UNIVERSAL METHOD FOR DETECTING VARIOUS ANALYTES

Abstract

The invention relates to a method for detecting various analytes, characterized by the following steps: a) providing separation particles containing, on their surface, firstly means of binding the analyte to be identified and secondly means of separating the analyte bound to the particles; b) providing identification particles firstly having, on their surface, means for binding the analyte to be identified and secondly containing on their surface or enclosed therein, means which are capable, after they have been detached or released from the particles, by virtue of their labeling, of generating a signal which serves for identification of the analyte; c) combining analyte, separation particles and identification particles; d) removing and washing the identification particles bound via the analyte by means of the separation particles; e) releasing the means which serve to identify the analyte, characterized in that the means which serve to identify the analyte are coupled reversibly to the identification particles and in that the identification molecules serve simultaneously for identification of the analyte and for detection.

Claims

1. A method for detection of at least one analyte comprising: a) providing separation particles or a solid phase containing on their surface on the one hand means of binding the analyte to be identified and on the other hand means of separating the analyte bound to the particles, b) providing identification particles having on their surface on the one hand means for binding the analyte to be identified and on the other hand containing on their surface or enclosed therein means, which are capable, after they have been detached or released from the particles, by virtue of their labeling of generating a signal which serves for identification of the analyte, c) combining analyte, separation particles and identification particles, d) removing and washing the identification particles bound via the analyte by means of the separation particles, e) releasing the means which serve to identify the analyte, thereby identifying the analyte coupled reversibly to the identification particles, wherein identification molecules serve simultaneously for identification of the analyte and for detection.

2. The method according to claim 1, wherein the identification molecules are labeled and serve as means for identification of the analyte, and wherein said molecules are either reversibly coupled to nucleic acids, which in turn are bound irreversibly to a functionalized surface of the identification particles or are enclosed in the identification particles.

3. The method according to claim 1, wherein the identification particles comprise siliceous particles and are bound via spacers, or the separation particles comprise magnetic particles with a magnetic core but a siliceous shell.

4. The method according to claim 1, wherein the surfaces of the identification particles do not carry any specific interaction partner and bind analytes nonspecifically or the surfaces bind several substances.

5. The method according to claim 1, wherein the identification particles comprise a COOH functionalized surface to which NH.sub.2 coupled nucleic acids are irreversibly bound.

6. The method according to claim 1, wherein identification molecules are labelled and comprise at least one labeling permitting direct identification of nucleic acid molecules.

7. The method according to claim 6, wherein the at least one labelling is FITC, biotin, digoxigenin or tamra.

8. The method according to claim 1, wherein the identification of the analyte occurs on a lateral flow strip or via a fluorescence measurement.

9. The method according to claim 1, wherein the identification molecules carry one or more fluorophores for identification.

10. The method according to claim 1, wherein the analyte is at least one immunogen or nucleic acid.

11. The method according to claim 1, wherein the analyte is at least one nucleic acid and the labeled identification molecules simultaneously serve as a means for binding the nucleic acid analyte to be identified to the identification particles, and a separate means for binding the nucleic acid analyte to be identified to the identification particles is not present.

12. The method according to claim 1, wherein the labeled identification molecules serve as an analyte for signal amplification.

13. The method according to claim 1, wherein the separation particles are magnetic or comprise a functionality permitting a separation process.

14. The method according to claim 1, wherein in a) a solid phase containing on their surface on the one hand means of binding the analyte to be identified and on the other hand means of separating the analyte bound to the particles is used instead of particles.

15. The method according to claim 1, wherein the size of the particles ranges between 1 nm and 10 m.

16. The method according to claim 1, wherein the identification particles comprise at least one of gold, latex, polymers or fullerenes.

17. Particles with a size ranging between 1 nm and 10 m that comprise a functionalized surface suitable for detection of at least one analyte said surface comprises on the one hand means for binding the analyte to be identified, and comprises irreversibly bound nucleic acids, wherein the particles further comprise labeled identification molecules which are reversibly coupled to the irreversibly bound nucleic acids.

