阅读说明：本技术 使用纳米金颗粒直接比色检测精胺 (Direct colorimetric detection of spermine using gold nanoparticles ) 是由 黄嘉咏 蔡迪雄 简振威 于 2020-05-06 设计创作，主要内容包括：本文提供了用于检测样品中的精胺的基于纳米金颗粒的比色法。这些方法基于从受试者获得的尿液样品中检测到的精胺的量,可用于诊断该受试者的前列腺癌。(Provided herein are nanogold particle-based colorimetric methods for detecting spermine in a sample. These methods are useful for diagnosing prostate cancer in a subject based on the amount of spermine detected in a urine sample obtained from the subject.)

1. A method for detecting spermine (Spm) in a sample, the method comprising:

providing a sample;

contacting the sample with a plurality of gold nanoparticles (MUA-aunps) comprising citrate and 11-mercaptoundecanoic acid (MUA), thereby forming a test sample having a test color; and

Detecting the presence of Spm in the sample based on the test color.

2. The method of claim 1, wherein the sample comprises urine obtained from a subject.

3. The method of claim 1, wherein the step of detecting based on the test color comprises a visual inspection of the test color or determining absorbance of the test sample using a spectrometer.

4. The method of claim 3, further comprising the steps of: comparing the test color to a color chart or color wheel prepared by using a correlation between colors of known concentrations of Spm in a standard sample comprising MUA-AuNP; or comparing the absorbance of the test sample obtained using a spectrometer with one or more calibration curves prepared using a correlation between the absorbance of Spm at known concentrations in a standard sample comprising MUA-AuNP; and determining the concentration of Spm in the sample.

5. The method of claim 2, wherein the method further comprises the steps of: comparing the test color to a color chart or color wheel prepared by using a correlation between known concentrations of Spm in a standard sample comprising MUA-AuNP; or comparing the absorbance of the test sample obtained using a spectrometer with one or more calibration curves prepared by using the correlation between known concentrations of Spm in a standard sample comprising MUA-AuNP; determining the concentration of Spm in the sample; and determining whether the subject has prostate cancer based on the concentration of Spm in the sample.

6. The method of claim 2, further comprising the steps of:

providing a urine sample obtained from the subject; and

diluting the urine sample 50-1000 fold using an aqueous solvent, thereby forming the sample.

7. The method of claim 1, wherein the aqueous solvent comprises Tris (hydroxymethyl) aminomethane (Tris) -HCl and NaCl.

8. The method of claim 1, wherein the plurality of MUA-aunps are prepared according to a process comprising:

make HAuCl₄Contacting with trisodium citrate, thereby forming a plurality of citrate-coated gold nanoparticles (citrate-AuNP); and

contacting the plurality of citrate-AuNPs with 11-mercaptoundecanoic acid, thereby forming the plurality of MUA-AuNPs.

9. The method of claim 8, wherein HAuCl₄And trisodium citrate in a molar ratio of 1:3 to 1:15, respectively.

10. The method of claim 8, wherein the citrate-AuNP and MUA are contacted at a molar ratio of 1:3 to 1:15, respectively.

11. The method of claim 2, wherein after the step of contacting the sample with the plurality of MUA-aunps, the test sample is incubated for at least 15 minutes prior to the step of detecting the presence of Spm in the sample based on the test color.

12. The method of claim 2, wherein after the step of contacting the sample with the plurality of MUA-aunps, the test sample is incubated for 15 and 30 minutes prior to the step of detecting the presence of Spm in the sample based on the test color.

13. The method of claim 1, wherein the method comprises:

providing a sample comprising urine at a concentration of 0.1-2% (v/v), wherein the urine is obtained from a subject;

contacting the sample with a plurality of MUA-aunps comprising citrate and MUA, thereby forming a test sample having a test color; and

detecting the presence of Spm in the sample based on the test color, wherein after the step of contacting the sample with the plurality of MUA-AuNPs, the test sample is incubated for 15 and 30 minutes prior to the step of detecting the presence of Spm in the sample based on the test color.

14. The method of claim 13, wherein the plurality of MUA-aunps have an average particle size of 1-20 nm.

15. The method of claim 13, wherein the sample comprises Tris-HCl and NaCl.

16. The method of claim 13, wherein the plurality of MUA-aunps are prepared according to a method comprising:

Make HAuCl₄With trisodium citrate in a molar ratio of 1:3 to 1:15, respectively, to form a plurality of citratescitrate-AuNP; and

contacting the plurality of citrate-AuNPs with MUA at a molar ratio of 1:3 to 1:15, respectively, thereby forming the plurality of MUA-AuNPs.

17. A method for diagnosing prostate cancer in a subject, the method comprising:

providing a sample comprising urine at a concentration of 0.1-2% (v/v), wherein the urine is obtained from the subject;

contacting the sample with a plurality of MUA-aunps comprising citrate and MUA, thereby forming a test sample having a test color; and determining whether the subject has prostate cancer based on the test color, wherein after the step of contacting the sample with the plurality of MUA-aunps, the test sample is incubated for 15 and 30 minutes prior to performing the step of determining whether the subject has prostate cancer based on the test color.

18. The method of claim 17, wherein the step of determining whether the subject has prostate cancer based on the test color comprises: comparing the test color to a color chart or color wheel prepared by using a correlation between known concentrations of Spm in a standard sample comprising MUA-AuNP; or comparing the absorbance of the test sample obtained using a spectrometer with one or more calibration curves prepared by using the correlation between known concentrations of Spm in a standard sample comprising MUA-AuNP; determining the concentration of Spm in the sample; and determining whether the subject has prostate cancer based on the concentration of Spm in the sample.

