CN120936877A - Rapid Spermine Detection Based on Lateral Immunoassay for Prostate Cancer Screening - Google Patents

Abstract

This invention relates to a testing device for detecting spermine in a liquid sample and a method of using the same, wherein the testing device comprises, 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.

Description

Spermine rapid detection based on lateral flow immunoassay for prostate cancer screening

Cross Reference to Related Applications

The present application claims priority from U.S. provisional patent application No. 63/453,491, filed on 3/21, 2023, which is hereby incorporated by reference in its entirety.

Background

The increasing incidence of Prostate Cancer (PC) has attracted considerable attention, which ranks men as the second most common cancer ¹. This number is expected to continue to grow ² due to aging and economic growth of the population. Although PC is a slowly spreading cancer, it shows no symptoms at an early stage. Current diagnostic methods for PC are mostly invasive. Rectal fingering is a simple test that involves the insertion of a gloved finger by a physician. The patient's rectum ^3,4 is examined by a paresthesia bump or enlargement. Biopsies are also considered a reliable method ^5,6 for PC diagnosis. The physician inserts a needle-like probe into the prostate and collects tissue for microscopic examination. Alternatively, prostate antigen (PSA) test 7 was developed as a less invasive method of PC diagnosis. PSA levels in blood were used as a reference for assessing PC risk. Although the above-described test methods are widely used by doctors, they can cause possible wounds, bleeding and pain to the patient.

Recent studies suggest the use of small molecules as biomarkers ^8-13 for cancer diagnosis. Among them, spm is one of the important biomarkers in the biogenic amine family. It has also proven useful for detection of PCs. A fluorescence sensor based on graphene quantum dots and organic dyes has been developed for Spm detection ¹⁴. The sensor is capable of detecting sub-micromolar levels of Spm in a human urine sample. In another report, the quenching of pepsin functionalized gold nanoclusters was used as a report signal ¹⁵ for Spm detection. In addition to optical sensing, spm can also be detected electrochemically. Recent work on impedance sensing of Spm using plastic electrodes has presented feasibility ¹⁶ for point-of-care (POC) sensing of PCs. The selectivity of Spm is due to boric acid on the electrode. Despite efforts, sensors have been developed that either require advanced instrumentation for reading readings or require electrical power. Therefore, it is critical to develop a low cost, portable and simple sensor for POC testing applications.

LFIA was used as POC device ^17-19 in sandwich format for rapid screening of patients in COVID-19 outbreaks. The test provides a simple readout in tens of minutes using cast lines on nitrocellulose membrane (NCM). The selectivity of LFIA is due to the coupling of species-specific antibodies to the nanoprobes. AuNP are used for readout results because they exhibit strong light absorption due to plasmonic absorption properties, which enables them to be promising candidates ^20-22 for colorimetric detection.

Thus, there is a need for a test device for detecting spermine in a liquid sample.

Disclosure of Invention

Provided herein is a test device for detecting spermine in a liquid sample comprising, in order, a sample pad, a binding pad, a membrane and an absorbent pad, wherein the binding pad comprises a spermine detection antibody conjugate comprising gold nanoparticles coupled to one or more spermine detection antibodies via one or more first linkers, wherein the one or more spermine detection antibodies selectively bind to spermine and the spermine detection antibody conjugate is mobile, the membrane comprising, in order from the binding pad, a test zone and a control zone, wherein the test zone comprises a spermine carrier protein conjugate comprising spermine 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 spermine detection antibody.

In certain embodiments, the gold nanoparticle is a citrate-capped gold nanoparticle.

In certain embodiments, each of the one or more first linkers has the formula-S (CH ₂)_m (c=o) NH-, where m is 1-16, represents the surface of the gold nanoparticle, and N represents the nitrogen present in the one or more spermine detection antibodies.

In certain embodiments, m is 6 to 12.

In certain embodiments, the carrier protein comprises bovine serum albumin, human serum albumin, keyhole limpet hemocyanin, bao Luoluo (Concholepas concholepas) hemocyanin, or an immunoglobulin Fc domain.

In certain embodiments, the secondary antibody is an immunoglobulin G (IgG) antibody.

