Cluster for the detection of an analyte

Abstract

The present invention relates to a cluster for the detection of an analyte, said cluster comprising a plurality of visually detectable colored particles and a plurality of luminescent particles, wherein (i) the particles are bound to each other, and (ii) at least one binding partner of an analyte is bound to the colored particles and/or the luminescent particles.

Claims

1. A cluster for the detection of an analyte, said cluster comprising: a plurality of visually detectable colored particles a plurality of luminescent particles, and at least one binding partner of an analyte, wherein (i) the visually detectable colored particles and the luminescent particles are covalently bound to each other through a covalent bond that is a peptide bond, wherein the convalent bond is a peptide bond, (ii) the binding partner of the analyte is bound to the colored particles, the luminescent particles, or both, and, (iii) the visually detectable colored particles are non-organometallic metal or metal oxide particles.

2. The cluster of claim 1, wherein the visually detectable colored particles are metal or metal oxide particles comprising Au, Ag, Ni, Pt, Cd, Fe, Cu, or an oxide thereof.

3. The cluster of claim 1, wherein the luminescent particles are fluorescent particles or phosphorescent particles.

4. The cluster of claim 1, wherein the at least one binding partner is selected from the group consisting of a protein, a DNA, a RNA, and an aptamer.

5. The cluster of claim 4, wherein the at least one binding partner is an antibody, and the analyte is a proteinaceous or peptidic antigen.

6. The cluster of claim 5, wherein the antibody is an antibody against the NS1 glycoprotein of the Dengue fever virus, and the proteinaceous or peptidic antigen is an antigen of the NS1 glycoprotein of the Dengue fever virus.

7. The cluster of claim 1, wherein the cluster has a diameter of 1 nm to 20 m.

8. The cluster of claim 1, wherein a ratio of the visually detectable colored particles to the luminescent particles is 50:50 to 80:20 by weight %.

9. The cluster of claim 1, wherein the colored particles and the luminescent particles are coated with an agent having at least bifunctionalities that allows binding of the colored particles and the luminescent particles together.

10. A device for the detection of an analyte in a liquid sample, said device comprising a plurality of the clusters of claim 1, wherein said device comprises a solid phase having a sample site, wherein at the sample site the plurality of clusters is disposed.

11. The device of claim 10, wherein the device additionally comprises a capture site, wherein at the capture site a plurality of capture agents binding to the analyte are immobilized, and wherein the capture site and the sample site are positioned such that capillary flow communication between the sample site and the capture site is allowed.

12. A kit for the detection of an analyte comprising (i) the cluster of claim 1; and (ii) information and/or instructions describing how to use the kit.

13. A method for the detection of an analyte in a liquid sample comprising (a) contacting the cluster of claim 1 with the liquid sample, and (b) determining the presence of the analyte by (i) visual color inspection, and optionally (ii) detection of luminescence.

14. A method for the detection of an analyte in a liquid sample by the device of claim 11, said method comprising (a) providing the device of claim 11, (b) contacting a liquid sample with the sample site, (c) allowing the liquid sample to flow to the capture site, and (d) determining the presence of the analyte at the capture site by (i) visual color inspection, and optionally (ii) detection of luminescence.

15. The cluster of claim 4, wherein the at least one binding partner is an L-ribonucleic acid aptamer.

Description

(1) The figures show:

(2) FIG. 1: Exemplary lateral flow immunoassay

(3) FIG. 2A-2I: Transmission electron microscopy of clusters after separation. Bright field images of (FIG. 2A) gold nanoparticles, (FIG. 2B) polystyrene nanoparticles and (FIG. 2C) low energy loss spectra of gold and polystyrene nanoparticles. Bright field (FIG. 2D) and 25 eV energy loss (FIG. 2E) from the same area showing better resolution for polystyrene nanoparticles observation. Cluster images (FIG. 2E-FIG. 2G) at 25 eV of the fraction used in lateral flow tests and images (FIG. 2H and FIG. 2I) of the removed fraction of clusters.

(4) FIG. 3A-3C: Lateral flow immunoassay for Dengue virus NS1 protein detection based on clusters (FIG. 3A) under UV light (FIG. 3B) and gold nanoparticles (FIG. 3C). The visible detection limits are shown by the red circles.

(5) FIG. 4A-4B: Immunospot assay for Dengue fever detection based on clusters (FIG. 4A-4B). Right image was taken under UV light (FIG. 4B).

