BIOMARKER SENSOR-BASED DETECTION OF DISEASE-SPECIFIC BIOMARKERS

Abstract

Biomarker detection sensors for detecting disease-specific biomarkers, kits based thereon, methods for determining disease-specific biomarkers in body fluids and uses are presented. The sensors include a substrate having bound thereon metal nanoparticles arranged in branched two-dimensional structures and/or a two-dimensional lattice, and anti-biomarker receptors bound to the metal nanoparticles.

Claims

1.-14. (canceled)

15. A biomarker detection sensor for detection of disease-specific biomarkers comprising a substrate, the substrate having bound thereon metal nanoparticles arranged in branched two-dimensional structures and/or a two-dimensional lattice, and anti-biomarker receptors bound to the metal nanoparticles.

16. The biomarker detection sensor according to claim 15, wherein the metal nanoparticles are gold nanoparticles.

17. The biomarker detection sensor according to claim 15, wherein the disease-specific biomarkers comprise PfLDH and the anti-biomarker receptors comprise anti-pLDH antibodies pre-treated with UV radiation.

18. The biomarker detection sensor according to claim 15, wherein a ratio R of diameter D of the metal nanoparticles to a distance d between individual metal nanoparticles in the two-dimensional lattice is >2.3.

19. The biomarker detection sensor according to claim 15, wherein a) the metal nanoparticles a1) are hexagonally or hexagonally-close packed, or a2) are arranged in branched band-like structures, or a3) form a combined two-dimensional structure wherein parts of the structure are hexagonally or hexagonally-close packed, and parts of the structure are arranged in branched, band-like structures; b) a diameter D of the metal nanoparticles is between 40 and 70 nm, and c) a distance K between individual metal nanoparticles is between 16 and 24 nm.

20. A biomarker detection sensor kit comprising A) at least one biomarker detection sensor according to claim 15, B0) a preparation comprising at least one biomarker-specific aptamer having a fluorophore bound thereto, optionally B1) at least one further preparation comprising at least one identical biomarker-specific aptamer with a different fluorophore bound thereto, and/or B2) at least one further preparation comprising at least one other biomarker-specific aptamer having another fluorophore bound thereto.

21. The biomarker detection sensor kit according to claim 20, wherein B2) is selected from B2i) at least one other biomarker-specific aptamer for detection of the same biomarker but binding to a different epitope of the biomarker with a different fluorophore bound thereto, and/or B2ii) at least one other biomarker-specific aptamer for detection of a further biomarker with a different fluorophore bound thereto.

22. The biomarker detection sensor kit according to claim 20, wherein in BO) the at least one biomarker-specific aptamer comprises a malaria 2008 aptamer.

23. The biomarker detection sensor kit of claim 22, further comprising cyanine 5 bound to the malaria 2008 aptamer as a fluorophore.

24. The biomarker detection sensor kit according to claim 20, wherein in B0) the preparation comprises 5-FAM fluorophore bound to the at least one biomarker-specific aptamer, and in B1) the at least one further preparation comprises Cy5-fluorophore bound to the at least one identical biomarker-specific aptamer.

25. A method for determination of disease-specific biomarkers in body fluids comprising i) providing the biomarker detection sensor according to claim 15, ii) applying to the sensor iia1) a body fluid sample, and iia2) at least one biomarker-specific aptamer having a fluorophore coupled thereto either after iia1) or simultaneously with iia1), or iib) a mixture of body fluid sample and at least one biomarker-specific aptamer with fluorophore coupled thereto, iii) irradiating the sample applied to the sensor with light source, iv) detecting fluorescence emission from the sample, v) optionally analysing the fluorescence emission, and vi) optionally storing and/or displaying the analysed fluorescence emission.

26. The method according to claim 25, wherein the body fluid sample is a blood sample.

27. The method according to claim 25, wherein the anti-biomarker receptors of the biomarker detection sensor comprise anti-pLDH antibodies and the at least one biomarker-specific aptamer comprises malaria-2008 aptamer.

28. The biomarker detection sensor of claim 15, being for qualitative or quantitative determination of disease-specific biomarkers.

