Method of immobilizing a nucleic acid probe to a solid support

Abstract

A nucleic acid probe, a method of immobilizing the nucleic acid probe to a solid support and the solid support including the immobilized probes using UV light. The nucleic acid probe includes a terminus anchor chain portion, and a capture portion wherein the terminus anchor chain portion includes a sequence of at least 18 nucleotides composed of stretches of up to 5 nucleotides of base type X with intermediate nucleotide(s) of base type Cytosine (C) and optionally one nucleotide of base type Guanine (G) or a sequence with at least 90% similarity thereto, wherein each base type X independently of each other designate base type Thymine (T) or base type Uracil (U).

Claims

1. A method of immobilizing a nucleic acid probe to a solid support, the method comprising providing the nucleic acid probe to comprise a terminus anchor chain portion, and a capture portion applying the nucleic acid probe onto a surface of the solid support, and anchoring only the anchor chain portion of the nucleic acid probe to the solid support by subjecting it to UV light, wherein the terminus anchor chain portion of the nucleic acid probe comprises a sequence of N nucleotides composed of at least two stretches of nucleotides of base type X with intermediate nucleotide(s) of base type Cytosine (C) and optionally one nucleotide of base type Guanine (G) between said stretches of base type X or a sequence with at least 90% similarity thereto of said sequence of N nucleotides, wherein the stretches of nucleotides of base type X independently of each other are at least 1 and no more than 5 nucleotides, wherein N is at least 18 and wherein each base type X independently of each other designate base type Thymine (T) or base type Uracil (U), wherein % similarity=100*(N−n)/N, wherein n is the number n of nucleotides in the sequence of N nucleotides which differs from the composition of the stretches of nucleotides of base type X with intermediate nucleotide(s) of base type C.

2. The method of claim 1, wherein the sequence of N nucleotides comprises less than 5% of nucleotides with purine nucleobases.

3. The method of claim 1, wherein the terminus anchor chain portion of the nucleic acid probe comprises a sequence of at least N nucleotides composed of at least two stretches of from 2 to 5 nucleotides of base type X with intermediate nucleotide(s) of base type C and wherein N is at least 20.

4. The method of claim 1, wherein the sequence of N nucleotides comprises repeating sub-sequences of nucleotides of base types according to the formula
(—(X).sub.Y—(C).sub.Z—).sub.M, wherein Y is an integer from 1 to 5, Z is an integer from 1 to 5, Y≥Z and M is an integer from 4 to 20.

5. The method of claim 4, wherein Y is an integer from 2 to 5, Z is an integer from 1 to 4, Y>Z and M is an integer from 4 to 20.

6. The method of claim 1, wherein the sequence of N nucleotides comprises repeating sub-sequences of nucleotides of base types according to the formula
(—(X).sub.Y2—(C).sub.Z—(X).sub.Y2—).sub.M, wherein Y.sub.2 is an integer from 1 to 4, Z is an integer from 1 to 4, and M is an integer from 4 to 20.

7. The method of claim 6, wherein Y.sub.2 is an integer from 2 to 3, Z is an integer from 1 to 3.

8. The method of claim 1, wherein the capture portion comprises a primer and/or a hybridization chain portion comprising a sequence of nucleotides adapted to hybridize to a complementary region of a target nucleic acid probe and/or adapted for performing a Polymerase Chain Reaction (PCR) assay.

9. The method of claim 1, wherein the nucleic acid probe is deposited onto the surface of the solid support by spotting.

10. The method of claim 1, wherein at least the surface of the solid support is a polystyrene PS surface.

11. The method of claim 1, wherein the solid support surface is essentially free of one or more of amine groups, methylene groups, thiol groups, epoxy groups, diazo groups or amide groups.

12. The method of claim 1, wherein the solid support is at least a part of a cartridge comprising a channel with a channel surface defining the channel, wherein the surface of the solid substrate forms at least a part of the channel surface and wherein the channel comprises a reaction section, wherein said nucleic acid probe immobilizes onto a surface within the reaction section of the channel.

13. The method of claim 12, wherein the reaction section comprises at least one optical element, wherein said optical element comprises a lens structure and/or a supercritical angle fluorescence structure (SAF structure), said SAF structure has a top surface, wherein said nucleic acid probe immobilizes onto said top surface.

14. A method of immobilizing a nucleic acid probe to a solid support, the method comprising providing the nucleic acid probe, wherein the nucleic acid probe comprises a terminus anchor chain portion and a capture portion, applying the nucleic acid probe onto a surface of the solid support, and anchoring the anchor chain portion of the nucleic acid probe to the solid support by subjecting it to UV light, wherein the terminus anchor chain portion of the nucleic acid probe comprises a sequence of N nucleotides composed of at least two stretches of nucleotides of base type X with intermediate nucleotide(s) of base type Cytosine (C) and optionally one nucleotide of base type Guanine (G) between said stretches of base type X or a sequence with at least 90% similarity thereto of said sequence of N nucleotides, wherein the stretches of nucleotides of base type X independently of each other are at least 1 and no more than 5 nucleotides, wherein N is at least 18 and wherein each base type X independently of each other designate base type Thymine (T) or base type Uracil (U), wherein % similarity=100*(N−n)/N, wherein n is the number n of nucleotides in the sequence of N nucleotides which differs from the composition of the stretches of nucleotides of base type X with intermediate nucleotide(s) of base type C, wherein the solid support is at least a part of a cartridge comprising a channel with a channel surface defining the channel, wherein the surface of the solid substrate forms at least a part of the channel surface and wherein the channel comprises a reaction section, wherein said nucleic acid probe immobilizes onto a surface within the reaction section of the channel, wherein the reaction section comprises at least one optical element, wherein said optical element comprises a lens structure and/or a supercritical angle fluorescence structure (SAF structure), said SAF structure has a top surface, wherein said nucleic acid probe immobilizes onto said top surface, wherein the optical element has a SAF structure with a conical, frustum shape with a frustum angle α, a top surface, a top diameter D and a height h, wherein the frustum angle α is from about 30° to about 70°, the top diameter to height aspect ratio D/h which is about 1.1 or less and the SAF structure comprises a top surface recess.

