Office in Surface Plasmon Resonance Detector

Abstract

A composition comprising gold nanoparticles linked to a polyclonal antibody. The composition can be used to detect the presence of a target that binds to the polyclonal antibody in a solution. Binding of the target to the polyclonal antibody triggers self-assembly of the polyclonal antibody and the gold nanoparticles. This self-assembly of the polyclonal antibody and the gold nanoparticles triggers a color change in the surface plasmons of the gold nanoparticles, indicating the presence of the target.

Description

[Technical field]

[0001] The present invention relates to compositions for detecting targets such as biomarkers, detectors for detecting targets such as biomarkers, and related methods. [Background technology]

[0002] The risk of a global pandemic caused by an infectious agent has been high for the last decade. The COVID-19 pandemic and its infection waves demonstrate the need for practical diagnostic tools. The low efficiency, high cost, and long test reaction times of existing diagnostic tests demonstrate the need for a change in diagnostic equipment, moving from expensive and slow laboratory-based techniques to accessible, reliable, and rapid diagnostic tools.

[0003] International health organizations are working hard to manage the pandemic by exploring all aspects of therapeutic development, focusing on innovative diagnostic tools for rapid and selective detection of COVID-19 infection. While the COVID-19 pandemic has highlighted the need for better diagnostic equipment for infectious diseases, non-communicable diseases such as cancer and dementia also require early diagnostic tools (and preferably tools that work during the pre-symptomatic or asymptomatic period) to improve clinical decision-making and medical treatment.

[0004] Better diagnostic tools are essential to deliver precision medicine, a medical model that proposes customizing health care by tailoring medical decisions, procedures, interventions, or products to subgroups of patients instead of a one-drug-fits-all model. In precision medicine, diagnostic tests can be used to select the appropriate and optimal treatment based on the patient's genetic makeup or other molecular or cellular analyses. Summary of the Invention

[0005] Among the objects of the invention are to provide products and methods for detecting targets such as biomarkers quickly, or inexpensively, or non-invasively, or with high sensitivity or specificity, preferably with all of these characteristics, and more generally for improved point-of-care diagnostic tests for infectious diseases, non-infectious diseases, or biomarkers.

[0006] Among the objectives of the present invention are to provide products and methods for detecting targets, such as biomarkers, that have a simple and / or low-cost development process from laboratory scale to pilot plant or commercial scale.

[0007] Among the objectives of the present invention are to provide products and methods for detecting targets, such as biomarkers, that do not require sophisticated laboratories or specialists, and / or are low cost and / or easy to use.

[0008] Among the objectives of the present invention is to provide products and methods for detecting targets, such as biomarkers, that overcome current limitations such as lack of precision, lack of compatibility with different biological fluids, low temperature stability or lack of portability.

[0009] Among the objects of the present invention are to provide products and methods for detecting targets, such as biomarkers, that improve upon current diagnostic capabilities in terms of specificity, speed, or cost, with enhanced sensitivity and / or reduced instrumentation size.

[0010] Among the objectives of the present invention is to provide products and methods for detecting targets, such as biomarkers, that allow for a reduction in sensor size, which is useful for providing portable, wearable, or implantable medical devices, and is also useful for integrating biosensors with other medical devices.

[0011] Among the objects of the present invention are to provide products and methods for detecting targets such as biomarkers at low concentrations and therefore potentially at an early stage of a condition. Early detection of biomarkers is particularly desirable for long-term conditions such as cardiovascular disease, cancer, chronic respiratory disease and diabetes.

[0012] The inventions disclosed herein improve the ability to detect infectious agents in major reservoirs such as animals and wastewater, and can improve comprehensive response planning for existing or potential health risk events.

[0013] In a first aspect, the present invention provides a composition according to claim 1.

[0014] The composition according to the invention scatters light differently when the gold nanoparticles and polyclonal antibodies are dispersed in a liquid compared to when they are self-assembled in the liquid. Plasmons exist on the surface of the gold nanoparticles. Plasmons are coherent delocalized electron vibrations. The plasmon band is an energy level associated with the plasmon. The transition from dispersed to self-assembled gold nanoparticles causes a shift in the energy of the plasmon band. Absorption by these different energy bands provides a visible color transition from red to blue and blue to red.

[0015] The composition according to the invention can be dispersed in a liquid having a red color. When the surfaces of the nanoparticles are brought into contact, short-range interactions allow for bonds and other interactions between the nanoparticles. When these bonds or interactions are formed, the solution changes color from red to blue, and sometimes purple. These color changes are plasmon-generated color changes.

[0016] The composition according to the invention can use the reaction between the polyclonal antibody (linked to the gold nanoparticle) and the target in the sample (to which the polyclonal antibody binds) as a physical process that brings the nanoparticle surface into contact or interaction range. This results in a color change from red to blue, indicating that the target, e.g., biomarker, is present in the sample. Figure 1 shows a schematic of how the composition according to the invention undergoes self-assembly in the presence of a biomarker. Gold nanoparticles 1 are preferably covalently linked to polyclonal antibodies 2. Biomarkers 3 from the sample that are added have several different regions 4 to which the antibody 2 can bind. Binding of the polyclonal antibody to the different regions of the biomarker brings the nanoparticles closer together (as shown on the right side of the reaction), resulting in a color change from red (left side of the reaction) to blue (right side of the reaction).

[0017] Figure 2 shows a schematic of how a polyclonal antibody binds to a biomarker as compared to the binding of a monoclonal antibody to a biomarker. The monoclonal antibody 5 binds only to one region 6 of the biomarker. This prevents the self-assembly effect provided by the distinct portions 7 and 7' of the polyclonal antibody binding to corresponding (distinct) portions 8 and 8' of the biomarker.

[0018] Figures 4A-D show the binding of nanoparticle-monoclonal antibody complexes to biomarkers compared to the binding of nanoparticle-polyclonal antibody complexes to biomarkers. Monoclonal antibodies do not result in self-assembly of nanoparticles (Figure 4A), whereas polyclonal antibodies result in self-assembly of nanoparticles and a subsequent color change from red to blue or purple. The color change is shown in Figure 4C. Biomarker concentration provides a color change from red to blue. Figure 4D shows that the degree of color change can provide an indication of the stage of infection in a patient. As biomarker concentration increases with increasing infection, the color change darkens until it reaches gray. At very high concentrations of biomarkers, the effect is washed out due to the rapid and strong response, which can result in precipitation of the components.