19. The method of claim 17, wherein the plurality of MUA-aunps are prepared according to a method comprising:

make HAuCl₄Contacting trisodium citrate at a molar ratio of 1:3 to 1:15, respectively, to form a plurality of citrate-aunps; and

contacting the plurality of citrate-AuNPs with MUA at a molar ratio of 1:3 to 1:15, respectively, thereby forming the plurality of MUA-AuNPs.

20. The method of claim 17 wherein the MUA-AuNP has an average particle size of 10-15 nm.

Technical Field

The present disclosure relates generally to gold nanoparticles (aunps) and their use in spermine (Spm) detection and cancer diagnosis.

Background

In recent years, research into a group of biological polyamines (such as putrescine, spermidine, and Spm) has received much attention. Spm, a polycation derived from amino acids, plays an important role in the regulation of immune responses, neuronal regulation and certain pathological events due to its positively charged nature, which readily binds cellular components and thereby regulates cellular physiology. In a recent study, it was demonstrated that urine Spm can be used as a biomarker to distinguish prostate cancer patients from non-malignant Benign Prostatic Hyperplasia (BPH) patients. In addition to prostate cancer, Spm has also been widely reported as a possible cancer biomarker for a range of cancers, which has led to an increased interest in developing improved Spm detection methods.

Conventional methods for quantitative detection of urine Spm include chromatography in combination with different detectors and electromigration methods, such as ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), chemiluminescence-based High Performance Liquid Chromatography (HPLC), and the like. However, the above techniques require expensive instruments, complex sample preparation and highly trained technician handling.

The application of known probes and methods for detecting urinary Spm is limited by the complex matrix (matrix) and composition of the urine, which presents challenges for sample preparation/purification for efficient detection. Recently, aunps have been used in a number of applications, including chemical sensing and cellular imaging based on their unique aggregation-induced photophysical properties. However, aggregation-based sensing strategies using AuNP remain challenging due to stability issues, where aggregation can easily occur in biological samples (e.g., urine), which can lead to false negative results.

Therefore, there is a need to develop improved methods for detection of uropolyamine in complex matrices (e.g., urine) based on AuNP.

SUMMARY

Provided herein are methods utilizing thiol-protected aunps that exhibit enhanced stability, high sensitivity, and excellent selectivity for Spm. The utility (practicality) of the methods described herein has been validated for the detection of Spm in clinical urine samples for cancer screening.

The present disclosure provides a series of biological probes (sensors) that can be used to determine the concentration of Spm in a sample and for cancer diagnosis. More specifically, provided herein are citrate-coated aunps that are stabilized and whose sensitivity can be modulated by the presence of a thiol-containing reagent, such as mercaptoundecanoic acid. Without being bound by theory, it is believed that the citrate ligand interacts with Spm through electrostatic forces, causing aunps to be pulled together and aggregate, which results in a red-shift of the absorption associated with Surface Plasmon Resonance (SPR) to longer wavelengths. Thus, because of the presence of Spm in the sample, Spm-induced aggregation of aunps as described herein can be observed by a distinct color change of the AuNP suspension from red to blue/violet, and provides a convenient and straightforward means for detection and quantification.

In a first aspect, provided herein is a method for detecting spermine (Spm) in a sample, the method comprising: providing a sample; contacting the sample with a plurality of gold nanoparticles (MUA-aunps) comprising citrate and 11-mercaptoundecanoic acid (MUA), thereby forming a test sample having a test color; and detecting the presence of Spm in the sample based on the test color.

In a first embodiment of the first aspect, provided herein is a method of the first aspect, wherein the sample comprises urine obtained from the subject.

In a second embodiment of the first aspect, provided herein is the method of the first aspect, wherein the step of detecting based on the test color comprises visual inspection of the test color or determining absorbance of the test sample using a spectrometer.

In a third embodiment of the first aspect, provided herein is the method of the second embodiment of the first aspect, further comprising the steps of: comparing the test color to a color chart or color wheel prepared by using correlations between colors of a known concentration of Spm in a standard sample comprising MUA-AuNP; or comparing the absorbance of the test sample obtained using the spectrometer with one or more calibration curves prepared using a correlation between the absorbance of Spm at known concentrations in a standard sample comprising MUA-AuNP; and determining the concentration of Spm in the sample.

In a fourth embodiment of the first aspect, provided herein is the method of the first embodiment of the first aspect, wherein the method further comprises the steps of: comparing the test color to a color chart or color wheel prepared by using the correlation between known concentrations of Spm in a standard sample comprising MUA-AuNP; or comparing the absorbance of the test sample obtained using the spectrometer with one or more calibration curves plotted using the correlation between Spm at known concentrations in a standard sample comprising MUA-AuNP; determining the concentration of Spm in the sample; and determining whether the subject has prostate cancer based on the concentration of Spm in the sample.

In a fifth embodiment of the first aspect, provided herein is the method of the first embodiment of the first aspect, further comprising the steps of: providing a urine sample obtained from a subject; and diluting the urine sample 50-1000 fold with an aqueous solvent, thereby forming the sample.

In a sixth embodiment of the first aspect, provided herein is the method of the first embodiment of the first aspect, wherein the aqueous solvent comprises Tris (hydroxymethyl) aminomethane (Tris) -HCl and NaCl.

In a seventh embodiment of the first aspect, provided herein is the method of the first embodiment of the first aspect, wherein the plurality of MUA-aunps are prepared according to a process comprising: make HAuCl₄Contacting with trisodium citrate, thereby forming a plurality of citrate-capped gold nanoparticles (citrate-AuNP); and contacting a plurality of citrate-aunps with 11-mercaptoundecanoic acid, thereby forming the plurality of MUA-aunps.

In an eighth embodiment of the first aspect, provided herein is the method of the seventh embodiment of the first aspect, wherein the HAuCl₄And trisodium citrate in a molar ratio of 1:3 to 1:15, respectively.

In a ninth embodiment of the first aspect, provided herein is the method of the seventh embodiment of the first aspect, wherein the citrate-AuNP and MUA are contacted at a molar ratio of 1:3 to 1:15, respectively.