15. The method of claim 1, wherein the anchor chain portion of the nucleic acid is anchored to the solid support by subjecting it to UV light comprising a wavelength in the range of from about 250 nm to 500 nm.

16. The method of claim 1, wherein the anchor chain portion of the nucleic acid is anchored to the solid support by subjecting it to UV light using an amount of energy from about 0.2 Joule/cm.sup.2 to about 15 Joule/cm.sup.2.

17. A solid support comprising a surface carrying dried nucleic acid probe, the nucleic acid probe comprising a terminus anchor chain portion, and a capture portion, wherein the terminus anchor chain portion of the nucleic acid probe comprises a sequence of N nucleotides composed of at least two stretches of nucleotides of base type X with intermediate nucleotide(s) of base type Cytosine (C) between said stretches of base type X or a sequence with at least 90% similarity thereto of said sequence of N nucleotides, wherein the stretches of nucleotides of base type X independently of each other are at least 2 and no more than 5 nucleotides, wherein N is at least 18 and wherein each base type X independently of each other designate base type Thymine (T) or base type Uracil (U), wherein % similarity=100*(N−n)/N, wherein n is the number n of nucleotides in the sequence of N nucleotides which differs from the composition of the stretches of nucleotides of base type X with intermediate nucleotide(s) of base type C, wherein only the terminal anchor portion is photochemically anchored to the solid support.

18. The suspension of claim 17, wherein the terminus anchor chain portion of the nucleic acid probe comprises a sequence of at least N nucleotides composed of at least two stretches of from 2 to 5 nucleotides of base type X with intermediate nucleotide(s) of base type C.

19. The suspension of claim 17, wherein the sequence of N nucleotides of the terminus anchor chain portion of the nucleic acid probe comprises repeating sub-sequences of nucleotides of base types according to the formula
(—(X).sub.Y—(C).sub.Z—).sub.M, wherein Y is an integer from 1 to 5, Z is an integer from 1 to 5, Y≥Z and M is an integer from 4 to 20.

20. The suspension of claim 19, wherein Y=2 and M≥10 or Y=3 and M≥6 or Y=4 and M≥4.

21. The suspension of claim 17, wherein the sequence of N nucleotides of the terminus anchor chain portion of the nucleic acid probe comprises repeating sub-sequences of nucleotides of base types according to the formula
(—(X).sub.Y2—(C).sub.Z—(X).sub.Y2—).sub.M, wherein Y2 is an integer from 2 to 4, Z is an integer from 1 to 4, and M is an integer from 4 to 20.

22. The suspension of claim 21, wherein Y.sub.2=2 and M≥10 or Y.sub.2=3 and M≥4.

23. The suspension of claim 17, wherein the capture portion comprises a chain of nucleotides having from about 4 to about 100 nucleotides.

24. A solid support comprising an immobilized nucleic acid probe, the nucleic acid probe comprising a terminus anchor chain portion, and a capture portion, wherein the terminus anchor chain portion of the nucleic acid probe comprises a sequence of N nucleotides composed of at least two stretches of nucleotides of base type X with intermediate nucleotide(s) of base type Cytosine (C) between said stretches of base type X or a sequence with at least 90% similarity thereto of said sequence of N nucleotides, wherein the stretches of nucleotides of base type X independently of each other are at least 2 and no more than 5 nucleotides, wherein N is at least 18 and wherein each base type X independently of each other designate base type Thymine (T) or base type Uracil (U), wherein % similarity=100*(N−n)/N, wherein n is the number n of nucleotides in the sequence of N nucleotides which differs from the composition of the stretches of nucleotides of base type X with intermediate nucleotide(s) of base type C, wherein only the terminal anchor portion is photochemically anchored to the solid support.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The above and/or additional objects, features and advantages of the present invention will be further elucidated by the following illustrative and non-limiting description of embodiments and examples of the present invention, with reference to the appended drawings.

(2) FIG. 1 is diagram showing a number of marked polytails tested in a first example a schematic top view of a microfluidic cartridge according to an embodiment of the invention.

(3) FIG. 2 is diagram showing the immobilization percent's of the respective marked polytails in the first example.

(4) FIG. 3 is a process diagram applied in a further example.

(5) FIG. 4 is diagram showing the immobilization percent's of a number of marked polytails tested in the further example.

(6) FIG. 5 are images of the spots of the polytails in the further example.