[0019] The use of polyclonal antibodies linked to gold nanoparticles can surprisingly provide a significant increase in the ability to detect targets at ultra-low concentrations that bind to the polyclonal antibodies. Thus, the present invention is useful for detecting targets such as specific biomarkers at ultra-low concentrations. The ability of polyclonal antibodies to self-assemble gold nanoparticles very effectively in response to target molecules that bind to the polyclonal antibodies is believed to be due to the complex structure of polyclonal antibodies.

[0020] Polyclonal antibodies (pAbs) are secreted by different B cell lineages in the body (whereas monoclonal antibodies are derived from a single cell lineage). Polyclonal antibodies are immunoglobulin molecules that react against a specific target (which may be a biomarker), each identifying a different region (epitope) of the target. Thus, a specific target is detected by more than one antibody. Multiple interactions occur between the target and the polyclonal antibodies. This draws the gold nanoparticles (to which they are bound) into a tightly packed self-assembled structure, triggering a change in plasmon band energy and, consequently, a visible color change.

[0021] Unexpectedly, the present invention is able to provide this technical effect without the natural aggregation of the nanoparticle-linked polyclonal antibody complex, which would prevent the color transfer. Natural aggregation in solution would prevent the sensitivity of the color transfer to the target (such as a biomarker) by initiating a color change in the absence of the target.

[0022] The prefix "nano" refers to the fact that at least one dimension of the material's structure is less than 100 nanometers (nm).

[0023] The compositions and associated methods according to the invention can be easy to manufacture / perform. The use of gold nanoparticles provides an acceptable and recognizable visual color change. Gold nanoparticles are also non-toxic.

[0024] A biomarker is a measurable indicator of a biological state. By definition, a biomarker is a "characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic intervention." In general, biomarkers can be physical biomarkers (e.g., proteins) and non-physical biomarkers (e.g., heart rate). Biomarkers referred to herein are physical biomarkers, e.g., those that can physically interact with polyclonal antibodies.

[0025] Furthermore, the scale-up process from laboratory to pilot plant or commercial scale is simple and low cost.

[0026] Preferably, each polyclonal antibody is covalently linked to a gold nanoparticle.

[0027] Preferably, each polyclonal antibody is linked to a gold nanoparticle by a linker. The linker may include an N-hydroxysulfosuccinimide-derived group, which provides robust binding of the polyclonal antibody to the nanoparticle.

[0028] The linker may include an ester bond, which provides an easy to generate and robust attachment of polyclonal antibodies to nanoparticles.

[0029] The polyclonal antibodies may each be conjugated to the gold nanoparticles via primary amide groups, which provides easy to generate and robust conjugation of the polyclonal antibodies to the nanoparticles.

[0030] The polyclonal antibody may be adapted to specifically bind to one of a peptide, a protein, a hormone, an antibody, and an organic compound. The entity that the polyclonal antibody is adapted to bind to is referred to herein as a target.

[0031] Preferred targets and "target and polyclonal antibody" combinations exemplified herein are as follows: 1. FLAG® peptide (DDDDK peptide) / Polyclonal anti-DDDDK tag (binds to FLAG® tag sequence) antibody (ab1162). 2. Recombinant human topoisomerase II alpha protein (ab159735) / Polyclonal anti-topoisomerase II alpha antibody (ab12318). 3. Recombinant anti-human topoisomerase II alpha antibody of rabbit origin (ab52934). 4. Polyclonal goat anti-rabbit IgG H&L (ab182016). 5. Avian CORT / anti-corticosterone antibody (Sigma C8784-100TST) produced in rabbits / used in birds. 6. Human CORT Corticosterone CORT 500mg (Sigma 27840). 7. Non-commercial avian stress hormone CORT extraction and detection in methanol. 8.Anti-human IgG antibody (ab2410). 9.Anti-human IgM antibody (ab26867). 10.Anti-human IgA antibody (ab2411).

[0032] In a preferred embodiment, the polyclonal antibody may be adapted to bind to one or more of amyloid beta peptides, CSF biomarkers, natriuretic peptides, IgG, IgM, IgA, CigG, ANA, anti-dsDNA (anti-double stranded DNA antibodies), VOCs (volatile organic compounds such as hormones), CEA, troponin T / C / I, CRP / CD27 / CD38, CA / GP120 / GP41, cortisol, adrenaline and norepinephrine. These biomarkers act as indicators of diseases selected from cancer, dementia, CVD, tuberculosis, malaria and HIV. Biomarkers of sepsis, cholera and pneumonia may also be detected by selecting an appropriate polyclonal antibody. Preferably, the polyclonal antibody is adapted to bind to a specific biomarker associated with any of the medical conditions described herein, including the following as shown in Table 1: [Table 1]

[0033] Table 1 shows examples of classes of targets detectable using the products and methods according to the invention. The examples in the detailed description below provide data showing that certain hormones, peptides, antibodies (both specific and non-specific) and proteins can be detected by the invention. It is believed that other targets that fall under these class headings, including the specific biomarkers and other targets listed herein, are also detectable by the invention due to their similar properties or structures and the common mechanism of action of polyclonal antibody binding.

[0034] Preferably, the polyclonal antibodies are adapted to bind to a virus such as influenza A, B or HIV-1 CA protein, HIV-1 envelope glycoprotein GP120 HIV-1 GP120, GP 41, or components thereof such as spike protein or nucleoprotein.

[0035] Preferably, the polyclonal antibody is adapted to bind to an antigen or capsid protein of a virus, such as a respiratory viral infection (RVS). For example, an IgG human anti-SARS-CoV-2 and any variants thereof can be linked to the nanoparticles. For example, an anti-SARS-CoV-2 (COVID-19) spike antibody raised against the recombinant SARS-CoV-2 (2019 nCoV) spike protein (S2) domain or an anti-SARS-CoV-2 (COVID-19) nucleocapsid antibody raised against the recombinant SARS-CoV-2 (2019 nCoV) nucleocapsid protein can be linked to the nanoparticles.

[0036] Preferably, the polyclonal antibodies are adapted to bind to biomarkers associated with Helicobacter pylori, poxvirus, tuberculosis (TB), pneumonia or Vibrio cholerae.