(6) FIG. 5: Pictures of two serum dilution series for each commercial kits (Standard Diagnostics, SD, and Biorad, BR). Each concentration has pictures of the tests following the order: (1) commercial test, SD or BR, (2) colorimetric, CI, and (3) fluorescence, FI, signal of the cluster based test. The circles indicate the lowest concentration that the observers could recognize by naked eyes.

(7) The examples illustrate the invention.

EXAMPLE 1- MATERIALS AND METHODS

(8) In order to illustrate the invention, gold and fluorescent nanoparticle clusters were used to detect Dengue fever. A protein non-structural from the virus, NS1, was chosen as target analyte. The result was compared to gold based LFIA.

(9) Chemicals

(10) Gold nanoparticles (mean diameter: 40 nm), bovine serum albumin (BSA) powder, biotin, streptavidin, anti-streptavidin IgG antibody, N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), dialysis membranes (MWCO 100 kDa and 130 kDa), sucrose, potassium phosphate mono- and dibasic were purchased from Sigma-Aldrich (Milwaukee, USA). Streptavidin-labeled gold nanoparticles (mean diameter: 40 nm) was purchased from British Biocell (Cardiff, United Kingdom). FluoSpheres (PS) carboxylate-modified microspheres (excitation: 580 nm/emission: 605 nm) 0.04 m, and nitrocellulose AC99 membrane were obtained from Invitrogen (Carlsbad, United States) and Whatman (Maidstone, United Kingdom), respectively. Sample and conjugate pad were obtained from Pall (Dreieich, Germany). Absorbent pad and backing card were provided from Millipore (Billerica, USA) and Lohmann (San Jose, USA), respectively. Dengue virus NS1 glycoprotein mouse monoclonal antibody (supernatant) and Melon gel IgG purification kit were obtained from Abcam (Cambridge, United Kingdom) and Thermo Scientific (Rockford, United States), respectively. Dengue NS1 Ag ELISA was purchased from Standard Diagnostics (Youngin, Korea).

(11) Preparation of Gold- and Polystyrene-Nanoparticle Protein Conjugates

(12) Gold nanoparticle dispersions at 15% were adjusted to pH 8 with NaOH 0.01M and 30 L of an albumin solution at a concentration of 1 mg/mL was added to 0.3 mL of the gold dispersion. The mixture was stirred for 30 min, and then, to remove the excess of proteins, it was centrifuged at 5000 rpm for 15 min at 4 C. The clear supernatant was carefully removed, and the precipitated gold conjugates were resuspended in 400 L of 0.01M phosphate buffer, pH 7.4, and stored at 4 C. Gold nanoparticles conjugated with NS1 antibody was produced by following the same procedure. Albumin coated polystyrene nanoparticles were prepared as described by Linares and coauthors (Linares, E. M. et al., 2013, Biosens. Bioelectron. 41, 180-185).

(13) Preparation of Gold-Polystyrene Nanoparticle (PS) Clusters

(14) Colloidal gold and fluorescent particles, previously coated with albumin, were covalently bound by forming peptide bonds between albumin molecules, using water-soluble carbodiimide to activate the surface carboxyl groups. To produce clusters with 80:20 (wt. %) of gold:PS, 300 L of gold nanoparticles at 15% of solids was mixed with 562 L of PS nanoparticles at 2% and incubated during 30 minutes in a shaker at RT. Subsequently, 2 mg of EDC (1-ethyl-3-(3-dimethylamino) propyl carbodiimide hydrochloride) was added to the suspension and incubated for 3 hours at RT. Thereafter, the suspension was centrifuged twice at 3000 rpm for 2 minutes and washed with 0.01 mol/L phosphate buffer, pH 7.4. Clusters were deposited on 1 mL of 1 mol/L sucrose solution in a centrifuge tube and centrifuged at 13000 rpm at 4 C. for 30 min. An aliquot of 100 L was removed and the rest was divided in two aliquots. The aliquot on the top was used for the assay, after passing through a 220 m filter.

(15) Cluster Functionalization with Biomolecules

(16) Clusters were conjugated to streptavidin and monoclonal NS1 Dengue antibody. An aliquot of 500 L of cluster dispersion 80:20 gold:PS was added to 500 L of a 0.5 mg/mL solution of protein dissolved in 0.01 mol/L phosphate buffer, pH 6. The suspension was incubated for 30 minutes at RT. Subsequently, 1 mg of EDC was added and mixed by vortexing and the pH were adjusted to 6.5 with diluted NaOH. The dispersion was incubated on a shaker for 3 hours at RT. To separate the protein-labeled clusters from unbound proteins, the suspension was centrifuged three times at 3000 rpm for 10 minutes at RT. The final suspension was kept in a phosphate buffer containing 1% BSA.