(7) FIG. 6 shows two nucleic acid probes of embodiments of the invention and one nucleic acid probe having a comparative polytail (terminus anchor chain portion)

(8) FIG. 7 shows images of different concentration of the nucleic acid probes of FIGS. 6a and 6b where the nucleic acid probes are marked.

(9) FIG. 8 is an image of a control probe and two different capture portions—a Flic gene that targeting salmonella and a Brfz gene that targeting Bordetella bacteria.

(10) FIG. 9a show a first process scheme for performing SP-PCR.

(11) FIG. 9b show a second process scheme for performing SP-PCR.

(12) FIG. 10a are images of a solid support with spotted nucleic acid probes subject to SP-PCR with and without washing.

(13) FIG. 10b is a plot of the average signal minus background of the images of FIG. 10a.

(14) FIG. 11 is a diagram showing the immobilization percent's of a number of marked polytails and nucleic acid probes where the polytails/nucleic acid probes are immobilized using different UV exposure time.

(15) FIG. 12 show the immobilization percent as a function of UV exposure time for a marked polytail.

(16) FIGS. 13a-13e are images of a number of immobilized polytails and nucleic acid probes obtained at different UV exposure time before and after wash wherein the UV emitter used was an 8 W UV emitter.

(17) FIGS. 14a-14c are images of a number of immobilized polytails and nucleic acid probes obtained at different UV exposure time before and after wash wherein the UV emitter used was a 16 W UV emitter.

(18) FIG. 15a is diagram showing the signal minus background for a number of marked nucleic acid probes having different polytails.

(19) FIG. 15b are images of the immobilized nucleic acid probes of FIG. 15a.

(20) FIG. 16a is diagram showing the signal minus background for a marked nucleic acid probes immobilized to the solid support using different time of UV exposure and thereby UV dosage, where the immobilized nucleic acid probe has been subjected to SP-PCR.

(21) FIG. 16b are images of the immobilized nucleic acid probes of FIG. 16a.

(22) FIG. 17 is a cross-sectional view of a SAF structure comprising immobilized nucleic acid probes.

(23) FIG. 18 is a perspective view of a section of a reaction channel of a cartridge, where the reaction section comprises SAP structures with immobilized nucleic acid probes, which have been subjected to SP-CPR.

(24) FIG. 19 is a perspective view of a SAF structure illustrated with a dotted top part to show the frustum angle α.

(25) FIGS. 19a-19d illustrate a standard SAF structure with a frustum angle of 60 degrees.

(26) FIGS. 20a-20e show a SAF structure with a top surface recess.

(27) FIGS. 21a-21d show another SAF structure with a top surface recess.

(28) FIG. 22 shows seven different SAF structures used in example 11.

(29) FIG. 23 shows the signal intensity result of example 11.

(30) FIG. 24 shows the coefficient of variation result of example 11.

(31) The figures are schematic and simplified for clarity. Throughout, the same reference numerals are used for identical or corresponding parts.

(32) Further scope of applicability of the present invention will become apparent from the description given hereinafter. However, it should be understood that the description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this description and examples.

(33) A simple UV cross-linking process scheme for attaching TC-tagged DNA oligonucleotides on various substrates was used. The process scheme used corresponds to the process scheme described in Sun Y, Perch-Nielsen I, Dufva M, et al. “Direct immobilization of DNA probes on non-modified plastics by UV irradiation and integration in microfluidic devices for rapid bioassay”. Anal Bioanal Chem. 2012; 402(2):741-748. doi:10.1007/s00216-011-5459-4.

(34) The technique has been showed to have not only high versatility but also high thermal stability comparable to other. In this study, this method was used to immobilize different marked polytails and marked nucleic acid probes to a PS solid support. The markers used in the below examples were fluorescence dyes. “Quasar 570” and “Cy3” were used as fluorescence dyes.

(35) A number of different marked polytails and nucleic acid probes were used in the experiments including the following listed in table 1.

