COMPOSITE PARTICLES FOR BIOLOGIC ASSAY

Abstract

Composite particles including nanoparticles dispersed in a matrix and their use in biologic assay. The nanoparticles selectively absorb or selectively emit light and have a size in at least one of its dimensions shorter than 20 nm. The weight fraction of the nanoparticles in the composite particles is greater than 0.5% and less than 50%, and the matrix of the composite particles is inorganic and includes less than 90% by weight of silica. Also, the composite particles are functionalized with a specific-binding component and have a mean size greater than 50 nm and less than 1000 nm.

Claims

1.-11. (canceled)

12. A composite particle comprising nanoparticles dispersed in a matrix wherein: the nanoparticles selectively absorb or selectively emit light; the nanoparticles have a size in at least one of its dimensions shorter than 20 nm; weight fraction of the nanoparticles in said composite particle is greater than 0.5% and less than 50%; matrix is inorganic and comprises less than 90% by weight of silica; the composite particle is functionalized with a specific-binding component; and the composite particle has a mean size greater than 50 nm and less than 1000 nm.

13. The composite particle according to claim 12, wherein the nanoparticles are metallic and absorbs selectively light by plasmonic effect.

14. The composite particle according to claim 12, wherein the nanoparticles are inorganic and emit selectively light by luminescence.

15. The composite particle according to claim 12, wherein the nanoparticles have one dimension shorter than 10 nm.

16. The composite particle according to claim 15, wherein the nanoparticles have one dimension shorter than 5 nm.

17. The composite particle according to claim 12, wherein the nanoparticles have a shape selected from nanocubes, nanospheres, nanorods, nanowires, nanorings, nanoplates, nanosheets, nanoribbons or nanodisks.

18. The composite particle according to claim 12, wherein a first fraction of the nanoparticles selectively absorbs or selectively emits light; and a second fraction of the nanoparticles selectively absorbs or selectively emits light differently from the first fraction of the nanoparticles.

19. The composite particle according to claim 12, wherein the matrix comprises SiO.sub.2, Al.sub.2O.sub.3, ZrO.sub.2, HfO.sub.2, Si.sub.1-xZr.sub.xO.sub.2, Al.sub.2-2xZr.sub.2xO.sub.(3+x), or Hf.sub.1-xZ.sub.xrO.sub.2, x being a rational number between 0 (excluded) and 1 (excluded).

20. a Method of detection of a target analyte in a sample comprising the steps of: i) providing a sample; ii) letting a composite particles according to claim 12 get in contact with said sample so that the specific-binding component of said composite particles binds with a target analyte; iii) separating said composite particles bound with said target analyte from composite particles not bound with said target analyte; and iv) measuring light absorption or light emission of said composite particles bound with said target analyte or composite particles not bound with said target analyte.

21. The method of detection according to claim 20, wherein steps ii) and iii) are performed on a strip.

22. The method of detection according to claim 20, wherein measure of step iv) is made with a portable device.

23. The method of detection according to claim 20, wherein measure of step iv) is made with a mobile phone or a smartphone.

24. An assay test strip, comprising: a porous substrate; a sample receiving zone; a composite particle according to claim 12 that specifically binds a target analyte; and a detection zone comprising a first immobilized reagent that specifically binds said target analyte and a second immobilized reagent that specifically binds said specific-binding component of said composite particle.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0120] FIG. 1 describes assay device and protocol used in example 1-a.

[0121] FIG. 2 is a series of photograph of assay results from example 1.

[0122] FIG. 3 is a series of fluorescence microscopy pictures of results from example 1-b.

[0123] FIG. 4 describes an assay test strip.

[0124] FIG. 5 is a series of photographs of assay results from example 2.

[0125] FIG. 6 is a series of photographs of assay results from example 3.

EXAMPLES

[0126] Table 1 below lists all composite particles used in experiments, as well as test results.

