SUBSTRATE FOR AMPLIFYING THE CHEMILUMINESCENCE

Abstract

The present invention relates to the use of a substrate for enhancing the chemiluminescence of one or more luminophores produced in a chemiluminescence reaction, wherein the substrate comprises a solid polymeric carrier with a plurality of depressions separated from one another and that the solid carrier is at least partially coated with a metal.

Claims

1. A method for enhancing the chemiluminescence of one or more luminophores produced in a chemiluminescence reaction, comprising: providing a substrate that comprises a solid polymeric carrier with a plurality of depressions separated from one another, wherein the solid polymeric carrier is at least partially coated with at least one metal.

2. The method according to claim 1, wherein the depressions in the plurality of depressions have a distance from each other of 0.2 μm to 2.5 μm.

3. The method according to claim 1, characterized wherein the depressions in the plurality of depressions have a length and a width, and wherein the ratio of the length to the width ranges from 2:1 to 1:2.

4. The method according to claim 1, wherein the depressions in the plurality of depressions have a length and a width, wherein the length is 0.1 μm to 2 μm and the width is 0.1 μm to 2 μm.

5. The method according to claim 1, wherein the depressions in the plurality of depressions have an essentially round shape.

6. The method according to claim 1, wherein the depressions in the plurality of depressions have a depth of 0.1 μm to 5 μm.

7. The method according to claim 1, wherein the at least one metal comprises at one or more than one at least partial metal layers arranged above one another; and optionally the at least one metal has a thickness of 10 nm to 200 nm.

8. The method according to claim 1, wherein the at least one metal is chosen from silver, gold, aluminum, chrome, indium, copper, nickel, palladium, platinum, zinc, tin, and alloys comprising one or more thereof.

9. The method according to claim 1, wherein the solid polymeric carrier comprises at least one material chosen from thermoplastic polymers, polycondensates, polyolefins, vinyl polymers, styrene polymers, polyacrylates, polyvinylcarbazole, polyacetal polymers, fluoropolymers, thermoplastic polycondensates, thermosetting polycondensates, and polyadducts, and optionally comprises one or more of TiO.sub.2, glass, carbon, pigments, lipids, and waxes.

10. The method according to claim 1, wherein the one or more luminophores are produced by an enzyme.

11. The method according to claim 10, wherein the enzyme is chosen from horseradish peroxidase, alkaline phosphatase, luciferase and hydrolytic enzymes.

12. The method according to claim 1, wherein the one or more luminophores are chosen from luminol and its derivatives, 1,2-dioxetane, acridinium esters and luciferins.

13. The method according to claim 10, wherein the enzyme is directly and/or indirectly bound to the substrate.

14. (canceled)

15. A method for determining or quantifying at least one analyte in an aqueous sample, comprising: a) contacting the aqueous sample with a substrate that comprises a solid polymeric carrier with a plurality of depressions separated from one another, wherein the solid polymeric carrier is at least partially coated with at least one metal; the substrate further comprising an analyte-binding molecule directly or indirectly bound to the substrate, b) adding at least one additional analyte-binding molecule to which at least one enzyme is directly or indirectly bound that produces one or more luminophores from one or more precursors in a chemiluminescence reaction, and c) measuring the light emission resulting from the chemiluminescence reaction.

16. The method according to claim 15, wherein the at least one enzyme is chosen from horseradish peroxidase, alkaline phosphatase and luciferase.

17. The method according to claim 15, wherein the one or more luminophores are chosen from luminol and its derivatives, 1,2-dioxetane, acridinium esters and luciferins.

18. The method according to claim 15, wherein the analyte-binding molecule and the at least one additional analyte-binding molecule are chosen from antibodies, antibody fragments, Fab, F(ab)′2 fragments, scFv fragments, nucleic acids, aptamers, and combinations thereof.

19. The method according to claim 15, wherein the at least one enzyme is indirectly bound via a carrier molecule chosen from antibodies and antibody fragments, Fab, F(ab)′2 fragments, scFv fragments, nucleic acids, aptamers, and combinations thereof.

20. The method according to claim 15, wherein the light emission is measured at a wavelength of 280 nm to 850 nm.

Description

[0068] The present invention will be explained in further details with reference to the following figures, without, however, being limited to these.

[0069] FIG. 1 shows the inventive substrate comprising a solid carrier that is coated with a metal layer. The solid carrier has depressions having a depth, a width and a length. The depressions are positioned on the solid carrier at a certain distance (period) from each other.