(36) TABLE-US-00001 TABLE 1 Different polytail labelled with fluorescence dye for washing and thermocycling experiments. Polytail optional # capture portion and 5′-3′  1 20T.sup.10C.sup.10 TTTTTTTTTTCCCCCCCCCC/3′cy3 (SEQ ID NO: 1)  2 30T.sup.15C.sup.15 TTTTTTTTTTTTTTTCCCCCCCCCCCCCCC/3′cy3 (SEQ ID NO: 2)  3 40T.sup.20C.sup.20 TTTTTTTTTTTTTTTTTTTTCCCCCCCCCCCCCCCCCCCC/3′cy3 (SEQ ID NO: 3)  4 60T.sup.30C.sup.30 TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCCCCCCCCCCCCCCCCCCCCCCCCC (SEQ ID ON: 4) CCCCC/3′cy3  5 40TC.sup.20 TCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTC/3′cy3 (SEQ ID NO: 5)  6 20T.sup.20 TTTTTTTTTTTTTTTTTTTT/3′cy3 (SEQ ID NO: 6)  7 20T.sup.10C.sup.10hilA TTTTTTTTTTCCCCCCCCCCCGGTTTAATCGTCCGGTCGTAGTGGTGTCTCCGCC (SEQ ID NO: 7) AGCGCCGCAACCTACGACTCATACA/3′cy3  8 20T.sup.10C.sup.10fliC TTTTTTTTTTCCCCCCCCCCACTTACGCTGCAAGTAAAGCCGAAGGTCACAACTT (SEQ ID NO: 8) TAAAGCACAGCCTGATCTGGCGGAA/3′cy3  9 20C.sup.20 CCCCCCCCCCCCCCCCCCCC/3′cy3 (SEQ ID NO: 9) 10 20A.sup.20 AAAAAAAAAAAAAAAAAAAA-3′-Cy3 (SEQ ID NO: 10) 11 20G.sup.20 GGGGGGGGGGGGGGGGGGGG-3′-Cy3 (SEQ ID NO: 11) 12 40CT.sup.20 CTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCT-quasar 570 (SEQ ID NO: 12) 13 40TTCC.sup.20 TTCCTTCCTTCCTTCCTTCCTTCCTTCCTTCCTTCCTTCC-quasar 570 (SEQ ID NO: 13) 14 42TTTCCC.sup.7 TTTCCCTTTCCCTTTCCCTTTCCCTTTCCCTTTCCCTTTCCC-quasar 570 (SEQ ID NO: 14) 15 40TTTTCCCC.sup.5 TTTTCCCCTTTTCCCCTTTTCCCCTTTTCCCCTTTTCCCC-quasar 570 (SEQ ID NO: 15) 16 42TTC.sup.14 TTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTC-quasar 570 (SEQ ID NO: 16) 17 40TTTC.sup.10 TTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTC-quasar 570 (SEQ ID NO: 17) 18 40TTTTC.sup.8 TTTTCTTTTCTTTTCTTTTCTTTTCTTTTCTTTTCTTTTC-quasar 570 (SEQ ID NO: 18) 19 39TCC.sup.13 TCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCCTCC-quasar 570 (SEQ ID NO: 19) 20 40TCCC.sup.10 TCCCTCCCTCCCTCCCTCCCTCCCTCCCTCCCTCCCTCCC-quasar 570 (SEQ ID NO: 20) 21 39CT.sup.13 TCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCT-quasar 570 (SEQ ID NO: 21) 22 42TTCCTT.sup.7 TTCCTTTTCCTTTTCCTTTTCCTTTTCCTTTTCCTTTTCCTT-quasar 570 (SEQ ID NO: 22) 23 40TA.sup.20 TATATATATATATATATATATATATATATATATATATATA-quasar 570 (SEQ ID NO: 23) 24 40TG.sup.20 TGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTG-quasar 570 (SEQ ID NO: 24) 25 40AG.sup.20 AGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG-quasar 570 (SEQ ID NO: 25) 26 40GC.sup.20 GCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGC-quasar 570 (SEQ ID NO: 26) 27 40AC.sup.20 ACACACACACACACACACACACACACACACACACACACAC-quasar 570 (SEQ ID NO: 27) 28 39CG.sup.13 TCGTCGTCGTCGTCGTCGTCGTCGTCGTCGTCGTCGTCG-quasar 570 (SEQ ID NO: 28) 29 40TTCG.sup.10 TTCGTTCGTTCGTTCGTTCGTTCGTTCGTTCGTTCGTTCG-quasar 570 (SEQ ID NO: 29) 30 40TAGC.sup.10 TAGCTAGCTAGCTAGCTAGCTAGCTAGCTAGCTAGCTAGC-quasar 570 (SEQ ID NO: 30)

(37) The nucleic acid probes or nucleic acid probes comprising polytails of numbers 5, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 and 29 are examples of the inventions. The remaining nucleic acid probes or nucleic acid probes comprising polytails are comparative examples.

EXAMPLE 1

(38) In this example, the nucleic acid probes or nucleic acid probes comprising polytails of numbers 6, 9, 10, 1, 2, 3, 4, 5, 7, 8 (in the order as shown in FIG. 1).

(39) The polytails/nucleic acid probes were diluted in 5× saline sodium citrate (SSC) buffer (Promega, WI, USA) with 0.04% Triton X-100 (Sigma-Aldrich, USA). The polytails/nucleic acid probes solutions were spotted onto a cleaned PS slides using a non-contact sciFLEXARRAYER S11 spotting machine (Scienion, Germany). Each polytails/nucleic acid probes solution was spotted in four consecutive spots. After drying, the slides were exposed to UV irradiation at 254 nm with energy of 1.8 Joule/cm.sup.2 in an Ultraviolet Crosslinkers (UVP, Fisher Scientific, Denmark) to immobilize the polytails/nucleic acid probes onto surface of the substrate.

(40) Thereafter the solid support (PS slide) was washed for 5 minutes using milliQ water obtained from Millipore Corporation. The MilliQ water was ‘ultrapure’ water of “Type 1”, as defined by various authorities (e.g. ISO 3696),

(41) After the UV exposure the immobilization efficiency (immobilization percent) was measured and determined as follows

(42) The immobilization efficiency was calculated as below equation:

(43) $\frac{\mathrm{Signal}\mathrm{obtained}\mathrm{after}\mathrm{washing}}{\mathrm{Signal}\mathrm{obtained}\mathrm{after}\mathrm{UV}\mathrm{crosslink}}\times 100\%=\mathrm{Immobilization}\mathrm{efficiency}\mathrm{of}\mathrm{washing}.$

(44) The results are shown in FIG. 2. It can be seen that the polytail 40TC.sup.20 (SEQ ID NO:5) (the number 5 polytail as listed above) has a much higher immobilization efficiency than the comparative polytails.