[0037] The gold nanoparticles may be provided with a surface layer of polyethylene glycol (PEG). The layer may be a complete or partial coating. The layer is preferably applied prior to linking the gold nanoparticles to the polyclonal antibodies. This increases the extent to which the polyclonal antibodies can be linked to the nanoparticles. This provides a composition that is easier to manufacture and is more sensitive to the target molecule due to the greater number of polyclonal antibodies that are linked to the gold nanoparticles. The PEG molecules also provide a more stable nanoparticle-polyclonal antibody complex that is less likely to self-aggregate prematurely or in the absence of a specific target.

[0038] Preferably, the PEG molecule contains a carboxylic acid group, preferably at or adjacent to the terminus of the PEG molecule, which enhances the above effects by providing a PEG molecule that is more readily conjugated to polyclonal antibodies and forms more robust bonds, especially when NHS / EDC conjugation reactions are used.

[0039] The chain length of the polyethylene glycol is preferably between 1000 and 3000, more preferably between 1000 and 2500. These ranges enhance the above-mentioned effects.

[0040] Preferably, the gold nanoparticles are substantially spherical. The gold nanoparticles may be spherical.

[0041] The diameter of the gold nanoparticles may be greater than 3.5 nm. This provides the composition with an acceptable level of surface plasmon and therefore an acceptable level of visible color. The diameter of the gold nanoparticles may be between 5-50 nm, preferably between 10-40 nm. These ranges provide the composition with an acceptable level of surface plasmon and therefore a strong visible color for easy identification of color changes.

[0042] The composition can be a powder. The composition may be lyophilized. This provides a convenient form for transporting the composition that can be added to a liquid sample or a different liquid to which the sample is added. The lyophilized or powder form of the composition is also useful for addition to a detection device that is shipped to a user in a dry form. The powder or lyophilized form can be adhered to the surface of the detection device. The user then adds the sample or a separate liquid to perform the detection test.

[0043] In a further aspect, the invention provides a liquid comprising a composition described herein (comprising gold nanoparticles and a polyclonal antibody) dispersed in the liquid, thereby providing a convenient medium into which a sample can be introduced for detection of targets in the sample.

[0044] The concentration of the composition containing gold nanoparticles and polyclonal antibodies in the liquid may be such that the absorbance of the liquid is between 0.1 and 1 AU (absorbance unit).

[0045] The liquid may include liquids selected from the group of water, methanol, plasma, saliva, serum, sodium chloride solution having a concentration between 50 mM and 200 mM, potassium chloride solution having a concentration between 50 mM and 200 mM, magnesium chloride solution (MgCl2) having a concentration between 5 mM and 100 mM, PBS (phosphate buffered saline) having a concentration of less than 10% (preferably about 0.1%), and SBB (sodium borate buffer) having a concentration of less than 1 M (preferably about 0.1 M). These liquids provide an acceptably low level of spontaneous aggregation of the gold nanoparticles in the composition.

[0046] Ethanol is less preferred for use in the present invention because it can lead to aggregation of dispersed gold nanoparticle / polyclonal antibody complexes in the absence of the target (to which the polyclonal antibody binds). In a preferred embodiment, the solution is substantially free of ethanol.

[0047] In a further aspect, the invention provides a method of making a composition described herein comprising the step of linking gold nanoparticles to a polyclonal antibody.

[0048] The method may further comprise attaching a linker to the gold nanoparticles prior to linking the gold nanoparticles to the polyclonal antibody. The linker may be an activating group that increases the ability of the gold nanoparticles to bind to the polyclonal antibody.

[0049] The step of attaching the linker to the gold nanoparticles may include reacting the gold nanoparticles with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, followed by reaction with N-hydroxysulfosuccinimide, known as the EDC,NHS reaction.

[0050] The step of linking the gold nanoparticles to the polyclonal antibodies may be carried out in the presence of polyethylene glycol (PEG). Similarly, PEG may be applied to the nanoparticles prior to linking any polyclonal antibodies to the nanoparticles. Preferably, the nanoparticles include a partial or complete coating on their surface that includes citrate groups or other carboxylic acid derived groups. These groups generate an activated partial or complete coating that is well suited for further linking reactions. Preferably, this activated partial or complete coating is formed by treating uncoated gold nanoparticles with sodium citrate or another carboxylate solution. Preferably, the nanoparticles with this activated partial or complete coating are mixed with a liquid that includes PEG, such that a partial or complete coating of PEG is provided on the activated partial or complete coating.

[0051] The partial or complete coating of PEG increases the degree to which polyclonal antibodies are linked to the nanoparticles. Thus, the number of polyclonal antibodies that can be bound to PEG-treated nanoparticles is greater than the number of polyclonal antibodies that can be bound to the corresponding non-treated nanoparticles. One mechanism by which PEG treatment achieves this result is the increased surface area provided by the PEG layer. The increased polyclonal antibody incorporation by PEG-treated nanoparticles provides a composition that is easier to manufacture and is more sensitive to target molecules because of the greater number of polyclonal antibodies that are linked to the gold nanoparticles. The partial or complete coating of PEG on the nanoparticles is also believed to prevent undesirable spontaneous aggregation (which can lead to undesirable discoloration).

[0052] Preferably, the chain length of the polyethylene glycol is between 1000 and 3000, preferably between 1000 and 2500. This enhances the above effects and ensures that the PEG treatment does not affect the plasmonic properties of the nanoparticles. The presence of a partial or complete PEG coating improves the detection of small targets such as hormones and small (e.g. molecular) biomarkers.

[0053] Preferably, prior to linking the gold nanoparticles to the polyclonal antibody (or linker), the gold nanoparticles are treated to have citrate groups disposed on their surface, preferably in the form of a layer (discussed above), providing a functionalized surface to which a linker, such as the product of the EDC-NHS reaction, or a polyclonal antibody can be readily and robustly linked.

[0054] In some embodiments, fluorescently labeled polyclonal antibodies can be used to indicate that binding between the gold nanoparticles and the polyclonal antibody has occurred. The fluorescence of the complex of linked gold nanoparticles and polyclonal antibodies can be detected using a fluorescent microscope.

[0055] In a further aspect, the invention provides a method of detecting a target in a sample, the method comprising combining a composition as described herein or a liquid as described herein with the sample. The sample may be a liquid.

[0056] The sample may have a volume of between 3 and 5 microliters. This is preferred because only a very small amount of the composition containing the nanoparticles and the polyclonal antibody is required. However, this small volume is still sufficient to give a discernible result signal.

[0057] The liquid or sample may contain a buffer having a pH in the range of 4 to 9, preferably 4 to 8.5, 4 to 8 or 4 to 7. This prevents undesired or premature aggregation of the nanoparticle and polyclonal antibody composition. A pH outside these ranges is less preferred as it may drive aggregation by altering the surface charge of the nanoparticles.

