BLOCKING METHOD

Abstract

A method of applying a blocking composition (116) on a substrate for an assay (20) comprises: providing a substrate for an assay (20), wherein the substrate for an assay comprises a solid substrate provided with a plurality of discrete spots of a biological material on a surface thereof; and spraying the blocking composition (116) onto the substrate (20) as particles or droplets having a diameter being less than the diameter of the printed spots of biological material.

Claims

1. A method of applying a blocking composition on a substrate for an assay, the method comprising: providing a substrate for an assay, wherein the substrate for an assay comprises a solid substrate provided with a plurality of discrete spots of a biological material on a surface thereof; and spraying the blocking composition onto the substrate as particles or droplets having a diameter being less than the diameter of the printed spots of biological material.

2. A method according to claim 1, wherein the method comprises spray coating the blocking composition onto the substrate.

3. A method according to claim 1, wherein the substrate is provided with an array of the biological material on a surface thereof.

4. A method according to claim 1, wherein the substrate for an assay comprises a microarray.

5. A method according to claim 1, wherein the size of the spots of biological material on the surface of the substrate is about 100 .Math.m - 300 .Math.m.

6. A method according to claim 1, wherein the blocking composition comprises, consists essentially of or consists of a blocking buffer.

7. A method according to claim 1, wherein the biological material comprises a peptide, a protein, an amino-acid, a nucleic acid, oligonucleotide, DNA, RNA, a lipid, a carbohydrate, an enzyme, a metabolite, an antibody, an antigen, a cell, red blood cells, plasma, or serum.

8. A method according to claim 1, comprising spraying the blocking composition onto the substrate using an air pressure-driven spray coating device.

9. (canceled)

10. A method according to claim 1, comprising spraying the blocking composition onto the substrate using an ultrasonic atomiser.

11. (canceled)

12. A method of applying a blocking composition on a substrate for an assay, the method comprising: providing a substrate for an assay, wherein the substrate for an assay comprises a solid substrate provided with a plurality of discrete spots of a biological material on a surface thereof, and wherein the surface of the substrate is substantially planar; and spraying the blocking composition onto the substrate.

13. A method according to claim 1, comprising at least one of: preserving the integrity of a/the biological material immobilised thereto; and/or improving specificity and/or sensitivity during subsequent analysis.

14. (canceled)

15. A method according to claim 1, wherein the method reduces, avoids and/or prevents the occurrence of aberrant results, optionally wherein the method reduces, avoids and/or prevents the occurrence of false positive and/or false negative results.

16. A method according to claim 1, further comprising: applying a sample on the blocked substrate, and analysing the substrate.

17. An apparatus for applying a blocking composition on a substrate for an assay, the apparatus comprising: a substrate support for receiving the substrate for an assay; and a spraying device configured to spray the blocking composition on the substrate, wherein the spraying device is configured to spray the blocking composition as particles or droplets having a diameter being less than the diameter of printed spots of biological material being provided on the substrate.

18. An apparatus according to claim 17, wherein the substrate is provided on a substrate holder, and wherein the substrate support has a receiving element configured to support the substrate holder.

19. An apparatus according to claim 17, wherein the substrate support comprises a masking element configured to be positioned over the substrate or substrate holder, wherein the masking element is configured to cover a peripheral region of the substrate which is devoid of any biological material on its surface, wherein the masking element has an opening near a central region thereof.

20. (canceled)

21. An apparatus according to claim 17, wherein the apparatus comprises an enclosure configured to at least partially surround or contain the spray coating device, wherein the enclosure includes a support frame configured to connect and secure the spraying device, wherein the support frame is configured to connect and secure a first mounting plate, wherein the first mounting plate is configured to support a first spraying device.

22-23. (canceled)

24. An apparatus according to claim 17, wherein the apparatus comprises a first connecting element configured to allow adjustment of the position of the spraying device or first spraying device.

25. An apparatus according to claim 17, wherein the spraying device or first spraying device comprises an ultrasonic atomiser.

26. An apparatus according to claim 17, wherein the apparatus comprises a movable platform configured to receive the substrate support, wherein the movable platform comprises one or more moving means to allow the movable platform to be moved in a first direction, and one or more moving means to allow the movable platform to be moved in a second direction transverse to the first direction.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0124] The present invention will now be further described in detail and with reference to the figures in which:

[0125] FIGS. 1 and 2 shows a conventional method of applying a blocking buffer on a microarray according to the prior art;

[0126] FIG. 3 shows an image of a reacted microarray illustrating a “Comet Tail” effect;

[0127] FIG. 4 illustrates a method of applying a blocking buffer on a microarray according to a first embodiment;

[0128] FIGS. 5a, 5b, 6a, 6b, 7a, 7b, 8a and 8b illustrate a comparison of the assay images obtained using a conventional blocking method and the method of FIG. 4, for four different samples;

[0129] FIG. 9 is a graph comparing the occurrence of comet tails obtained using a conventional blocking method and the method of FIG. 4, for different samples;

[0130] FIG. 10 shows a front view of an apparatus according to an embodiment;

[0131] FIG. 11 shows a part-exploded perspective front view of the enclosure of the apparatus of FIG. 10;

[0132] FIG. 12 shows a rear perspective view of the enclosure of the apparatus of FIG. 10;

[0133] FIG. 13 shows a front view of the apparatus of FIG. 10, showing an ultrasonic atomiser and a substrate support;

[0134] FIGS. 14 to 16 show illustrations of an embodiment of the substrate support of FIG. 13;

[0135] FIG. 17 shows a schematic view of another embodiment of a spraying apparatus;

[0136] FIG. 18 shows a schematic view of another embodiment of a spraying apparatus.