29. The biomarker detection sensor of claim 28, wherein the biomarkers comprise malaria biomarkers in blood fluid samples.

30. The biomarker detection sensor kit of claim 20, being for qualitative or quantitative determination of disease-specific biomarkers.

31. The biomarker detection sensor kit of claim 30, wherein the biomarkers comprise malaria biomarkers in body fluid samples.

32. The method according to claim 25, being for qualitative or quantitative determination of disease-specific biomarkers.

33. The method according to claim 32, wherein the biomarkers comprise malaria biomarkers.

34. The biomarker detection sensor of claim 15, wherein the substrate is a substrate of an automated multititer plate.

Description

FIGURE DESCRIPTION

[0128] FIG. 1 is a schematic representation of the procedure for preparing a biomarker detection sensor according to the present invention.

[0129] The preparation of these arrays of gold nanoparticles was initially carried out via nanolithography based on the self-assembly of block copolymers, as illustrated in section A). For this purpose, block copolymers 7 are used, which have a hydrophilic chain (shown by dash) and a hydrophobic chain (shown by block). By mixing with a non-polar solvent, in particular toluene T, inverse micelles 8 are formed, i.e. micelles in which the core is hydrophilic and the outer shell is hydrophobic. A gold salt Au-S (for example HAuCl.sub.4) is then added to this mixture containing the inverse micelles, causing the inverse micelles to take up gold precursors 9 in their centre, the hydrophilic core. These micelles can then be deposited on a substrate 1, for example by means of dip coaters. Due to their structure, the micelles then arrange themselves in regular structures on the surface of the substrate. Afterwards, the micelles around the gold precursors are removed, for example by means of plasma furnace treatment. By this, the gold precursors 9 are also reduced to the gold nanoparticles 2. A two-dimensional lattice of gold nanoparticles 2 (unfilled circles) then remains on the surface.

[0130] In Section B) it is illustrated how a solution of anti-pLDH antibodies 10 is pre-treated by UV radiation (shown as a flash) and then subsequently this treated aqueous solution 10a is poured onto the substrate with gold nanoparticles thereon prepared in section A), whereby the antibodies bind to the gold nanoparticles.

[0131] As a result, the substrate 1 illustrated in section C) is obtained with gold nanoparticles 2 thereon to which the anti-biomarker receptors are bound, which is shown here by filled circles. This fabrication method is also described, for example, in Lohmueller, T., Aydin, D., Schwieder, M. et al, “Nanopatterning by block copolymer micelle nanolithography and bioinspired applications”, Biointerphases 6, MR1-MR12 (2011), https://doi.org/10.1116/1.3536839.

[0132] FIG. 1 illustrates the arrangement of the particles on the substrate only schematically and is not limited to any particular arrangement. The production method illustrated in this figure can be used for the production of substrates with nanoparticles arranged hexagonally thereon (as illustrated), as well as for the production of branched nanoparticles or also for mixed forms.

[0133] FIG. 2a is a scanning electron microscope image of a substrate provided with ordered gold nanoparticles according to a variant of the present invention. From this image it can be seen that defects can certainly occur during the manufacture of the sensors. However, these defects are not detrimental to the overall performance of the biomarker detection sensor, as they are averaged out, so to speak, in the context of the plasmon resonance or dual amplification.

[0134] FIG. 2b are two scanning electron micrographs of another substrate provided with ordered gold nanoparticles according to another variant of the present invention. From these images it can be seen that in this case hexagonal structures are still present in the near order, but the far order shows essentially branched structures as well as isolated nanoparticles. The upper image shows a structure before and the lower image one after the AuNP growth.

[0135] FIG. 3 is a diagram in which the two narrower curves represent the excitation spectrum (left, dashed curve) and the emission spectrum (right, dotted curve) of cyanine 5 (Cy5) and the broad curve represents the experimentally determined extinction spectrum of a malaria biomarker detection sensor (prepared according to example A) described below). It can be seen that the broad extinction spectrum allows the overlapping of the plasmonic resonance over both extinction as well as emission spectra of cyanine 5. The wavelength in nm is plotted on the x-axis and the extinction (left) and normalised excitation/emission (right) on the y-axis.

