Rapid detection of spermine for prostate cancer screening based on lateral flow immunoassay

Abstract

The present invention relates to a test device for detecting agmatine in a liquid sample and a method of use thereof, wherein the test device comprises in order: a sample pad, a conjugate pad, a membrane, and an absorbent pad, wherein: the conjugate pad comprises an agmatine detection antibody conjugate comprising gold nanoparticles coupled to one or more agmatine detection antibodies via one or more first linkers, wherein the one or more agmatine detection antibodies selectively bind to agmatine, and the agmatine detection antibody conjugate is mobile; the membrane comprises in order from the conjugate pad: a test zone and a control zone, wherein the test zone comprises an agmatine carrier protein conjugate comprising agmatine coupled to a carrier protein via a second linker, and the control zone comprises a secondary antibody, wherein the secondary antibody is immobilized on the membrane, and the secondary antibody binds to the agmatine detection antibody.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 453,491, filed March 21, 2023, which is incorporated herein by reference in its entirety. Background Technology

[0003] The increasing incidence of prostate cancer (PC) has attracted widespread attention, ranking as the second most common cancer in men. 1 This number is expected to continue to grow due to population aging and economic growth. 2 Although PC is a slowly spreading cancer, it does not show symptoms in its early stages. Currently, most diagnostic methods for PC are invasive. A digital rectal exam is a simple test that involves a doctor inserting a gloved finger to examine the patient's rectum by feeling for any unusual lumps or enlargements. 3,4 Biopsy is also considered a reliable method for diagnosing PC. 5,6 Doctors insert a needle probe into the prostate and collect tissue for microscopic examination. Alternatively, the prostate antigen (PSA) test has been developed as a less invasive diagnostic method for PC. The PSA level in the blood is used as a reference for assessing PC risk. Although these tests are widely used by doctors, they can cause patients potential wounds, bleeding, and pain.

[0004] Recent studies suggest using small molecules as biomarkers for cancer diagnosis. 8-13 Spm is an important biomarker in the biogenic amine family. It has also been shown to be useful for the detection of PC. A fluorescent sensor based on graphene quantum dots and organic dyes has been developed for the detection of Spm. 14 This sensor is capable of detecting submicromolar levels of Spm in human urine samples. In another report, the quenching of pepsin-functionalized gold nanoclusters was used as a reporting signal for Spm detection. 15 Besides optical sensing, Spm can also be detected by electrochemical methods. Recent work on impedance sensing of Spm using plastic electrodes demonstrates the feasibility of point-of-care (POC) sensing for PCs. 16 The selectivity of Spm is attributed to boric acid on the electrode. Despite efforts, the developed sensors either require advanced instrumentation for readings or electrical power. Therefore, developing low-cost, portable, and simple sensors for POC testing applications is crucial.

[0005] LFIA was used as a sandwich-type point-of-care device for rapid patient screening during the COVID-19 outbreak.17-19 The assay, using cast lines on a nitrocellulose membrane (NCM), provides simple readout results within tens of minutes. The selectivity of LFIA is attributed to the conjugation of species-specific antibodies to the nanoprobe. AuNPs were used for readout because they exhibit strong light absorption due to their plasmon resonance absorption properties, making them promising candidates for colorimetric detection. 20-22 .

[0006] Therefore, there is a need for testing devices for detecting spermine in liquid samples. Summary of the Invention

[0007] This document provides a testing device for detecting spermine in liquid samples, the testing device comprising, in sequence: a sample pad, a conjugate pad, a membrane, and an absorbent pad, wherein: the conjugate pad contains a spermine detection antibody conjugate, the spermine detection antibody conjugate containing gold nanoparticles coupled to one or more spermine detection antibodies via one or more first connectors, wherein the one or more spermine detection antibodies selectively bind to spermine, and the spermine detection antibody conjugate is mobile; the membrane, starting from the conjugate pad, comprises, in sequence: a test region and a control region, wherein the test region contains a spermine carrier protein conjugate, the spermine carrier protein conjugate containing spermine coupled to a carrier protein via a second connector, and the control region contains a secondary antibody, wherein the secondary antibody is immobilized on the membrane, and the secondary antibody binds to the spermine detection antibody.

[0008] In some embodiments, the gold nanoparticles are citrate-terminated gold nanoparticles.

[0009] In some embodiments, each of the one or more first connectors has the formula: *-S(CH2) m (C=O)NH**-, where m is 1-16, * represents the surface of the gold nanoparticles, and N** represents nitrogen present in the one or more spermine detection antibodies.

[0010] In some implementations, m is 6-12.

[0011] In some embodiments, the carrier protein includes bovine serum albumin, human serum albumin, keyhole hemocyanin, concholepas concholepas hemocyanin, or the Fc domain of an immunoglobulin.