DETAILED DESCRIPTION OF THE DRAWINGS

[0137] FIGS. 1 and 2 show a conventional (wet) method (10) for applying a blocking buffer to a microarray (20). As shown in FIG. 1, the method comprises a dispensing tip 31 which (as shown in FIG. 2), dispenses a blocking buffer 16 in a dispensing phase 33. The blocking buffer 16 spreads over the surface of the microarray 20 and excess blocking buffer is aspirated during an aspiration phase 34 to yield a blocked microarray 22.

[0138] FIG. 3 shows an image of a microarray 40 used to assay reactions between an antibody printed on the microarray and red cells from a sample. FIG. 3 illustrates the “Comet Tail” effect, where it can be seen that the areas showing positive reaction exceed the original printed spot area. In this example, three cell lines were impacted, namely Positive Controls (41), Cell Line “A” (42), and Cell Line “B” (43).

[0139] FIG. 4 shows an embodiment of a method according to a first embodiment. In this method, a microarray 20′ was paced on a support surface 30′. The peripheral surface of the microarray 20′ (the “waste area” not printed with any biological material) was protected by a masking element 26. The microarray way was printed with cell lines previously identified as having propensity to cause Comet Tail effect, and printed following an Immunohematology (IH) layout.

[0140] The exposed (printed) area of microarray 20 was then sprayed with a blocking buffer (as specified in Table 1 below) using a manual spray device 50, with the nozzle of the device 50 being aligned at approximately 0° (i.e. substantially parallel to the microarray 20), in order to minimise the pressure exerted on the printed spots during the spraying process, and thus reduce the occurrence of “blast spots”.

TABLE-US-00001 Blocking buffer composition (in water) Components Bovine Serum Albumin (BSA) Sodium chloride Potassium dihydrogen orthophosphate (anhydrous) Disodium hydrogen orthophosphate (anhydrous)

[0141] FIGS. 5a, 5b, 6a, 6b, 7a, 7b, 8a and 8b illustrate a comparison of the images obtained using a conventional blocking method and by the method of FIG. 4, for four different samples.

[0142] It can be observed that, for each sample, the conventional blocking method led to a number of comet tails 45a,45b,45c,45d, whilst applying the blocking buffer with the spray coating method described in connection with FIG. 4 eliminated the “comet tail” effect entirely, thus improving specificity of the results.

[0143] FIG. 9 is a graph comparing the occurrence of Comet Tails obtained using a conventional blocking method and the method of FIG. 4, for different samples.

[0144] It can be seen that comet tails occurred in the vast majority of samples prepared using a conventional “spreading” blocking method, whereas comet tails rarely occurred in microarrays blocked using the spray coating method of FIG. 4. Without wishing to be bound by theory, it is believed that the occurrence of comet tails in the samples blocked using the spray coating method of FIG. 4 may be attributed to a non-uniform coverage of the spray, particularly around the peripheral areas of the substrate.

[0145] FIG. 10 shows apparatus 60 for spraying a blocking buffer on a microarray, according to an embodiment. The apparatus has an enclosure 61 which in this embodiment is made of metal, but could in other embodiments be made of any other suitable material. As best shown in FIGS. 11 and 12, the enclosure includes a cuboid frame 62 which provides support and structural integrity. The frame 62 is supported on feet 63 for stability. Conveniently, the apparatus 60 is provided with transparent or translucent panels including a rear panel 65 and two side panels 66 so as to allow a user to view and/or monitor the inside of the enclosure 61, in use, whilst confining any sprayed material to the inside of the enclosure 61. There is also provided a front door 64 which is also made of a transparent or translucent material such as glass or plastic in order to allow a user to view and/or monitor inside the enclosure 61.

[0146] The enclosure 61 has a support frame 67 which includes struts or rails attached to the frame 62 (but may in other embodiments form an integral part of the frame 62), and which is configured to connect and secure a first mounting plate 71. Advantageously, the rails of the support frame 67 are movable relative to the frame 62, e.g. in two dimensions and/or in a vertical plane, so as to allow adjustment of height and width thereof. The first mounting plate 71 is configured to support a first spraying device 81 via connecting arms 72 which are attached to apertures 73 in the first mounting plate 71. In this embodiment, the first mounting plate 71 is disposed substantially vertically, and the first spraying device 81 is an ultrasonic spray coating device, e.g. an ultrasonic atomiser.