In a tenth embodiment of the first aspect, provided herein is the method of the first embodiment of the first aspect, wherein after the step of contacting the sample with the plurality of MUA-aunps, the test sample is incubated for at least 15 minutes prior to the step of detecting the presence of Spm in the sample based on the test color.

In an eleventh embodiment of the first aspect, provided herein is the method of the first embodiment of the first aspect, wherein after the step of contacting the sample with the plurality of MUA-aunps, the test sample is incubated for 15 and 30 minutes, followed by the step of detecting the presence of Spm in the sample based on the test color.

In a twelfth embodiment of the first aspect, provided herein is a method of the first aspect, wherein the method comprises: providing a sample comprising urine at a concentration of 0.1-2% (v/v), wherein the urine is obtained from a subject; contacting the sample with a plurality of MUA-aunps comprising citrate and MUA, thereby forming a test sample having a test color; and detecting the presence of Spm in the sample based on the test color, wherein after the step of contacting the sample with the plurality of MUA-aunps, the test sample is incubated for 15 and 30 minutes, and then the step of detecting the presence of Spm in the sample based on the test color is performed.

In a thirteenth embodiment of the first aspect, provided herein is the method of the twelfth embodiment of the first aspect, wherein the plurality of MUA-aunps have an average particle size of 1-20 nm.

In a fourteenth embodiment of the first aspect, provided herein is the method of the twelfth embodiment of the first aspect, wherein the sample comprises Tris-HCl and NaCl.

In a fifteenth embodiment of the first aspect, provided herein is the method of the twelfth embodiment of the first aspect, wherein the plurality of MUA-aunps is prepared according to a process comprising: make HAuCl₄With trisodium citrateContacting at a molar ratio of 1:3 to 1:15, respectively, to form a plurality of citrate-aunps; and contacting a plurality of citrate-aunps with MUAs at a molar ratio of 1:3 to 1:15, respectively, thereby forming the plurality of MUA-aunps.

In a second aspect, provided herein is a method of diagnosing prostate cancer in a subject, the method comprising: providing a sample comprising urine at a concentration of 0.1-2% (v/v), wherein the urine is obtained from a subject; contacting the sample with a plurality of MUA-aunps comprising citrate and MUA, thereby forming a test sample having a test color; and determining whether the subject has prostate cancer based on the test color, wherein after the step of contacting the sample with the plurality of MUA-aunps, the test sample is incubated for 15 and 30 minutes, and then the step of determining whether the subject has prostate cancer based on the test color.

In a first embodiment of the second aspect, provided herein is a method of the second aspect, wherein the step of determining whether the subject has prostate cancer based on the test color comprises: comparing the test color to a color chart or color wheel prepared by using the correlation between known concentrations of Spm in a standard sample comprising MUA-AuNP; or comparing the absorbance of the test sample obtained using the spectrometer with one or more calibration curves prepared by using the correlation between known concentrations Spm in a standard sample comprising MUA-AuNP; determining the concentration of Spm in the sample; and determining whether the subject has prostate cancer based on the concentration of Spm in the sample.

In a second embodiment of the second aspect, provided herein is the method of the second aspect, wherein the plurality of MUA-aunps is prepared according to a method comprising: make HAuCl₄Contacting trisodium citrate at a molar ratio of 1:3 to 1:15, respectively, to form a plurality of citrate-aunps; and contacting a plurality of citrate-aunps with MUAs at a molar ratio of 1:3 to 1:15, respectively, thereby forming the plurality of MUA-aunps.

In a third embodiment of the second aspect, provided herein is the method of the second aspect, wherein the MUA-AuNP has an average particle size of 10-15 nm.

Brief Description of Drawings

The above and other objects and features of the present disclosure will become apparent when the following description of the present disclosure is considered in conjunction with the accompanying drawings.

Fig. 1 shows table 1, table 1 shows the results of Spm detection (μ M) of clinical urine samples by MUA-AuNP (n ═ 100) and LC-MS/MS of fig. 4A.

FIG. 2 shows a representative schematic representation of colorimetric detection of Spm using MUA-AuNPs as described herein.

FIG. 3 shows a representative schematic representation of the preparation of MUA-AuNP and aggregation based on electrostatic interactions between MUA-AuNP and Spm.

Fig. 4A shows a Transmission Electron Microscope (TEM) image of spherical MUA-AuNP that appears red in the absence of Spm.

FIG. 4B shows a TEM image of the MUA-AuNP of FIG. 4A in the presence of Spm (250nM), and the color of the MUA-AuNP is purple.

Figure 5 shows a typical response of MUA-AuNP of figure 4A at different incubation times to urine samples containing different concentrations of Spm.

FIG. 6 shows a color scheme of different concentrations (0-500nM) of Spm in water (2.5uL) added to 235uL of MUA-AuNP solution (5mM Tris 50mM NaCl pH 7.2).

FIG. 7 shows a typical UV-visible absorption spectrum (hereinafter "spectrum") of spherical MUA-AuNP having SPR absorption of about 525nM in red, which undergoes a spectral shift upon addition of Spm (0-200 nM).

FIG. 8A shows a graph of the specific absorbance change when MUA-AuNP interacts with Spm (0-200 nM).

FIG. 8B shows a graph of Abs610/Abs527 as a function of Spm concentration.

FIG. 9 shows typical spectra of MUA-AuNPs for different polyamines (250 nM). MUA-AuNP showed SPR absorption shift only in the presence of Spm.

FIG. 10 shows a graph of normalized Abs610/Abs527 versus SPM concentration (60-160nM) for MUA-AuNPs as described herein.

Figure 11 shows a picture of an optimized sample dilution study.

Figure 12 shows the UV-visible absorption spectra of citrate-AuNP prepared from different concentrations of trisodium citrate.