(17) Strip Tests and Immunospot Assay

(18) Before setting up the strip test for clusters, all used membranes received different treatments: the sample pad was dipped into 0.01 mol/L phosphate buffer, pH 7.4, containing with 5% BSA and 0.05% Tween20 and dried for 2 hour at 60 C.; the conjugate pad was previously immersed in 1 mmol/L borate buffer, pH 9, with 10% of sucrose, and then clusters at 5% (w/v) were deposited and dried at RT; anti-NS1 protein antibody and biotinylated-albumin at a concentration of 1 mg/mL in 0.01 mol/L phosphate buffer, pH 7.4, were spotted onto nitrocellulose to form the detection and control lines by using Dimatix printer from Fujifilm (Santa Clara, USA). BSA was biotinylated according to Guesdon, J. L. et al., 1979. J. Histochem. Cytochem. 27, 1131-1139. Ho, J. A. A., Zeng, S. C., Tseng, W. H., Lin, Y. J., Chen, C. H. 2008. Anal. Bioanal. Chem. 391, 479-485. The detection pad was dried at RT; and the absorbent pad was used as received. Subsequently, all membranes were laminated on the backing card with an overlap of 2 mm between them. The membranes were cut at 4 mm wide. Serum samples were analyzed by adding 100 L on the sample pad. When the flow stopped, 100 L of 0.01 mol/L phosphate buffer at pH 7.4 was added. The same buffer was used as blank. Analysis was performed after 25 min.

(19) Immunospotting Assay

(20) For mutiple tests, 4 L of serum was deposited on the nitrocellulose membrane (7 cm10 cm) with 15 mm spacing between each spot to avoid contamination and checking the alignment between them to fit on the wells of an ELISA microplate. This sample volume was the minimum volume necessary to observe a clear result. After 10 minutes, a blocking solution containing BSA 3% in phosphate buffer 0.01 mol/L, pH 7.4 was added to cover the entire membrane for 15 minutes at RT. Subsequently, the blocking solution was removed and 3 mL fluorescent conjugates 1% in phosphate buffer 0.01 mol/L, pH 7.4 was added and incubated for 30 minutes.

(21) Comparison with Commercial Kits

(22) Two kits, Standard Diagnostics Bioline Dengue Duo and Biorad Strip, were used to compare their performance with the proposed assay. The kits were chosen based on their performance described by Blacksell S. D. J. Biomed. Biotechnol. 2012, 1-12. Eight infected serum samples were diluted with phosphate buffer to produce solutions with lower NS1 concentration. The dilution varied with the initial concentrations of NS1, which were determined using enzyme linked immunosorbent assay. A panel of 14 observers analyzed the tests and verified the minimum dilution that could be observed by them. The visual detection limit was established by the concentration which at least half of the observers could detect. The tests were performed in duplicate and the observers could visualize the same signal in the strip tests with the same concentration. Pictures were recorded and the signals of the test lines (area: 1 mm4 mm) were converted to gray scale and then compared with graphs of gray scale vs. NS1 concentration. The acquired images under UV lamp were converted to gray scale and the signal was inverted in the scale to facilitate the comparison with the colorimetric signal.

EXAMPLE 2-USE OF CLUSTERS OF THE INVENTION TO DETECT NS1

(23) Cluster Characterization

(24) Spectra from EELS (Electron energy loss spectroscopy) were obtained for gold (FIG. 2A) and polystyrene (FIG. 2B) nanoparticles from the areas indicated in the images and are depicted in the FIG. 1c. Polystyrene spectrum has higher intensity at the low energy loss than gold, and hence it will appear brighter in the energy image. The bright field (FIG. 1D) and its respective image at 25 eV (FIG. 2E) show clearer that polystyrene particles are poorly observed at bright field but better observed in the low energy loss image. The first fraction (from up to down in the centrifuge tube) concentrates smaller clusters as observed in the FIG. 2E-G, where it is possible to observe cluster up to 5 particles with mixed composition. The images from the second fraction indicate the presence of bigger clusters with variable composition and format. Tests with each fraction showed that the first fraction produces better signal and reproducibility.