EXAMPLE 2

(45) This example was conducted following the process diagram shown in FIG. 3.

(46) Different lengths and configurations of TC polytails/nucleic acid probes with different polytails were used. The marked polytails/nucleic acid probe used was as follows (mentioned in the order from left to right as shown in FIG. 4) Numbers 12, 5, 13, 14, 15, 16, 17, 18, 22, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 30.

(47) The polytails/nucleic acid probes were immobilized using the same procedure as described in example 1. Thereafter the solid support was washed.

(48) The signals of different polytails/probes were obtained by microscope after spotted and UV crosslink. Next, the slides were washed with 0.1× saline sodium citrate (SSC) buffer for 5 minutes and another 5 minutes in MilliQ water to remove un-attached probe and fluorescence signal.

(49) The solid support was imaged and the immobilization efficiency was calculated as below equation:

(50) $\frac{\mathrm{Signal}\mathrm{obtained}\mathrm{after}\mathrm{washing}}{\mathrm{Signal}\mathrm{obtained}\mathrm{after}\mathrm{UV}\mathrm{crosslink}}\times 100\%=\mathrm{Immobilization}\mathrm{efficiency}\mathrm{of}\mathrm{washing}.$

(51) The immobilization efficiency after wash for each polytails/nucleic acid probe is shown as the first columns on FIG. 4.

EXAMPLE 3

(52) The immobilized and washed polytails/nucleic acid probes were thereafter subjected to treatment conditions corresponding to harsh SP-PCR thermocycler treatment conditions.

(53) The immobilized polytails/probes were subjected different temperature by the PCR program of 94° C. for 2 minutes follow by 30 cycles of 94° C. for 10 seconds, 60° C. for 20 seconds, 72° C. for 20 seconds, then another 15 PCR cycles of 94° C. for 10 seconds, 65° C. for 20 seconds, 72° C. for 20 seconds. The polytail were tested in a flat-bed PCR thermocycler (Proflex, Thermo fisher) and fluorescence signal were obtained.

(54) The immobilized efficiency after PCR thermocycler treatment was calculated as follows:

(55) $\begin{array}{cc}\frac{\mathrm{Signal}\mathrm{obtained}\mathrm{after}\mathrm{thermo}\mathrm{cycler}}{\mathrm{Signal}\mathrm{obtained}\mathrm{after}\mathrm{UV}\mathrm{crosslink}}\times 100\%=\mathrm{Immobilization}\mathrm{efficiency}\mathrm{of}\mathrm{thermo}\mathrm{cycler}.& \left(2\right)\end{array}$

(56) The immobilization efficiency after PCR thermocycler treatment for each polytails/nucleic acid probe is shown as the second columns on FIG. 4.

(57) It can be seen that the immobilization efficiency both after washing and in particular after the PCR thermocycler treatment is much higher for the nucleic acid probes of the present invention. In particular, the above mentioned preferred nucleic acid probes show an extraordinary high immobilization efficiency.

(58) The images acquired of the PS slides solid support in examples 2 and 3 are shown in FIG. 5. Clearly, the nucleic acid probes with polytails having more base type T have an exceptional high immobilization efficiency.

EXAMPLE 4

(59) 3 different nucleic acid probes were synthesized comprising a) a first nucleic acid probe according to an embodiment of the invention had a polytail of the nucleotide sequence 40TTTC.sup.8 (SEQ ID NO:18) and a capture portion targeting a hilA gene, b) a second nucleic acid probe according to an embodiment of the invention had a polytail of the nucleotide sequence 42TTCCTT.sup.7 (SEQ ID NO:22) and a capture portion targeting the hilA gene and c) a comparative nucleic acid probe with a polytail of the nucleotide sequence 20T.sup.10C.sup.10 (SEQ ID NO:1) and a capture portion with hilA gene for detecting Salmonella spp. The nucleic acid probes are shown in FIG. 6.

(60) The nucleic acid probes were spotted to a solid support (PS substrate) in different concentrations ranging from 1 μM to 60 μM.

(61) A 25 μL of SP-PCR reaction mixture was prepared. The SP-PCR mixture consists of 1× Phusion® Human Specimen PCR Buffer (Thermo Fisher Scientific), 400 nM of hilA forward and 1600 nM hilA reverse primers, and 0.05 U/μL Phusion Hot Start II High-Fidelity DNA polymerase (Thermo Fisher Scientific). A Gene Frame (Thermo Fisher Scientific) was used to create a 25 μL reaction chamber surrounding the solid support primer array. The PCR master mix was loaded by pipette into the gene frame and sealed with a cover slip. The PS slide was spotted with the nucleic acid probes. The SP-PCR was conducted in a flat-bed PCR thermocycler, where a piece of 1 cm thick polystyrene insulation foam was used to separate the slides from the lid of the PCR thermocycler. The SP-PCR conditions were: 94° C. 2 minutes follow by 30 cycles of 94° C. for 10 seconds, 60° C. for 20 seconds, 72° C. for 20 seconds, then another 15 PCR cycles of 94° C. for 10 seconds, 65° C. for 20 seconds, 72° C. for 20 seconds. A higher annealing temperature was used in the later 15 PCR cycles to enhance the SP-PCR. After the SP-PCR, the chamber was washed with 0.1×SSC and 0.1% of Sodium dodecyl sulphate (SDS) (Promega, WI, USA) for 5 minutes then rinsed with deionized water and dried at room temperature. The slide was ready for scanning.