Figure 13 shows UV-visible absorption spectra of MUA-AuNP prepared from different concentrations of trisodium citrate.

FIG. 14 shows pictures of MUA-AuNPs prepared using different concentrations of MUA in a MUA-citrate ligand exchange reaction.

Detailed description of the invention

The methods described herein for detecting Spm rely on aunps that have unique optical properties due to size and morphology (e.g., having sizes in the range of 1-100 nm). The bead-shaped AuNP (about 30nm) absorbs light in the blue-green region of the visible spectrum (450 nm and 550nm) while reflecting red light (about 700nm) to produce a strong red color. As the AuNP increases in size or AuNP aggregation occurs, larger particles may exhibit increased scattering and broadening of the absorption peak and a red-shift of SPR-related absorption to longer wavelengths. This phenomenon can be observed by the color change of AuNP suspensions caused by aggregation.

The present disclosure provides AuNP-based high sensitivity probes for rapid and direct detection of Spm in a sample, with the advantage of chromaticity, allowing detection to be determined either visually or alternatively using low cost spectrometers.

The methods provided herein are capable of selectively detecting Spm in a sample (e.g., urine) within 15 to 30 minutes with nanomolar sensitivity, which is an improvement over existing methods. In addition, the detection can be easily carried out without training, and any non-technical personnel can operate the detection device. The probes can be readily prepared in a two-step process from commercially available starting material reagents. MUA-AuNP is highly stable in the buffer system described herein and can be conveniently stored.

The methods described herein utilize MUA-AuNP and MUA or a conjugate base thereof comprising citrate or a conjugate acid thereof.

Citric acid and MUA contain 4 and 2 acidic protons, respectively, and thus can exist in multiple proton states. MUA-AuNP as described herein may include citrate and MUA in any protonic state and mixtures thereof. The proton state may depend on the pH of the composition comprising MUA-AuNP.

The MUA-aunps described herein can be prepared using a number of methods well known in the art. All of these methods are contemplated by the present disclosure. In certain embodiments, the MUA-AuNP is prepared in a two-step process comprising reducing a gold salt with citric acid or a salt thereof to form citrate-AuNP, followed by ligand exchange with a thiol-containing compound to produce MUA-AuNP.

citrate-AuNP can be prepared by reducing a gold salt using a citrate reducing agent selected from citric acid or a salt thereof. In certain embodiments, the gold salt is Au (i), Au (ii), Au (iii), or mixtures thereof. The gold salt may include an anion selected from the group consisting of: chloride ion, bromide ion, iodide ion, nitrate radical, hydroxyl radical and PF₆、BF₄Mesylate, triflate, cyanide, acetate, and the like, and combinations thereof. In certain embodiments, gold salts suitable for use in preparing the citrate-AuNP described herein may be represented by the formula: MAuX₄Wherein M is H, Li, Na and NH₄(ii) a And for each instance, X is independently Cl, Br, or I. Exemplary gold salts suitable for preparing the citrate-AuNP described herein include, but are not limited to, HAuCl₄And HAuBr₄。

In certain embodiments, the citrate reducing agent may be represented by the formula: (CO)₂M)CH₂C(OH)(CO₂M)CH₂(CO₂M), wherein for each instance, M is independently selected from H, Li, Na, and NH₄. In certain embodiments, the citrate reducing agent is trisodium citrate, trilithium citrate, magnesium citrate, calcium citrate, or mixtures thereof.

In the following examples, citrate-AuNP used in the methods described herein is produced directly by a wet chemistry method involving the reduction of chloroauric acid (HAuCl) with trisodium citrate in an aqueous solvent (e.g., water) ₄) Thereby producing monodisperse citrate-AuNP.

In reducing gold salts, citric acid reducing agent is generally used in excess. The citrate reducing agent may be used in a molar ratio of 1 to 20 with respect to the gold salt according to the oxidation state of the gold salt.

In certain embodiments, the molar ratio of citrate reducing agent to gold salt is from 2:1 to 20: 1; 2:1 to 19: 1; 2:1 to 18: 1; 2:1 to 17: 1; 2:1 to 16: 1; 2:1 to 15:1(ii) a 3:1 to 15: 1; or 3:1 to 13: 1. In certain embodiments, 155-1,240 mol% or 310-1,240 mol% trisodium citrate is used to reduce HAuCl₄。

The reduction of the gold salt may be carried out in an aqueous solvent. The reduction may be carried out at a temperature of 23-100 ℃. In certain embodiments, the reduction is carried out at a temperature of 60-100 ℃, 40-100 ℃, 80-100 ℃, 90-100 ℃ or 100 ℃.

The average size of citrate-AuNP prepared according to the methods described herein may be in the range of 1 to 20 nm. In certain embodiments, the citrate-AuNP has an average size of 5 to 20nm, 7 to 20nm, 10 to 17nm, or 10 to 15 nm. In certain embodiments, the citrate-AuNP has an average size of about 13 nm. In certain embodiments, the suspension of citrate-AuNP may have an average size of about 13nm, with the possibility that the suspension includes citrate-AuNP in the range of 10 to 50 nm.

MUA-AuNP can be prepared from citrate-AuNP by a ligand exchange reaction in which one or more citrate ligands bound to the surface of the citrate-AuNP are replaced with one or more MUA ligands, thereby forming a plurality of MUA-aunps comprising citrate and MUA ligands. Figure 3 depicts a representative reaction sequence showing the preparation of MUA-aunps and the displacement of surface-bound citrate ligands with MUA, thereby forming a plurality of MUA-aunps.

The citrate-AuNP ligand exchange reaction may be carried out in an aqueous solvent (e.g., water).