[0127] NS stands for nanosphere. Dimensions are diameter of the sphere or diameter of the core and thickness of the successive shells.

[0128] NPL stands for nanoplate. Dimensions are length, width and thickness. Thickness is always the smallest dimension in nanoplates.

[0129] (1) stands for intensity of light measured in a fluorescence test (arbitrary unit) using an inverted fluorescence microscope LEICA DMi8, with HXP 120 lamp (Gain=1, water objective 63×, excitation with a UV-violet band of 20 nm centered at 390 nm), with an integration of signal over a time of n milliseconds (n varies from 1 to 500 ms).

[0130] (2) Stands for Intensity of Light Absorption.

[0131] Composite particles (#4 to #11) are obtained by a spray process: 500 μL of CdSe.sub.0.72S.sub.0.28/CdS/ZnS nanoplates suspended in water (10 mg/mL) were mixed with 20 mL of water, tetraethoxysilane (TEOS) and ammonia, then loaded on a spray-drying set-up. The liquid was sprayed towards a tube furnace heated at a temperature ranging from the boiling point of the solvent to 1000° C., with a nitrogen flow. The composite particles CdSe.sub.0.72S.sub.0.28/CdS/ZnS in SiO.sub.2 matrix were collected at the surface of a filter.

[0132] Composite particles (#12 to #24) are obtained by a simultaneous spray process: 500 μL of nanoplates or nanospheres suspended in heptane (10 mg/mL) were mixed with aluminum tri-sec butoxide and 20 mL of cyclohexane, then loaded on a spray-drying set-up. On another side, an aqueous solution was prepared and loaded the same spray-drying set-up, but at a different location than the first heptane solution. The two liquids were sprayed simultaneously towards a tube furnace heated at a temperature ranging from the boiling point of the solvent to 1000° C., with a nitrogen flow. The composite particles comprising nanoplates or nanospheres in Al.sub.2O.sub.3 were collected at the surface of a filter.

[0133] Composite particles (#25) are obtained by a spray process: 500 μL of CdSe.sub.0.72S.sub.0.28/CdS/ZnS nanoplates suspended in water (10 mg/mL) were mixed with 20 mL of water, tetraethoxysilane (80 w %), Zirconium propoxide (20 w %) and ammonia, then loaded on a spray-drying set-up. The liquid was sprayed towards a tube furnace heated at a temperature of 300° C., with a nitrogen flow. The composite particles CdSe.sub.0.72S.sub.0.28/CdS/ZnS in Si.sub.0.89Zr.sub.0.11O.sub.2 matrix were collected at the surface of a filter.

[0134] Composite particles (#26) are obtained by a simultaneous spray process: 500 μL of nanoplates or nanosphere suspended in heptane (10 mg/mL) were mixed with aluminium tri-sec butoxide (70 w %), zirconium propoxide (30 w %) and 20 mL of cyclohexane, then loaded on a spray-drying set-up. On another side, an aqueous solution was prepared and loaded the same spray-drying set-up, but at a different location than the first heptane solution.

[0135] The two liquids were sprayed simultaneously towards a tube furnace heated at a temperature of 300° C., with a nitrogen flow. The composite particles comprising nanoplates or nanospheres in a matrix comprising 70 mol % Al.sub.2O.sub.3 and 30 mol % ZrO.sub.2 were collected at the surface of a filter.

[0136] Composite particles (#27) are obtained by a simultaneous spray process: 500 μL of nanoplates or nanosphere suspended in heptane (10 mg/mL) were mixed with Zirconium propoxide and 20 mL of cyclohexane, then loaded on a spray-drying set-up. On another side, an aqueous solution was prepared and loaded the same spray-drying set-up, but at a different location than the first heptane solution. The two liquids were sprayed simultaneously towards a tube furnace heated at a temperature of 300° C., with a nitrogen flow. The composite particles comprising nanoplates or nanospheres in ZrO.sub.2 were collected at the surface of a filter.