[0012] In some implementations, the secondary antibody is an immunoglobulin G (IgG) antibody.

[0013] In some embodiments, the sample pad further comprises bovine serum albumin and a nonionic surfactant, and the binding pad further comprises a nonionic surfactant.

[0014] In some implementations, the second connector is Where n is an integer selected from 2 to 6; This indicates the nitrogen present in the spermine, and N # This indicates the nitrogen present in the carrier protein.

[0015] In some embodiments, the gold nanoparticles are citrate-terminated gold nanoparticles, the carrier protein is bovine serum albumin, and the second linker is... Where n is an integer selected from 2 to 5; This indicates the nitrogen present in the spermine, and N # The nitrogen present in the carrier protein is represented by the formula: *-S(CH2), each of the one or more first linkers having the formula: m (C=O)NH**-, where m is 8-12, * represents the surface of the gold nanoparticles, and N** represents the nitrogen present in each of the one or more spermine detection antibodies.

[0016] In some embodiments, the test region is prepared by depositing a solution containing the spermine-carrier protein at a concentration of 0.5-1.0 mg / mL.

[0017] In some embodiments, the control region is prepared by depositing a solution containing an immunoglobulin G (IgG) antibody at a concentration of at least 0.05 mg / mL.

[0018] In some embodiments, the test region is prepared by depositing a solution containing the spermine-carrier protein at a concentration of 0.5-1.0 mg / mL; and the control region is prepared by depositing a solution containing immunoglobulin G (IgG) antibody at a concentration of at least 0.05 mg / mL.

[0019] In some implementations, m is 10 and n is 3.

[0020] In a second aspect, this document provides a method for detecting spermine in a liquid sample, the method comprising: providing the testing apparatus described herein; applying the liquid sample onto the sample pad such that the liquid sample flows from the sample pad through the conjugation pad and the membrane to the absorbent pad; detecting the presence or absence of a visual signal at the test area; and optionally detecting the presence or absence of a visual signal at the control area, wherein the detection of the presence or absence of a visual signal at the test area indicates the presence of spermine in the liquid sample.

[0021] In some implementations, the detection of the presence or absence of a visual signal at the test area indicates a concentration of spermine in the liquid sample above a threshold.

[0022] In some implementations, the liquid sample comprises a urine sample obtained from the subject.

[0023] In some embodiments, the liquid sample also contains phosphate-buffered saline.

[0024] In some embodiments, the liquid sample also contains bovine serum albumin and nonionic surfactants.

[0025] In some embodiments, the sample pad further comprises bovine serum albumin and a nonionic surfactant, and the binding pad further comprises a nonionic surfactant.

[0026] In some embodiments, the liquid sample comprises a urine sample obtained from a human subject, and the method further includes determining whether the human subject has prostate cancer based on whether the sample contains spermine at a concentration above the threshold.

[0027] Brief description of the attached diagram

[0028] The foregoing aspects and many accompanying advantages of the invention will become more readily apparent and understood when taken in conjunction with the accompanying drawings and by referring to the following detailed description.

[0029] Figure 1 Schematic diagram of the LFIA device used for SpM detection

[0030] Figure 2 (a) TEM image of the original Cit-AuNP after synthesis (scale bar of inset is 10 nm), (b) UV-vis absorption of Cit-AuNP and MUA-AuNP, (c) DLS size of AuNP at each modification stage, and (d) gel electrophoresis of AuNP.

[0031] Figure 3 The concentrations of BSA-Spm were optimized using values ​​of 0.125, 0.25, 0.5, 0.75, and 1 mg / mL.

[0032] Figure 4 Detection of spiked Spm at 2, 1, 0.2, 0.1 and 0.02 ppm in run buffer (RB).

[0033] Figure 5 LFIA specificity was tested using PUT and 1,6-diaminohexane as interferants, with both targets at 0.2 ppm.

[0034] Figure 6The test results show the optimized concentrations of anti-rabbit IgG antibodies at 0.05, 0.125, 0.25, and 0.5 mg / mL in the control area.

[0035] Figure 7 This shows the LFIA results using 48 clinical urine samples. *Indicates PCa urine sample. Detailed Implementation

[0036] definition

[0037] Throughout this disclosure, unless the context otherwise requires, the word “comprising” or variations thereof (such as “including”) should be understood to imply inclusion of the stated integer or group of integers, but not to exclude any other integer or group of integers. It should also be noted that in this disclosure, particularly in the claims and / or paragraphs, terms such as “comprising” may have the meaning they are given under U.S. patent law; for example, they may mean “including”; and terms such as “consistently of” have the meaning they are given under U.S. patent law, for example, they allow for elements not explicitly stated, but exclude elements found in the prior art or affecting the essential or novel features of the invention.