MUA may be used in a molar ratio between 1 and 20 relative to citrate-AuNP (stoichiometry is based on the assumption that 100% of the gold salt is converted to citrate-AuNP and relative to gold). In certain embodiments, the molar ratio of MUA to citrate-AuNP is between 2:1 and 20: 1; 2:1 to 19: 1; 2:1 to 18: 1; 2:1 to 17: 1; 2:1 to 16: 1; 2:1 to 15: 1; 3:1 to 15: 1; or between 3:1 and 13: 1. In certain embodiments, between 155-1,240 mol% or 310-1,240 mol% MUA is used to perform a ligand exchange reaction with citrate-AuNP.

In certain embodiments, the average size of the MUA-AuNP is in the range of 1 to 20 nm. In certain embodiments, the average size of the MUA-AuNP is 5 to 20nm, 7 to 20nm, 10 to 17nm, or 10 to 15 nm. In certain embodiments, the citrate-AuNP has an average size of about 13 nm.

The method of detecting Spm in a sample may comprise: providing a sample; contacting the sample with a plurality of MUA-aunps comprising citrate and MUA, thereby forming a test sample having a test color; and detecting the presence of Spm in the sample based on the test color.

The sample may contain a Spm target. Such samples may include water samples, food samples or biological samples obtained from plants or animals or from bodily fluids (e.g., urine) of a subject (including mammals, such as humans). The sample may be a "clinical sample", i.e. a sample from a patient, e.g. urine. In certain embodiments, the sample comprises urine obtained from a human subject.

The sample may comprise urine obtained from a subject that has been diluted 5 to 1,000 times with an aqueous solvent (i.e., by adding 1 part urine to 4 parts aqueous solvent to 1 part urine to 999 parts aqueous solvent). In certain embodiments, urine may be diluted 5 to 1000 times with an aqueous solvent; 5 to 500 times; 10 to 500 times; 10 to 400 times; 10 to 300 times; 10 to 200 times; 20 to 200 times; 30 to 200 times; 40 to 200 times; 50 to 200 times; 50 to 150 times; 50 to 125 times; 50 to 100 times; 70 to 120 times; 80 to 120; 90 to 120 times; or 90 to 110 times. In certain embodiments, the sample comprises 0.1-10%; 0.1-9%; 0.1 to 8 percent; 0.1-7%; 0.1-6%; 0.1-5%; 1 to 5 percent; 1 to 4 percent; 1 to 3 percent; 1 to 2 percent; 1 to 1.5 percent; 1 to 1.4 percent; 1 to 1.3 percent; 1 to 1.1 percent; 0.2-1.1%; 0.3-1.1%; 0.4-1.1%; 0.5-1.1%; 0.6-1.1%; 0.7-1.1%; 0.8 to 1.1 percent; or 0.9-1.1% urine (v/v).

To dilute the urine sample, the aqueous solvent may be buffered. Any buffer system commonly used to buffer clinical samples can be used to buffer urine samples. In certain embodiments, the sample comprises Tris-HCl or (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) (HEPES) buffer. In certain embodiments, the sample has a molecular weight of 7.0 to 7.5; 7.0-7.4; 7.0-7.3; or a pH between 7.1 and 7.3.

The aqueous solvent may also contain NaCl in a concentration of 1-100 mM. In certain embodiments, the concentration of NaCl in the sample is 10-100mM, 10-90mM, 10-80mM, 20-80mM, 30-70mM, or 40-60 mM.

The sample may also contain a preservative, such as NaN₃For preventing microbial growth. NaN in the sample₃The concentration of (C) may range between 0.01 and 0.1% (w/w). In certain embodiments, NaN is present in the sample₃The concentration of (a) is in the range of 0.01 to 0.1%; 0.01 to 0.09%; 0.01 to 0.08%; 0.02 to 0.08%; 0.03 to 0.08%; 0.03 to 0.07%; 0.03 to 0.06%; or 0.04 to 0.06% (w/w).

As described herein, MUA-AuNP can comprise citrate salts of different proton states and/or MUA (e.g., the conjugate base of MUA and the conjugate acid of citrate salt). All of these forms are contemplated in the methods described herein. In the example where the MUA-AuNP comprises the conjugate acid of citrate, one or both of the carboxylic acids present in the citrate may be present in protonated form. In examples where the MUA-AuNP comprises a conjugate base of MUA, the conjugate may be Na, Li, Mg, Ca, NH ₄And the like in salt form.

Detecting the presence of Spm in the sample can be done by visual inspection and/or using a spectrometer. Any conventional spectrometer is capable of measuring the absorbance of a test sample, which may fall between about 450 to 700 nm. In certain embodiments, the spectrometer is a visible light spectrometer or an ultraviolet-visible light spectrometer capable of measuring absorbance of a test sample of 450 to 700, 500 to 650nm, 550 to 650nm, 600 to 650nm, or 600 to 630 nm.

Detection of Spm in a sample can also be performed by simple visual inspection of the sample due to the apparent difference in color of the MUA-AuNP aggregates formed in the presence of Spm. From the color of the test sample, it can be determined whether Spm is present.

Detecting the presence of Spm in the sample may also include determining the concentration of Spm in the sample. In such an example, the method may further comprise the steps of: comparing the test color to a color chart or color wheel prepared by using correlations between colors of a known concentration of Spm in a standard sample comprising MUA-AuNP; or comparing the absorbance of the test sample obtained using the spectrometer with one or more calibration curves prepared using a correlation between the absorbance of Spm at known concentrations in a standard sample comprising MUA-AuNP; and determining the concentration of Spm in the sample.

The correlation between the colour of the test sample and the MUA-AuNP in a standard sample with a known concentration of Spm can be determined by preparing a series of standard samples, preferably comprising a similar analyte matrix, containing MUA-AuNP and a known concentration of Spm (e.g. from 50 to 300nM of Spm), and determining the colour of each standard sample with a different concentration of Spm. The concentration of the test sample can then be determined by simply comparing the color of the test sample with the color of each standard sample having a different concentration Spm, and determining the concentration of Spm in the standard sample based on which standard sample Spm concentration has the closest color. To simplify this process, a color chart, a color wheel, or the like indicating the correlation between the test/standard sample color and the Spm concentration may be prepared in advance.

