immobilised slides and plugs prepared above were placed into the first reservoir, left for 2 minutes, then agitated by lifting up and down before being left in the reservoir for a further 2 minutes. The slides and plugs were then placed into the second reservoir containing clean ultrapure water, in a similar fashion (i.e. being left for 2 minutes, agitated, then left for 2 minutes). The slides and plugs were then placed into the third reservoir containing the blocking solution where they were left for 15 minutes. The slides and plugs were then placed into the fourth solution, i.e. a clean 0.2% SDS solution where they were left for 2 minutes, then agitated by lifting up and down, before being left in the reservoir for two minutes, and then they were placed into the fifth reservoir, which contained fresh ultrapure water, where they were again left for 2 minutes, then agitated by lifting up and down, before being left in the reservoir for two minutes. The slides and plugs were then dried using nitrogen. The slides or plugs were not allowed to dry between each step, and clean solutions were used for each reservoir.
A warfarin assay was printed using the procedure outlined above. Three array plugs were hybridised with a PCR amplified swab sample. This swab sample is wild type homozygous in the *2 SNP and heterozygous in the *3 and VK SNPs.
A standard DNA hybridisation assay was carried out using ssDNA immobilised on polypropylene according to the invention, with cyanine-labelled ssDNA used as a control. Tests were performed both for immobilised ssDNA which had not subsequently been subjected to a blocking step, and for immobilised ssDNA which had been subjected to a blocking step after 7 days incubation as outlined in Example 1.6. Measurements were taken at two exposure times, 600 ms and 800 ms. The results of this analysis are shown in
The experimental results demonstrate that the immobilisation technique according to the invention allows for selective and sensitive detection to take place, with minimal background signal for fluorescence measurements. The optional blocking step results in increased clarity for the microarray, as evidenced by the microarray spots in
The method of the invention therefore represents a convenient technique for the immobilisation of amine-terminated oligonucleotides onto polypropylene, using readily available instrumentation and reagents, and which results in sufficient reproducible immobilisation for use in hybridisation techniques.
All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. Each feature disclosed in this specification (including any accompanying claims, abstract and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features. The invention is not restricted to the details of the foregoing embodiment(s). The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.
With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity.
It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations).
It will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope being indicated by the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1900260.9 | Jan 2019 | GB | national |
This application is the U.S. National Phase Application under 35 U.S.C. § 371 of International Application No. PCT/GB2019/053639, filed Dec. 19, 2019, designating the U.S. and published in English as WO 2020/144456 A1 on Jul. 16, 2020, which claims the benefit of Great Britain Application No. GB 1900260.9, filed Jan. 8, 2019. Any and all applications for which a foreign or a domestic priority is claimed are identified in the Application Data Sheet filed herewith and are hereby incorporated by reference in their entireties under 37 C.F.R. § 1.57.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2019/053639 | 12/19/2019 | WO | 00 |