[0038] Furthermore, throughout this disclosure and claims, unless the context otherwise requires, the word “comprising” or variations thereof (such as “including” or “containing”) should be understood to imply inclusion of the said integer or group of integers, but not to exclude any other integer or group of integers.

[0039] Unless otherwise expressly stated, the use of the singular in this document includes the plural, and vice versa. Furthermore, unless otherwise expressly stated, when the term “about” is used before a quantitative value, this teaching also includes the specific quantitative value itself. As used herein, unless otherwise specified or inferred, the term “about” means a variation of ±10%, ±7%, ±5%, ±3%, ±1%, or ±0% from the nominal value.

[0040] As used herein, the term "antibody" encompasses antibodies and antibody fragments thereof derived from any antibody-producing mammal (e.g., mice, rats, rabbits, and primates, including humans) that specifically bind to a target antigen. Exemplary antibodies include polyclonal antibodies, monoclonal antibodies, and recombinant antibodies; multispecific antibodies (e.g., bispecific antibodies); humanized antibodies; mouse antibodies; chimeric mouse-human, mouse-primate, and primate-human monoclonal antibodies; and anti-idiotype antibodies. Antigen-binding molecules can be any complete antibody molecule or fragment thereof (e.g., having a functional antigen-binding domain).

[0041] Antibody fragments are portions derived from or related to full-length antibodies, preferably including their complementarity-determining regions (CDRs), antigen-binding regions, or variable regions. Illustrative examples of antibody fragments that may be used in this disclosure include Fab, Fab′, F(ab)2, F(ab′)2, and Fv fragments, scFv fragments, diabody, linear antibody, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. A “single-chain Fv” or “scFv” antibody fragment contains the V of the antibody. H and V L Domains, wherein these domains are present within a single polypeptide chain. Fv polypeptides may also contain V... H and V L The polypeptide linkers between the domains allow scFv to form the desired structure for antigen binding. Antibody fragments can be recombined or produced via enzymatic digestion.

[0042] This document provides a testing device for detecting spermine in liquid samples, the testing device comprising, in sequence: a sample pad, a conjugate pad, a membrane, and an absorbent pad, wherein: the conjugate pad contains a spermine detection antibody conjugate comprising gold nanoparticles coupled to one or more spermine detection antibodies via one or more first connectors, wherein the one or more spermine detection antibodies selectively bind to spermine, and the spermine detection antibody conjugate is mobile; the membrane, starting from the conjugate pad, comprises, in sequence: a test region and an optional control region, wherein the test region contains a spermine carrier protein conjugate comprising spermine coupled to a carrier protein via a second connector, and the optional control region contains a secondary antibody, wherein the secondary antibody is immobilized on the membrane and the secondary antibody binds to the spermine detection antibody.

[0043] Figure 1 An exemplary testing apparatus is shown. The testing apparatus (100) has a sample pad (101), a binding pad (102), a membrane (103) comprising a test area (103A) and a control area (103B), and an absorbent pad (104) mounted on an optional backing card (105). All these components are continuous and / or overlapping (e.g., overlapping by 2 mm), allowing the liquid sample to move through the test strip via capillary action.

[0044] The sample pad comprises a material capable of receiving the liquid sample to be measured and allowing the liquid sample to migrate to the binding pad. The sample may comprise materials selected from: fibrous paper; microporous membranes composed of cellulose materials, cellulose, cellulose derivatives (such as cellulose acetate, cellulose nitrate), glass fibers, textiles (such as natural cotton and nylon), porous gels, and combinations thereof. In some embodiments, the sample pad also comprises additional reagents, such as proteins, nonionic surfactants, and buffer salts. In some embodiments, the sample pad also comprises one or more additional reagents selected from: bovine serum albumin, 20. Triton TM X-100, glycerin, and polyethylene glycol.

[0045] The conjugate pad contains a spermine detection antibody conjugate (typically in a dried and immobilized form). When the RB containing a liquid sample flows into the conjugate pad, the spermine detection antibody conjugate is moved, i.e., it rises from the conjugate pad material and migrates into the membrane along with the RB. During this process, if spermine is present in the liquid sample, a spermine-spermine detection antibody conjugate complex is formed.

[0046] The spermine detection antibody conjugate comprises gold nanoparticles coupled to one or more spermine detection antibodies via one or more first linkers.

[0047] One or more spermine detection antibodies may be the same or different. One or more spermine detection antibodies selectively bind to spermine. There are no particular limitations on the type of spermine detection antibody used in the testing apparatus and methods described herein, and this disclosure covers all types of spermine detection antibodies capable of selectively binding spermine. Spermine detection antibodies may be derived from any host species. In some embodiments, the host species is a rat, mouse, guinea pig, hamster, rabbit, goat, sheep, chicken, donkey, horse, cattle, dog, cat, pig, monkey, human, or any other species. In some embodiments, the host species is a rabbit.