[0136] FIG. 3b for a substrate prepared according to example B) described below shows the experimental (solid line) extinction spectrum. In addition, the plasmon fluorophore overlap with 5-FAM (emission spectrum, small dashed line//excitation spectrum, large dashed line) and Cy5 (emission spectrum, dash-dot line//excitation spectrum, dotted line) dyes is shown. It can be seen that the experimental extinction spectrum shows two plasmonic resonances at 524 nm and 675 nm. Isolated AuNPs contribute to the resonance at lower wavelength, as expected from 30 nm gold nanospheres in air, whereas AuNPs arranged along the branches lead to a collective resonance at higher wavelength. The extinction is plotted on the left axis, excitation/emission on the right axis. The wavelength in nm is plotted on the horizontal axis.

[0137] FIG. 4 is a diagrammatic representation of the particle size distribution of gold nanoparticles prepared according to example A) described below. The mean diameter D of the gold nanoparticles in this example is about 48 nm (47.67±0.14 nm).

[0138] FIG. 5 is a diagrammatic representation of the interparticle distances in nanometres relative to the respective centres of the nanometres. The average interparticle distance, measured centre to centre, is about 68 nm (68.47±0.19 nm)), resulting in an interparticle distance, measured particle edge to particle edge, of about 20 nm (produced according to example A) described below).

[0139] FIG. 6 schematically illustrates the mode of operation of the present invention by way of a single gold nanoparticle (AuNP). The basis is a gold nanoparticle 2 arranged on a substrate 1, to which antibodies 3 (shown here as Y) are bound. In this figure, it becomes clear from the way the antibodies 3 are shown that they are bound to the gold nanoparticles 2 in a conformation that makes the binding epitopes of the antibodies readily accessible to the respective biomarker. This is illustrated in that one “arm” of each antibody 3 is oriented away from the gold nanoparticle 2. Furthermore, the biomarker 4 (P<DH) under investigation is shown (only one, for clarity, in the form of a cloud), which is shown here as binding to the binding epitope of the middle antibody 3. On the other side of the biomarker 4, an aptamer 5 is then shown, which thus, so to speak, grips the biomarker 4 together with the antibody 3 (sandwich). On the side of the aptamer 5 facing away from the biomarker 4, the fluorophore 6 is shown as a star which is intended to represent its fluorescence emission. The distance between the fluorophore 6 and the surface 1 is here about 10 nm, whereby with this arrangement both a high sensitivity as well as a high selectivity are achieved.

[0140] FIG. 7 shows an image of the fluorescence dots of a sensor assembly with detected biomarker according to example B), below. The image shows a reproduction in which the red and green channels resulting from detection with two colour channels, i.e. two different fluorophores (5-FAM and Cy5), are shown united (here in black and white for reproduction purposes). One can clearly see the fluorescence dots and thus the good detectability that can be achieved with the present invention. A corresponding detectability also applies to the variant according to example A) (not shown).

LIST OF REFERENCE SIGNS

[0141] In the figures, same reference signs mean same materials, substances, etc.
1 substrate
2 gold nanoparticle
2a gold nanoparticles with bound antibodies
3 antibody
4 biomarker
5 aptamer
6 fluorophore
7 block copolymer
8 invers micelle
9 gold nanoparticle precursor
10 solution of anti-pLDH antibodies (untreated)
10a solution of anti-pLDH antibodies (after UV treatment)
T toluene
Au-S gold salt, in particular HAuCl.sub.4

[0142] The present invention will now be explained in more detail with reference to the following non-limiting examples. The following non-limiting examples serve to illustrate the embodiments embodied therein. It is known to person skilled in the art that variations of these examples are possible within the scope of the present invention.

