Kidney injury detection reagent, preparation method thereof and kidney injury detection equipment

Abstract

The invention discloses a kidney injury detection reagent, a preparation method thereof and kidney injury detection equipment, and belongs to the technical field of biomedical materials. The renal injury detection reagent comprises a gold cluster nano-material, wherein the gold cluster nano-material is gold cluster nano-particles terminated by sulfydryl-beta-cyclodextrin; the particle size of the gold cluster nano material is 1.9 nm to 2.11 nm; the gold cluster nano material is used for carrying out near-infrared two-region fluorescence imaging in a kidney region and / or carrying out enzyme-like activity chromogenic reaction in urine. The kidney injury detection reagent can realize the strategy of accurately, early, non-invasively and real-timely detecting acute kidney injury, and reveals the tiny change of the acute kidney injury in the early stage.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular, to a reagent for detecting kidney injury, a preparation method thereof, and a device for detecting kidney injury. Background Art

[0002] Acute kidney injury is a common clinical disease with a high incidence and mortality rate, characterized by a rapid decline in renal function. This abnormality is mainly manifested as a decrease in glomerular filtration rate, leading to the accumulation of metabolic waste, electrolyte imbalance, and decreased urine output. The occurrence of acute kidney injury is relatively insidious but progresses rapidly. Therefore, rapid early diagnosis is crucial for timely intervention and improving the treatment effect. Currently, the clinical diagnosis of acute kidney injury mainly relies on two indicators: elevated serum creatinine and blood urea nitrogen, and decreased urine output. However, these indicators are easily affected by various factors and thus cannot reflect the early changes in renal function. These limitations often lead to delayed diagnosis, and by this time, irreversible kidney damage has often occurred. Although imaging techniques such as enhanced CT and MRI have certain effects in obtaining renal anatomical information, their widespread application in the early diagnosis of acute kidney injury is severely limited due to problems such as low spatio-temporal resolution, radiation exposure, technical complexity, and high cost. In addition, these imaging techniques cannot provide molecular-level information, making it difficult to detect the subtle pathological changes in the early stage of acute kidney injury, limiting the understanding of the pathogenesis of acute kidney injury, and thus hindering the development of effective treatment and prevention measures.

[0003] In view of this, the present invention is specifically proposed. Summary of the Invention

[0004] The purpose of the present invention is to provide a reagent for detecting kidney injury, a preparation method thereof, and a device for detecting kidney injury to solve or improve the above technical problems.

[0005] The present invention can be implemented as follows:

[0006] In a first aspect, the present invention provides a reagent for detecting kidney injury, which comprises gold cluster nanomaterials, and the gold cluster nanomaterials are gold cluster nanoparticles capped with mercapto-β-cyclodextrin;

[0007] The particle size of the gold cluster nanomaterials is 1.9 nm to 2.11 nm;

[0008] The gold cluster nanomaterials are used for near-infrared second-region fluorescence imaging in the kidney area and / or enzymatic activity color reaction in urine.

[0009] In an optional embodiment, the gold cluster nanomaterials are used for the combined detection of near-infrared second-region fluorescence imaging and enzymatic activity color reaction in the kidney area and urine.

[0010] In an alternative embodiment, the second near-infrared region fluorescence imaging includes: using a long-pass filter with a wavelength above 1000 nm, an exposure time of 500 ms to 1000 ms, an excitation laser of 808 nm, and a power of 200 mW / cm 2 .

[0011] In an alternative embodiment, the chromogenic reagent used in the enzyme-like activity chromogenic reaction includes 3,3’,5,5’-tetramethylbenzidine.

[0012] In a second aspect, the present invention provides a renal injury detection device, which includes a detection module and an analysis module;

[0013] The detection module is configured to detect the fluorescence intensity and its change rate of the renal injury detection reagent in the target kidney region and the normal kidney; and / or, the detection module is configured to detect the enzyme-like activity chromogenic reaction of the renal injury detection reagent in the target urine and the normal urine;

[0014] The analysis module is configured to compare the fluorescence intensity and its change rate of the renal injury detection reagent in the target kidney region and the normal kidney region, and / or, the analysis module is configured to compare the enzyme-like activity chromogenic reaction of the renal injury detection reagent in the target urine and the normal urine;

[0015] The renal injury detection reagent is the renal injury detection reagent of any one of the foregoing embodiments.