In certain embodiments, the sample pad further comprises bovine serum albumin and a nonionic surfactant, and the conjugate pad further comprises a nonionic surfactant.

In certain embodiments, the second linker isWherein n is an integer selected from 2-6; represents nitrogen present in the spermine and N ^# represents nitrogen present in the carrier protein.

In certain embodiments, the gold nanoparticle is a citrate-capped gold nanoparticle, the carrier protein is bovine serum albumin, and the second linker isWherein n is an integer selected from 2-5; Represents nitrogen present in the spermine and N ^# represents nitrogen present in the carrier protein, each of the one or more first linkers has the formula-S (CH ₂)_m (c=o) NH-, where m is 8-12, represents the surface of the gold nanoparticle, and N represents nitrogen present in each of the one or more spermine detection antibodies.

In certain embodiments, the test zone is prepared by depositing a solution comprising the spermine-carrier protein at a concentration of 0.5-1.0 mg/mL.

In certain embodiments, the control zone is prepared by depositing a solution comprising immunoglobulin G (IgG) antibodies at a concentration of at least 0.05 mg/mL.

In certain embodiments, the test zone is prepared by depositing a solution comprising the spermine-carrier protein at a concentration of 0.5-1.0mg/mL, and the control zone is prepared by depositing a solution comprising immunoglobulin G (IgG) antibodies at a concentration of at least 0.05 mg/mL.

In certain embodiments, m is 10 and n is 3.

In a second aspect, provided herein is a method of detecting spermine in a liquid sample comprising providing a test device as described herein, applying the liquid sample on the sample pad such that the liquid sample flows from the sample pad through the conjugate pad and the membrane to the absorbent pad, detecting the presence or absence of a visual signal at the test zone, and optionally detecting the presence or absence of a visual signal at the control zone, wherein the detection of the presence or absence of a visual signal at the test zone is indicative of spermine in the liquid sample.

In certain embodiments, the detection of the presence or absence of a visual signal at the test zone is indicative of a concentration of spermine in the liquid sample above a threshold concentration.

In certain embodiments, the liquid sample comprises a urine sample obtained from a subject.

In certain embodiments, the liquid sample further comprises phosphate buffered saline.

In certain embodiments, the liquid sample further comprises bovine serum albumin and a nonionic surfactant.

In certain embodiments, the sample pad further comprises bovine serum albumin and a nonionic surfactant, and the conjugate pad further comprises a nonionic surfactant.

In certain embodiments, the liquid sample comprises a urine sample obtained from a human subject, and the method further comprises determining that the human subject is suffering from prostate cancer based on whether the sample contains spermine above the threshold concentration.

Brief description of the drawings

The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings.

FIG. 1 schematic diagram of an LFIA device for Spm detection

FIG. 2 (a) TEM image of as-synthesized Cit-AuNP (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.

FIG. 3 BSA-Spm concentration was optimized using 0.125, 0.25, 0.5, 0.75 and 1 mg/mL.

FIG. 4 detection of 2, 1, 0.2, 0.1 and 0.02ppm of labelled Spm in Running Buffer (RB).

FIG. 5 is a test for LFIA specificity using PUT and 1, 6-diaminohexane as interferents, both targets being 0.2ppm.

FIG. 6 shows the results of the test optimized for anti-rabbit IgG antibody concentrations of 0.05, 0.125, 0.25 and 0.5mg/mL in the control zone.

Fig. 7 shows LFIA results using 48 clinical urine samples. * PCa urine samples are indicated.

Detailed Description

Definition of the definition

Throughout this disclosure, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It is also noted that in this disclosure, particularly in the claims and/or paragraphs, terms such as "comprising" and the like may have the meanings given in U.S. patent laws, e.g., they may mean "including" and terms such as "consisting essentially of" have the meanings given in U.S. patent laws, e.g., they allow elements not explicitly recited, but exclude elements found in the prior art or that affect essential or novel features of the invention.

Furthermore, throughout the present disclosure and claims, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

The use of the singular herein includes the plural and vice versa unless explicitly stated otherwise. Furthermore, unless explicitly stated otherwise, when the term "about" is used prior to a quantitative value, the present teachings also include the specific quantitative value itself. As used herein, unless otherwise indicated or inferred, the term "about" refers to a change from a nominal value of ±10%, ±7%, ±5%, ±3%, ±1% or ±0%.