[0058] The concentration of target in the sample is 5 x 10 -18 Mole ~ 1 x 10 -9 This provides an ultra-low concentration detection method.

[0059] The sample can be a saliva sample, a urine sample, a soft tissue sample, a serum sample, a plasma sample, or an aqueous sample. This provides a wide range of flexibility for the sample to be tested. In a preferred embodiment, the liquid and the sample are of the same type. This prevents undesired or premature self-assembly. For example, if the sample to be run is a plasma sample, the liquid (containing the complex of nanoparticles and polyclonal antibodies with which the sample is combined) can include plasma.

[0060] The method may include a diagnostic step in which a color change indicates the presence of a target that is a biomarker for disease, thereby diagnosing the disease.

[0061] The combination of the composition or liquid and the sample may undergo a color change from red to blue, which occurs if the sample contains a target that is bound by the polyclonal antibodies in the composition or liquid.

[0062] Preferably, if the sample contains a target to which the polyclonal antibody binds, the solution undergoes a color change in less than 3 minutes, preferably less than 2 minutes, preferably less than 1 minute after combining the sample with the solution.

[0063] The target can be a biomarker, and when the composition described herein or the liquid described herein is combined with a sample, there are gold nanoparticles that are linked to probes that bind to the biomarker.The advantages of this method are discussed and illustrated in Figure 9C and Figure 11 and the corresponding detailed description below.This method is useful for detecting certain types of antibodies that act as biomarkers for certain conditions.

[0064] The target can be an antibody that binds to the probe. The target can be a specific antibody that specifically binds to the probe. For example, the method can detect a specific type of IgG or IgM or IgA antibody.

[0065] The probe may be a protein, enzyme or peptide, such as a disease antigen or portion thereof. The probe may be a spike protein or a nucleocapsid protein associated with any of the conditions described herein.

[0066] In a further aspect, the invention provides a detection test comprising the first liquid or the first composition described herein. The test may be a device.

[0067] The detection assay may be adapted to carry out the methods described herein.

[0068] The detection test may be adapted to transmit the detection results to a remote location. It may do so via a wireless network and / or the Internet. The detection test may be connected to a mobile device, such as a phone or tablet or computer, to display the detection results or to transmit the detection results to a remote location.

[0069] The detection test may include a second composition as described herein or a second liquid as described herein, A first composition as described herein or a first liquid as described herein, and The second composition described herein or the second liquid described herein may comprise: The first and second compositions or liquids described herein contain different polyclonal antibodies such that they are adapted to detect different targets, i.e. the test may include a combination of two different liquids, two different compositions or any combination of liquids and compositions, provided that the polyclonal antibodies associated with each liquid / composition are different from the polyclonal antibodies associated with the other liquid / composition.

[0070] The different targets may both be biomarkers associated with a particular disease. For example, one biomarker may be the presence of a specific IgM antibody, and the other biomarker (to which the relevant polyclonal antibody binds) may be a specific IgG antibody. These embodiments may provide patients with a rapid and accurate method of assessing the stage of infection by the presence of IgM and IgG antibodies or otherwise. A hypothetical example of such a detector and method is provided in Example 8, discussed below.

[0071] The first and second compositions or liquids can be spaced apart so that they can react independently with different samples, allowing a user to run, for example, blood samples as well as urine samples on the same detection test.

[0072] The first and second compositions or liquids can be adapted to react with a single sample or different samples simultaneously, providing a rapid test that is still capable of detecting multiple targets.

[0073] Embodiments of the invention will now be described with reference to the drawings and the following examples. [Brief description of the drawings]

[0074] [Figure 1] FIG. 1 is a schematic diagram of how a composition according to the present invention undergoes self-assembly in the presence of a biomarker. [Diagram 2] FIG. 1 is a schematic diagram of how polyclonal antibodies bind to biomarkers compared to monoclonal antibody binding to biomarkers. [Diagram 3] FIG. 1 shows reaction steps that can be used to link polyclonal antibodies to gold nanoparticles. [Figure 4A] FIG. 1 shows the binding of nanoparticle-monoclonal antibody complexes to biomarkers and the subsequent self-assembly and color change compared to the binding of nanoparticle-polyclonal antibody complexes to biomarkers. [Figure 4B] FIG. 1 shows the binding of nanoparticle-monoclonal antibody complexes to biomarkers and the subsequent self-assembly and color change compared to the binding of nanoparticle-polyclonal antibody complexes to biomarkers. [Figure 4C] FIG. 1 shows the binding of nanoparticle-monoclonal antibody complexes to biomarkers and the subsequent self-assembly and color change compared to the binding of nanoparticle-polyclonal antibody complexes to biomarkers. [Figure 4D] FIG. 1 shows the binding of nanoparticle-monoclonal antibody complexes to biomarkers and the subsequent self-assembly and color change compared to the binding of nanoparticle-polyclonal antibody complexes to biomarkers. [Figure 5A]FIG. 1 shows reaction steps that can be used to link fluorescent polyclonal antibodies to gold nanoparticles and the fluorescence microscopy images obtained in Example 1. [Figure 5B] FIG. 1 shows reaction steps that can be used to link fluorescent polyclonal antibodies to gold nanoparticles and the fluorescence microscopy images obtained in Example 1. [Figure 5C] FIG. 1 shows reaction steps that can be used to link fluorescent polyclonal antibodies to gold nanoparticles and the fluorescence microscopy images obtained in Example 1. [Figure 5D] FIG. 1 shows reaction steps that can be used to link fluorescent polyclonal antibodies to gold nanoparticles and the fluorescence microscopy images obtained in Example 1. [Figure 5E] FIG. 1 shows reaction steps that can be used to link fluorescent polyclonal antibodies to gold nanoparticles and the fluorescence microscopy images obtained in Example 1. [Figure 5F] FIG. 1 shows reaction steps that can be used to link fluorescent polyclonal antibodies to gold nanoparticles and the fluorescence microscopy images obtained in Example 1. [Figure 6A] FIG. 1 shows the results of detection of protein / enzyme biomarkers as described in Example 2. [Figure 6B] FIG. 1 shows the results of detection of protein / enzyme biomarkers as described in Example 2. [Figure 6C] FIG. 1 shows the results of detection of protein / enzyme biomarkers as described in Example 2. [Figure 6D] FIG. 1 shows the results of detection of protein / enzyme biomarkers as described in Example 2. [Figure 7A] FIG. 1 shows the results of detection of peptide biomarkers as described in Example 3. [Figure 7B] FIG. 1 shows the results of detection of peptide biomarkers as described in Example 3. [Figure 7C] FIG. 1 shows the results of detection of peptide biomarkers as described in Example 3. [Figure 8A] FIG. 1 shows the results of the detection of cortisone as described in Example 4. [Figure 8B] FIG. 1 shows the results of the detection of cortisone as described in Example 4. [Figure 8C] FIG. 1 shows the results of the detection of cortisone as described in Example 4. [Figure 8D] FIG. 1 shows the results of the detection of cortisone as described in Example 4. [Figure 9A] FIG. 1 shows the detection results of general and specific types of antibodies. [Figure 9B] FIG. 1 shows the detection results of general and specific types of antibodies. [Figure 9C] FIG. 1 shows the detection results of general and specific types of antibodies. [Figure 9D] FIG. 1 shows the detection results of general and specific types of antibodies. [Figure 10] FIG. 1 shows the reaction occurring in the viral antigen sensor described in Example 6. [Figure 11] FIG. 1 shows the reactions occurring in the viral serological sensor described in Example 7. [Figure 12] Graph of typical antibody production versus days post-infection. The array also shows a summary result when a multi-biomarker sensor according to the invention is used to identify different targets, as described in Example 8. [Figure 13] FIG. 1 shows an array and schematic results when a multi-biomarker sensor according to the invention is used to identify different targets, as described in Example 9. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0075] Example 1 describes a method for making a composition according to one embodiment of the invention.

