DNA printing methods and compositions

Abstract

A composition for printing a biological material on a substrate for an assay comprises a biological material, wherein the biological material comprises a DNA-containing material; and a viscosity modifier, wherein the viscosity modifier is at a concentration of less than about 30% wt% in the composition.

Description

[0001] DNA printing methods and compositions

[0002] Field

[0003] The present disclosure relates to a composition for printing a biological material, for example dsDNA, on a substrate for an assay such as a microarray.

[0004] A microarray is a two-dimensional array of a biological material, such as proteins, DNA, antigens or antibodies, lipids, or peptides, deposited and immobilised on a solid and typically flat substrate, typically made of functionalized glass or plastic. The substrate is generally selected to provide adequate binding with the biological material, but also to avoid any modification of the material, such as denaturation in the case of a protein.

[0005] Typically, once the microarray of biological material has been deposited on the substrate, a blocking buffer is applied to the microarray surface to prevent unwanted materials binding to the surface of the substrate. The microarray is then reacted with a sample, and detection is carried by a suitable microarray analysis technique, for example dark field microscopy. An example of such microarray-based technology used for blood grouping and donor disease screening is MosaiQ™ by AliveDx.

[0006] Deposition of the biological material is typically carried out by printing, for example using a system such as MosaiQ™ Manufacturing System (MMS), Arrayjet’s Inkjet Microarray Technology or Scienion’s sciFLEXARRAYER. However, printing requires the printed solution to have a viscosity within a workable range, typically a dynamic viscosity of at least 1 mPa.s, e.g. at least 2 mPa.s, and / or of about 1-20 mPa.s, e.g about 1-10 mPa.s, e.g. about2-9 mPa.s, e.g. about 4-9 mPA.s, e.g. about 6-9 mPa.s. To that end, a viscosity modifier is typically included in the printing composition. It will be appreciated, however, that the optimum viscosity for a given printed solution may depend on the type of printing equipment used.

[0007] In order to ensure that the deposited biological material is immobilized on the substrate upon printing, it is desirable for the biological material to interact with, e.g. bind to, the substrate. Factors influencing binding of the biological material to the substrate include: the type of biological material; the type of substrate; pH; ionic strength; viscosity.

[0008] The pH of the biological material composition is typically controlled using a buffering agent, such as a phosphate, a carbonate or an acetate buffer. The buffering agent may typically be provided at a concentration of about 5-300 mM in the composition.

[0009] The ionic strength of the biological material composition is typically controlled using a salt, such as NaCI. The salt may typically be provided at a concentration between about 0 and 5M. A typical salt concentration may be around 150 mM, as this may correspond to the osmolality of cell cytoplasm.

[0010] The viscosity of the biological material composition is typically controlled using a viscosity modifier, such as sucrose, trehalose, or glycerol. In particular, printing of proteinic molecules typically requires the presence of a viscosity modifier to avoid quick drying of the liquid material deposited on the substrate, which could have a negative impact on the protein integrity. The viscosity modifier may typically be provided at a concentration of about 30-60 % w / v or w / w, depending on the type of viscosifier.

[0011] Typical substrates have a coating that includes reactive groups which react, e.g. covalently, with the biological material, in order to promote binding. An example of such a substrate is a substrate, e.g. glass, coated with an epoxy-containing material such as an epoxysilane material. Such substrates as commonly referred to as “epoxy glass”. An advantage of these substrates is that the epoxy groups in the coating may react, typically covalently, with a number of chemical groups that are typically present in many biological materials, such as amino (secondary amines), hydroxyl, or thiol moieties, thereby enabling immobilization upon printing. For example, molecules of protein origin are capable of binding to epoxy groups via positively charged amino acids such as Arginine, Histidine or Lysine.

[0012] However, certain biological materials either do not include free reactive moieties capable of reacting with the reactive groups, e.g. epoxy, in the coating, or have reactive moieties whose reactivity is diminished or inhibited due to the conformation of the biological material. For example, double-stranded DNA (dsDNA) displays only phosphate negative charges on the surface of its helical structure, which tends to limit potential covalent binding to epoxy groups in the coating.