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

An antibody fragment is derived from or related to a full length antibody, preferably including its Complementarity Determining Regions (CDRs), antigen binding regions, or variable regions. Illustrative examples of antibody fragments that can be used in the present disclosure include Fab, fab', F (ab) ₂、F(ab′)₂, and Fv fragments, scFv fragments, diabodies (diabodies), linear antibodies, single chain antibody molecules, multispecific antibodies formed from antibody fragments, and the like. A "single chain Fv" or "scFv" antibody fragment comprises the V _H and V _L domains of an antibody, wherein these domains are present in a single polypeptide chain. Fv polypeptides may also comprise a polypeptide linker between the V _H and V _L domains, which enables the scFv to form the desired structure for antigen binding. Antibody fragments may be produced recombinantly or by enzymatic digestion.

Provided herein is a test device for detecting spermine in a liquid sample comprising, in order, a sample pad, a binding pad, a membrane, and an absorbent pad, wherein the binding pad comprises a spermine detection antibody conjugate comprising gold nanoparticles coupled to one or more spermine detection antibodies via one or more first linkers, wherein the one or more spermine detection antibodies selectively bind to spermine and the spermine detection antibody conjugate is mobile, the membrane comprising, in order from the binding pad, a test zone and an optional control zone, wherein the test zone comprises a spermine carrier protein conjugate comprising spermine coupled to a carrier protein via a second linker, and the optional control zone comprises a secondary antibody, wherein the secondary antibody is immobilized on the membrane and the secondary antibody binds to the spermine detection antibody.

An exemplary testing apparatus is shown in fig. 1. The test device (100) has a sample pad (101), a conjugate pad (102), a membrane (103) comprising a test zone (103A) and a control zone (103B), and an absorbent pad (104) mounted on an optional backing card (105). All of 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.

The sample pad comprises a material capable of receiving a liquid sample to be assayed and allowing the liquid sample to migrate to the conjugate pad. The sample may comprise a material selected from the group consisting of fibrous paper, microporous membranes composed of cellulosic materials, cellulose derivatives (such as cellulose acetate, cellulose nitrate), glass fibers, textiles (such as natural cotton and nylon), porous gels, and combinations thereof. In certain embodiments, the sample pad further comprises additional reagents, such as proteins, nonionic surfactants, and buffer salts. In certain embodiments, the sample pad further comprises one or more additional reagents selected from the group consisting of bovine serum albumin,20. Triton ^TM X-100, glycerol and polyethylene glycol.

The conjugate pad comprises a spermine detection antibody conjugate (typically in dried and immobilized form). As the RB comprising the 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 with the RB into the membrane, during which time a spermine-spermine detection antibody conjugate complex is formed if spermine is present in the liquid sample.

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

The one or more spermine detection antibodies may be the same or different. One or more spermine detection antibodies selectively bind to spermine. The type of spermine detection antibody used in the test devices and methods described herein is not particularly limited, and the present disclosure encompasses all types of spermine detection antibodies that are capable of selectively binding spermine. The spermine detection antibody may be derived from any host species. In certain embodiments, the host species is a rat species, a mouse species, a guinea pig species, a hamster species, a rabbit species, a goat species, a sheep species, a chicken species, a donkey species, a horse species, a cow species, a canine species, a cat species, a pig species, a monkey species, a human species, or any other species. In certain embodiments, the host species is a rabbit species.

In certain embodiments, the gold nanoparticle is a citrate-capped gold nanoparticle 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 (CH ₂)_m (c=o) NH-, where m is 1-16, represents the surface of the gold nanoparticle, and N represents the nitrogen present in each of the one or more spermine detection antibodies.

In certain embodiments, m is 1-16, 2-16, 3-16, 4-16, 5-16, 6-16, 7-16, 8-14, 10-16, 10-15, 10-14, 10-13, 10-12, 6-14, 7-13, 8-12, or 9-11. In certain embodiments, m is 10. Citrate-capped gold nanoparticles coupled to one or more linkers having the formula x-S (CH ₂)₁₀CO₂ H, useful in preparing 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).