[0076] Examples 2-5 describe exemplary compositions and related targets that can be detected using methods or compositions according to the invention.

[0077] Examples 6-9 describe exemplary compositions and methods for detecting viruses, as well as exemplary compositions, methods and devices for detecting multiple targets.

[0078] Example 1 This example provides a method for covalently linking polyclonal antibodies to gold nanoparticles. This method demonstrates the advantage of a step of providing a polyethylene glycol (PEG) full or partial coating on the nanoparticles prior to linking the polyclonal antibodies to the nanospheres. The general method used was as follows: Prepare Sodium Borate Buffer 1M (SBB) pH 8.5. Add 5 microliters of AuNPs into the tube (optionally add polyethylene glycol (PEG) to coat the nanoparticles before linking to the antibody). Add 10 microliters of NHS (N-hydroxysulfosuccinimide) 0.2M. · Add 5 microliters of EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) 0.2 M. This step and the previous step constitute the NHS-EDC ligation reaction. Add 1 microliter of fluorescent polyclonal antibody (2mg / ml). Add 494 microliters of 0.1M SSB solution. Keep overnight at room temperature (20-25°C) for the ligation reaction to occur. Centrifuge at 10,000 rpm for 10 minutes to separate the nanoparticle-polyclonal antibody complex from the liquid. · The AuNPs were then washed twice with 0.1 M (SBB) and resuspended in 50 μl of sodium borate buffer 1 M (SBB, pH 8.5).

[0079] This method is shown diagrammatically in Figure 3 and at the top of Figure 5. The conjugation reaction is known as the EDC-NHS reaction. This provides a covalent bond between the polyclonal antibody and the gold nanoparticle surface.

[0080] Spherical AuNPs with diameters of 10 nm and 40 nm, with and without PEG treatment (see Figures 5A-D), were covalently linked with a fluorescently labeled antibody, goat anti-mouse IgG H&L (Alexa Fluor® 594). The antibody was linked to the gold nanoparticles using the EDC-NHS reaction. The complexes formed were analyzed by a ZOE Cell Imager fluorescence microscope. The parameters used were as follows: red channel excitation: 556 / 20 nm emission: 615 / 61 nm.

[0081] Figures 5A-5D show the results. Figure 5A shows 10 nm AuNP+antibody conjugated without PEG. Figure 5B shows 40 nm AuNP+antibody conjugated without PEG. Figure 5C shows 10 nm AuNP+antibody conjugated with PEG. Figure 5D shows 40 nm AuNP+antibody conjugated with PEG.

[0082] Figure 5F shows 40 nm AuNPs covalently linked to goat anti-mouse IgG H&L (Alexa Fluor® 488 nm) fluorescently labeled antibody. Analysis is performed by using a ZOE Cell Imager fluorescent microscope. The parameters used were: green channel excitation: 488 nm emission: 515 / 45 nm.

[0083] The fluorescent markers here show the difference in efficiency of polyclonal antibody uptake between PEG-treated gold nanoparticles (Figures 5C and 5D - high fluorescence) and non-PEG-treated nanoparticles (Figures 5A and 5B - low fluorescence).

[0084] Example 2 In this example, the target is a protein / enzyme biomarker. To test the reaction efficiency and specificity of the present invention for protein / enzyme detection, human anti-topoisomerase II antibody and human DNA topoisomerase 2-alpha protein were used. The present invention has been found to be accurate and sensitive when scaled down to the final volume required. The present invention is specific to the selected antigen.

[0085] The results are shown in Figure 6. A) Schematic diagram of protein detection. B) shows the results of a reaction in an Eppendorf tube with a final volume of 100 microliters: Sample tube 1 is the positive control: this tube exhibits a blue color induced (from the starting red liquid) by creating a pH change (to a high pH) or by adding large amounts of salt. Sample tube 2 is a 10 nanometer diameter AuNP linked to a monoclonal antibody after addition of DNA topoisomerase 2-alpha protein. The color is red. Sample tube 3 is a 40 nanometer diameter AuNP linked to a monoclonal antibody after addition of DNA topoisomerase 2-alpha protein. The color is red. Sample tube 4 is a 10 nanometer diameter AuNP linked to a polyclonal human anti-topoisomerase II antibody after addition of DNA topoisomerase 2-alpha protein. The color is blue. Sample tube 5 is a 40 nanometer diameter AuNP linked to a polyclonal human anti-topoisomerase II antibody after addition of DNA topoisomerase 2-alpha protein. The color is blue. Sample tube 6 is a negative control with only AuNPs linked to a polyclonal human anti-topoisomerase II antibody. It is red in color.

[0086] Reactions were performed in serum as reaction buffer. In all cases, a protein / biomarker concentration of 1 nM (nanomolar) (DNA topoisomerase 2-alpha protein) was used.