[0013] US 2004 / 0054160 A1 (Pal) discloses a medium or ink solution containing nucleic acid, which contains 30%-80% by volume of an organic solution comprising DMSO, ethylene glycol, formamide, or a combination thereof. Deng et al (Viscosity effects on micro bubble actuator in ssDNA solutions, Micro Electro Mechanical Systems, 2004) discloses an investigation of the viscosity effects on dynamic behavior of the micro bubble actuator under pulse heating, by adding sucrose in a single-stranded DNA (ssDNA) solution to change its viscosity.

[0014] WO 02 / 059372 A2 (Fagnani et al discloses a biochip formed with optically clear hydrogel cells on the surface of a solid substrate.

[0015] It is an object of the present invention to obviate and / or mitigate the limitations and / or disadvantages associated with the prior art and / or with conventional methods and systems.

[0016] It is an object of the present invention to provide a printing composition, e.g. a printing buffer, that promotes immobilisation of a biological material, e.g. of a DNA material such as dsDNA, on a substrate, e.g. functionalised glass.

[0017] As explained above, the binding between a deposited biological material and a substrate depends on a number of factors, including for example the type of biological material, the type of substrate, pH, ionic strength, and viscosity. In addition, viscosity also influences the printability of the composition comprising the biological material being printed.

[0018] The present invention is based on the surprising findings that the binding between a DNA material, e.g. dsDNA, and a substrate, e.g. a functionalised substrate such as epoxy glass, may be significantly improved by reducing the concentration of the viscosity modifier in the printing composition below concentration levels typically used in printing buffers. This is surprising because, as explained above, viscosity modifiers are considered to be essential to achieve a viscosity in the printing composition adequate to undergo printing. Without wishing to be bound by theory, it is believed that the macromolecule nature of DNA materials, e.g. dsDNA, allows the composition to remain printable despite a much lower viscosifier concentration in the composition than in conventional samples.

[0019] According to a first aspect there is provided a composition for printing a biological material on a substrate for an assay, the composition comprising: a biological material; and a viscosity modifier, wherein the viscosity modifier is at a concentration of less than about 30 wt% in the composition. The concentration of the viscosity modifier may be up to about 25 wt%, e.g. up to about 20 wt%, e.g. up to about 15 wt%, e.g. up to about 10 wt%, e.g. up to about 5% wt, e.g. up to about 9 wt%, e.g. up to about 8 wt%, in the composition.

[0020] The concentration of the viscosity modifier may be in the range of about 0.1-25 wt%, e.g. 0.1-20 wt%, e.g. 0.1-15 wt%, e.g. 0.1-10 wt%, e.g. 0.1-5 wt%, in the composition. Typically, the concentration of the viscosity modifier may be in the range of about 0.1-10 wt%, e.g. about 1-8 wt%, e.g. about 1-7.5 wt%, e.g. about 2.5-7.5 wt%, in the composition.

[0021] The viscosity modifier may comprise a carbohydrate, e.g. a monosaccharide, disaccharide, or oligosaccharide. Typically, the viscosity modifier may comprise a disaccharide, e.g. one or more compounds selected from the list consisting of sucrose, trehalose, lactose, maltose, cellobiose, and chitobiose. In an embodiment, the viscosity modifier may comprise or may consist of sucrose and / or trehalose.

[0022] The viscosity modifier may comprise a polyol such as glycerol.

[0023] The weight concentrations of the viscosifier may be expressed as %w / v or %w / w in the composition. For example, when the viscosity modifier comprises a carbohydrate such as sucrose or trehalose, the concentration may be in %w / w or %w / v. When the viscosity modifier comprises a polyol such as glycerol, the concentration may typically be in %w / v.

[0024] In an embodiment, the viscosity modifier may comprise a carbohydrate, e.g. a disaccharide such as sucrose or trehalose.