The membrane may comprise any material through which the liquid sample may diffuse by capillary action. For example, the membrane may comprise a material selected from naturally occurring materials, synthetic materials, or naturally occurring materials deformed by synthesis, such as polysaccharides (e.g., cellulosic materials, paper, cellulose derivatives such as cellulose acetate and nitrocellulose), polyethersulfones, polyethylene, nylon, polyvinylidene fluoride, polyesters, polypropylene, silica, inorganic materials such as deactivated alumina, diatomaceous earth, mgSO ₄, or other inorganic fine powder materials uniformly dispersed in a porous polymer matrix along with vinyl chloride, vinyl chloride-propylene copolymers, and vinyl chloride-vinyl acetate copolymers, 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 films such as polyacrylamide, and the like. In certain embodiments, the film comprises nitrocellulose, polyethersulfone, polyethylene, nylon, polyvinylidene fluoride, polyester, polypropylene, or combinations thereof.

The membrane includes a test zone and a control zone in that order from the conjugate pad.

The test zone comprises a spermine carrier protein conjugate comprising spermine coupled to a carrier protein via a second linker. The carrier protein helps to immobilize the spermine in the test zone. The present disclosure is not particularly limited to carrier proteins. Thus, the present disclosure encompasses any carrier protein used in the art to immobilize an analyte in an LFIA test device. In certain embodiments, the carrier protein is bovine serum albumin, KLH, thyroglobulin, bao Luoluo-like hemocyanin (CCH) or ovalbumin. In certain embodiments, the carrier protein is bovine serum albumin.

The second linker is not particularly limited and may be any bifunctional linker known in the art capable of covalently coupling spermine to a carrier protein. In certain embodiments, the second linker isWherein n is an integer selected from 2-5, 2-4, 2-3 or 3-4; represents nitrogen present in spermine and N ^# represents nitrogen present in carrier protein. In certain embodiments, n is 3.

As shown by the dilution study results in fig. 3, no visual signal was observed when the test zone was prepared by depositing spermine carrier protein conjugate at a concentration below 0.5 mg/mL. Thus, the test zone may be prepared by depositing a solution of the spermine carrier protein conjugate at a concentration of at least 0.5 mg/mL. In certain embodiments, the test zone may be prepared by depositing a solution of the spermine carrier protein conjugate at a concentration of 0.5-1.0 mg/mL. In certain embodiments, the test zone may be prepared by depositing a solution of the spermine carrier protein conjugate at a concentration of about 0.75 mg/mL.

The control zone comprises a secondary antibody immobilized on the surface of the control zone. The secondary antibody may be a species-specific anti-immunoglobulin antibody specific for the spermine detection antibody. Secondary antibodies may belong to any antibody class (e.g., igG, igA, igD, igE and IgM) or isotype. In certain embodiments, the secondary antibody is an IgG antibody, such as IgG1, igG2, igG3, or IgG4.

As shown in the dilution study results in fig. 6, no visual signal was observed when the control zone was prepared by depositing secondary antibodies at a concentration below 0.05 mg/mL. The control zone may be prepared by depositing a solution of the secondary antibody at a concentration of at least 0.05mg/mL, at least 0.125mg/mL, at least 0.25mg/mL, or at least 0.5 mg/mL. In certain embodiments, the control is prepared by depositing a solution of the secondary antibody at a concentration of 0.05mg/mL to 0.5 mg/mL. In certain embodiments, the control is prepared by depositing a solution of the secondary antibody at a concentration of about 0.25 mg/mL.

In general, the test and control zones can have any shape, including rectangular, non-rectangular, circular, half-moon, oval, plus, minus, one line, multiple lines, symbols, geometric, alphanumeric, or any combination thereof. In certain embodiments, the test zone and the control zone may be present in the form of lines. Thus, they may also be referred to herein as "test lines" and "control lines," respectively.