[0016] In an alternative embodiment, the detection module includes a first detection module, a second detection module, a third detection module, and a fourth detection module;

[0017] The first detection module is configured to detect the fluorescence intensity and its change rate of the renal injury detection reagent in the target kidney region; the second detection module is configured to detect the fluorescence intensity and its change rate of the renal injury detection reagent in the normal kidney region;

[0018] The third detection module is configured to detect the enzyme-like activity chromogenic reaction of the renal injury detection reagent in the target urine; the fourth detection module is configured to detect the enzyme-like activity chromogenic reaction of the renal injury detection reagent in the normal urine.

[0019] In an alternative embodiment, the analysis module includes a first analysis module and a second analysis module;

[0020] The first analysis module is configured to compare the fluorescence intensity and its change rate of the renal injury detection reagent in the target kidney region and the normal kidney region;

[0021] The second analysis module is configured to compare the enzyme-like activity chromogenic reaction of the renal injury detection reagent in the target urine and the normal urine.

[0022] Thirdly, the present invention provides a preparation method of a renal injury detection reagent according to any one of the foregoing embodiments, comprising the following steps: performing a reduction reaction on a gold-sulfur coordination complex formed by a gold source and mercapto-β-cyclodextrin under alkaline conditions.

[0023] In an alternative embodiment, it further comprises: dialyzing the reaction mixture obtained from the reduction reaction.

[0024] In an alternative embodiment, the preparation of the gold-sulfur coordination complex comprises: performing a first vortex stirring on a gold source solution and a mercapto-β-cyclodextrin solution;

[0025] Wherein, the concentration of the gold source solution is 1 mmol / L to 100 mmol / L, and the concentration of the mercapto-β-cyclodextrin solution is 0.5 mmol / L to 50 mmol / L; the volume ratio of the gold source solution to the mercapto compound solution capped with β-cyclodextrin is 1:1 to 1:10; the time of the first vortex stirring is 5 min to 60 min.

[0026] In an alternative embodiment, the gold source is chloroauric acid.

[0027] In an alternative embodiment, the reduction reaction comprises: performing a second vortex stirring on a solution of the gold-sulfur coordination complex and a reducing agent solution under alkaline conditions;

[0028] Wherein, the alkaline condition is provided by an alkaline solution; the alkaline solution comprises a sodium hydroxide solution with a concentration of 0.5 mol / L to 5 mol / L; the reducing agent solution comprises a sodium borohydride solution with a concentration of 0.05 mol / L to 0.5 mol / L; the time of the second vortex stirring is 1 h to 10 h.

[0029] The beneficial effects obtained by the present invention include:

[0030] The renal injury detection reagent provided by the present invention not only has strong photostability, but also has good biocompatibility. It can be used to reflect the metabolic capacity and filtration capacity of the kidney, and can realize the strategy of early, rapid, effective, real-time, low-cost and non-invasive detection of acute kidney injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0032] Figure 1 It is a graph showing the results of cytotoxicity test of the gold cluster material in Test Example 1;

[0033] Figure 2 It is a graph showing the test results of the photo-stability of the gold cluster material in Test Example 1;

[0034] Figure 3 It is a graph showing the imaging results in the second near-infrared region in Test Example 2;

[0035] Figure 4 It is a graph showing the chromogenic reaction results in Test Example 2;

[0036] Figure 5 It is a graph showing the test results of serum creatinine and serum urea nitrogen in Test Example 2;

[0037] Figure 6 It is a graph showing the imaging results in the second near-infrared region in Test Example 3;

[0038] Figure 7 It is a graph showing the chromogenic reaction results in Test Example 3;

[0039] Figure 8 It is a graph showing the imaging results in the second near-infrared region in Test Example 4;

[0040] Figure 9 It is a graph showing the chromogenic reaction results in Test Example 4. Detailed implementation manners

[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0042] The renal injury detection reagent provided by the present invention, its preparation method, and the renal injury detection device will be specifically described below.

[0043] The present invention provides a renal injury detection reagent, which includes a gold cluster nanomaterial, and this material is a gold cluster nanoparticle capped with mercapto-β-cyclodextrin.

[0044] In the above gold cluster nanoparticles, the sulfur atom in the mercapto forms a coordination bond with the gold ion to control the size and dispersibility of the gold cluster nanoparticles.