Description

EMBODIMENTS

Example 1

Production of the Identification Particles and Checking of their Purity and Functionality

[0052] As identification particles, COOH functionalized polylactic acid particles with the size 500 nm have been used. A NH.sub.2 coupled oligonucleotide (NH.sub.2-GTG TTC GTG TCA TCT AGG AG) and an antibody against yellow fever virus are covalently bound to said particles. Binding occurred with a PolyLink-Protein Coupling Kit for COOH microparticles according to the instructions of the kit.

[0053] Subsequently the particles were washed and blocked with Tris-BSA.

[0054] As a next step, hybridization of the covalently bound oligonucleotide to the double-labeled identification molecule (FITC-TGC AGC AGG TGA TAA CCT TTG GCT CCT AGA TGA CAC GAA CAC-digoxigenin) followed. The bases shown in italics of the identification molecule are complementary to the covalently bound oligonucleotide. The identification particle functionalized with the antibodies and the oligonucleotides is schematically shown in FIG. 3.

[0055] The particles have been washed twice and subsequently chromatographically purified. Application of the particle supernatant onto a lateral flow strip with the binding domains for FITC and digoxigenin should supply information on the purity of the particles of the unbound, double-labeled identification oligonucleotide. On FIG. 4 (strip A) the strip is shown. The signal on the strip shows that the particles despite several washing and a chromatographic purification are not free of unbound identification molecules. The particles were continued to be washed with large volumes of Tris-BSA buffer until the supernatant showed no positive result on the lateral flow strip (FIG. 4; strip B).

[0056] Subsequently, it had to be shown that the identification particles despite several washings remain functional, hence that the double-labeled oligonucleotide is still hybridized to the oligonucleotide covalently bound to the particle. For this purpose, the purified identification particles were heated up to 70 C. in order to detach hybridization between the two oligonucleotides. The supernatant after heating was again applied onto a lateral flow strip. In that case, the lateral flow strip showed a positive result (FIG. 4; strip C) which proves that the identification particles on the one hand are clean and do not contain any more identification molecules (which leads to false positive results), on the other hand are functionally active for further identification.

[0057] The experiment demonstrated moreover the importance of the double-labeled identification molecules for process monitoring during production of the identification molecules.

Example 2

Vaccinia Virus Identification by Means of Functionalized Polymer Detection Particles with Enclosed Identification Molecules

[0058] Nanocapsules, which do not present the double-labeled identification molecules on the surface but have these enclosed, serve as identification particles. Said polyester nanoparticles are produced by a direct miniemulsion by using the monomer 5,6-benzo-2-methylene-1,3-dioxepane (BMDO) and refunctionalized by carbodiimidazole couplings. Said production permits, apart from encapsulation of the DNA molecules, also an antibody binding on the surface of the nanoparticles. The identification particles differ from the other identification particles described only in that the identification molecules are now inside the particles and not on the surface. On the surface there is now only one antibody left. The identification particles and the separation particles are functionalized with anti-vaccinia virus antibodies. Identification of the vaccinia viruses occurs as described below. Detection particles and identification particles are introduced into the sample containing the vaccinia viruses:
Step 1: Formation of a complex from separation particles-virus-identification particles. A complex between the identification particles and separation particles and the vaccinia viruses is created. In the case of a target negative sample, no complex formation occurs and the identification particles as well as separation particles remain separated from each other.
Step 2: Magnetic separation. In the case of a positive sample, the complex formed (separation particles-virus-identification particles) is separated from the sample solution via magnetic separation, and subsequently washed twice in PBS, and briefly dried after a renewed separation.
Step 3: Release of the double-labeled molecules, identification on a lateral flow strip. A 15 minutes incubation at 85 C. generates a steam pressure within the identification particles which causes bursting of the particle walls and release of the double-labeled molecules. After the addition of water and a short vortex step, the particle supernatant is applied onto a lateral flow strip where the identification of the double-labeled molecules, and thus the identification of the existence of vaccinia viruses in the sample occurs.
The embodiment is schematically shown again in FIG. 4.