[0070] FIG. 2 shows a plan view (A) and a cross section (B) of an inventive solid carrier. The depressions on the solid carrier are characterized by a width, a length and a depth and are at a certain distance (period) from each other.

[0071] FIG. 3 shows the MEC enhancement as a function of the antibody concentration.

[0072] FIG. 4 shows the signal-to-noise ratios in a 2-step assay setup on MEF and standard MTPs. Table 2 and the following graphics show the achieved SNRs or MEC enhancements as a function of the concentration of the coated goat antibody.

EXAMPLES

[0073] By means of the examples described below it was examined whether the substrates developed for metal-enhanced fluorescence are also suitable for improving the signal-to-noise ratio of chemiluminescence measurements.

[0074] For the following examples, microtiter plates having the structure disclosed in AT 517 746 at their bottom were used. Particularly, silver-coated polymeric carriers with a plurality of depressions separated from one another and having a diameter of 0.4 μm, a period (i.e. distance between two depressions) of 1 μm, and a depth of 0.7 μm were applied to the bottom of the microtiter plates. Commercially available microtiter plates of the company Greiner (Austria) were used for comparison purposes in order to determine the extent of the enhancement effect achieved.

Example 1:Direct Detection of Adsorbed, Enzyme-Labeled Antibodies

[0075] The simplest method for detecting an MEC (“metal-enhanced chemiluminescence”) enhancement effect adsorbing an enzyme-labeled antibody to the bottom of a microtiter plate described above and, after a washing step, detecting the bound antibody by means of a chemiluminescence substrate for the respective enzyme.

[0076] Procedure [0077] 50 μl of a donkey-anti-goat-antibody (Sigma, SAB3700287, 1 mg/ml) dilution in 50 mM phosphate buffer/100 mM NaCl with concentrations of 10.sup.−9 to 10.sup.−15 mol/L were incubated for 2 h at RT in the dark on the MEF or Greiner 1×8 HB strip MTPs (VWR, 737-0195). [0078] The content of the wells (depressions on microtiter plates) was discarded and the plates were washed 3×with 200 μl 50 mM phosphate buffer/100 mM NaCl/0.1% TritonX100. [0079] According to the manufacturer specifications (BM Chemiluminescence ELISA Substrate Kit, Sigma, 11759779001), 10 μl of the chemiluminescence substrate were mixed with 100 μl of the enhancer and 890 μl of the assay buffer, both included in the kit. The substrate contained in the kit is CSPD (disodium 3-(4-methoxyspiro{1,2-dioxetan-3,2′-(5′-chloro)tricyclo[3.3.1.1.sup.3,7]decan}-4-yl)phenyl phosphate), which is converted into an instable dioxetane by the ALP and has its emission maximum at 477 nm. The enhancer used for enhancing the quantum yield in this kit was Emerald II. [0080] 150 μl of this reaction mixture were pipetted onto the MTPs and the emerging chemiluminescence signal was monitored with a SPARK microtiter plate reader by TECAN.

[0081] Results

[0082] Temporal Evolvement of the Signal-To-Noise Ratio (SNR, Chemiluminescence of a Well with Antibodies/Chemiluminescence without Antibodies)

[0083] As may be seen in Table 1, the SNR only increases over time in the microtiter plates with a structured bottom as described at the beginning (MEF-MTP), while it stagnates or even decreases on a standard microtiter plate (“Greiner MTP”) by Greiner.

TABLE-US-00001 TABLE 1 SNR over time Signal-to-noise ratio-MEC enhancement t = 30 sec t = 300 sec Antibody MEF- Greiner MEF- Greiner (M) MTP MTP MEC MTP MTP MEC 0 1.0 1.0 1.0 1.0 1.0 1.0 10{circumflex over ( )}-15 1.9 1.0 1.9 3.2 1.0 3.2 10{circumflex over ( )}-14 4.5 2.0 2.3 10.2 1.9 5.5 10{circumflex over ( )}-13 18.4 4.2 4.4 50.2 3.8 13.1 10{circumflex over ( )}-12 147.4 8.6 17.1 427.9 8.8 48.7 10{circumflex over ( )}-11 1385.8 35.3 39.2 3564.6 36.7 97.2 10{circumflex over ( )}-10 8144.8 211.5 38.5 16237.9 176.3 92.1 10{circumflex over ( )}-9  16176.0 441.0 36.7 25781.6 309.8 83.2

[0084] This may be explained by the fact that due to the MEC, which increases the quantum yield, quenching of the bulk solution enriching itself with luminophores is prevented. Consequently, the detection of the adsorbed antibody on the MEF-MTP becomes more and more sensitive over time compared to a standard MTP and allows a reduction of the detection limit by at least a power of ten.