EXAMPLES

0. Materials and Chemicals

[0143] Diblock copolymers (P18226-S2VP and P3807-S2VP, respectively) were purchased from Polymer Source Inc (Dorval, Canada) and were prepared from polystyrene(x)-b-2-polyvinylpyridine(y) (PS(x)-b-P2VP(y)), wherein x=30000 and y=8500, or x=325000 and y=92000 indicate the respective molecular weight of polystyrene (PS) and poly(2-vinylpyridine) (P2VP) (The number average (M.sub.N) of the distribution is indicated in each case. The ratio of M.sub.w:M.sub.N for P18226-S2VP is 1.06 and indicates a very monodisperse distribution). Toluene (99.8%), gold(III) chloride trihydrate (HAuCl.sub.4*3H.sub.2O), silver nitrate (AgNO.sub.3) and ascorbic acid were purchased from Sigma-Aldrich; acetone (≥99.0%), 2-propanol (≥99.5%) and ethanol (≥99.5%) were purchased from Merck Millipore; Hexadecyltrimethylammonium bromide (CTAB) (≥99.0%) was purchased from Fluka; bovine serum albumin (BSA) (fraction V IgG-free, low in fatty acids) came from Gibco. High purity deionised water used for all aqueous solutions was dosed from a Milli-Q® system (18.2 megaohm specific resistance).

[0144] 10 mM phosphate buffered saline (PBS) (NaCl 10 mM, NaH2PO4 10 mM, Na.sub.2HPO.sub.4 10 mM, MgCl.sub.2 1 mM, pH 7.1) and 25 mM Tris-HCI buffer (NaCl 100 mM, imidazole 20 mM, Tris (=Tris(hydroxymethyl)aminomethane) 25 mM, HCI 25 mM, pH 7.5) were prepared by dissolving the reagents (purchased from Sigma-Aldrich) in high purity water. Pan-malaria antibody (anti-pLDH monoclonal antibody clone 19 g7) was prepared by Vista Laboratory Services (Langley, USA). Recombinant Plasmodium falciparum lactate dehydrogenase (P<DH) and P<DH were obtained by bacterial expression. Malaria 2008s aptamers labelled with 5-FAM or cyanine 5-tag (5′-5-FAM(Cy5)-CTG GGC GGT AGA ACC ATA GTG ACC CAG CCG TCT AC-3′) were produced by Friz Biochem GmbH (Neuried, Germany). Millex® syringe filters (pore size 0.20 μm) with hydrophobic polytetrafluoroethylene membrane were purchased from Merck Millipore; Superslip® coverslips (borosilicate glass, thickness 0.13-0.17 mm) were purchased from Thermo Fisher Scientific and cut by a diamond-tipped glass cutter.

EXAMPLE A

[0145] 1. fabrication of an ordered array of AuNPs for the detection of PfLDH in blood

[0146] Nanolithography based on self-assembly of block copolymers was used to prepare arrays of ordered AuNPs with adjustable density, size and interparticle distance. 29.2 mg of diblock copolymers P18226-S2VP were added to 15 ml of toluene under vigorous stirring and controlled conditions (argon inert gas, O.sub.2<1 ppm, H.sub.2O<0.1 ppm) and kept for 72 h to obtain a homogeneously dispersed invers micelles with hydrophilic core and outer hydrophobic shell (spherical). Then, 15.7 mg HAuCl.sub.4*3H.sub.2O were added to the solution for 72 hours to incorporate the gold precursor into the hydrophilic core of the micelles, resulting in the formation of AuNPs encased in a hydrophobic shell. Once the gold powder was fully dispersed, the yellowish solution was filtered to remove micelle aggregates and impurities. While maintaining agitation, this solution could be stored for at least six months.

[0147] Before the PS-AuNPs were deposited on the glass coverslips (which had a size of 10 ×8 mm.sup.2), the substrates were cleaned by ultrasound for five minutes successively in acetone, 2-propanol and pure ethanol and dipped in toluene to “make” the surface non-polar so that the hydrophobic shells could adhere to it. Subsequently, the substrates were immersed in the solution containing PS-AuNPs using an immersion coater with careful adjustment of the immersion speed. It was found that an immersion speed of 0.6 mm/s enabled a particularly good coating of the glass surface, both in terms of the density of the arrangement of the AuNPs as well as in terms of the large-area uniformity of the deposition. The PS-AuNPs were transferred to the non-polar glass surface by hydrophobic interaction, resulting in a hexagonal arrangement by self-assembly. A corresponding process is also illustrated in FIG. 1. Afterwards, the copolymers were etched by oxygen plasma treatment (0.8 mbar pressure, 200 W, 30 minutes), immobilising the AuNPs at prefixed positions on the glass surface.