[0025] Typically, the weight percentage of the viscosity modifier may be expressed as w / v.

[0026] The concentration of the viscosity modifier, e.g. disaccharide, may be up to about 10% w / v, e.g. up to about 9 %w / v, up to about 8% w / v, e.g. up to about 7% w / v, e.g. up to about 6% w / v, e.g. up to about 5% w / v, e.g. up to about 4% w / v, e.g. up to about 3% w / v, e.g. up to about 2% w / v, e.g. up to about 1 % w / v, e.g. up to about 0.1 % w / v, e.g. up to about 0.01 % w / v. The concentration of the viscosity modifier may be in the range of about 0.01-9% w / v, e.g. about 0.01-8% w / v, e.g. about 0.1-8% w / v, e.g. about 1-7.5% w / v, e.g. about 1-5% w / v.

[0027] It will be understood that w / v concentrations can be converted to w / w concentrations and vice versa. For example, for sucrose, conversions are illustrated in

[0028] Table 1 below.

[0029] Table 1 : w / w to w / v conversions for an aqueous sucrose solution

[0030] The composition may comprise a buffering agent, such as a phosphate, a carbonate or an acetate buffer. The buffering agent may typically be provided at a concentration of about 5-300 mM, e.g. about 10-250mM, e.g. about 20-200 mM in the composition.

[0031] The composition may comprise a salt, typically an inorganic salt. By such provision, the salt may affect the ionic strength of the composition, but may have limited or no reactivity with the biological material. Typically, the salt may comprise one or more compounds selected from the list consisting of sodium chloride (NaCI), calcium chloride (CaCh), magnesium chloride (MgCh), sodium bicarbonate (NaHCCh), potassium chloride (KCI), sodium sulphate (Na2SO4), calcium carbonate (CaCCh), and calcium phosphate (Cas(PO4)2. Conveniently, the last may comprise or may consist of NaCI.

[0032] The salt may typically be provided at a concentration between about 0 and 5M, e.g. between about 0.025M and 4M, e.g. between about 0.1 and 4M, e.g. between about 0.5 and 4M, e.g. between about 1 and 2M. For example, when the biological material comprises a DNA material, the salt may typically be provided at a concentration between about 1 and 4M, e.g. between about 1.5 and 2M, in the composition. It was surprisingly found that the binding between a DNA material, e.g. dsDNA, and a substrate, e.g. a functionalised substrate such as epoxy glass, may be significantly improved by increasing the concentration of the salt in the printing composition, e.g. above about 0.5, 1 or 1.5M. The composition may comprise a carrier or solvent, typically water such as distilled water, deionised water or the like.

[0033] The composition comprises a biological material. The term “biological material” will be herein understood as encompassing any biological material, whether naturally or synthetically made.

[0034] Typically, the biological material may comprise a peptide, a protein (including recombinant proteins), an amino-acid, a nucleic acid (DNA and / or RNA), an oligonucleotide, a lipid, a carbohydrate, an enzyme, a metabolite, an antibody (including monoclonal and / or polyclonal antibodies and any (antigen binding) fragments thereof), an antigen, cells, red blood cells, plasma, serum or the like.

[0035] The biological material may comprise a DNA-containing material.

[0036] The DNA-containing material may comprise or may consist of a DNA material, which may optionally further comprise a non-DNA material such as a protein or the like. The DNA-containing material may comprise or may consist of a DNA complex.

[0037] The biological material may comprise or may consist of chromatin.

[0038] The biological material may comprise or may consist of a DNA material.

[0039] The biological material may comprise or may consist of dsDNA. The biological material may comprise or may consist of plasmid dsDNA and / or linear dsDNA. Advantageously, it was surprisingly discovered that, when the biological material comprises or is a DNA material such as dsDNA or chromatin, the binding between the DNA material and the substrate may be enabled and / or improved, despite the concentration of the viscosity modifier being much lower than in conventional printing buffers, particularly in certain types of printing equipment such as MMS. Without wishing to be bound by theory, it is believed that this effect may be due to the non-Newtonian characteristics of DNA in relation to viscosity.