Example 3

Identification of a Small Amount of Yellow Fever Virus by Means of the Particles Produced According to the Invention

[0059] As identification particles the particles produced in embodiment 1 have been used. As separation particles the COOH functionalized dextran-iron oxide-particles have been used. Said particles by means of the PolyLink-Protein Coupling Kit for COOH microparticles according the instructions of the kit were coupled with the same antibody as the identification particles. Yellow fever viruses from a viral culture were used as analytes. The concentration of the viruses was 1000 PFU/ml.

Reaction Batch:

[0060]

TABLE-US-00001 Positive Sample Negative Sample Viral culture 10 l~100 PFU 1xPBS 100 l 200 l Identification particles 5 l 5 l Separation particles 5 l 5 l

Work Steps:

[0061] 1. The reaction batch was incubated in 2 ml reaction vessels for 10 minutes at room temperature.
2. The reaction vessels were placed in a magnetic trap.
3. The sample supernatant was discarded.
4. The samples were washed twice in the magnetic trap.
5. After removal of the washing solution, 100 l water were added to each sample.
6. The samples were removed from the magnetic trap and incubated for 10 minutes at 70 C.
7. The samples were placed again into a magnetic trap.
8. 5 l of the supernatant were applied onto a lateral flow strip for detection of the released identification particles.

[0062] Total expenditure of time including incubation times 30 minutes max.

[0063] The results of the identification for a positive and a negative sample are shown in FIG. 5. As the result shows, direct identification of the yellow fever viruses could be carried out successfully with the process being characterized by its simplicity (no device is required) as well as by its high sensitivity and rapid performance.

Example 4

Comparison of the Identification Sensitivity of a Lateral Flow Test with a Fluorescence Measurement

[0064] For the experiment, FAM-biotin labeled identification molecules were used where the FAM labeling can serve not only as an immunogen for an antibody binding a lateral flow strip but also as a fluorescent substance for a fluorescence measurement.

[0065] The identification molecules were measured in a dilution series (see table and FIGS. 7A and B). The fluorescence measurement occurred with a real time PCR device as an end-point measurement.

TABLE-US-00002 Number of identification End-point fluorescent molecules Lateral Flow Strip measurement .sup.5 10.sup.11 positive positive .sup.5 10.sup.10 positive positive 5 10.sup.9 positive questionable 5 10.sup.8 positive negative 5 10.sup.7 positive negative 5 10.sup.6 positive negative 5 10.sup.5 positive negative 5 10.sup.4 negative negative

[0066] The experiment showed that not only a detection without a device but also a detection of the identification particles based on the fluorescence measurement is possible. Moreover it could be demonstrated that the fluorescence measuring instruments common in laboratories have an approximately 10000-fold lower sensitivity than a lateral flow test without a device. Hence, the samples in other bio barcode based identification methods where fluorescence of the identification molecules is measured must operate with very expensive and sensitive technology in order to achieve the same sensitivity.

Example 5

An Example for a Lateral Flow Based Multiplex Detection of the Identification Particles

[0067] In this experiment the identification particles were labeled with different labels. The results of the experiment and a schematic representation of a multiplex lateral flow strip are shown in FIGS. 8 A and B.

Explanation on the Figures

[0068] FIG. 1 shows the principle of identification for nucleic acids and antigens:

1. Combining analyte, identification particles and separation particles.
2. Magnetic separation and washing of the unbound particles and molecules. Subsequent detachment of the identification particles.
3. Direct identification of the double-labeled identification particles on a lateral flow strip.
4. Cascade-like amplification reaction by a second identification cycle.

[0069] In FIG. 2 a schematic representation of the binding a nucleic acid analyte to the identification particles is shown. A schematic representation of the functionalized identification particle is shown in FIG. 3. The oligonucleotide complexes and antibodies shown are bound on the surface of the particle several times. FIG. 4 is a representation according to example 2.

[0070] Results of the lateral flow test in the process of production of the functionalized identification particles are shown in FIG. 5. FIG. 6 shows the identification of a lower amount of yellow fever virus. The comparison of the detection sensitivity of a lateral flow strip (A) with a fluorescence measurement (B) is the subject matter of FIG. 7. FIG. 8 shows the example of a multiplex detection of the identification particles.