(62) After the SP-PCR, the slides were scanned using a microscope (ZEISS Axiovert 200, Germany). Microarray image was analysed using ImageJ software (Molecular devices). A circle was drawn and adjusted to the size of the spot and the mean light intensity value was determined as signal. Another circle was drawn nearby was used as the background. The signal in this study was defined as the signal of the 4 spots on the array, subtracting the mean background.

(63) FIG. 7 shows the resulting immobilization efficiency at different nucleic acid probe concentrations after the PS-PCR. It can easily be seen that the nucleic acid probes of the invention has a much higher immobilization efficiency than the comparative nucleic acid probe.

(64) FIG. 8 is an image of a control probe and two different capture portions—a Flic gene that targeting salmonella and a Brtz gene that targeting Bordetella bacteria. As control probe the polytail 42TTCCTT.sup.7 (SEQ ID NO:22) was used

(65) As shown in FIG. 7, the polytail 42TTCCTT.sup.7 (SEQ ID NO:22) that targeting Salmonella spp. showed about the same round shape after SP PCR than before, which means that the 42TTCCTT.sup.7 (SEQ ID NO:22) polytail help the entire probe to be immobilized on the surface with a very high bonding efficiency.

(66) The first process scheme for performing SP-PCR shown in FIG. 9a is a standard process scheme

(67) The second process scheme for performing SP-PCR shown in FIG. 9b is a novel SP-PCR process, which has been made available due to the present invention.

(68) Thanks to the high immobilization efficiency provided by the nucleic acid probes and the method of the invention the SP-PCR may now be performed without washing after the UV crosslinking (immobilization) and/or without washing after the PS-PCR procedure.

(69) FIG. 10a are images of a solid support with spotted nucleic acid probes subject to SP-PCR with and without washing.

EXAMPLE 5

(70) In example 5 the two nucleic acid probes of example 5 which represent embodiments of the invention namely the nucleic acid probe a) a first nucleic acid probe according to an embodiment of the invention had a polytail of the nucleotide sequence 40TTTC.sup.8 (SEQ ID NO:18) and a capture portion targeting a hilA gene and b) a second nucleic acid probe according to an embodiment of the invention had a polytail of the nucleotide sequence 42TTCCTT.sup.7 (SEQ ID NO:22) and a capture portion targeting the hilA gene were used.

(71) The spotting and the SP-PCR procedure was performed following the procedure of example 4 using a nucleic acid probe concentration of 60 μM.

(72) The result is shown in FIG. 10a prior to washing and after washing. FIG. 10b shows the average of the signal minus background with washing and without washing and it can be seen that there is a relatively low amount a false positive in the non-washed samples.

EXAMPLE 6

(73) A number of different marked polytail/nucleic acid probes were subjected to different UV exposure time and different UV dose for immobilization. The polytail/nucleic acid probes used were as shown in FIG. 1.

(74) The polytail/nucleic acid probes were spotted to the solid support as described in example 1 but with different UV exposure.

(75) Four spots of each polytails/nucleic acid probe were subjected to an UV exposure from an 8 W UV emitter for 3 minutes. Four spots of each polytails/nucleic acid probe were subjected to an UV exposure from a 16 W UV emitter for 8 minutes.

(76) The result is shown in FIG. 11 where the left plot for each polytail/nucleic acid probe is the 8 W UV emitter for 3 minutes treatment and the right left plot for each polytail/nucleic acid probe is the 16 W UV emitter for 8 minutes treatment. It appears that the 8 W UV emitter for 3 minutes treatment is better than the 16 W UV emitter for 8 minutes treatment.

EXAMPLE 7

(77) A marked polytail with the sequence 40 T/C (also called 40TC.sup.20) (SEQ ID NO:5) was used in this test. Samples of the polytail were spotted to the solid support as described in example 1 but with different UV exposure.

(78) For some samples the 8 W UV emitter was used and for other the 16 W UV emitter was used. The exposure time was varied as shown in FIG. 12 where the immobilization efficiency after wash is plotted as a function of the exposure time for each of the two emitters.

(79) It can be seen that the lower watt (8 Watt UV emitter) is better than the higher watt emitter. Further, the 8 W emitter has an immobilization optimum around 3 minutes which means that the nucleic acid probe can be immobilized a rather low UV dosage, which is highly advantageous since the risk of damaging the capture portion thereby may be reduced or even avoided.

(80) In an embodiment of the invention the nucleic acid probe had a polytail of the nucleotide sequence 40TTTTC.sup.8 (SEQ ID NO: 18) and a capture portion targeting a hilA gene was used.

(81) 3 different nucleic acid probes was synthesized and c) a comparative nucleic acid probe with a polytail of the nucleotide sequence 20T.sup.10C.sup.10 (SEQ ID NO:1) and a capture portion with hilA gene for detecting Salmonella spp. The nucleic acid probes are shown in FIG. 6.