[0087] The monoclonal antibody sample tube does not show a color change from red to blue. The polyclonal antibody sample shows a color change from a red starting solution to a blue solution upon addition of DNA topoisomerase 2-alpha protein.

[0088] C) shows the plasmonic color change using 40 nanometer diameter AuNPs and the polyclonal antibody mentioned above with decreasing biomarker concentrations. These decreasing concentrations are represented by the tapering wedges and the concentrations listed below the figure. A final reaction volume of 3 microliters was used. The top row of this figure is a negative control with only AuNPs linked to a polyclonal human anti-topoisomerase II antibody. The color is red in all cases. The second row shows various concentrations of DNA topoisomerase 2-alpha protein added to a liquid containing AuNPs linked to a polyclonal human anti-topoisomerase II antibody. In the bottom row, all concentrations show blue color except for the 1 aM (attomolar) concentration at the far right of the array, which remained red.

[0089] Therefore, 10 x 10 -18 Even at target concentrations as low as 10 attomolar (10 aM), the polyclonal antibody-nanoparticle conjugates result in a detectable color change.

[0090] D) Reaction efficiency (%) as a function of biomarker concentration. 100% positive individual results were those in which the color change was clear and occurred in less than 5 minutes. Individual results that did not achieve these characteristics (e.g., unclear color change or took longer than 5 minutes) were assigned a reaction efficiency of 0%. Bars show the average efficiency obtained over 25 individual tests performed for each set of conditions. Results show that even biomarker concentrations as low as 10 aM show good reaction efficiency.

[0091] Example 3 The target in this example is a peptide biomarker. The structure of the peptide and the results of this example are shown in Figure 7. Peptides are short chains of amino acids. Peptides are generally considered to be short chains of two or more amino acids. Due to their small size, peptides are very difficult to detect. To test the reaction efficiency and specificity for peptide detection, the FLAG® expression system was used. We covalently linked a polyclonal antibody (anti-DDDDK tag (binding to the FLAG® tag sequence) (ab1162), which specifically interacts with the FLAG® peptide (ddddk peptide) biomarker) onto gold nanoparticles with diameters of 10 nanometers and 40 nanometers.

[0092] The results are shown in Figures 7A to 7C.

[0093] FIG. 7A shows a schematic of peptide detection and the associated red to blue color change.

[0094] Figure 7B shows the color change indicating detection. On the right is a photograph showing the color change of the three samples. On the left is a schematic of the color change shown in the photograph.

[0095] Sample C in the photograph and the corresponding array in the schematic diagram is a control sample with no peptide added. The liquid has a red color.

[0096] Sample 1 (and the corresponding array in the schematic diagram) shows the reaction of 10 nanometer diameter AuNPs + anti-DDDDK polyclonal antibody in the presence of 5 pM peptide. The liquid has a light purple color.

[0097] Sample 2 (and the corresponding array in the schematic diagram) shows the reaction of 40 nanometer diameter AuNPs + anti-DDDDK polyclonal antibody in the presence of 5 pM peptide. The liquid has a dark blue color.

[0098] Figure 7C shows the reaction efficiency (%) as a function of biomarker concentration. The reaction efficiency was calculated as follows: if the color change was clear and occurred in less than 5 minutes, a reaction efficiency of 100% was assigned for each individual result. Individual results that did not achieve these characteristics (i.e., unclear color change or took longer than 5 minutes) were assigned a reaction efficiency of 0%. The bars on the chart show the average reaction efficiency obtained for 25 individual tests performed for each set of conditions. The results show that peptide detection has excellent efficiency even down to concentrations as low as 50 aM (attomolar).

[0099] Example 4 It has been shown that parasitic / viral / bacterial infections in animals result in elevated stress hormone levels. To investigate this type of biomarker, a sensor was prepared to detect the presence of corticosterone (CORT), a stress hormone isolated from avian samples. Commercially available avian anti-CORT antibodies were covalently linked to 10 nm and 40 nm AuNPs. Stress hormones from feather samples were obtained by methanol extraction and quantified using a NanoDrop spectrophotometer (A280). Small amounts of the resulting CORT were added to a liquid according to the present invention. The results show a color change even after using ultra-small amounts of sample, demonstrating the efficiency of the present invention. Efficiency was also shown with methanol samples, and no non-specific (i.e., off-target) aggregation of the "nanoparticle + polyclonal antibody complex" was detected. To prove specificity, human commercially available corticosterone (CORT) was also tested. If the test was specific, no color change would be expected when using avian anti-CORT antibodies on human CORT samples. No color change was detected using human CORT. This demonstrates the specificity of the invention (ie, no cross-reactivity with the human version of the biomarker - in this case hormones).

[0100] The results are shown in Figures 8A to 8D.

[0101] FIG. 8A shows a schematic of the hormone detection reaction and the associated red to blue color change.

[0102] FIG. 8B shows the results of the test using CORT hormone and "anti-avian CORT antibody". A schematic version of each array summarizes the color changes shown in the photographic version for clarity. CORT hormone was extracted with methanol from three feathers (samples A, B and C) for 20 minutes. The resulting CORT concentrations were measured by a NanoDrop spectrophotometer (A280). Array (i) shows the liquid according to the invention before any CORT was added. All liquids are red in color. Array (ii) shows the liquids after reaction with avian CORT and human CORT.

[0103] Row A (negative control), row B (human CORT added) and row C (very low avian CORT) show no color change. Rows D-F, corresponding to various concentrations of avian CORT, all show a red to blue color change, except for column 8, which shows the negative control row.

[0104] FIG. 8C shows an array to demonstrate the reaction of human CORT with gold nanoparticles linked to anti-avian CORT antibodies. Row i) shows a control containing gold nanoparticles and antibodies in serum. Row ii) shows a control containing gold nanoparticles and antibodies in methanol. Column iii) shows gold nanoparticles and antibodies mixed with human cortisone. In the array, columns 1-4 relate to nanoparticles with a diameter of 10 nm. Columns 5-8 relate to nanoparticles with a diameter of 40 nm. The wedge labeled C shows the qualitative concentration of human cortisone in row iii). The concentrations range from 0.025 ng / microliter to 0.005 ng / microliter. Both row i) and row ii) are red in color as expected. Row iii) shows that no color change is shown (the liquid remains red) across the various concentrations of human cortisone for both 10 and 40 nanometer diameter nanoparticles. These results indicate that any discolouration is specifically related only to the detection of avian cortisone and not to the detection of human cortisone, regardless of concentration.