In certain embodiments, the test device further comprises a backing card, wherein the sample pad, the conjugate pad, the membrane, and the absorbent pad are disposed on a surface of the backing card. The backing card may comprise any material so long as the backing card is capable of supporting the sample pad, conjugate pad, membrane, and absorbent pad. In general, it is preferred that the backer card be liquid impermeable so that liquid sample fluid that diffuses through the membrane does not leak. Materials for the backer card may include, but are not limited to, glass, polymeric materials such as polystyrene, polypropylene, polyester, polybutadiene, polyvinylchloride, polyamide, polycarbonate, epoxy, methacrylate, and polymelamine.

The present disclosure also provides a method of detecting spermine in a liquid sample comprising providing a test device as described herein, applying the liquid sample on the sample pad such that the liquid sample flows from the sample pad through the conjugate pad and the membrane to the absorbent pad, detecting the presence or absence of a visual signal at the test zone, and optionally detecting the presence or absence of a visual signal at the control zone, wherein detection of the presence or absence of the visual signal at the test zone is indicative of spermine in the liquid sample.

The test device operates in a competitive test assay mode which involves binding of spermine to the spermine detection antibody conjugate in the binding pad, if present, thereby forming a spermine-spermine detection antibody conjugate complex that moves, i.e., migrates, with RB. Once the liquid sample in RB contacts the test zone, any free spermine detection antibody conjugate in the liquid sample will form a spermine detection antibody conjugate-spermine carrier protein conjugate complex, which results in a visual signal at the test zone. Once the liquid sample in RB contacts the control zone, the secondary antibody immobilized on the surface of the control zone forms a complex with the free spermine detection antibody conjugate and/or spermine-spermine detection antibody conjugate complex, resulting in a visual signal at the control zone whether or not spermine is present in the liquid sample. As shown in fig. 1, when a visual signal is present in the control zone but not in the test zone, this indicates the presence of spermine (or a concentration of spermine above the threshold concentration), and when a visual signal is present in both the control zone and the test zone, this indicates the absence of spermine (or a concentration of spermine below the threshold concentration).

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

The liquid sample may include biological samples, environmental samples, food samples, and the like. Exemplary biological samples include body fluids such as urine, whole blood, saliva, sweat, plasma, and serum. In certain embodiments, the liquid sample comprises urine.

The liquid sample may further comprise phosphate buffered saline, and optionally bovine serum albumin and a non-ionic surfactant, such as20. Triton ^TM X-100, etc.

Alternatively, the liquid sample may be diluted prior to addition to the sample pad. In certain 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). Exemplary dilution buffers may include phosphate buffered saline.

In the following examples, BSA andSample pads and conjugate pads are pre-treated 20 to ensure smooth flow and inhibit non-specific binding. The optimum BSA content was set at 1m/v%. Competitive detection of Spm in LFIA is facilitated by AuNP-Spm-Ab at the conjugate pad and BSA-Spm conjugate at the test line. AuNP-Spm-Ab first binds to Spm in RB, then unreacted AuNP-Spm-Ab binds to 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 two lines, indicating insufficient Spm concentration and risk of PC in the liquid sample.

Synthesis, modification and coupling of gold nanoparticles

The Cit-AuNP produced exhibited a size of about 13nm and a spherical morphology in TEM images (FIG. 2 (a) and inset). Ligand exchange from citrate to carboxylic acid is achieved by thiol-gold chemisorption of MUA. Fig. 2 (b) indicates successful ligand exchange by monitoring the displacement of the absorbance maximum. The curve did not broaden, indicating that MUA exchange did not cause aggregation to AuNP. The amine groups on the molecule were targeted by one pot EDC/NHS coupling in borate buffer, spm-Ab coupling to carboxylic acid groups. Thereafter, the prepared AuNP-Spm-Ab was dispersed in a solution containing sucrose and20 To prevent precipitation. To gain a deeper understanding of the actual size of the AuNP-Spm-Ab in the buffer, the DLS size of the AuNP at each modification step is shown in fig. 2 (c). The increase in AuNP size was consistent with the coupling step, nor was the size distribution significantly broadened. Furthermore, the gel electrophoresis image in FIG. 2 (d) provides more evidence of successful coupling of Spm-Ab to AuNP. The first two lanes correspond to Cit-AuNP and MUA-AuNP, while the lane labeled with AuNP-Anti consisted of Spm-Ab. It is apparent that AuNP-Spm-Ab migrates a shorter distance than Cit-AuNP and MUA-AuNP. This is due to the increased load of the Spm-Ab.