(25) Detection Limit for LFIA Based on the Clusters and Compared to Gold Nanoparticles

(26) LFIA based on clusters (FIG. 3A-B) shows a visible signal up to 10 ng/mL, but when the strip tests are under UV light, the detection limit achieves lower values, up to 2.5 ng/mL. In order to compare the clustering effect on the detection limit, LFIA based on gold nanoparticles were built and depicted in the FIG. 3C. It shows a visible test line until 500 ng/mL, once the signal at 250 ng/mL is barely observable. The results demonstrate that clusters provide a detection limit 50 times lower than gold nanoparticles and it decreases to 200 times under UV lamp.

(27) Thus, if the colored sign is slightly positive, indicating uncertain result, a lamp/LED can be used to excite the fluorescent particles and the fluophores will emit in the visible spectrum. It will provide sensitivity 4 times better than the colorimetric signal.

(28) Clusters Used to Detect NS1 in an Immunospotting Assay

(29) A panel with 48 samples of Dengue infected patients was deposited on nitrocellulose and detected with gold-fluorescent nanoparticles cluster. FIG. 4 indicates that the fluorescent nanoparticles provide an additional signal that contributes to a more reliable assay. Based on the exposed results, nanoparticle clusters represent a powerful option to overcome sensitivity limitations of LFIA.

(30) Comparison with Commercial Kits

(31) FIG. 5 shows the comparison of the two kits with the proposed assay, showing the pictures for two serum dilutions per kit. FIG. 5 indicates the serum dilution series and the performance of the commercial kits in comparison to the cluster based test. The lowest concentrations indicated as visible by the observers are shown by the circles. The cluster based tests showed better performance than the commercial kits.

(32) TABLE-US-00001 TABLE 1 Visual detection limits for the proposed method and the Biorad strip. Detection limit (ng/mL) Biorad Cluster Colorimetry Colorimetry Fluorometry Sample 1 50 25 10 Sample 2 100 50 10 Sample 3 100 50 25 Sample 4 200 100 50

(33) The detection limit obtained for the Biorad Strip is 200 ng/mL and the detection limit observed for the tests based on cluster is 100 ng/mL for the colorimetric signal and 50 ng/mL for the fluorometric signal.

(34) The Biorad strip tests showed better performance than the Standard Diagnostics Bioline Dengue Duo, but it is has lower sensitivity than the tests based on the clusters.

(35) Label Comparison with Other Labels for LFIA

(36) Different labels for Dengue fever detection were compared. The results indicate that the cluster of particles shows better performance that the most used labels for LFIA or immunspotting assay.

(37) TABLE-US-00002 TABLE 2 Comparison of labels for Dengue fever detection. Detection limit Duration Detection system Assay type (ng/mL) (min) Gold nanoparticles Lateral flow 500 20 (gold standard) immunoassay Carbon black Lateral flow 10 25 immunoassay Fluorescent Immunospotting Optical fiber 45-60 nanoparticles assay reader: 5 ELISA reader: 15 UV lamp: 200 Phospho- Immunospotting Microscope: 190 25 rescentPSHEMA- assay Ru particles Gold-fluorescent Lateral flow Visible: 10 25 nanoparticle clusters immunoassay Under UV: 2.5
Optimization During Cluster Preparation

(38) Numerous reactions were performed to develop and optimize the clusters, including the items: Particle functionalization: It is preferred to avoid the direct immobilization of the streptavidin/antibody on the particle surface Spacer: the biofunctionalization is preferably enhanced by using a spacer based on BSA coating Cluster composition: depending on the specific application of the cluster of the invention, the composition of particles was optimized, e.g., by changing the particle concentration, reaction time and activator concentration in the production process of the clusters.

(39) TABLE-US-00003 TABLE 3 Summary of the advantages of cluster based assays for Dengue fever detection. Gold nanoparticles Cluster (standard) Colorimetric NS1 100 200-500 Detection limit (ng/mL) Fluorescence NS1 50 Detection limit (ng/mL) Capability to detect the 1.sup.st-2.sup.nd 3.sup.rd disease from the outbreak of the symptoms (day) Capacity to recognize Enhanced by the Limited by the low the analyte highest number of number of recognition available molecules recognition immobilized on the molecules particles (hindrance) Capacity to determine Enhanced by Limited by the visual the analyte in low fluorescence color provided by single concentration colored particles