[0148] Because the plasmonic resonance strongly depends on the ratio R between the AuNP diameter D and the interparticle distance d, higher values of R guarantee a larger coupling between the AuNPs. For R>2/3, the plasmonic resonance is dominated by the collective behaviour of the AuNPs, and several advantages for metal-enhanced fluorescence (MEF) result. To enhance R, the substrates were incubated with 2 ml of an Au deposition solution (CTAB 190 mM, HAuCl.sub.4*3H.sub.2O 42 mM, AgNO.sub.3 8 mM, ascorbic acid 100 mM) for 2 h in the absence of light. Afterwards, the substrates were rinsed abundantly with high purity water and stored in the absence of light until use. The characterisation of the nanostructured substrates was done by UV-VIS spectroscopy and scanning electron microscopy (SEM).

[0149] The results of the measurements are shown in FIGS. 2 to 5.

2. functionalisation

[0150] The functionalisation of the AuNPs with pan-malaria antibodies was performed by photochemical immobilisation technique (PIT). For this purpose, 1 ml aqueous solution of anti-pLDH (25 μg/ml) were irradiated by a UV lamp for 30 seconds and then poured onto the substrate. The UV source consisted of two U-shaped low-pressure mercury lamps (2 W at 254 nm) into which a standard quartz cuvette could be inserted. Taking into account the geometry of the lamps and the proximity to the cuvette, the radiation intensity used to generate the thiol groups was about 1 W/cm .sup.2. This intensity was so weak that a direct photolysis of the disulphide bridges, which absorb only weakly at 254 nm, is avoided. Only the disulphide bridge of the Cys-Cys-Trp triad (where the tryptophan “activates” the disulphide bridge) was cleaved by this. Functionalisation via PIT ensured that the antibodies (Abs) were covalently linked to the gold surface and their binding sites were accessible to the environment.

3. washing

[0151] The samples were rinsed with high purity water (dosed using a Milli-Q® system) to remove unbound antibodies.

4. blocking

[0152] 1 ml bovine serum albumin (BSA) solution (50 μg/ml) was applied to cover the free gold surface and protect it from non-specific adsorption.

5. washing

[0153] The samples were rinsed copiously with high purity water. Storage until use was in PBS solution (10 mM) at room temperature.

6. analyte detection (P<DH fixation by immobilised Abs)

[0154] The desired amount of Plasmodium falciparum lactate dehydrogenase (P<DH) was added to 1 ml of a diluted solution of uninfected human blood (dilution 1:100 in 25 mM Tris buffer). The functionalised substrates were incubated with 1 ml of contaminated blood solution of the same dilution for 2 hours at room temperature. A rocking shaker was used to accelerate binding kinetics and improve analyte diffusion. Concentrations between 0.01 femtomoles and 10 nanomoles were measured.

7 washing

[0155] The samples were rinsed copiously with Tris buffer (25 mM) and with high purity water to remove unbound proteins.

8. fluorescence aptamer-based assay

[0156] The samples were transferred to 1 ml of 10 mM phosphate-buffered saline (PBS), wherein here the PBS still contains 0.1 μM malaria 2008s aptamers labelled with Cy5 tag (5′-Cy5-CTG GGC GGT AGA ACC ATA GTG ACC CAG CCG TCT AC-3′) (i.e. the malaria2008 aptamer with fluorophore bound to it. The solution was gently shaken in the absence of light for 2 hours using a rocking shaker. The result was a sandwich arrangement. Such an arrangement is exemplary also shown in FIG. 6.

9 washing

[0157] The samples were rinsed abundantly with PBS and high purity water to remove unbound aptamers.