[0137] Composite particles (#28) are obtained by a simultaneous spray process: 500 μL of nanoplates or nanosphere suspended in heptane (10 mg/mL) were mixed with Hafnium n-butoxide and 20 mL of cyclohexane, then loaded on a spray-drying set-up. On another side, an aqueous solution was prepared and loaded the same spray-drying set-up, but at a different location than the first heptane solution. The two liquids were sprayed simultaneously towards a tube furnace heated at a temperature of 300° C., with a nitrogen flow. The composite particles comprising nanoplates or nanospheres in HfO.sub.2 were collected at the surface of a filter.

[0138] Composite particles (#29) are obtained by a simultaneous spray process: 500 μL of nanoplates or nanosphere suspended in heptane (10 mg/mL) were mixed with Hafnium butoxide (50 w %) and zirconium propoxide (50 w %) and 20 mL of cyclohexane, then loaded on a spray-drying set-up. On another side, an aqueous solution was prepared and loaded the same spray-drying set-up, but at a different location than the first heptane solution. The two liquids were sprayed simultaneously towards a tube furnace heated at a temperature of 300° C., with a nitrogen flow. The composite particles comprising nanoplates or nanospheres in Hf.sub.0.4Zr.sub.0.6O.sub.2 were collected at the surface of a filter.

[0139] Functionalization with streptavidin was made differently for nanoparticles not included in a matrix (used in control experiments) and for composite particles.

[0140] For nanoparticles not included in a matrix (#3): CdSe/Cd.sub.0.3Zn.sub.0.7S/ZnS nanoplates where complexed with a copolymer comprising 30 mol % of (5, 7-dimercapto)-N-(3-methacrylamidopropyl)heptanamide, 40% of 3-[N,N,N-(3-methacrylamidopropyl)-dimethyl-ammonio]propane-1-sulfonate and 30 mol % of methacrylic acid so as to provide a dispersing agent for the nanoparticles. The nanoparticles were then suspended in 210 μL of a buffer solution at pH 6.0 (50 mmol/L of 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid and 100 mmol/L NaCl). 200 μL of this suspension were then added to 80 μL of streptavidine-maleimide complex solution (C=9.47 10.sup.−5 mol/L in buffer at pH 6.0-50 mmol/L of 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid and 100 mmol/L NaCl). The resulting mixture was gently stirred for 2 hours at room temperature. Purification was performed on Vivaspin 100 kDa thus yielding nanoparticles functionalized with streptavidin.

[0141] For composite particles, a dispersion of composite particles in ultrapure water (500 μL of a 2 mg/mL stock) was mixed with approximately 1 μL of a 0.5 mol/L NaOH solution (to ensure a pH between 9 and 10) and 10 μL of streptavidin solution (6 mg/mL in ultrapure water). After stirring for 90 minutes at room temperature, 100 μL of BSA solution (0.1 g/mL in ultrapure water) was added and the resulting mixture was again stirred for 1 hour at room temperature. After purification, functionalized composite particles are suspended in 200 μL of following solution: borate buffer (pH=10) containing triton X-100 (0.1% v/v), sucrose (10% w/v) and sodium azide (0.05% w/v).