[0048] In some embodiments, the gold nanoparticles are citrate-terminated gold nanoparticles coupled to one or more spermine detection antibodies via one or more first linkers, wherein each of the one or more first linkers has the formula: *-S(CH2). m (C=O)NH**-, where m is 1-16, * represents the surface of the gold nanoparticle, and N** represents the nitrogen present in one or more spermine detection antibodies.

[0049] In some embodiments, m is 1-16, 2-16, 3-16, 4-16, 5-16, 6-16, 7-16, 8-16, 8-14, 10-16, 10-15, 10-14, 10-13, 10-12, 6-14, 7-13, 8-12, or 9-11. In some embodiments, m is 10. Coupling to one or more formulas *-S(CH2) 10 CO2H, citrate-terminated gold nanoparticles that can be used to prepare the linkers of the spermine detection antibody conjugates described herein, the synthesis of which is described in U.S. Patent Application No. 15 / 929,495, which is incorporated herein by reference in its entirety.

[0050] The membrane may contain any material through which a liquid sample can diffuse via capillary action. For example, the membrane may contain materials selected from: naturally occurring materials, synthetic materials, or naturally occurring materials modified through synthesis, such as polysaccharides (e.g., cellulose materials, paper, cellulose derivatives, such as cellulose acetate and cellulose nitrate); polyethersulfone; polyethylene; nylon; polyvinylidene fluoride; polyester; polypropylene; silica; inorganic materials uniformly dispersed in a porous polymer matrix together with vinyl chloride, vinyl chloride-propylene copolymers, and vinyl chloride-vinyl acetate copolymers, such as inactivated alumina, diatomaceous earth, MgSO4, or other inorganic fine powder materials; naturally occurring textiles (e.g., cotton) and synthetic textiles (e.g., nylon or rayon); porous gels, such as silica gel, agarose, dextran, and gelatin; polymer membranes, such as polyacrylamide; etc. In some embodiments, the membrane comprises cellulose nitrate, polyethersulfone, polyethylene, nylon, polyvinylidene fluoride, polyester, polypropylene, or combinations thereof.

[0051] The membrane consists of test and control areas, starting from the conjugate pad.

[0052] The test area contains a spermine carrier protein conjugate comprising spermine coupled to a carrier protein via a second linker. The carrier protein facilitates the immobilization of spermine in the test area. This disclosure does not specifically limit the carrier protein. Therefore, this disclosure covers any carrier protein in the art for immobilizing analytes in an LFIA assay device. In some embodiments, the carrier protein is bovine serum albumin, KLH, thyroglobulin, borospirol hemocyanin (CCH), or ovalbumin. In some embodiments, the carrier protein is bovine serum albumin.

[0053] The second linker is not particularly limited and can be any bifunctional linker known in the art capable of covalently coupling spermine to a carrier protein. In some embodiments, the second linker is Where n is an integer selected from 2-5, 2-4, 2-3, or 3-4; This represents nitrogen present in spermine, and N# This represents nitrogen present in the carrier protein. In some implementations, n is 3.

[0054] like Figure 3 The dilution studies showed that no visual signal was observed when the test area was prepared by depositing a spermine carrier protein conjugate at a concentration below 0.5 mg / mL. Therefore, the test area can be prepared by depositing a solution of spermine carrier protein conjugate at a concentration of at least 0.5 mg / mL. In some embodiments, the test area can be prepared by depositing a solution of spermine carrier protein conjugate at a concentration of 0.5–1.0 mg / mL. In some embodiments, the test area can be prepared by depositing a solution of spermine carrier protein conjugate at a concentration of about 0.75 mg / mL.

[0055] The control area contains a secondary antibody immobilized on its surface. The secondary antibody may be a species-specific anti-immunoglobulin antibody specific to the spermine detection antibody. The secondary antibody may belong to any antibody class (e.g., IgG, IgA, IgD, IgE, and IgM) or isotype. In some embodiments, the secondary antibody is an IgG antibody, such as IgG1, IgG2, IgG3, or IgG4.

[0056] like Figure 6 The dilution studies showed that no visual signal was observed when the control area was prepared by depositing secondary antibody at a concentration below 0.05 mg / mL. The control area can be prepared by depositing solutions of secondary antibody at concentrations of at least 0.05 mg / mL, at least 0.125 mg / mL, at least 0.25 mg / mL, or at least 0.5 mg / mL. In some embodiments, the control is prepared by depositing a solution of secondary antibody at a concentration of 0.05 mg / mL to 0.5 mg / mL. In some embodiments, the control is prepared by depositing a solution of secondary antibody at a concentration of about 0.25 mg / mL.