[0040] Typically, the substrate may be made of glass, silicon, or a polymer such as nitrocellulose.

[0041] The substrate may optionally be coated with a coating layer which may be selected so as to improve or alter properties of or interaction with the biological material, including adhesion, immobilisation, stabilisation, etc. The coating layer may comprise, may consist essentially of or may consist of a metal such as aluminium or gold, or a polymer such as hydrophilic polymers or hydrophilic polymer, e.g. polyacrylamide, epoxysilane, or the like. Preferably, the substrate may comprise or may be glass coated with an epoxycontaining material, e.g. an epoxysilane material. This type of substrate may be referred to as ‘epoxy glass’.

[0042] The substrate may be referred to as a ‘chip’.

[0043] The assay may comprise or may be a microarray.

[0044] Typically, the size, e.g. diameter, of the discrete amounts, e.g. spots, of biological material immobilised to the surface of the substrate may be approximately 100pm - 300pm, e.g. about 150pm - 250pm, typically about 210pm + / -40 pm.

[0045] According to a second aspect there is provided a method of printing a biological material on a substrate for an assay, the method comprising: providing a composition comprising a biological material; and a viscosity modifier, wherein the viscosity modifier is at a concentration of less than about 30 wt% in the composition; and printing the composition on the substrate.

[0046] The features relating to the composition described in the first aspect may equally apply here, and are not repeated, merely for brevity.

[0047] Typically, the substrate may comprise or may be glass coated with an epoxycontaining material, e.g. an epoxysilane material. This type of substrate may be referred to as ‘epoxy glass’.

[0048] The method may comprise drying the composition.

[0049] The method may comprise printing the composition at one or more discrete locations on the substrate, e.g. on a surface thereof.

[0050] The method may comprise printing the composition at one or more discrete locations defining an array, e.g. a microarray.

[0051] The array may be or may define a one-dimensional, two-dimensional, or three dimensional array. Typically, the array may be or may define a two-dimensional array on a surface thereof.

[0052] Typically, a surface of the substrate, e.g. a surface on which the composition is printed and / or immobilized, is flat or planar or is substantially flat or planar. The terms “flat” and “planar” will be understood to refer to the shape of the surface at a macroscopic level, i.e., it be herein understood to mean that the surface does not include any wells or grooves that may be configured to receive a substance or composition. Rather, the substance or composition is printed and / or immobilised at different and / or discrete locations of the substrate on an otherwise substantially continuous surface. At least a portion of the substrate, e.g. a portion of the surface of the substrate configured to receive the composition or other substances, may be free of wells or recesses.

[0053] The method may further comprise blocking materials printed on the substrate, e.g. blocking the biological material of the composition, and / or any other substances printed and / or immobilised on the substrate.

[0054] The method may comprise spraying a blocking composition onto at least a portion of the substrate, e.g. on the biological material.

[0055] The blocking composition may comprise, may consist of or may consist essentially of a blocking buffer.

[0056] According to a third aspect, there is provided the use of a composition for printing on a substrate for an assay, wherein the composition comprises: a biological material; and a viscosity modifier, wherein the viscosity modifier is at a concentration of less than about 30 wt% in the composition.

[0057] The features relating to the composition described in the first aspect may equally apply here, and are not repeated, merely for brevity.

[0058] The biological material may comprise a DNA-containing material, e.g. dsDNA and / or chromatin.

[0059] The viscosity modifier may comprise a carbohydrate, e.g. a disaccharide such as sucrose or trehalose.

[0060] The substrate may comprise or may be glass coated with an epoxy-containing material, e.g. an epoxysilane material. This type of substrate may be referred to as ‘epoxy glass’.