TABLE-US-00001 TABLE 1 Weight λ λ fraction of Mean absorption emission Intensity nanoparticles size (nm)/FWHM (nm)/FWHM Migration Separation of light # Nanoparticle Matrix (%) (nm) (nm) (nm) (Y/N) (Y/N) (1) or (2) 1 Au NS - 25 nm None NA NA 525/100 Y Y diameter 2 PbS NS - 3 nm Al.sub.2O.sub.3 5 180 895/200 Y Y diameter 3 CdSe/Cd.sub.0.3Zn.sub.0.7S/ZnS None NA NA 665/31 Y Y 18 NPL 24 × 21 × 9 nm @50 ms 4 CdSe.sub.0.72S.sub.0.28/CdS/ZnS SiO.sub.2 5 250 653/25 Y Y >255 NPL 24 × 21 × 9 nm @ 1 ms 5 CdSe.sub.0.72S.sub.0.28/CdS/ZnS SiO.sub.2 0.5 220 653/25 Y Y NPL 24 × 21 × 9 nm 6 CdSe.sub.0.72S.sub.0.28/CdS/ZnS SiO.sub.2 1 290 653/25 Y Y NPL 24 × 21 × 9 nm 7 CdSe.sub.0.72S.sub.0.28/CdS/ZnS SiO.sub.2 2 250 653/25 Y Y NPL 24 × 21 × 9 nm 8 CdSe.sub.0.72S.sub.0.28/CdS/ZnS SiO.sub.2 12.5 230 653/25 Y Y NPL 24 × 21 × 9 nm 9 CdSe.sub.0.72S.sub.0.28/CdS/ZnS SiO.sub.2 25 270 653/25 Y Y NPL 24 × 21 × 9 nm 10 CdSe.sub.0.72S.sub.0.28/CdS/ZnS SiO.sub.2 50 300 653/25 Y Y NPL 24 × 21 × 9 nm 11 CdSe.sub.0.72S.sub.0.28/CdS/ZnS SiO.sub.2 5 740 653/25 N NPL 24 × 21 × 9 nm 12 CdSe.sub.0.72S.sub.0.28/CdS/ZnS Al.sub.2O.sub.3 5 160 661/30 Y Y NPL 31 × 18 × 9 nm 13 CdSe.sub.0.72S.sub.0.28/CdS/ZnS Al.sub.2O.sub.3 5 810 661/30 N NPL 31 × 18 × 9 nm 14 CdSe.sub.0.72S.sub.0.28/CdS/ZnS Al.sub.2O.sub.3 5 460 661/30 Y Y NPL 31 × 18 × 9 nm 15 CdSe/CdS/ZnS Al.sub.2O.sub.3 5 300 620/45 Y Y core/shell/shell NS 4.5-1.5-1 nm 16 Cd.sub.0.10Zn.sub.0.90Se.sub.0.10S.sub.0.90/ZnS Al.sub.2O.sub.3 5 300 540/37 Y Y core/shell NS - 4-1 nm 17 InP/ZnSe.sub.0.50S.sub.0.50/ZnS Al.sub.2O.sub.3 5 300 630/45 Y Y core/shell/shell NS - 3.5-2-1 nm 18 InP/ZnS Al.sub.2O.sub.3 5 300 630/40 Y Y core/shell NS - 3.5-2 nm 19 InP/ZnSe Al.sub.2O.sub.3 5 300 635/40 Y Y core/shell NS - 3.5-2 nm 20 CuInSe.sub.2/ZnS Al.sub.2O.sub.3 5 300 625/40 Y Y 21 CuInS.sub.2/ZnS Al.sub.2O.sub.3 5 300 625/40 Y Y 22 Carbon dots Al.sub.2O.sub.3 5 300 625/40 Y Y 23 Mn doped Al.sub.2O.sub.3 5 300 635/35 Y Y CdSe/CdS 24 Ag doped Al.sub.2O.sub.3 5 300 645/35 Y Y CdSe/CdS 25 CdSe.sub.0.72S.sub.0.28/CdS/ZnS Si.sub.0.89Zr.sub.0.11O.sub.2 50 300 653/25 Y Y NPL 24 × 21 × 9 nm 26 CdSe.sub.0.72S.sub.0.28/CdS/ZnS 70 w % Al.sub.2O.sub.3 50 300 653/25 Y Y NPL 24 × 21 × 9 nm and 30 w % ZrO.sub.2 27 CdSe.sub.0.72S.sub.0.28/CdS/ZnS ZrO.sub.2 50 300 653/25 Y Y NPL 24 × 21 × 9 nm 28 CdSe.sub.0.72S.sub.0.28/CdS/ZnS HfO.sub.2 50 300 653/25 Y Y NPL 24 × 21 × 9 nm 29 CdSe.sub.0.72S.sub.0.28/CdS/ZnS Hf.sub.0.4Zr.sub.0.6O.sub.2 50 300 653/25 Y Y NPL 24 × 21 × 9 nm

[0142] The present invention is further illustrated by the following examples.