[0085] Dependence on Concentration and Extent of MEC

[0086] The simplest way to conduct a quantification of the enhancement extent is by comparing the SNRs on MEF and standard MTPs.

[0087] This showed a clear dependence of the extent of MECs on the concentration (data after 300 sec measurement time), as shown in FIG. 3. This enhancement increased up to a concentration of 10.sup.−11 of molar antibody adsorption solutions to almost the 100-fold, but then starts decreasing again, which is probably also due to increasing quenching. Even with low concentrations in the sub-picomolar range, there is still observable a clear 3- to 13-fold enhancement of the SNR of a standard MTP.

Example 2: Indirect Detection of Adsorbed, Non-Labeled Antibodies

[0088] The extent of the MEF (“metal-enhanced fluorescence”) effect strongly depends, inter alia, on the distance of the fluorophores from the nanostructure (s. also Hawa et al. Analytical Biochemistry 549(2018): 39-44).

[0089] In the case of an enzyme-catalyzed MEC test, contrary to MEF tests with e.g. directly fluorescently labeled antibodies, the produced luminophore diffuses away from the surface and may thus only be enhanced as long as it is on the surface. In order to show that a luminescence enhancement is also possible in an assay setup over two protein layers (at least approx. 10-15 nm), the experiment described in Example 1 was modified in that first, a goat antibody was applied to the MEF-MTP, which was then detected after a blocking step with an ALP-labeled anti-goat antibody.

[0090] Procedure: [0091] 50 μl of a goat-antibody (Jackson, 111-005-008, 2 mg/ml) dilution in 50 mM phosphate buffer/100 mM NaCl with concentrations of 0-1 μg/ml were incubated over night at 4° C. in the dark on the MEF or Greiner 1×8 HB strip MTPs (VWR, 737-0195). [0092] The content of the wells was discarded and the plates were washed 3× with 200 μl 50 mM phosphate buffer/100 mM NaCl/0.1% TritonX100. [0093] Blocking of unspecific binding was conducted by incubation for 2 hours at RT with 100 μl of 50 mM phosphate buffer/100 mM NaCl/0.1% TritonX100 with 5% polyvinylpyrrolidone (5% PBSPTx) [0094] After a further washing step, the plates were incubated with 50 μl of a 30 ng/ml solution of the ALP-labeled anti-goat antibody also used in item 2 for 2 h at room temperature. [0095] After a final washing step, again 150 μl of the luminophore reaction mixture described in Example 1 were added and the emerging chemiluminescence signal was monitored with a SPARK microtiter plate reader by TECAN.

[0096] Results

[0097] A quantification of the extent of enhancement was again conducted by comparing the SNRs on MEF and standard MTPs (Greiner MTP). Table 2 and FIG. 4 show the obtained SNR values and MEC enhancements as a function of the concentration of the coated goat antibody.

TABLE-US-00002 TABLE 2 Signal-to-noise ratio in 2-step assay setup Signal-to-noise ratio Antibody (μg/ml) MEF-MTP Std-MTP MEC 0 1.0 1.0 n.a. 0.01 24.4 1.0 24.4 0.1 35.2 1.6 22.0 1 77.5 2.9 26.7

[0098] The enhancement effect is even observable over two protein layers. This is surprising because compared to the MEF with surface-bound fluorophores, the enzyme-produced luminophores diffuse and move further away from the surface. In any case, sensitivity increases by a factor of 20-30 are to be considered analytically relevant since they seem to increase with increasing coating concentrations.

[0099] Discussion

[0100] The structures developed for metal-enhanced fluorescence are thus also suitable for metal-enhanced chemiluminescence.

[0101] This is very surprising because the reach of the effect is only 40-50 nm (distance from the surface) and the chemiluminescent substrate produced by the enzyme diffuses away from the surface. Apparently enhancement is so strong that averaged over time, enough molecules are present close to the surface. Also the observed extent of the MEC is unexpected and has not been described in the literature so far.