[0061] For the avoidance of doubt, any feature described in respect of any aspect of the invention may be applied to any other aspect of the invention, in any appropriate combination. For example, method or use features may be applied to composition features and vice versa. Brief Description of the Drawings

[0062] The present invention will now be further described in detail and with reference to the figures in which:

[0063] Figure 1 is a graph showing the effect of the nature of the viscosifier in a printing buffer on signal differentiation between a sample negative for dsDNA antibodies (‘Pool NegT) and three samples positive for dsDNA antibodies (HM060, HM061 , WHO), when printing on Scienion;

[0064] Figure 2 is a graph showing the effect of the nature of the viscosifier in a printing buffer on signal differentiation between three samples negative for dsDNA antibodies (‘NEG’ in green) and three samples positive for dsDNA antibodies (HM060, HM061 , WHO - ‘POS’ in orange), when printing on MMS;

[0065] Figure 3 is a graph showing differentiation between a sample negative for dsDNA antibodies (Neg 1) and a sample positive for dsDNA antibodies (WHO), for various concentrations of glycerol viscosifier in a printing buffer;

[0066] Figures 4a and 4b are graphs illustrating POS / NEG discrimination, and SNR (Positive to Negative Ratio), respectively, for various samples positive or negative for dsDNA antibodies using different concentrations of trehalose viscosifier in a printing buffer;

[0067] Figure 5 is a graph showing differentiation in signal between samples negative for chromatin antibodies (‘NEG’ in green) and samples positive for chromatin antibodies (‘HIGH POS’ in orange), for different types of buffer;

[0068] Figure 6 is a graph showing differentiation in signal between samples negative for chromatin antibodies (‘NEG’ in green) and samples positive for chromatin antibodies (‘POS’ in orange), for different concentrations of buffer;

[0069] Figure 7 is a graph showing differentiation in signal between samples negative for chromatin antibodies (‘NEG’ in green) and samples positive for chromatin antibodies (‘POS’ in orange), for high concentrations of salt at 2M and 4M;

[0070] Figures 8a and 8b are graphs illustrating signal intensity, and SNR (Positive to Negative Ratio) respectively, for samples characterised for chromatin antibodies using lower concentrations of salt at 275mM and 2M;

[0071] Figures 9a and 9b are graphs illustrating signal intensity, and SNR respectively when detecting dsDNA antibodies using plasmid dsDNA or linear dsDNA as printed capture agent. Detailed Description of the Drawings

[0072] In the present disclosure, reference is made to a number of terms, which have the meanings provided below, unless a context indicates to the contrary. The nomenclature used herein for defining compounds, in particular the compounds according to the invention, is in general based on the rules of the IIIPAC organisation for chemical compounds, specifically the “IIIPAC Compendium of Chemical Terminology (Gold Book)”. For the avoidance of doubt, if a rule of the IIIPAC organisation is in conflict with a definition provided herein, the definition herein is to prevail. Furthermore, if a compound structure is in conflict with the name provided for the structure, the structure is to prevail.

[0073] The term “comprising” or variants thereof is to be understood herein to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0074] The term “consisting” or variants thereof is to be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, and the exclusion of any other element, integer or step or group of elements, integers or steps.

[0075] The term “about” herein, when qualifying a number or value, is used to refer to values that lie within ± 5% of the value specified. For example, if a temperature is specified to be about 5 to about 13 °C, temperatures of 4.75 to 13.65 °C are included.

[0076] Reference to physical states of matter (such as liquid or solid) refer to the matter’s state at 25 °C and atmospheric pressure unless the context dictates otherwise.

[0077] As explained above, the present inventors have discovered that the binding between a DNA material, e.g. dsDNA, and a substrate, e.g. a functionalised substrate such as epoxy glass, may be significantly improved by reducing the concentration of the viscosity modifier in the printing composition below concentration levels typically used in printing buffers. This is surprising because viscosity modifiers are considered to be essential to achieve a viscosity adequate to undergo printing. Without wishing to be bound by theory, it is believed that the macromolecule nature of DNA materials, e.g. dsDNA, allows the composition to remain printable despite a much lower viscosifier concentration in the composition than in conventional samples. Examples

[0078] Experimental

[0079] Materials Table 2: Antigens

[0080] Table 3: Reference samples

[0081] Table 4: Consumables

[0082] Methods

[0083] Scienion rapid prototyping

[0084] The sciFLEXARRAYER S3 is an automated instrument, using ultra-low volume liquid handling of various types of samples in diagnostics, genomic, proteomics, and technical applications.