Example 1-a: Device and Protocol for Assay

[0143] As shown on FIG. 1, a pad (1) comprises a nitrocellulose FF80HP porous substrate (2) in the form of a strip (1 cm×7 cm). 3 μL of Bovine Serum Albumin (BSA)-Biotin complex (1 mg/mL, Bovine Serum Albumin, Biotinylated supplied by Thermofisher) is deposited at 1 cm from the higher end of the porous membrane and let for drying 120 minutes at room temperature, thus forming a test zone (3).

[0144] Then, a 5 μL droplet containing composite particles functionalized with streptavidin (borate buffer (pH=10) containing triton X-100 (0.1% v/v), sucrose (10% w/v) and sodium azide (0.05% w/v) solution) is laid on the lower part of a porous membrane (4). After 600 seconds drying at room temperature, the lower end of the porous membrane is dipped in a solution (borate buffer (pH=10) containing 1% BSA (w/v), 1% tween 20 (v/v) and triton X-100 (0.1% v/v)) that flows by capillarity along the porous membrane (2) and transport composite particles towards test zone (3). Upon binding of streptavidin with biotin, composite particles attach to the test zone (3).

[0145] Pad is held vertically. After 600 seconds at room temperature, pad (1) is removed from solution and optical measurements is run on the test zone (3).

[0146] In this protocol, the amount of BSA—Biotin complex is the same on each pad and the concentration of composite particles is varied.

[0147] Same experiment was run with nanoparticles functionalized with streptavidin, but not included in a matrix.

[0148] Samples #1 to #17 were evaluated according to this protocol. Table 1 reports results of migration and separation for all type of composite nanoparticles used.

[0149] In a variant of this protocol, composite particles functionalized with streptavidin are simply mixed with the borate buffer instead of being deposited on the porous membrane and the concentration of BSA—Biotin complex is varied in test zone. Other features of the protocol are unchanged.

[0150] FIGS. 2a-2d shows the result of some of these experiments obtained with the main protocol.

[0151] FIG. 2a shows fluorescence pictures of pads loaded with CdSe/Cd.sub.0.3Zn.sub.0.7S/ZnS nanoplates #3 of dimensions 24×21×9 nm not included in a matrix, with concentration of nanoparticle in the ratio 1, ½, ¼, ⅛ and 1/16 from left to right. Emitted light is red at a wavelength of 653 nm. This experiment is a control showing expected result of migration and separation on nanoparticles. In this experiment, concentration of Bovine Serum Albumin (BSA)-Biotin complex solution deposited on the membrane is 0.2 mg/mL.

[0152] FIG. 2b shows fluorescence pictures under UV illumination of pads loaded with composite particles #4, with concentration of composite particles in a ratio 1 (left) and 1/10 (right). Emitted light is red at a wavelength of 653 nm. One can observe bright red lines around the test zone, demonstrating both migration and separation of composite particles.

[0153] FIG. 2c shows pictures of pads loaded with composite particles #2, with concentration of composite particles in a ratio 1 (left) and ¼ (right). As nanoparticles here are absorbent, they are identified by a black color on a picture in visible light. One can observe clear black lines around the test zone, demonstrating both migration and separation of composite particles.

[0154] FIG. 2d shows fluorescence pictures under UV illumination of pads loaded with composite particles #12, with concentration of composite particles in a ratio 1, ¼, ⅛ and 1 from left to right. Emitted light is red at a wavelength of 653 nm. One can observe bright red lines around the test zone, demonstrating both migration and separation of composite particles. Picture on the right is a control: pad does not contain biotin, but only BSA in the test zone. This demonstrates that no fluorescence signal is observed in the test zone. Finally, composite particles are separated by immobilization on biotin in the test zone, as expected.