[0147] The apparatus 60 also has a second mounting plate 76 which in this embodiment also acts as an upper panel and is attached to an upper portion of the frame 61. The second mounting plate 76 is configured to support a second spraying device 82 via connecting arms 77 which are attached to apertures 78 in the second mounting plate 76. In this embodiment, the second mounting plate 76 is disposed substantially horizontally, and the second spraying device 82 is an air pressure-driven spray coating device, e.g. spray coating device having a spray valve.

[0148] The connecting arms 72,77 allow adjustment of the position, height, and spraying angle of a respective spraying device 81,82.

[0149] In FIG. 10, both the first spraying device 81 and the second spraying device 82 are shown for ease of understanding. However, it will be appreciated that, in use, a user may choose to select and connect only one type of spraying device, i.e. either first spraying device 81 or the second spraying device 82, depending on the most appropriate type of device required for a specific application.

[0150] Typically, the enclosure 61, e.g. frame 62, has a height of about 75 cm, a width of about 50 cm, and a depth of about 50 cm which keeps the overall size of the apparatus 60 relatively compact, whilst permitting a suitable level of adjustment of the spray devices 81,82 in X, Y, and/or Z directions.

[0151] Typically the apparatus 60 allows the nozzle of the spraying devices 81,82, to be positioned at a distance of about 0 mm to about 750 mm, from the substrate.

[0152] When the spray coating device is a pressure-driven spray coating device 82, the apparatus 60 allows the nozzle of the spraying device 82 to be positioned at a distance of about 500 mm to about 750 mm form the substrate.

[0153] When the spray coating device is an ultrasonic atomiser 81, the apparatus 60 allows the nozzle of the spraying device 81 to be positioned at a distance of about 0 mm to about 500 mm from the substrate.

[0154] FIG. 13 shows a front view of the apparatus 60 of FIG. 10, showing only the ultrasonic atomiser 81 for clarity, and a substrate support 90 configured to receive a substrate which in this embodiment is a microarray 20 provided on microarray holder 32. As can be seen, the microarray 20 is substantially flat and does not include any wells or recesses.

[0155] As shown in FIGS. 14-16, the substrate support 90 has a receiving element 91 configured to support the microarray holder 32. In this embodiment, the receiving element 91 is provided on a lower portion of the frame 62.

[0156] Advantageously, the substrate support 90 has a holding element 92 configured to hold the microarray in position, in use. In this embodiment, the holding element 92 is in the form of a pair of guides attached to the receiving element 91. The holding element 92 may be sized so as to receive a microarray slide of a conventional size. In other embodiments, the holding element 92 may be adjustable so as to receive microarrays of different sizes.

[0157] As shown in FIG. 15, there is provided a masking layer 93 configured to be positioned over the microarray holder 32 and to cover part thereof, and in particular to cover a peripheral region of the microarray 20 which is devoid of any biological material on its surface. Thus, in use, the microarray slide may be sandwiched between the receiving element 91 and the masking layer 93.

[0158] The masking layer has an opening 95 near a central region thereof. The opening 95 is sized so as to match the area of the microarray 20 to be coated with a blocking buffer. By such provision, the masking layer 93 protects the areas of the microarray slide 20 which should remain free of blocking buffer. The opening typically has a size of about 128 mm × 128 mm (+/- 1 mm).

[0159] The location of the masking layer 93 may be secured, in use, by pins 94 which engage an upper surface of the receiving element 91 and engage a lower surface of the masking layer 93.

[0160] FIG. 16 shows a view of the assembled substrate support 90 in which the masking layer 93 is depicted in a see-through appearance for ease of visualisation.

[0161] FIG. 17 shows a schematic view of another embodiment of a spraying apparatus 160. The apparatus 160 of FIG. 17 is generally similar to the apparatus 60 of FIG. 10, like parts denoted by like numerals, incremented by ‘100’. In this embodiment, the apparatus 160 has a substrate support 190 which is provided with or connected to wheels 196 to allow the substrate support 190 to be moved in a first direction represented by arrow A. The substrate support 190 is also provided with or connected with bearings (not shown) to allow the substrate support 190 to be moved in a second direction represented by arrow B, which is transverse to the first direction. In this embodiment, the substrate support 190 is connected to a handle 197 that allows a user to control movement of the substrate support 190, e.g. in direction A and/or B.

[0162] FIG. 18 shows a schematic view of another embodiment of a spraying apparatus 260. The apparatus 260 of FIG. 18 is generally similar to the apparatus 160 of FIG. 17, like parts denoted by like numerals, incremented by ‘100’. In this embodiment, the apparatus 260 includes a tray 298 below the substrate support 290, configured to collect excess blocking buffer dispensed by the spraying device 281. The apparatus 260 further comprises a permeable sheet 299 configured to support the substrate support 290 and to allow drainage of excess blocking buffer 216 through the sheet 299. The permeable sheet 299 also provides the movable substrate support 290 with a suitable support surface for the wheels 296 allowing movement of the substrate support 290 whilst permitting excess liquid therethrough, e.g. into the tray 298.

[0163] It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as described herein without departing from the scope of the present invention. The present embodiments are therefore to be considered for illustrative purposes and are not restrictive, and are not limited to the extent of that described in the embodiment.