[0085] This printer provides, in a relatively fast turnaround time, printed slides with the ability to evaluate several conditions at once. Printing using Scienion is performed on glass with dimensions similar to a microscope slide.

[0086] The Scienion printer does not allow for a higher glycerol content than 30% (w / v). This is different from MMS print line which typically requires a level of glycerol of 30-50% (w / v).

[0087] No preservative layer is added on the slide after a print. A blocking (using a blocking fluid) occurs only during a manual assay.

[0088] Results

[0089] A. Printing buffer

[0090] As mentioned above, printing of a biological material composition onto a microarray typically requires the printed composition to have a viscosity within a workable range for the printing equipment to function as intended, typically a dynamic viscosity above 1 mPa.s, typically between about 1 and 10 mPa.s, e.g. between about 2 and 9 mPa.s. To that end, a viscosity modifier is typically added to the printing composition. In particular, printing of proteinic molecules typically requires the presence of a viscosity modifier to avoid quick drying of the liquid material deposited on the substrate, which could have a negative impact on the protein integrity. The viscosity modifier may typically be provided at a concentration of about 30-60 % w / v.

[0091] A1 Type of viscosifier

[0092] A dsDNA composition was printed using Scienion, using three different viscosity modifiers (glycerol, sucrose, and trehalose), at the maximum concentration possible for each viscosifier when printing on Scienion (30% for glycerol, 40% for each of sucrose and trehalose). Each sample contained the same concentration of linear dsDNA (100ug / mL) and the same phosphate printing buffer containing 2M NaCI and 1 % w / v sucrose.

[0093] The results are shown in Figure 1. Figure 1 shows that positive / negative discrimination (i.e. , the difference between the signal for negative samples vs the signal for positive samples) is greater with sucrose and with trehalose, compared to glycerol. Therefore, it can be seen that selecting a carbohydrate compound such as sucrose or trehalose, may be advantageous, compared to a polyol compound such as glycerol. A dsDNA composition was then printed using MMS followed by MosaiQ testing, using three different viscosity modifiers (glycerol, sucrose, and trehalose), at the (40% w / v for glycerol, 60% w / v for each of sucrose and trehalose). Each sample contained the same concentration of plasmid dsDNA (100ug / mL) and the same phosphate printing buffer containing 2M NaCI and 1 % w / v sucrose.

[0094] The results are shown in Figure 2. Figure 2 shows that there was no signal and no Pos / Neg discrimination with glycerol. Positive / negative discrimination was observed with sucrose or trehalose. This confirms that selecting a carbohydrate compound such as sucrose or trehalose, may be advantageous, compared to a polyol compound such as glycerol.

[0095] A2 Concentration of viscosifier

[0096] A dsDNA composition was printed using Scienion followed by manual assay, using three different concentrations of glycerol. Each sample contained the same concentration of linear dsDNA (100ug / mL) and the same phosphate printing buffer containing 2M NaCI and 1 % w / v sucrose.

[0097] The results are shown in Figure 3. Figure 3 shows that there was no pos / neg differentiation using a 30% w / v glycerol concentration. This indicates that 30% glycerol condition is not reliably reproducible. However, lower concentration of 5, 1 and 0% w / v glycerol led to satisfactory pos / neg discrimination.

[0098] A dsDNA composition was then printed using MMS followed by MosaiQ testing, using different concentrations of trehalose. Each sample contained the same concentration of plasmid dsDNA (100ug / mL) and the same phosphate printing buffer containing 2M NaCI and 1 % w / v sucrose.

[0099] The results are shown in Figures 4a and 4b.