(82) In FIGS. 13a-13e the images of the immobilized polytails obtained at the different UV exposure time before and after wash using the 8 W UV emitter are shown.

(83) In FIGS. 14a-14c the images of the immobilized polytails obtained at the different UV exposure time before and after wash using the 16 W UV emitter are shown.

EXAMPLE 8

(84) Nucleic acid probes with different length of polytails were tested. The nucleic acid probes comprised the Brtz gene that targets Bordetella bacteria.

(85) The nucleic acid probes were spotted, immobilized and washed according to the process described in example 1. FIG. 15a show the signal minus background for the various nucleic acid probes. It can be seen that the nucleic acid probes with very short polytails are difficult to immobilize and that nucleic acid probes of embodiments of the invention with polytails of 18 nucleotides or more show an effective immobilization. FIG. 15b are images of the immobilized nucleic acid probes.

EXAMPLE 10

(86) Samples of a nucleic acid probe of an embodiment of the invention having the polytail 42TTCCTT.sup.7 (SEQ ID NO:22) and the capture portion that targeting Bordetella bronchiseptica bacteria were tested.

(87) The nucleic acid probe samples were spotted, immobilized and washed according to the process described in example 1 but using a different UV exposure time. After the immobilization the samples were subjected to PS-PCR treatment as described in example 2.

(88) After the PS-PCR treatment the signal minus background signal for each sample were determined. FIG. 16a show the results and it can be seen that an effect immobilization of the nucleic acid probes if embodiments of the invention may be obtained using very low UV dosage.

(89) FIG. 16b are images of the immobilized nucleic acid probes.

(90) The SAF structure 1 corresponds to the SAF structures disclosed in WO17133741 and further details may be found in this document. The SAF structure is mounted to a bottom 2 of a reaction section of a channel of a microfluidic cartridge. The nucleic acid probes 3 marked with fluorophores and of an embodiment of the invention are mounted to a top surface of the SAF structure 1.

(91) The SAF structure 1 has a conical frustum shape with the top surface The SAF structure 1 has a protruding height, a top surface diameter, and a bottom diameter.

(92) The excited fluorophores emit light anisotropically into the SAF structure—which has a higher refractive index than the sample, the air or the liquid in the reaction section—with an angle above a supercritical angle (θc). The emitted light is collimated and can be read out by a reader as a circle of light.

(93) FIG. 18 is a perspective view of a section of a reaction channel with the edges 12 of a cartridge, where the reaction section comprises SAP structures 11 with immobilized nucleic acid probes, which have been subjected to SP-CPR.

(94) FIG. 19 shows a SAF structure illustrated with a dotted top part to show the frustum angle α. The SAF structure 21 has a bottom periphery 24 where it in mounted to or integrated with the remaining part of the solid support. At its bottom periphery, the SAF structure has a bottom diameter d.sub.b. The SAF structure has a top surface 23 with a diameter D. From the bottom to the top surface, the SAF structure has the height h. The illustrated top part is an imaginary top, shown to illustrate the frustum angle.

(95) FIGS. 19a-19d illustrate a standard SAF structure 31, comprising top surface 33. The SAF structure is integrated with a remaining part of the solid support 32. Only some of the remaining part of the solid support is shown. As explained above the solid support may form a cartridge with a channel or it may be a part thereof.

(96) FIG. 19a is a perspective view of the SAF structure 31.

(97) FIG. 19b is a top view of the SAF structure 31.

(98) FIG. 19c is a side view of the SAF structure 31.

(99) FIG. 19d is a cross sectional view of the SAF structure 31 seen in the section A-A of FIG. 19c.

(100) The SAF structure has a height h and a top diameter D. D and h may individually of each other be as disclosed elsewhere herein. The SAF structure 31 is illustrated with a frustum angle of 60 degrees. It should be understood that the SAF structure may have another frustum angle as disclosed elsewhere herein.

(101) It can be seen that the top surface is flat.

(102) In an embodiment, the SAF structure 31 has the following dimensions:

(103) D=0.2 mm; h=0.25 mm and the SAF frustum angle α is 60 degrees.

(104) FIGS. 20a-20d illustrate a preferred SAF structure 41, comprising top surface 43 with a recess 44. The SAF structure is integrated with a remaining part of the solid support 42.

(105) FIG. 20a is a perspective view of the SAF structure 41.

(106) FIG. 20b is a top view of the SAF structure 41.

(107) FIG. 20c is a side view of the SAF structure 41.

(108) FIG. 20d is a cross sectional view of the SAF structure 41 seen in the section A-A of FIG. 20c.

(109) FIG. 20e show a part of the FIG. 20a, where the recess edge width W is marked. The recess edge width W is advantageously at least about 0.008, such as at least about 0.01, such as at least about 0.015.

(110) The SAF structure has a height h and a top diameter D. D and h may, individually of each other, be as disclosed elsewhere herein. The SAF structure height h is determine from the top surface 43 without the recess 44.

(111) The recess has a height h1, which may be as disclosed elsewhere herein.

(112) The recess is substantially round and is located such that its center axis is coincident with the center axis of the SAF. The recess has a height h1, which may be as disclosed elsewhere herein.

(113) As it can be seen, the recess floor is substantially flat. The recess diameter d is determined at the floor of the recess and may be as disclosed elsewhere herein.