10. measurement of the fluorescence signal

[0158] Fluorescence images were using a Zeiss Axio Observer Z1 phase-inverted contrast fluorescence microscope equipped with Zeiss Colibri.2 LED light source (module 625 nm), Zeiss Plan-Apochromat lens 10×/0.45 Ph 1 M27 (FWD=2.1 mm), Kubus 50 Cy5 filter (excitation 625-655 nm/emission 665-715 nm) and pco.edge 5.5 sCMOS photodetector (scale 0.650 μm×0.650 μm per pixel, image size 2560×2160 pixels, scaled image size 1.66 mm×1.40 mm, 16 bit dynamic range, 2 seconds exposure time for each image). The taken fluorescence images were processed with ImageJ software. The “rolling ball” algorithm was used to calculatively remove the difference in brightness between the centre and the edge of the image caused by the optics. The background was measured locally for each pixel by average forming over a circle around the pixel. Such a value is then subtracted from the original image, smoothing out spatial variations of the background. The “rolling ball” radius was set to 10 pixels, a size sufficiently larger than the size of the largest objects that were not part of the background. A threshold slightly higher than the smoothed background was set to segment the image, the overall intensity of which was measured by summing the signal components from all spots. To obtain a good and reliable analysis of the fluorescence signals, ten images were taken randomly from each sample and their intensity average was determined.

11. simulation of the E-field around the gold nanoparticles

[0159] The optical response of the 2D AuNP frameworks was simulated by the “FDTD Solutions” tool implemented in Lumerical software. A linearly polarised electromagnetic radiation that ran along the z-direction was used to study the system. Virtual (simulated) photo-detectors (PDs), sensibly placed in the working space, could measure the intensity of the electromagnetic field as a function of time. A photo detector was assigned to measure the excitation spectrum of the nanostructure. Symmetric/antisymmetric boundary conditions (BCs boundary conditions), set along the x and y directions, extend the plasmonic excitation over an infinite 2D framework and at the same time reduce the simulation time by a factor of 8 without degrading the accuracy of the results. Bloch BCs (periodic boundary conditions) were used only for polarisation studies to compensate for the phase shift that occurs when an electromagnetic disturbance when a non-zero angle reintroduced on the opposite side of the working space. Perfectly adjusted layer BCs in the z-direction ensure perfect absorption of the electromagnetic waves backscattered by the plane containing the light source and incident through the opposite side of the working environment. The working environment was resolved over a grid with a spatial resolution of 0.5 nm, ensuring high accuracy while keeping the simulation time within a few hours. The AuNPs were modelled as homogeneous gold hemispheres, whereas the substrate was represented as a thick dielectric layer of silicon dioxide (SiO.sub.2).

EXAMPLE B

[0160] Essentially corresponding to Example A) above, the following procedure was followed:

1. preparation of an array of AuNPs

[0161] 24.3 mg of the diblock copolymers P3807-S2VP were added to 15 ml of toluene with vigorous stirring and kept for 72 hours to obtain a homogeneous, monodisperse solution of invers micelles. Then 13.1 mg HAuCl.sub.4*3H.sub.2O were added to the solution for 72 hours to allow the formation of gold nuclei encased in polystyrene shells (PS-AuNPs). The yellowish solution was then filtered to remove possible copolymer aggregates. Diblock copolymers and gold(III) chloride trihydrate were handled in a glovebox under inert gas (argon) and controlled conditions (O.sub.2<1 ppm, H.sub.2O<0.1 ppm).

[0162] Before the PS-AuNPs were deposited on the glass coverslips (10×8 mm.sup.2), the substrates were cleaned by ultrasound for five minutes successively in acetone, 2-propanol and ethanol and then immersed in a non-polar solvent to allow the adhesion of hydrophobic polystyrene shells. Subsequently, the substrates were immersed in the solution containing PS-AuNPs using an immersion coater and an immersion speed of 0.8 mm/s. This immersion speed allows a good coating of the substrate surface while preventing maximum packing density (cf. FIG. 2b, from which it can be seen that in the near order hexagonal structures are still given, but the far order shows branched structures). Afterwards, the copolymers were etched by oxygen plasma treatment (0.8 mbar pressure, 200 W, 30 minutes), which immobilised the AuNPs at prefixed positions on the glass surface. Subsequently, the substrates were incubated with 2 ml of an Au deposition solution (CTAB 190 mM, HAuCl.sub.4*3H.sub.2O 42 mM, AgNO.sub.3 8 mM, ascorbic acid 100 mM) for 2 hours in the absence of light.