[0057] Typically, the test area and control area can have any shape, including rectangular, non-rectangular, circular, crescent-shaped, elliptical, plus sign, minus sign, single line, multiple lines, symbols, geometric shapes, alphanumeric shapes, or any combination thereof. In some embodiments, the test area and control area may exist in the form of lines. Therefore, they may also be referred to herein as "test line" and "control line," respectively.

[0058] In some embodiments, the testing apparatus further includes a backing card, on which the sample pad, conjugate pad, membrane, and absorbent pad are disposed on the surface of the backing card. The backing card can contain any material, as long as it can support the sample pad, conjugate pad, membrane, and absorbent pad. Typically, the backing card is preferably liquid-impermeable, so that liquid sample fluid diffused through the membrane will not leak. Materials used for the backing card may include, but are not limited to, glass; polymeric materials, such as polystyrene, polypropylene, polyester, polybutadiene, polyvinyl chloride, polyamide, polycarbonate, epoxide, methacrylate, and melamine.

[0059] This disclosure also provides a method for detecting spermine in a liquid sample, the method comprising: providing the testing apparatus described herein; applying the liquid sample onto the sample pad such that the liquid sample flows from the sample pad through the conjugation pad and the membrane to the absorbent pad; detecting the presence or absence of a visual signal at the test area; and optionally detecting the presence or absence of a visual signal at the control area, wherein the detection of the presence or absence of the visual signal at the test area indicates the presence of spermine in the liquid sample.

[0060] The testing device operates in a competitive assay mode, which involves, if spermine is present, spermine binding to a spermine detection antibody-conjugate in the conjugate pad to form a spermine-spermine detection antibody-conjugate complex. This complex moves with the RB, i.e., migrates. Once the liquid sample in the RB contacts the test area, any free spermine detection antibody-conjugate in the liquid sample forms a spermine detection antibody-conjugate-spermine carrier protein conjugate complex, resulting in a visual signal at the test area. Once the liquid sample in the RB contacts the control area, secondary antibodies immobilized on the surface of the control area form complexes with free spermine detection antibody-conjugate and / or spermine-spermine detection antibody-conjugate complexes, resulting in a visual signal at the control area regardless of the presence of spermine in the liquid sample. Figure 1 As shown, when the visual signal is present in the control area but not in the test area, this indicates the presence of spermine (or a spermine concentration above the threshold concentration), and when the visual signal is present in both the control and test areas, this indicates the absence of spermine (or a spermine concentration below the threshold concentration).

[0061] In some embodiments, the liquid sample comprises a urine sample obtained from a human subject, and the method further includes determining whether the human subject has prostate cancer, an increased likelihood of having prostate cancer, and / or should undergo additional testing (e.g., digital rectal examination, prostate biopsy, etc.) based on whether the liquid sample contains spermine at a concentration above a threshold.

[0062] Liquid samples may include biological samples, environmental samples, food samples, etc. Exemplary biological samples include bodily fluids such as urine, whole blood, saliva, sweat, plasma, and serum. In some embodiments, the liquid sample includes urine.

[0063] Liquid samples may also contain phosphate-buffered saline, and optionally bovine serum albumin and nonionic surfactants, such as... 20. Triton TM X-100, etc.

[0064] Optionally, the liquid sample may be diluted before being added to the sample pad. In some embodiments, the liquid sample is prepared by diluting the sample with a dilution buffer at a volume ratio of 1:1-100 (sample:dilution buffer). An exemplary dilution buffer may include phosphate-buffered saline.

[0065] In the example below, BSA and 20. Pre-treat the sample pad and conjugation pad to ensure smooth flow and inhibit non-specific binding. The optimal BSA concentration is set at 1 mg / v%. Competitive detection of Spm in LFIA is facilitated by the AuNP-Spm-Ab at the conjugation pad and the BSA-Spm conjugate at the test line. The AuNP-Spm-Ab first binds to Spm in the RB, then the unreacted AuNP-Spm-Ab binds to the BSA-Spm conjugate at the test line. Finally, the fluid passes through the control line with IgG and is absorbed by the absorbent pad. A positive result corresponds to the appearance of both lines, indicating insufficient Spm concentration in the liquid sample and the risk of PC.