(114) The recess has a conical, frustum shape with a top surface formed by the floor.

(115) In the shown embodiment, the frustum angle θ and the frustum angle α are both 60 degrees. It should be understood that the recess frustum angle θ and the SAF frustum angle α may have other value(s) as disclosed elsewhere herein.

(116) The recess increases the spotting robustness and increase the read out signal intensity.

(117) In an embodiment, the SAF structure 41 has the following dimensions:

(118) D=0.2 mm, h=0.25 mm, d=0.05 mm, h1=0.01 mm, the SAF frustum angle α is 60 degrees and the recess frustum angle θ is 60 degrees.

(119) In another embodiment, the SAF structure 41 has the following dimensions:

(120) D=0.2 mm, h=0.3 mm, d=0.05 mm, h1=0.01 mm, the SAF frustum angle α is 60 degrees and the recess frustum angle θ is 60 degrees.

(121) FIGS. 21a-21d illustrate another preferred SAF structure 51, comprising top surface 53 with a recess 54. The SAF structure is integrated with a remaining part of the solid support 52.

(122) FIG. 21a is a perspective view of the SAF structure 51.

(123) FIG. 21b is a top view of the SAF structure 51.

(124) FIG. 21c is a side view of the SAF structure 51.

(125) FIG. 21d is a cross sectional view of the SAF structure 51 seen in the section A-A of FIG. 21c.

(126) The SAF structure has a height h and a top diameter D. D and h may, individually of each other, be as disclosed elsewhere herein. The SAF structure height h is determine from the top surface 53 without the recess 54.

(127) The recess 54 has a height h1, which may be as disclosed elsewhere herein.

(128) The recess is substantially round and is located such that its center axis is coincident with the center axis of the SAF. The recess has a height h1, which may be as disclosed elsewhere herein.

(129) As it can be seen, the recess floor is substantially flat. The recess diameter d is determined at the floor of the recess and may be as disclosed elsewhere herein.

(130) The recess has rounded recess edge a rounding radius R, which may be as disclosed elsewhere herein.

(131) In an embodiment, the SAF structure 51 has the following dimensions:

(132) D=0.2 mm; h=0.25 mm, d=0.08 mm, h1=0.01 mm, the recess edge is rounded with a radius R=0.05 mm and the SAF frustum angle α is 60 degrees.

(133) In another embodiment, the SAF structure 51 has the following dimensions:

(134) D=0.2 mm; h=0.3 mm, d=0.08 mm, h1=0.01 mm, the recess edge is rounded with a radius R=0.05 mm and the SAF frustum angle α is 60 degrees.

EXAMPLE 11

(135) Seven cartridges were produced from polystyrene. Each cartridge had a microfluidic channel with a reaction section and eight identical SAF structures protruding from the wall in the reaction section.

(136) The SAF structures of the first cartridge were shaped as the SAF structure no. 1 in FIG. 22; the structures of the second cartridge were shaped as the SAF structure no. 2 in FIG. 22 and so on.

(137) SAF structure no. 3 was of the type shown in FIGS. 19a-19d.

(138) SAF structure no. 5 was of the type shown in FIGS. 21a-21d and SAF structure no. 6 was of the type shown in FIGS. 20a-20e.

(139) An equal amount of Cy3-labelled oligo was spotted onto the top surface of each of the respective SAF structures and allowed to dry.

(140) In the first test round, the reaction chambers was maintained filled with air. The Cy3-labels were subjected to light at the excitation wavelength (˜550 nm) and the signal intensities of the SAF structure emission signals were detected.

(141) For each cartridge, the average SAF structure air/PS interface intensity signal was determined.

(142) In the second test round, the reaction chambers of the respective cartridges were filled with water. The Cy3-labels were subjected to light at the excitation wavelength (˜550 nm) and the signal intensities of the SAF structure emission signals were detected.

(143) For each cartridge, the average SAF structure water/PS interface intensity signal was determined.

(144) The results are shown in FIG. 23.

(145) The coefficient of variation (CoV) was determined for the SAF structures of the respective cartridges.

(146) It can be seen that the SAF structures nos. 1 and 2 had relatively high light intensities for the air/PS interface signals. Both for the air/PS interface signal intensities and the water/PS interface signals, the CoV were however, relatively high.

(147) The SAF structures no. 6 had the highest light intensity for the water/PS interface signals and the CoV. Both for the air/PS interface signal intensities and the water/PS interface signals, the CoV were however, relatively high.

(148) The CoV for sample 6 was however relatively high. It is believed that the reason for this relatively high CoV is that the some of the SAF structures at their bottom periphery where they are integrated with the remaining solid support, had small surface corrugations and/or protrusions, which may result in loss of signal. Hence, it is expected that by decreasing the top diameter to height aspect ratio D/h, this possibly loss of light signal may be mitigated. It is believed that by increasing the height e.g. to about 0.3 mm, the signal may be increased and the CoV may be decreased.

(149) The SAF structures no. 5 had both a highest light intensity and a low CoV for the water/PS interface signals.

(150) It is believed that the rounded edges of the recess ensure a very effective and robust spotting, which add to the low CoV.

(151) The SAF structures no. 6 had the highest light intensity for the water/PS interface signals and the CoV. Both for the air/PS interface signal intensities and the water/PS interface signals, the CoV were however, relatively high.