Optimization of control and test zones

The concentration of BSA-spm and IgG at the test and control lines is critical for visible signal. Insufficient concentration results in a slow response of the immobilized receptor to the flow and therefore no observable signal.

IgG did not produce a clear signal until concentrations >0.05mg/mL and showed strong interaction with AuNP-Spm-Ab at 0.25mg/mL (FIG. 6). Thus, this concentration was maintained throughout the experiment.

On the other hand, no signal was observed until the concentration of BSA-Spm reached 0.5 mg/mL. 1mg/mL BSA-Spm provided a clear signal at the test line, and thus this concentration was chosen for subsequent LFIA manufacture and testing.

Detection of spermine using lateral flow immunoassay

Spermine was spiked into RB at different concentrations (2, 1, 0.2, 0.1 and 0.02 ppm) to test the cutoff concentrations for screening normal and potential PC patients. The test was run for 10 minutes and figure 3 shows an image of each concentration band. At 1 and 2ppm, a single red line is shown at the control line of LFIA. This observation is attributed to the AuNP-Spm-ab reacted in RB and the spiked Spm. As a result, there is no bond at the test line. When the Spm concentration was reduced to 0.2ppm, two lines were observed. The faint red line at the test line was due to the binding of unreacted AuNP-Spm-ab to BSA-Spm at the test line, which also indicates an insufficient amount of Spm in the liquid sample. Since the amount of unreacted AuNP-Spm-Ab was increased, the strength of the test line was further enhanced at 0.1 and 0.02ppm Spm.

Specificity of lateral flow immunoassay

LFIA was tested with different amines to investigate its specificity. FIG. 5 shows the results of the specificity test with Putrescine (PUT) and 1, 6-diaminohexane. PUT is biogenic amine ^25-27 in the biogenic amine family similar to Spm, whereas 1, 6-diaminohexane has a structure similar to Spm. The amine concentration was maintained at 0.2ppm for comparison with the Spm test results. LFIA indicates an insufficient amount of Spm in the liquid sample, which indicates good selectivity of LFIA.

Conclusion(s)

Competitive LFIA assays for Spm were developed using AuNP-Spm-Ab as a biological probe and BSA-Spm as a carrier protein-conjugate. The AuNP-Spm-Ab, control and test lines were optimized to provide a cut-off concentration of 0.2ppm for rapid screening of potential PC patients. In addition, LFIA was tested with different concentrations of spiked Spm liquid samples. Positive results indicate low Spm concentration and suggest risk of PC. The sensor is capable of determining the normal Spm concentration of a liquid sample with high specificity in about 10 minutes. The LFIA developed for Spm is expected to be used for future clinical PC screening applications.

Examples

Material

Gold (III) chloride trihydrate (HAuCl ₄), trisodium citrate (Na ₃ -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), sulfo-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 glutaraldehyde linker) was purchased from Creative Diagnostics. The purity of the chemicals was analytical grade and used without purification. All dilutions and liquid samples were prepared using ultrapure water from the MilliQ water purification system. For LFIA device assemblies, the backing card was purchased from Kenosha Tapes, while the fiberglass liquid sample and conjugate pad were obtained from Millipore. Absorbent pads were purchased from Whatman, and NCM, hi-Flow Plus, HF180 were purchased from Merck.

Apparatus and method for controlling the operation of a device

The morphology and size of the gold nanoparticles were examined using a transmission electron microscope (JEM-210F, jeol, japan). UV-vis absorption spectra were recorded by using an Agilent 8453 diode array spectrophotometer (Agilent Technologies, USA) and the hydrodynamic size of AuNP at each modification stage was monitored by a Zetasizer (Malvern, UK). Test and control lines were distributed over NC-M using a side stream printer (AGISMART RP-2000,Rega Biotechnology Inc). LFIA was cut using a lateral flow tape cutter (LFST 0007, lateral Flow Strip Cutter, china).