The correlation between the colour of the test sample and the MUA-AuNP in a standard sample with a known concentration of Spm can be determined by preparing a series of standard samples, preferably comprising a similar analyte matrix, containing MUA-AuNP and a known concentration of Spm (e.g. from 50 to 300nM of Spm), and determining the absorbance of each standard sample with a different concentration of Spm using a spectrometer. Using the correlation between the absorbance of a known concentration of Spm in a standard sample comprising MUA-AuNP, one or more calibration curves can be prepared. The concentration of Spm in the test sample can then be determined by comparing the absorbance of the test sample to a calibration curve.

Referring to fig. 2, the method of detecting Spm includes: (a) adding MUA-AuNP suspension 2 into buffer solution 1; and (b) adding sample 3 or 4 suspected of containing Spm to buffer 1, wherein sample 5 containing Spm causes a color change (red to purple). Also, where no color change was observed (remaining red), sample 6 contained no or only low levels of Spm.

In certain embodiments, the method of detecting Spm comprises the steps of: the MUA-aunps described herein are contacted with a sample suspected of containing Spm to form a test sample, and the absorption of the test sample is measured. In certain embodiments, the step of measuring the absorption is accomplished visually by comparison to a color wheel or chart, or by measurement using a spectrometer. In certain embodiments, the step of measuring the absorption of the test sample comprises detecting the absorption intensity of the test sample in the range of 450 to 700 nm. In certain embodiments, the step of measuring the absorption comprises detecting the absorption intensity of the sample in the range of 500 to 650nm, 550 to 650nm, 600 to 650nm, or 600 to 630 nm.

In certain embodiments, the step of contacting the sample with the plurality of MUA-aunps comprises contacting an aqueous solution comprising the plurality of MUA-aunps with the sample. The concentration of MUA-AuNP in the aqueous solvent may vary depending on the concentration of Spm in the sample. The determination of the necessary concentration of MUA-AuNP in an aqueous solvent can be determined based on the guidance described herein and general knowledge known to those skilled in the art. The concentration of multiple MUA-aunps in aqueous solvent can be calculated according to analytical approximation of spherical gold nanoparticles developed by Haiss (2007) and assuming that all citrate-aunps are converted to MUA-aunps. The concentration of the plurality of MUA-aunps in the aqueous solvent may be in the range of 0.742 to 56.6 nmol/L. In certain embodiments, the concentration of the plurality of MUA-aunps in the aqueous solvent may be in the range of 0.742 to 1.42nmol/L (0.25X MUA), 1.484 to 2.84nmol/L (0.5X MUA), 2.97 to 5.66nmol/L (1X MUA), 5.94 to 11.32nmol/L (2X MUA), 11.88 to 22.64nmol/L (4X MUA), or 29.7 to 56.6nmol/L (10X MUA). In certain embodiments, the concentration of the plurality of MUA-aunps in the aqueous solvent may be in the range of 29.7 to 56.6nmol/L (10X MUA).

In certain embodiments, the step of contacting the sample with the plurality of MUA-aunps comprises adding the plurality of MUA-aunps to the sample or adding the sample to an aqueous solution comprising the plurality of MUA-aunps. In certain embodiments, an aqueous solvent comprising a plurality of MUA-aunps is added to the sample.

In certain embodiments, the incubation time after contacting the MUA-AuNP with the sample suspected of containing Spm is 30 minutes or less, at which point the test color of the test sample is examined. In certain embodiments, the incubation time after contacting the MUA-AuNP with the sample suspected of containing Spm to check the test color of the test sample is at least 15 minutes; or 15-30 minutes, 15-25 minutes, 15-20 minutes, 20-30 minutes, or 25-30 minutes. As shown in fig. 5, the methods provided herein can advantageously provide test results in as little as 5-15 minutes, as evidenced by the time entries (time entries) of samples 3, 4, and 5 at 5, 10, and 15 minutes, where only the color intensity of the test sample continues to change, rather than the absorption wavelength of the test sample.

MUA-AuNP showed a highly selective red-shift in the presence of Spm, which was not observed in other biogenic amines, as shown in figure 9. This high selectivity can increase the sensitivity of the method for detecting Spm in complex analyte matrices, such as urine.

Methods of detecting and quantifying Spm in a test sample comprising urine obtained from a subject can be used to diagnose whether the subject has prostate cancer. In such instances, the methods described herein may further comprise the steps of: comparing the test color to a color chart or wheel prepared by using the correlation between Spm at known concentrations in the standard sample; or comparing the absorbance of the test sample obtained using the spectrometer with one or more calibration curves prepared by using the correlation between Spm at known concentrations in standard samples; determining the concentration of Spm in the sample; and determining whether the subject has prostate cancer based on the concentration of Spm in the sample.

In certain embodiments, a method for diagnosing prostate cancer in a subject comprises: providing a sample comprising urine at a concentration of 1-2% (v/v), wherein the urine is obtained from a subject; contacting the sample with a plurality of MUA-aunps comprising citrate and MUA or a conjugate base thereof for 15 to 30 minutes, thereby forming a test sample having a test color; and determining whether the subject has prostate cancer based on the test color.

In certain embodiments, the step of determining whether the subject has prostate cancer based on the test color comprises: comparing the test color to a color chart or color wheel prepared by using the correlation between known concentrations of Spm in a standard sample comprising MUA-AuNP; or comparing the absorbance of the test sample obtained using the spectrometer with one or more calibration curves prepared by using the correlation between Spm at known concentrations in standard samples; determining the concentration of Spm in a sample comprising MUA-AuNP; and determining whether the subject has prostate cancer based on the concentration of Spm in the sample.