[0100] Figure 4a shows the data presented as average signal minus background, and shows that pos / neg discrimination is increased at lower concentrations of trehalose, particularly at concentrations of trehalose of 20% w / v and lower.

[0101] Figure 4b shows data presented as positive to negative ratio (the higher pos / neg ratio is the better the assay performance). Again, this graph illustrates improved performance at lower concentrations of trehalose, particularly at concentrations of trehalose of 20% w / v and lower. B. Buffer

[0102] B1 type of buffer

[0103] In this section, the effect of the buffer selection was investigated.

[0104] A DNA composition was printed using Scienion followed by manual assay, using three different types of buffer, namely acetate, phosphate and carbonate. Each sample contained the same concentration of nucleosome (chromatin) (100ug / mL) and the same viscosity modifier (10% w / v sucrose), with 150 mM NaCI.

[0105] The results are shown in Figure 5. Figure 5 shows that all three types of buffer provided adequate pos / neg discrimination, and that the choice of buffer therefore is not a critical parameter for performance of the dsDNA antibody detection assay, when printing a DNA material as capture agent.

[0106] B2 Concentration of buffer

[0107] A DNA composition was printed using Scienion followed by manual assay, using two different concentrations of buffer, namely 10mM and 190mM carbonate buffer. Each sample contained the same concentration of nucleosome (chromatin) (100ug / mL) and the same viscosity modifier (1% w / v sucrose). Both buffers contained 300 mM NaCI.

[0108] The results are shown in Figure 6. Figure 6 shows that both concentrations led to adequate pos / neg discrimination, and that the concentration of buffer therefore is not a critical parameter for performance of the dsDNA antibody detection assay, when printing a DNA material as capture agent.

[0109] C. Salt concentration

[0110] The potential effect of salt concentration on DNA binding was investigated.

[0111] A first experiment was carried out on the Scienion prototyping system, using samples of nucleosome (chromatin) (50ug / mL) and a printing buffer containing 24mM phosphate, 1 % w / v sucrose 10%w / v trehalose and NaCI at a concentration of either 2M or 4M.

[0112] The results are shown in Figure 7. Figure 7 shows that both 2M and 4M NaCI concentrations led to adequate pos / neg discrimination.

[0113] Another experiment was carried out, also on the Scienion prototyping system, using samples of dsDNA (100ug / mL) and a printing buffer containing 24mM phosphate, 1 % w / v trehalose and NaCI at a concentration of either 275mM or 2M. The results are shown in Figures 8a and 8b.

[0114] Figure 8a shows the data presented as average signal minus background, and shows that pos / neg discrimination is increased at higher concentrations of NaCI (i.e. at higher ionic strength).

[0115] Figure 8b shows data presented as positive to negative ratio (the higher pos / neg ratio is the better the assay performance), confirming an improved performance at a concentration in NaCI of 2M compared to 275mM.

[0116] D. dsDNA Type (linear and plasmid)

[0117] The aim of this study was to compare the results obtained between plasmid and linear dsDNA.

[0118] The following probes were used:

[0119] - Linear dsDNA 100 ug / ml in a printing buffer containing 24mM phosphate, 2M NaCI and 1% w / v sucrose, with 0% trehalose added.

[0120] - Plasmid dsDNA 100 ug / ml in a printing buffer containing 24mM phosphate, 2M NaCI and 1% w / v sucrose, with 0% trehalose added.

[0121] The results are shown in Figures 9a and 9b.

[0122] Figure 9a shows the data presented as average signal minus background, and shows that although both types provide satisfactory results, the signals of linear dsDNA were higher than those of plasmid dsDNA for the positive samples (Figure 8).

[0123] Figure 9b shows data presented as positive to negative ratio (the higher pos / neg ratio is the better the assay performance), confirming that linear dsDNA generated slightly greater SNR than plasmid dsDNA, although both generated adequate SNR results.