[0066] Synthesis, modification and coupling of gold nanoparticles

[0067] The prepared Cit-AuNPs exhibit a size of approximately 13 nm and a spherical morphology in TEM images. Figure 2 (a) and illustration). Ligand exchange from citrate to carboxylic acid was achieved via thiol-gold chemisorption through MUA. Figure 2 (b) Successful ligand exchange was indicated by monitoring the shift of the maximum uptake value. The lack of curve broadening indicates that MUA exchange did not induce aggregation to AuNP. Spm-Ab was coupled to the carboxylic acid group via one-pot EDC / NHS coupling in borate buffer, targeting the amine group on the molecule. Subsequently, the prepared AuNP-Spm-Ab was dispersed in a solution containing sucrose and... To prevent precipitation, add 20% PB. To gain a deeper understanding of the actual size of AuNP-Spm-Ab in the buffer, [further details are needed]. Figure 2 (c) shows the DLS size of AuNPs in each modification step. The increase in AuNP size is consistent with the coupling step, and the size distribution does not widen significantly. Furthermore, Figure 2The gel electrophoresis image in (d) provides further evidence of successful Spm-Ab coupling to AuNP. The first two lanes correspond to Cit-AuNP and MUA-AuNP, while the lane labeled AuNP-Anti consists of Spm-Ab. Clearly, the AuNP-Spm-Ab migration distance is shorter compared to Cit-AuNP and MUA-AuNP. This is attributed to the increased Spm-Ab loading.

[0068] Optimization of control and test areas

[0069] The concentrations of BSA-spm and IgG at the test and control lines are crucial for the visible signal. Insufficient concentrations result in a slow response to fixed receptor convection, thus producing no observable signal.

[0070] IgG did not produce a clear signal until the concentration was >0.05 mg / mL, and showed a strong interaction with AuNP-Spm-Ab at 0.25 mg / mL. Figure 6 Therefore, this concentration was maintained throughout the experiment.

[0071] On the other hand, a signal was not observed until the BSA-Spm concentration reached 0.5 mg / mL. A clear signal was provided at the test line at 1 mg / mL BSA-Spm, therefore this concentration was chosen for subsequent LFIA fabrication and testing.

[0072] Spermine detection using lateral flow immunoassay

[0073] Spermine was spiked into RB at different concentrations (2, 1, 0.2, 0.1, and 0.02 ppm) to test the cutoff concentration for screening normal and potential PC patients. The test was performed over 10 minutes. Figure 3 Images of the bands at each concentration are shown. At 1 and 2 ppm, a single red line appears at the control line for LFIA. This observation is attributed to the reaction of AuNP-Spm-ab in RB with the spiked Spm. As a result, no binding occurs at the test line. When the Spm concentration is reduced to 0.2 ppm, two lines are observed. The faint red line at the test line is due to the binding of unreacted AuNP-Spm-ab with BSA-Spm at the test line, which also indicates insufficient Spm in the liquid sample. The intensity of the test lines further increases at 0.1 and 0.02 ppm Spm due to the increased amount of unreacted AuNP-Spm-Ab.

[0074] Specificity of lateral flow immunoassay

[0075] Different amines were used to test LFIA to study its specificity. Figure 5Results of specificity tests using putrescine (PUT) and 1,6-diaminohexane are shown. PUT is a biogenic amine similar to Spm in the biogenic amine family. 25-27 1,6-Diaminohexane has a structure similar to Spm. The amine concentration was maintained at 0.2 ppm for comparison with Spm detection results. LFIA consistently indicated insufficient Spm levels in the liquid samples, demonstrating the good selectivity of LFIA.

[0076] in conclusion

[0077] A competitive LFIA assay for Spm was developed using AuNP-Spm-Ab as the biological probe and BSA-Spm as the carrier protein conjugate. The AuNP-Spm-Ab, control, and test lines were optimized to provide a cutoff concentration of 0.2 ppm for rapid screening of potential PC patients. Furthermore, the LFIA was tested with liquid samples spiked with different concentrations of Spm. A positive result indicates low Spm concentrations and suggests a risk of PC. The sensor is capable of determining the normal Spm concentration in liquid samples with high specificity within approximately 10 minutes. The developed LFIA for Spm holds promise for future clinical PC screening applications.

[0078] Example

[0079] Material

[0080] Gold(III) chloride trihydrate (HAuCl4), trisodium citrate (Na3-Cit), 11-mercaptoundecanoic acid (MUA), sucrose, 20 (polyoxyethylene (20) sorbitan monolaurate), borate buffer powder, and hydrochloric acid were purchased from Sigma-Aldrich. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl), sulfonyl-NHS (N-hydroxysulfosuccinimide) (S-NHS), BSA powder, and phosphate buffer (PB, pH 7.4) powder were obtained from Thermofisher. Anti-spermine rabbit polyclonal IgG (Spm-Ab) and goat anti-rabbit polyclonal IgG (HRP) were purchased from Abcam. BSA-Spm conjugate (spermine coupled to BSA via a glutaraldehyde linker) was purchased from Creative Diagnostics. All chemicals were of analytical grade purity and used without purification. All diluents and liquid samples were prepared using ultrapure water from a MilliQ water purification system. For LFIA device components, backing cards were purchased from Kenosha Tapes, while glass fiber liquid samples and conjugate pads were obtained from Millipore. The absorbent pads were purchased from Whatman, and the NCM, Hi-Flow Plus, and HF180 were purchased from Merck.