Also provided herein is a device for carrying out the method of detecting Spm described herein, the device comprising a compartment suitable for containing an incubation solution; and a second compartment containing a mixture comprising MUA-AuNP, the second compartment being punctured such that a probe mixture comprising MUA-AuNP can fall into the compartment containing the incubation buffer; a third compartment for transferring a sample suspected of containing the Spm target to the first compartment containing the MUA-AuNP and an incubation buffer, which is capable of exhibiting a visible color change in the presence of the Spm target.

The first compartment of the test kit may be any alternative device capable of carrying a liquid, such as simply a glass vial or an Eppendorf tube. The second compartment may be any device capable of delivering fluid to the system, such as a syringe, pipette, or any other suitable device known in the art. Likewise, the third compartment may be any suitable duct shown in this embodiment. In general, a test kit comprising all three compartments may be assembled or designed as one single unit, such as a cup or any substitute.

In certain embodiments, the kit further comprises a color chart or color wheel that can be used to correlate the color of the sample with at least one of the concentration of Spm in the sample or the likelihood that the patient has cancer.

In certain embodiments, the kit further comprises instructions for performing a method of detecting Spm in a sample described herein.

Reagent and apparatus

Spm, chloroauric acid (HAuCl)₄) Trisodium citrate, MUA, hydrochloric acid (HCl), nitric acid (HNO)₃) TRIS (hydroxyethyl) aminomethane (TRIS), sodium chloride (NaCl), sodium azide (NaN)₃) And hydrogen hydroxideSodium (NaOH) was obtained from Sigma-Aldrich (hong kong, china). Ethanol (EtOH) was purchased from ACROS (usa). All chemicals and reagents were of analytical grade and were used without further purification. Water was purified in a MilliQ direct water purification system (Millipore, usa). UV-Vis absorption spectra were recorded using a Cary 8453UV-Vis spectrometer (Agilent, hong Kong, China). Dynamic Light Scattering (DLS) was measured using a Zetasizer Nano-ZS90 system (Malvern Instruments, Shanghai, China).

All glassware was freshly prepared 3:1(v/v) HNO₃-HCl bath and then rinsed thoroughly with Milli-Q water. First through sodium citrate mediated HAuCl₄Reduction to prepare 13nm citrate-AuNP, as described previously but with minor modifications. Briefly, HAuCl was added₄(4.25mg, 12.5. mu. mol) was dissolved in 25.0mL Milli-Q water (0.50mM) to form a pale yellow aqueous solution. The solution was heated to reflux with stirring and then reacted with 1.0mL of a 1.0mL aqueous solution (2% w/w) of sodium citrate dihydrate (0.02g, 68. mu. mol) to form a dark purple solution. The resulting solution was stirred at 100 ℃ for an additional 20 minutes until the solution became wine red in color, indicating complete formation of citrate-AuNP. By controlling the amount of citrate used in the synthesis, the size of the obtained nanoparticles can be controlled in the range of 13 to 50 nm.

Based on the synthesis of citrate-AuNP as exemplified above, the effect of different equivalents of trisodium citrate on the photophysical properties of the citrate-AuNP prepared was further investigated. Various equivalents of trisodium citrate (0.25X, 0.5X, 1X, 1.25X, 1.5X and 2X corresponding to 17. mu. mol, 34. mu. mol, 68. mu. mol, 85. mu. mol, 102. mu. mol and 136. mu. mol, respectively) were combined with HAuCl₄citrate-AuNP was prepared by reacting 25.0mL of an aqueous Milli-Q solution (0.50mM) (4.25mg, 12.5. mu. mol). The prepared citrate-AuNP was diluted and checked using UV-Vis absorption measurements. As shown in fig. 12, when 0.25x (17 μmol) citrate was used during the reduction, a red shift in absorbance of the citrate-AuNP product was observed (absorbance maximum at 532 nm), indicating a relative increase or aggregation of citrate-AuNP. In contrast, citrate concentrations greater than 0.25x resulting in only slight changes in absorbance indicate little or no aggregation and/or relatively littlecitrate-AuNP.

After citrate-AuNP preparation was complete, the dark wine red solution was allowed to cool to room temperature and transferred to a clean 50mL erlenmeyer flask. The volume of the mixture was adjusted to 25.0mL by adding water.

In certain embodiments, the pH of the citrate-AuNP suspension is adjusted (e.g., using NaOH) to 11, and then 1-mercaptoundecanoic acid in 200 μ Ι _ of ethanol is added dropwise to generate MUA-AuNP by ligand exchange between citrate and thiol-containing reagent. The mixture was then stirred overnight, the residue was collected by centrifugation and redispersed using a minimal amount of supernatant to give a concentrated (10X) MUA-AuNP suspension.

citrate-AuNP obtained from different concentrations of trisodium citrate (except 0.25 x) was further reacted with the same amount of MUA (i.e., 0.5x, 1x, 1.25x, 1.5x and 2x corresponding to 34 μmol, 68 μmol, 85 μmol, 102 μmol and 136 μmol, respectively). UV-Vis absorption measurements were performed after dilution of MUA-AuNP. The mean absorbance shift after MUA modification was about 5nm and the absorbance shift variation for the same batch of citrate-AuNP was in the 1nm range, showing the importance of obtaining the desired citrate-AuNP (fig. 13).

MUA-AuNPs were prepared using different concentrations of MUA (0.25X, 0.5X, 1X, 2X, 4X corresponding to 17. mu. mol, 34. mu. mol, 68. mu. mol, 85. mu. mol, 136. mu. mol, 272. mu. mol, respectively). MUA was added directly to the prepared citrate reduction reaction product mixture. As shown in figure 14, doubling or halving the amount of MUA had no effect on the absorbance of the product MUA-AuNP (in 5mM Tris, 50mM NaCl), indicating no difference in aggregation and/or particle size.