[0105] FIG. 8D shows the reaction efficiency (%) as a function of biomarker concentration. The reaction efficiency was calculated as follows: if the color change was clear and occurred in less than 5 minutes, a reaction efficiency of 100% was assigned for the individual result. Individual results that did not achieve these characteristics (i.e., indistinct color change or took longer than 5 minutes) were assigned a reaction efficiency of 0%. The bars on the chart show the average reaction efficiency obtained over 25 individual tests performed for each set of conditions. The results demonstrate excellent detection (90% reaction efficiency) of avian cortisone even down to concentrations as low as 50 aM (attomoles). These results also demonstrate the specificity of detection for avian cortisone compared to human cortisone. The human cortisone "bars" are shown as either 0% or 1% for all cortisone concentrations.

[0106] Example 5 In this example, the target is an antibody biomarker. In the first part of this example, anti-IgG, anti-IgM or anti-IgA polyclonal antibodies are linked to AuNPs and used to detect the presence of IgG, IgM or IgA antibody biomarkers. The results are shown in Figures 9A and 9B.

[0107] Figure 9A: Anti-rabbit IgG polyclonal antibodies were covalently linked to 40 nanometer diameter AuNPs. 2.5 AU / ml (activity units per ml) and 10 AU / ml samples of rabbit IgG were used as biomarkers for detection in reaction buffer. A 10 AU / ml rabbit IgG sample in saliva was also tested. In all samples, a color change from red to blue is induced by the presence of the biomarker IgG. The top row in red is the negative control. Rows 1-4 of the array results show replicate samples.

[0108] Figure 9B: This figure shows reaction efficiency (%) as a function of biomarker concentration. Reaction efficiency was calculated as follows: if the color change was clear and occurred in less than 5 minutes, a reaction efficiency of 100% was assigned for each individual result. Individual results that did not achieve these characteristics (i.e., indistinct color change or took longer than 5 minutes) were assigned a reaction efficiency of 0%. The bars on the chart show the average reaction efficiency obtained over 25 individual tests performed for each set of conditions. The results demonstrate excellent reaction efficiency even down to concentrations as low as 50 aM (attomolar).

[0109] In the second part of this example, Figures 9C and 9D show the detection of a specific type of IgG or IgM or IgA antibody. The antibody detected is an IgG anti-human DNA topoisomerase II.

[0110] Figure 9C: Specific antibody detection. DNA topoisomerase II was covalently linked to AuNPs with a diameter of 40 nanometers. The liquid is red in color. Human DNA topoisomerase II alpha can be considered as a probe that is linked to gold nanoparticles to provide the starting material.

[0111] In step A, anti-human topoisomerase II IgG polyclonal antibodies from rabbits were added to the starting material (nanoparticles + probes) as a biomarker. The liquid remains red. The added antibodies specifically bind to DNA topoisomerase II bound to the nanoparticle surface without causing nanoparticle aggregation. Here the AuNPs bound with anti-IgG polyclonal antibodies recognize the presence of IgG anti-human topoisomerase II alpha.

[0112] In step B, anti-rabbit IgG polyclonal antibody was added to the reaction as a color change inducer. The array shows the top row in red (negative control). Columns 1-4 show replicate samples. The first, second and third rows are all red. The color change from red to blue is only induced in the bottom row by the presence of specific anti-human DNA topoisomerase II IgG. Color change occurs only in the presence of this specific IgG. In this example, it was possible to detect specific antibodies in the range of 1.5-10 AU / ml in serum.

[0113] FIG. 9D shows reaction efficiency (%) as a function of biomarker concentration. Reaction efficiency was calculated as follows: if the color change was clear and occurred in less than 5 minutes, a reaction efficiency of 100% was assigned for each individual result. Individual results that did not achieve these characteristics (i.e., unclear color change or took longer than 5 minutes) were assigned a reaction efficiency of 0%. The bars on the chart show the average reaction efficiency obtained over 25 individual tests performed for each set of conditions. The results demonstrate excellent reaction efficiency even down to concentrations as low as 50 aM (attomolar) or 1 fM (femtomolar).

[0114] The results shown in Figures 9C and 9D demonstrate that the two-step technique can be used to detect specific antibodies (which themselves bind to specific targets associated with particular diseases).

[0115] In summary, a biomarker associated with a disease can be bound to the surface of a nanoparticle. A test sample believed to contain the corresponding specific antibody can be added to the liquid. The liquid will remain red regardless of whether the sample contains the target antibody. The presence of a specific antibody (if present) can then be detected by adding a polyclonal antibody (linked to gold nanoparticles) that binds only to the specific antibody to be detected. If the specific antibody is present, a color change from red to blue is induced.

[0116] Example 6 This example provides a viral antigen sensor as shown diagrammatically in FIG.

[0117] As an example for viral detection, anti-SARS-CoV-2 (COVID-19) spike (S2) antibodies raised against recombinant SARS-CoV-2 (2019-nCoV) spike protein (S2) domain or anti-SARS-CoV-2 (COVID-19) nucleocapsid protein (N) antibodies raised against recombinant SARS-CoV-2 (2019-nCoV) nucleocapsid protein (N) can be covalently linked to gold nanoparticles using EDC-NHS reaction (see Example 1).

[0118] The in vitro system can use the SARSCoV-2 spike glycoprotein (S2) and nucleocapsid (N) proteins, both of which are commercially available recombinant antigens. The complex morphology can be characterized by scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDX) prior to its use.

[0119] Alternatively, the detector can be designed to detect the presence of other viruses, such as influenza A, B or HIV-1 CA protein, HIV-1 envelope glycoprotein GP120 HIV-1 GP120, GP41, etc.

[0120] Antigen detection or capsid protein detection approaches can be used to detect the presence of other viruses, such as viral respiratory tract infections (RVS). IgG human anti-SARS-CoV-2 can be linked to gold nanoparticles.

[0121] Example 7 This example provides a viral serological IgG / IgM detector, which is shown diagrammatically in FIG.

[0122] Two sets of nanoparticles are prepared by the EDC-NHS conjugation reaction described herein. First, a set of nanoparticles is conjugated with COVID antigens as described above in Example 6. The COVID antigens form the probes. Second, a set of nanoparticles is conjugated with anti-human IgG or human IgM polyclonal antibodies. Both of these anti-human antibodies are commercially available and do not require laboratory preparation. The purpose of this sensor is to detect specific human IgG or IgM in a sample generated as an immune response to the COVID-19 virus.