[0081] equipment

[0082] The morphology and size of gold nanoparticles were examined using a transmission electron microscope (JEM-2100F, Jeol, Japan). UV-vis absorption spectra were recorded using an Agilent 8453 diode array spectrophotometer (Agilent Technologies, USA), and the hydrodynamic size of AuNPs at each modification stage was monitored using a Zetasizer (Malvern, UK). Test and control lines were dispensed onto the NC-M using a side-flow printer (Agismart RP-2000, Rega Biotechnology Inc.). LFIA was cut using a side-flow strip cutter (LFST0007, Lateral Flow Strip Cutter, China).

[0083] Example 1 - Preparation of citrate-stabilized AuNP (Cit-AuNP)

[0084] Based on our previous work, we synthesized Cit-AuNP. 22,24 In short, under magnetic stirring, 1 mL of 2 wt% Na3-Cit solution was added to 25 mL of MilliQ in a 125 mL Erlenmeyer flask. Subsequently, under vigorous stirring, 1.25 mL of 10 mM HAuCl4 solution was rapidly transferred to a boiling citrate solution. The colorless mixture gradually turned black, purple, and red, which is attributed to the formation of AuNP. The citrate-AuNP colloidal solution was cooled to room temperature and stored at 4 °C for further modification.

[0085] Example 2 - Preparation of AuNP-Spm-Ab

[0086] 200 μL of 25 mM MUA solution was added to the Cit-AuNP colloid and kept for 24 h with stirring at room temperature. The MUA-AuNP colloid was maintained at 4 °C until further use. Spm-Ab was then coupled to MUA-AuNP via carbodiimide chemistry. Simply put, 2.5 μL of EDC·HCl (50 mM) and S-NHS (50 mM) were added to 200 μL of MUA-AuNP in 10 mM borate buffer (pH 8) with shaking. Then, 5 μL of 100-fold diluted Spm-Ab was added to the mixture and reacted for 2 h. The resulting Au-Spm-Ab was purified by centrifugation and dispersed in PB (5% sucrose and 0.5% sodium hydroxide). 20)

[0087] Example 3 - Preparation of LFIA

[0088] The liquid sample and binding pad were pretreated before assembly onto the backing card. The sample pad was treated with a mixture of 1% BSA and 0.5%... The 20-component PB buffer solution was soaked for 1 hour, while the conjugate pad was soaked in a solution containing 5% sucrose and 0.5% PB buffer. 20 μL of 10 mM PB was applied to the conjugation pad for 1 h. The pretreated pad was then dried at 37 °C for 4 h. Meanwhile, 1 nM AuNP-Spm-Ab was sprayed onto the conjugation pad and dried at 37 °C overnight. The dispensing rate was set to 0.100 μL / mm to fabricate the bands. BSA-Spm conjugate (1 mg / mL) and goat anti-rabbit IgG (0.25 mg / mL) were dispensed onto the nitrocellulose membrane as the test and control areas, respectively. The components were assembled onto a backing card with a 1 mm overlap and dried at 37 °C overnight. Subsequently, the LFIA was cut into 3 mm wide pieces and stored in a desiccant oven.

[0089] Example 4 - Sideflow determination using spiked Spm liquid samples

[0090] Spm solutions were added at concentrations of 2, 1, 0.5, 0.2, and 0.1 ppm to a concentration containing 10 mM PB, 1% BSA, and 0.5% PB. Add 20 μL of RB to the total volume of LFIA and perform the assay over 10 minutes.

[0091] Example 5 – Clinical Urine Sample Testing and Diagnosis of Prostate Cancer

[0092] A total of 48 clinical urine samples were obtained from male patients. Fifteen samples were diagnosed with prostate cancer (PCa), and the remaining 33 samples were assessed as having no signs of malignancy (NEM). Spm levels in these clinical samples were quantified by liquid chromatography-tandem mass spectrometry (LC-MS / MS). Spm levels from PCa ranged from 18.64–2287.08 ppb, with a median of 156.19 ppb, while Spm levels from NEM ranged from 54.20–2790.33 ppb, with a median of 366.11 ppb.