[0155] FIGS. 2e-2g shows the result of some of these experiments obtained with the variant protocol.

[0156] A set of 12 pads are prepared with increasing dilution of BSA—Biotin complex: 0.5 mg/ml for pad 1 then twice diluted for each successive pad, yielding BSA—Biotin complex at 5 μg/ml for pad 7 and 0.16 μg/ml for pad 12.

[0157] A comparative experiment is run with gold nanoparticles not included in a matrix. FIG. 2e shows pictures under natural light of pads dipped in a buffer solution comprising 1 μg/ml gold nanoparticles grafted with streptavidin, diameter 40 nm, supplied by Nanocomposix. A very low signal (pointed by an arrow and evidenced in magnified view) is detected for pad 7: the limit of detection in this protocol is about 5 μg/ml of BSA—Biotin complex.

[0158] An experiment according to present disclosure is run. FIG. 2f shows fluorescence pictures under UV illumination of pads dipped in a buffer solution comprising 1 μg/ml composite nanoparticles #12. A very low signal (pointed by an arrow and evidenced in magnified view) is detected for pad 12: the limit of detection in this protocol is about 0.16 μg/ml of BSA—Biotin complex, about 30 to 35 times more sensitive than gold nanoparticles used at the same concentration in comparative experiment.

[0159] FIG. 2g is a TEM picture of composite nanoparticles #12. Average diameter is about 160 nm and one can see darks spots inside composite particles: each spot corresponds to a single nanoplate.

Example 1-b: Comparison of Composite Particles and Nanoparticles Alone

[0160] Microscope glass slides (diameter=13 mm) were first cleaned with a piranha solution (7 mL of 35% hydrogen peroxide solution into 20 mL of 96% concentrated sulfuric acid), then washed, and finally dried for 300 minutes in an oven at 70° C.

[0161] The glass slides were individually placed into small vials and completely coated with 200 μL of Bovine Serum Albumin (BSA)-Biotin complex (1 mg/mL in 0.05 mol/L NaCl solution, Bovine Serum Albumin, Biotinylated supplied by Thermofisher). The coated glass slides were incubated at room temperature for 2 days at 4° C. The glass slides were then washed with ultrapure water.

[0162] Control glass slides were coated with a 200 μL BSA solution (1 mg/mL in 0.05 mol/L NaCl solution) instead of the BSA-biotin solution.

[0163] One glass slide was immersed in 100 μL of BSA in a borate buffer solution (pH=10, BSA concentration: 10 mg/mL). After 10 minutes, 100 μL of the streptavidin functionalized nanoparticles or streptavidin functionalized composite particles suspension was added. After 1 h at room temperature, the glass slide was carefully washed with borate buffer (pH=10) and dried overnight at room temperature. An inverted fluorescence microscope LEICA DMi8, with HXP 120 lamp (Gain=1, water objective 63×, excitation with a UV-violet band of 20 nm centered at 390 nm), was used to observe the glass slide surface.

[0164] In this experiment, functionalized nanoparticles or functionalized composite particles are in large excess as compared to available biotin on the glass slide. One can expect that all biotin sites on glass slides will capture nanoparticles or composite particles. Optical measurements enable to compare sensitivity of test with nanoparticles or composite particles.

[0165] FIG. 3a shows glass slides image for acquisition times of 50, 100, 200 and 500 ms from left to right for nanoparticles #3, not included in a matrix. Mean intensity measured were respectively 18, 35, 70 and 176 (arbitrary units).

[0166] FIG. 3b shows glass slides image for acquisition times of 1 ms (left) and 2 ms (right) for composite particles #4. Even at 1 ms, mean intensity could not be measured because of saturation of fluorescence microscope (saturation corresponds to mean intensity of 255 in same arbitrary units).