EXAMPLE 1 preparation of citrate-stabilized AuNP (Cit-AuNP)

Cit-AuNP ^22,24 was synthesized according to our previous work. Briefly, 1mL of a 2wt% Na ₃ -Cit solution was added to 25mL MilliQ in a 125mL Erlenmeyer flask with magnetic stirring. Thereafter, the 1.25mL 10mM HAuCl ₄ solution was quickly pipetted into the boiling citrate solution with vigorous stirring. The colorless mixture gradually turned black, violet and red due to the formation of AuNP. The citrate-AuNP colloidal solution was cooled to room temperature and stored at 4 ℃ for further modification.

Example 2 preparation of AuNP-Spm-Ab

200. Mu.L of 25mM MUA solution was added to the Cit-AuNP colloid with stirring at room temperature for 24h. The MUA-AuNP gel was kept at 4℃until further use. Then, spm-Ab was coupled to MUA-AuNP via carbodiimide chemistry. Briefly, 2.5. Mu.L EDC. HCl (50 mM) and S-NHS (50 mM) were added to 200uL MUA-AuNP in 10mM borate buffer (pH 8) with shaking. Then, 5. Mu.L of 100-fold diluted Spm-Ab was added to the mixture and reacted for 2 hours. The Au-Spm-Ab obtained was purified by centrifugation and dispersed in PB (5% sucrose and 0.5%)20 A) is provided.

EXAMPLE 3 preparation of LFIA

The liquid sample and conjugate pad are pre-treated prior to assembly onto the backer card. The sample pad was incubated with 1% BSA and 0.5%20 In 10mM PB buffer for 1h, while the conjugate pad was incubated with 5% sucrose and 0.5%20 In 10mM PB for 1h. The pretreated pad was dried at 37 ℃ for 4h. On the other hand, 1nM AuNP-Spm-Ab was sprayed onto the conjugate pad and dried overnight at 37 ℃. The dispensing rate was set to 0.100. Mu.L/mm to produce a tape. BSA-Spm conjugate (1 mg/mL) and goat anti-rabbit IgG (0.25 mg/mL) were dispensed onto nitrocellulose membrane as test and control zones, respectively. The assemblies were assembled onto a backing card with a 1mm overlap and dried overnight at 37 ℃. Thereafter, LFIA was cut to 3mm width and stored in a dry box.

Example 4-lateral flow assay with a spiked Spm liquid sample

Spm solutions were added at 2, 1, 0.5, 0.2 and 0.1ppm to 10mM PB, 1% BSA and 0.5%20 RB. The total volume added to LFIA was 60 μl and the assay was completed for 10 minutes.

Example 5 clinical urine sample test and diagnosis of prostate cancer

A total of 48 clinical urine samples were obtained from male patients, 15 were diagnosed with prostate cancer (PCa), and the remaining 33 samples were evaluated as having no signs of malignancy (NEM). The Spm levels in these clinical samples were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The Spm level from PCa ranges from 18.64 to 2287.08ppb, the median 156.19ppb, while the Spm level from NEM ranges from 54.20 to 2790.33ppb, the median 366.11ppb.

By mixing 25. Mu.L of clinical urine sample with 25. Mu.L of running buffer (10 mM Phosphate Buffer (PB), 1% BSA,0.5%20, Ph 7.4) was thoroughly mixed to prepare a sample for LFIA. LFIA is used as a tool to screen for prostate cancer by rapidly sensing Spm. The sample was applied to the sample pad and passed for 15 minutes to ensure stable visualization of the control zone and/or the test zone. The results are shown in fig. 7, and the kit has a sensitivity of 86.7% and a specificity of 36.3%.

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Claims (20)

1. A testing device for detecting spermine in a liquid sample, the testing device comprising, in sequence: a sample pad, a conjugate pad, a membrane, and an absorbent pad, wherein: 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; The nitrogen present in the spermine is indicated by N, and N ^# 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; The nitrogen present in the spermine is represented by N, and N ^# represents the nitrogen present in the carrier protein. Each of the one or more first linkers has the formula: *-S( _CH2 ) _m (C=O)NH**-, where m is 8-12, * represents the surface of the gold nanoparticle, 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; Detecting the presence or absence of visual signals in the test area; and 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.