2. functionalisation

[0163] The functionalisation of the AuNPs with pan-malaria antibodies was performed by photochemical immobilisation technique (PIT). For this purpose, 1 ml aqueous solution of anti-pLDH (50 μg/ml) was irradiated by a UV lamp for 30 seconds and then poured onto the substrate. The UV source (Trylight, Promete S.r.l) consisted of two U-shaped low-pressure mercury lamps (6 W at 254 nm) into which a 10 mm standard quartz cuvette could be inserted. Taking into account the geometry of the lamps and the proximity to the cuvette, the radiation intensity used to generate the thiol groups was approximately 0.3 W/cm.sup.2.

3. washing as above
4. blocking as above
5. washing as above
6. analyte detection (P<DH fixation by immobilised Abs)

[0164] Uninfected human blood was diluted 1:100 in 25 mM Tris buffer to reduce sample turbidity. The desired amount of Plasmodium falciparum lactate dehydrogenase (P<DH) was added to 1 ml of a diluted solution of the sample to obtain analyte concentrations ranging from 1 fM to 1 μm (based on the undiluted blood). The functionalised substrates were incubated with 1 ml of contaminated blood solution for 2 hours at room temperature.

7. washing as above
8. fluorescence aptamer-based assay

[0165] 5-FAM and Cy-5 labelled malaria aptamers were transferred at a 1:1 ratio to 1 ml of 10 mM phosphate buffered saline (PBS) to obtain an aptamer concentration of 0.1 μM (i.e. the Malaria2008 aptamer with fluorophore bound to it). The solution was gently shaken in the absence of light for 2 hours using a rocking shaker. The result was a sandwich arrangement. Such an arrangement is also exemplary shown in FIG. 6.

9. washing as above
10. measurement of the fluorescence signal

[0166] Fluorescence images were with a Zeiss Axio Observer Z1 phase-inverted contrast fluorescence microscope equipped with Zeiss Colibri.2 LED light source (modules 470 and 625 nm), Zeiss Plan-Apochromat lens 10×/0.45 Ph 1 M27 (FWD=2.1 mm), 38 HE filter (excutation 450-490 nm/emission 500-550 nm) and Kubus 50 Cy5 filter (excitation 625-655 nm/emission 665-715 nm) and pco.edge 5.5 sCMOS photodetector (scale 0.650 μm×0.650 μm per pixel, image size 2560×2160 pixels, scaled image size 1.66 mm×1.40 mm, 16 bit dynamic range, 2 seconds exposure time for each image). The taken fluorescence images were processed using ImageJ software to process the full intensity emanating from the bright spots. First, the RGB images were split into two channels containing the red and green components to analyse the portions of the two fluorophores separately. Again, the “rolling ball” algorithm was used. FIG. 7 shows the resulting image in which the red and green channels are shown reunited (shown here in black and white for reproduction purposes).

11. testing of the specificity

[0167] The specificity of the apta immunosensor was tested against lactate dehydrogenase of Plasmodium vivax (P<LDH), which is 90% identical to P<LDH. The P<LDH was added to uninfected human blood (diluted 1:100 in 1 mL 25 mM Tris buffer) to obtain the highest analyte concentration tested in the calibration curve (1 μM based on undiluted whole blood). The measurement of the fluorescence intensity showed that although the lower bioreceptor layer—consisting of pan-malaria anti-PLDH—can detect any Plasmodium malaria marker, in the case of P<LDH no significant cross-reaction could be detected due to the extremely high specificity of the aptamers used as the upper bioreceptor layer, but the fluorescence for the desired target (P<LDH) was orders of magnitude higher, thus a very high specificity was proven.