[0167] Comparison of FIGS. 3a and 3b demonstrates that composite particles yield a much more intense optical signal than nanoparticles not included in a matrix, in similar conditions of saturation of sites (biotin) to be detected. Using the linear proportion of integration time, one could evaluate fluorescence intensity of sample #3 about 0.35 @ 1 ms, to be compared with at least 255 @ 1 ms (saturation) for sample #4 in FIG. 3b. Thus, amplification of fluorescence signal obtained using composite particles is about 255/0.35˜730, and thus sensitivity of assay would be improved by the same factor 730.

[0168] On control glass slide, covered only with BSA, no fluorescence at all was observed.

[0169] Table 1 clearly shows that intensity of optical signal is dramatically increased when using composite nanoparticles having a mean size greater than 50 nm and a weight fraction of nanoparticles greater than 0.5%, demonstrating that assay is more sensitive. For the same density of analytes, optical signal measured from a dispersion of large and dense composite particles comprising a given number of nanoparticles is more intense than optical signal measured from a dispersion comprising the same number of isolated nanoparticles or composite particles of small size or small weight fraction.

Example 2

[0170] The same pad is used as in example 1-a. However, test zone 3 is prepared by dispensing with a lateral flow reagent dispenser 1 μL/cm of a solution of antibody at 1 mg/mL in a phosphate saline buffer. The antibody is a capture antibody for ovalbumin (AB-Capture-Ova-35).

[0171] Besides, composite particles are grafted with another antibody for ovalbumin (AB-Tracker-Ova-1): 10 μg antibody are brought in contact with 100 μg composite particles in a borate 20 mM buffer solution (pH 9). The solution is then incubated during 1 h. After multiple washings, the grafted composite particles are dispersed in borate 10 mM buffer containing 0.1% Na-Casein (pH 9).

[0172] Then, 10 μg grafted composite particles are brought in contact with 100 μl of a solution of Ovalbumin in a buffer (phosphate saline 100 mM and NaCl 150 mM buffer (pH 7.4) containing tween (0.5% v/v) and BSA (0.1% w/v) solution) and let for incubation during 200 s under gentle stirring. Ovalbumin is thus able to complex AB-Tracker-Ova-1 grafted on composite particles.

[0173] Last, pad is dipped in the solution of grafted composite particles, as in variant of example 1-a, allowing for migration of composite particles along the pad. When complex of Ovalbumin and grafted composite particles reach test zone 3, ovalbumin is able to form a complex with AB-Capture-Ova-35, thus forming a structure usually known as “sandwich”: Ovalbumin acts as a linker between both antibodies and finally, composite particle is anchored to the test zone 3.

[0174] After completion of migration (5-10 mn), test zone 3 is illuminated so as to reveal the presence of composite particles by light absorption or light emission.

[0175] With this protocol, the sensibility of the assay is determined by varying the concentration of Ovalbumin during incubation. The lowest concentration of Ovalbumin during incubation yielding a detectable optical signal defines the sensibility limit.

[0176] Composite particles #12 to #17 have been grafted according to the protocol described above and brought in contact with solutions of Ovalbumin with varying concentrations. Solution 1 has a concentration of 1 ng/ml. Then, solution 1 is diluted twice to obtain next solution (solution 2), until a dilution of 1024 is obtained (10 successive dilutions yielding solution 11).

[0177] FIG. 5 shows fluorescence pictures under UV illumination of pads after experiment with composite particles #12. Pad number 0 is a comparative experiment, in which incubation is performed without Ovalbumin: no fluorescence is observed. Pad 1 is dipped in solution 1 and shows a very strong fluorescence signal. After dilution of 512 (Ovalbumin concentration of 2 pg/ml-pad 10 dipped in solution 10), fluorescence is still detected—indicated by arrow—whereas after 1024 dilution (pad 11 dipped in solution 11, not shown), no more fluorescence could be detected: sensibility of assay is about 2 pg/ml.