Claims

CLAIMS:1 . A method of printing a biological material on a substrate for an assay, the method comprising: providing a composition comprising a biological material, wherein the biological material comprises a DNA- containing material; and a viscosity modifier, wherein the viscosity modifier is at a concentration of less than about 30 wt% in the composition; and printing the composition on the substrate, wherein the substrate comprises or is made of glass coated with an epoxycontaining material.

2. A method according to claim 1 , comprising drying the composition.

3. A method according to claim 1 or claim 2, comprising printing the composition at one or more discrete locations on the substrate.

4. A method according to any one of claims 1 to 3, comprising printing a microarray on the substrate.

5. A method according to any one of claims 1 to 4, further comprising blocking the biological material of the composition printed and / or immobilised on the substrate.

6. A method according to any one of claims 1 to 5, wherein the biological material comprises or consists of DNA.

7. A method according to claim 6, wherein the biological material comprises or consists of dsDNA.

8. A method according to claim 7, wherein the dsDNA comprises or consists of plasmid dsDNA and / or linear dsDNA.

9. A method according to any one of claims 1 to 8, wherein the substrate for an assay comprises a microarray.

10. A method according to any one of claims 1 to 9, wherein the concentration of the viscosity modifier is up to about 25 wt%, optionally up to about 20 wt%, optionally up to about 15 wt%, optionally up to about 8 wt%, optionally up to about 5 wt%, in the composition.

11. A method according to any one of claims 1 to 10, wherein the concentration of the viscosity modifier is in the range of about 0.01-8 % w / v, optionally 0.1-8 % w / v, optionally 0.1 -7.5 % w / v, optionally 0.1-5% w / v, optionally 1-5 % w / v, in the composition.

12. A method according to any one of claims 1 to 11 , wherein the viscosity modifier comprises a disaccharide.

13. A method according to claim 12, wherein the viscosity modifier is selected from the list consisting of sucrose, trehalose, lactose, maltose, cellobiose, and chitobiose.

14. A composition for printing a biological material on a substrate for an assay, the composition comprising: a biological material, wherein the biological material comprises a DNA-containing material; and a viscosity modifier, wherein the viscosity modifier comprises a carbohydrate and is at a concentration of less than about 9 wt% in the composition.

15. A composition according to claim 14, wherein the biological material comprises or consists of DNA.

16. A composition according to claim 15, wherein the biological material comprises or consists of dsDNA, optionally wherein the dsDNA comprises or consists of plasmid dsDNA and / or linear dsDNA.

17. A composition according to any one of claims 14 to 16, wherein the concentration of the viscosity modifier is up to about 8 wt%, optionally up to about 5 wt%, in the composition.

18. A composition according to any one of claims 14 to 17, wherein the concentration of the viscosity modifier is in the range of about 0.01-8 % w / v, optionally 0.1-8 % w / v, optionally 0.1 -7.5 % w / v, optionally 0.1-5% w / v, optionally 1-5 % w / v, in the composition.

19. A composition according to any one of claims 14 to 18, wherein the viscosity modifier comprises a disaccharide.

20. A composition according to claim 19, wherein the viscosity modifier is selected from the list consisting of sucrose, trehalose, lactose, maltose, cellobiose, and chitobiose.21 . A composition according to any one of claims 14 to 20, wherein the composition further comprises a buffering agent.

22. A composition according to claim 21 , wherein the buffering agent is provided at a concentration of about 5-300 mM, in the composition.

23. A composition according to any one of claims 14 to 22, wherein the composition further comprises an inorganic salt.

24. A composition according to claim 23, wherein the salt is typically provided at a concentration between about 1 and 4M, optionally between about 1.5 and 2M, in the composition.

25. A composition according to any one of claims 14 to 24, further comprising a carrier or solvent, optionally water.

26. Use of a composition for printing on a substrate for an assay, wherein the composition comprises: a biological material; and a viscosity modifier, wherein the viscosity modifier is at a concentration of less than about 30 wt% in the composition, and wherein the substrate comprises or is made of glass coated with an epoxycontaining material.

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