[0123] Addition of a sample containing IgG or IgM antibodies to the test solution (containing the probe / antigen-linked nanoparticles and the "anti-human IgG / IgM polyclonal antibody + nanoparticle" complex) induces a color change from red to blue because the IgG / IgM in the sample binds to the probe / antigen-linked nanoparticles, and the bound complex then associates with the polyclonal antibody / nanoparticle complex to give a self-assembly.

[0124] An example of this is depicted diagrammatically in Figure 11, which shows an IgG human anti-SARS-CoV-2 N protein detection system.

[0125] Example 8 This example provides a multi-biomarker detector (antigen / IgG / IgM / ) on a single chip.

[0126] The design is shown in Figure 12. By providing a chip containing a composition according to the invention for detecting IgM and IgG antibodies, the user can be informed about the stage of the disease. The graph in Figure 12 shows the abundance of various antibodies in the virus (COVID infection) versus time. The earliest curve shows the increase and subsequent decrease of SARS-CoV-2 RNA and antigens present in the body. The second earliest curve shows the production of IgM antibodies at the early stage of the infection. The third curve shows that the production of IgG antibodies replaces IgM antibodies as this curve declines. Thus, the presence of IgG antibodies detected by the test can indicate that the infection is at a later stage, while the detection of only IgM antibodies indicates that the patient is still in the early stages of the infection. This information can be combined with a test for the presence of specific disease antigens provided by the fourth row of the array / device. This information is provided in a single test.

[0127] The array shown has a top row of negative controls that remain red. Rows 1-4 on the left are clear colored samples only. Rows 4-8 on the right show the blue color induced when a red composition or liquid according to the invention is added to a sample containing the biomarkers listed on the right side of the array.

[0128] Example 9 This example describes a multi-pathogen detector in a single device. The device, which can be a chip, is shown in FIG.

[0129] Due to the ability to adapt the present invention to different biomarkers, methods and devices according to the present invention are capable of detecting two or more biomarkers from a single sample (eg, a single blood, urine, saliva sample).

[0130] The method and device may also be capable of testing 10 or more samples. Infectious agents suitable for this methodology include Helicobacter pylori, poxvirus, tuberculosis (TB), pneumonia, and Vibrio cholerae. This multi-array detector can be used to test one or more selected biomarkers specific to each infectious pathogen. The same sensor can be adapted for non-infectious diseases such as cancer and dementia by selecting appropriate polyclonal antibodies to bind to the biomarker or biomarkers associated with these diseases. These embodiments of the invention are useful because two or more biomarkers are often required for proper diagnostic validation in cancer and dementia.

[0131] Figure 13 shows a schematic diagram of one chip multi-biomarker nanosensor detection. This embodiment can detect one or more biomarkers to simultaneously detect different infectious diseases and from different samples. This multi-biomarker nanosensor can be used as a first round broad spectrum diagnostic tool for infectious agent identification.

[0132] The array on the left shows the samples before addition of a liquid or composition according to the invention (all clear color). The array in the middle shows the samples immediately after addition of a composition or liquid according to the invention (all red color). The array on the right shows the color change when a composition / liquid according to the invention reacts with the sample to provide blue or purple wells in the array. The negative controls in the bottom row remain red.

Claims

1. A composition comprising gold nanoparticles, polyethylene glycol, and a polyclonal antibody, 1. A composition wherein the gold nanoparticles comprise a partial or complete coating on their surface comprising carboxylic acid-derived groups, the polyethylene glycol provides a partial or complete coating over the partial or complete coating of carboxylic acid-derived groups, and the polyclonal antibody is linked to the gold nanoparticles.

2. The composition of claim 1 , wherein the polyclonal antibody is adapted to specifically bind to one of a peptide, a protein, a hormone, and an antibody.

3. 3. The composition of claim 1 or 2, wherein the polyclonal antibody is adapted to bind to one or more of amyloid beta peptide, CSF biomarker, natriuretic peptide, IgG, IgM, IgA, CigG, ANA, anti-dsDNA, VOC, CEA, troponin T / C / I, CRP / CD27 / CD38, CA / GP120 / GP41, cortisol, adrenaline and norepinephrine.

4. 4. The composition according to any one of claims 1 to 3, wherein the chain length of the polyethylene glycol is between 1000 and 3000, preferably between 1000 and 2500.

5. 5. The composition of claim 1, wherein the gold nanoparticles have a diameter greater than 3.5 nm.

6. A liquid comprising the composition described in any one of claims 1 to 5, wherein the composition comprising the gold nanoparticles and the polyclonal antibody is dispersed in the liquid.

7. 7. The liquid of claim 6, comprising a liquid selected from the group of water, methanol, plasma, saliva, serum, a sodium chloride solution having a concentration between 50 mM and 200 mM, a potassium chloride solution having a concentration between 50 mM and 200 mM, a magnesium chloride solution having a concentration between 5 mM and 100 mM, 0.1 M SSB, and PBS having a concentration of less than 10%.

8. 6. A method for producing the composition of claim 1, comprising the step of linking the gold nanoparticles to a polyclonal antibody.

9. 9. The method of claim 8, wherein the step of linking the gold nanoparticles to the polyclonal antibody comprises adding polyethylene glycol to the gold nanoparticles prior to linking the gold nanoparticles to the polyclonal antibody.

10. 10. A method for detecting a target in a sample, comprising combining a composition according to any one of claims 1 to 5 or a liquid according to claim 6 or 7 with the sample.

11. The concentration of the target in the sample is 5×10 －１８ moles ~ 1 x 10 －９ The method of claim 10, wherein the molar ratio is between 1:1 and 1:

2.

12. 12. The method of claim 10 or 11, wherein the target is a biomarker, and when the composition of any one of claims 1 to 5 or the liquid of claim 6 or 7 is combined with the sample, gold nanoparticles are present, each linked to a probe that binds to the biomarker.

13. The method of claim 12 , wherein the target is an antibody that binds to the probe.

14. 14. The method of claim 12 or 13, wherein the probe is a protein, an enzyme, a hormone, or a peptide.

15. A method for producing a liquid composition comprising: a first composition according to any one of claims 1 to 5 or a first liquid according to claim 6 or 7; 10. A detection test comprising a second composition according to any one of claims 1 to 5 or a second liquid according to claim 6 or 7, A detection test wherein the first composition or first liquid and the second composition or second liquid comprise different polyclonal antibodies, and the first and second compositions or liquids are adapted to detect different targets.

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