[0178] As a comparison, nanoparticles from composite particles #12 were used without alumina encapsulation matrix, as single nanoparticles, with the same protocol. Fluorescence is observed on pad 5, but not on pad 6. Thus, the sensibility of assay is about 50 pg/ml, much lower with single nanoparticles as compared to composite particles, by a factor of about 30.

Example 3

[0179] The same pad is used as in example 1-a. However, test zone 3 is prepared by grafting of a capture antibody for ricin (AB-Capture-Rb-37). A solution of antibody at 1 mg/mL in a phosphate saline buffer is printed on test zone 3 using a lateral flow reagent dispenser (1 μL antibody/cm). In addition, a control zone 4 is prepared by grafting a AffiniPure Goat Anti-Mouse IgG+IgM (H+L). A solution of antibody at 0.5 mg/mL in a phosphate saline buffer is printed on control zone 4 using a lateral flow reagent dispenser (1 μL antibody/cm).

[0180] Besides, composite particles are grafted with another antibody for ricin (AB-Tracker-Rb-35). Antibody Rb35 −1 (5 μg) are brought in contact with composite particles (100 μg) in a borate 20 mM buffer solution (pH 9). The solution is then incubated during 1 h. After multiple washings, the grafted composite particles are dispersed in borate 10 mM buffer containing 0.1% NaCaseine (pH 9).

[0181] Then grafted composite particles (10 μg) are brought in contact with 100 μl of a solution of ricin in a buffer (phosphate saline 100 mM and NaCl 150 mM buffer (pH 7.4) containing tween (0.5% v/v) and BSA (0.1% w/v) solution) and let for incubation during 200 s. Ricin is thus able to complex AB-Tracker-Rb-35 grafted on composite particles.

[0182] Last, pad is dipped in the solution of grafted composite particles, as in variant of example 1-a, allowing for migration of composite particles along the pad. When complex of ricin and grafted composite particles reach test zone 3, ricin is able to form a complex with AB-Capture-Rb-37, thus forming a structure usually known as “sandwich”: Ricin acts as a linker between both antibodies and finally, composite particle is anchored to the test zone 3.

[0183] After completion of migration (30 mn), test zone 3 is illuminated so as to reveal the presence of composite particles by light absorption or light emission.

[0184] With this protocol, the sensibility of the assay is determined by varying the concentration of ricin during incubation. The lowest concentration of ricin during incubation yielding a detectable optical signal defines the sensibility limit.

[0185] Composite particles #12 to #29 have been grafted according to the protocol described above and brought in contact with solutions of ricin with varying concentrations. Solution 1′ has a concentration of 0.5 ng/ml. Then, solution 1′ is diluted twice to obtain next solution (solution 2′), solution 2′ is diluted 2.5 times to obtain next solution (solution 3′), solution 3′ is diluted twice to obtain next solution (solution 4′), solution 4′ is diluted twice to obtain next solution (solution 5′), and solution 5′ is diluted twice to obtain next solution (solution 6′), thus a dilution of 40 is obtained with these 5 successive dilutions.

[0186] FIG. 6 shows fluorescence pictures under UV illumination of pads after experiment with composite particles #12. Pad number 0′ is a comparative experiment, in which incubation is performed without Ricin: no fluorescence is observed. Pad 1′ shows a very strong fluorescence signal. After dilution of 16 (ricin concentration of 25 pg/ml-pad 5′), fluorescence is still detected—indicated by arrow—whereas after 32 dilutions (pad 6′), no more fluorescence could be detected: sensibility of assay is about 25 pg/ml.

[0187] As a comparison, colloidal gold nanoparticles, having a diameter of 40 nm, were used without Alumina encapsulation matrix, as single nanoparticles, in the same protocol. Fluorescence is observed on pad 2′, but not on pad 3′. Thus, the sensibility of assay is much lower with single gold nanoparticles as compared to composite particles, by a factor of 10.