[0093] By mixing 25 μL of clinical urine sample with 25 μL of running buffer (10 mM phosphate buffer (PB), 1% BSA, 0.5%)... Mix thoroughly (pH 7.4, 20) to prepare the sample for LFIA. LFIA is used as a tool for screening prostate cancer by rapidly sensing Spm. Apply the sample to the sample pad and allow 15 minutes to ensure stable visualization of the control and / or test areas. Results are shown in... Figure 7 The kit demonstrated a sensitivity of 86.7% and a specificity of 36.3%.

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Claims

1. A testing apparatus for detecting spermine in a liquid sample, the testing apparatus comprising, in sequence: Sample pads, conjugate pads, membranes, and absorbent pads, among which: The binding pad comprises a spermine detection antibody conjugate, the spermine detection antibody conjugate comprising gold nanoparticles coupled to one or more spermine detection antibodies via one or more first connectors, wherein the one or more spermine detection antibodies selectively bind to spermine, and the spermine detection antibody conjugate is mobile. The membrane, starting from the conjugate pad, sequentially comprises a test region and a control region, wherein the test region contains a spermine carrier protein conjugate containing spermine coupled to a carrier protein via a second linker, and the control region contains a secondary antibody immobilized on the membrane and bound to the spermine detection antibody.

2. The testing apparatus according to claim 1, wherein the gold nanoparticles are citrate-terminated gold nanoparticles.

3. The testing apparatus according to claim 1, wherein each of the one or more first connectors has the formula: *-S(CH2) m (C=O)NH**-, where m is 1-16, * represents the surface of the gold nanoparticles, and N** represents nitrogen present in the one or more spermine detection antibodies.

4. The testing apparatus according to claim 3, wherein m is 6-12.

5. The testing apparatus according to claim 1, wherein the carrier protein comprises bovine serum albumin, human serum albumin, keyhole hemocyanin, concholepas concholepas hemocyanin, or an immunoglobulin Fc domain.

6. The testing apparatus according to claim 1, wherein the secondary antibody is an immunoglobulin G (IgG) antibody.

7. The testing apparatus according to claim 1, wherein the sample pad further comprises bovine serum albumin and a nonionic surfactant, and the conjugate pad further comprises a nonionic surfactant.

8. The testing apparatus according to claim 1, wherein the second connector is Where n is an integer selected from 2 to 6; This indicates the nitrogen present in the spermine, and N # This indicates the nitrogen present in the carrier protein.

9. The testing apparatus according to claim 1, wherein the gold nanoparticles are citrate-terminated gold nanoparticles, the carrier protein is bovine serum albumin, and the second linker is... Where n is an integer selected from 2 to 5; This indicates the nitrogen present in the spermine, and N # The nitrogen present in the carrier protein is represented by the formula: *-S(CH2), each of the one or more first linkers having the formula: m (C=O)NH**-, where m is 8-12, * represents the surface of the gold nanoparticles, and N** represents the nitrogen present in each of the one or more spermine detection antibodies.

10. The testing apparatus of claim 9, wherein the test region is prepared by depositing a solution containing the spermine-carrier protein at a concentration of 0.5-1.0 mg / mL.

11. The test apparatus of claim 9, wherein the control region is prepared by depositing a solution containing an immunoglobulin G (IgG) antibody at a concentration of at least 0.05 mg / mL.

12. The testing apparatus of claim 9, wherein the test region is prepared by depositing a solution containing the spermine-carrier protein at a concentration of 0.5-1.0 mg / mL; and the control region is prepared by depositing a solution containing an immunoglobulin G (IgG) antibody at a concentration of at least 0.05 mg / mL.

13. The testing apparatus according to claim 12, wherein m is 10 and n is 3.

14. A method for detecting spermine in a liquid sample, the method comprising: Provide the testing apparatus according to claim 1; The liquid sample is applied to the sample pad, such that the liquid sample flows from the sample pad through the binding pad and the membrane to the absorbent pad; Detect the presence or absence of visual signals in the test area; as well as Optionally, the presence or absence of visual signals at the control area can be detected. The detection of the presence or absence of the visual signal at the test area indicates the presence of spermine in the liquid sample.

15. The method of claim 14, wherein the detection of the presence or absence of the visual signal at the test area indicates a concentration of spermine above a threshold in the liquid sample.

16. The method of claim 14, wherein the liquid sample comprises a urine sample obtained from the subject.

17. The method of claim 14, wherein the liquid sample further comprises phosphate-buffered saline.

18. The method of claim 17, wherein the liquid sample further comprises bovine serum albumin and a nonionic surfactant.

19. The method of claim 17, wherein the sample pad further comprises bovine serum albumin and a nonionic surfactant, and the binding pad further comprises a nonionic surfactant.

20. The method of claim 15, wherein the liquid sample comprises a urine sample obtained from a human subject, and the method further comprises determining whether the human subject has prostate cancer based on whether the sample contains spermine at a concentration above the threshold concentration.

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