The colloidal MUA-AuNP suspension was dropped onto a carbon coated copper mesh and allowed to stand to dry at room temperature. The copper grid was then analyzed on a FEI Tecnai G220S-Twin transmission electron microscope (thermo fisher, oregon, usa) with an acceleration voltage of 200 kV. TEM experiments performed demonstrated Spm-induced aggregation characteristics of MUA-AuNP (fig. 4a and 4 b).

To determine the robustness of this assay, different incubation times after addition of MUA-AuNP to the sample were tested. This is to ensure that the truly positive samples exhibit a colorimetric change at the optimal incubation time and therefore do not result in false negatives. Visual measurements were performed at different incubation times (fig. 5). The assays were plotted at 7 time points (0, 5, 10, 15, 20, 30 and 60 minutes). MUA-AuNP was found to aggregate within 15 minutes and saturate within 30 minutes. This indicates that the assay should be performed and recorded within 30 minutes for optimal accuracy.

The final sample had a volume of 250. mu.L, containing a buffer comprising 10mM Tris-HCl, 50mM NaCl, pH7.2 and 12.5. mu.L of 10 XMUA-AuNP. MUA-AuNP (10X, 12.5. mu.L) was diluted with 235. mu.L of 10mM Tris-HCl (50mM NaCl, pH7.2) buffer to give a 0.5X MUA-AuNP suspension. Standard solution (Spm) or clinical urine samples (2.5. mu.L) were added and diluted 100-fold with 0.5 XMUA-AuNP suspension. The solution was incubated for 30 minutes at rest, then the color change was observed and the UV-Vis absorption was measured (FIGS. 6 and 7).

The MUA-aunps of the present disclosure can be used to detect the concentration of Spm in a sample (e.g., in a urine sample). In certain embodiments, the method for colorimetric detection of Spm levels in a urine sample requires the following components: (a) a urine sample; (b) stable MUA citrate-AuNP (MUA-AuNP) for visual detection of color change, which indicates the level of Spm in a urine sample; and (c) buffer for diluting the NUA-AuNP and urine samples.

The level of Spm in urine can be measured and detected by visual color change, as shown in figure 2. Urine Spm levels can be used to distinguish prostate cancer patients from benign prostate hyperplasia patients. For optimal sensitivity and specificity, the detection threshold was determined to be 3.5 μ M. The detection limit of Spm of the present invention can be varied by adjusting the ion concentration of the buffer system 1. The amount of Spm in the urine sample can be determined by visual color change. First, the concentrated probe must be introduced into a buffer system. Second, the sample is added to the diluted probe solution and the amount of Spm in the sample can be quantified, for example, by using a calibration curve established by titration (fig. 8).

Advantageously, the compositions and methods described herein are capable of exhibiting a linear relationship of 60-160nM Spm. The Spm calibration curve was fitted with a least squares linear regression. The equation is-0.05886 +0.00654x, and the limit of detection (LOD) is calculated to be 60.54nM (fig. 10) from the equation LOD 3.3 x (standard deviation of intercept/slope).

To optimize the conditions for Spm detection, a series of experiments were performed on sample clarification by dilution, aimed at reducing the effect of interfering analytes present in human urine samples. Tris buffers (5mM Tris, 50mM NaCl, 0.05% (w/w) NaN) at different Spm concentrations (0. mu.M, 0.2. mu.M, 0.4. mu.M, 0.5. mu.M, 1.0. mu.M) were used ₃) And MUA-AuNP A Spm colorimetric calibration solution was prepared. 7 clinical urine samples were diluted with Tris buffer at different dilution times (10-fold, 50-fold, 100-fold) and then incorporated into SPM to obtain visual color changes (FIG. 11). For the 10-fold dilution, most samples remained red even after spiking with Spm, indicating that detection of Spm was limited by the sample matrix effects. For the 50-fold dilution, 6 of the 7 samples were purple in color after spiking, indicating improved Spm detection. For the 100-fold dilution, all samples turned purple after SPM addition, indicating that the 100-fold dilution shows the best sample purification efficiency to more accurately detect SPM in urine.

To test the selectivity and specificity of MUA-aunps to Spm targets as described herein, a selectivity experiment was performed to test MUA-aunps through a series of polyamines and related molecules, followed by UV-Vis absorbance measurements to investigate the aggregation characteristics of MUA-aunps in the presence of different polyamines (all at 250nM) (fig. 9). The results show that only the presence of Spm leads to a red-shift in absorbance, thus demonstrating the high selectivity of MUA-AuNP for the target Spm.

The following clinical urine samples were tested and used as examples to describe and illustrate the invention. Accordingly, this example should not be construed as limiting the scope of the invention.

Examples

Example 1 detection of urine Spm in cancer patients: 90 clinical urine samples from cancer patients were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify urine Spm. These clinical urine samples (5. mu.L) were added to 470. mu.L Tris-HCl buffer (5mM, 50mM NaCl, 0.05% (w/w) NaN₃) To prepare a sample. From HAuCl₄(4.25mg, 12.5. mu. mol) and trisodium citrate dihydrate (0.02g, 68. mu. mol) and MUA (1mg, 4.58. mu. mol)) An aqueous MUA AuNP solution (calculated as XX. mu.M, 25. mu.L) was prepared for determination of Spm levels in urine. After 100-fold dilution of the urine in the buffer system, the urine-matrix interference corresponds to 250nM Spm. All assays were performed in 5mM Tris-HCl buffer, 50mM NaCl, 0.05% NaN₃And at pH 7.2. The mixed sample was allowed to stand for 30 minutes to obtain a saturated color change (if any). The cross-check results are listed in table 1 (fig. 1) with an accuracy of 80/90 for cancer discrimination using the threshold point reported at 35 nM. Samples that did not give the expected color change were marked with an asterisk (see table 1).