[0045] It should be noted that not all gold cluster nanoparticles are applicable to this application, nor are any compounds with mercapto applicable to this application. This application specifically uses gold cluster nanoparticles capped with mercapto-β-cyclodextrin as the renal injury detection reagent, which not only has strong photo-stability but also has good biocompatibility, which is conducive to obtaining accurate detection results.

[0046] In the present invention, the particle size of the gold cluster nanomaterial can be 1.9nm to 2.11nm, which is smaller than the kidney filtration diameter. After entering the blood, the gold cluster nanomaterial can be quickly metabolized by the kidney and then excreted in urine as the gold cluster nanomaterial prototype, and the excretion rate can intuitively reflect the metabolic capacity and filtration capacity of the kidney. In other words, in the present invention, the gold cluster nanomaterial can be used to detect, reflect or characterize the metabolic capacity and filtration capacity of the kidney.

[0047] In some optional embodiments, the gold cluster nanomaterials can be used for near-infrared second-zone fluorescence imaging in the kidney region and / or for colorimetric reactions of enzyme-like activity in urine.

[0048] That is, gold cluster nanomaterials can be used alone for near-infrared zone II fluorescence imaging in the kidney area, or used alone for enzyme-like activity colorimetric reaction in urine; they can also be used for combined detection of near-infrared zone II fluorescence imaging and enzyme-like activity colorimetric reaction in the kidney area and urine.

[0049] In some optional embodiments, near-infrared second-zone fluorescence imaging includes: using a long-pass filter of more than 1000nm, an exposure time of 500ms to 1000ms (such as 500ms, 600ms, 700ms, 800ms, 900ms or 1000ms, etc.), an excitation laser of 808nm, and a power of 200mW / cm 2 .

[0050] If the exposure time is too short or too long, it will be detrimental to the quantification of the imaging signal and subsequent comparison.

[0051] In some optional embodiments, the colorimetric reagent used for the enzyme-like activity colorimetric reaction may exemplarily include 3,3',5,5'-tetramethylbenzidine, etc., wherein the enzyme-like activity may correspond to a catalase-like activity. By using 3,3',5,5'-tetramethylbenzidine (TMB) as a colorimetric reagent, a blue color may be displayed by the colorimetric reaction. The specific colorimetric reaction intensity may be known by measuring the absorbance at 652 nm.

[0052] The above-mentioned kidney injury detection reagent can detect or reflect or characterize the metabolic capacity of the kidney by combining the change of the fluorescence intensity of the kidney region with the color reaction of the enzyme activity of the gold cluster nanomaterial in urine through near-infrared second-zone fluorescence imaging, which is conducive to the early, rapid, sensitive, non-invasive and low-cost prediction, screening or detection of acute kidney injury. Among them, acute kidney injury can include prerenal acute kidney injury and renal acute kidney injury.

[0053] In some alternative embodiments, the kidney region for near-infrared second near-infrared fluorescence imaging in the kidney region includes the target kidney region and the normal kidney region. By comparing the fluorescence intensity and its change rate of the target kidney region and the normal kidney region, the kidney metabolic capacity of the target kidney region can be obtained; if the fluorescence intensity and its change rate of the target kidney region are less than those of the normal kidney region, it indicates that the kidney metabolic capacity of the target kidney region is low.

[0054] In some alternative embodiments, the urine for the enzyme-like activity color reaction in urine includes target urine and normal urine. By comparing the color development of the target urine and the normal urine, the metabolic capacity of the target kidney can also be obtained. Specifically, the amount of gold cluster nanomaterials excreted per unit time can be measured through the color development, and thus the quality of the filtration function of the kidney can be obtained and reflected. If the blue color after the color reaction of the target urine is lighter than that of the normal urine after the color reaction, it indicates that the filtration function of the target kidney is impaired.

[0055] If the fluorescence intensity and its change rate of the target kidney region are less than those of the normal kidney region, and the blue color after the color reaction of the target urine is lighter than that of the normal urine after the color reaction, it can be known that the blood flow of the target kidney is impaired and the renal function is impaired.

[0056] Correspondingly, the present invention also provides a renal injury detection device, which includes a detection module and an analysis module.

[0057] Among them, the detection module is used to detect the fluorescence intensity and its change rate of the renal injury detection reagent in the target kidney region and the normal kidney; and / or, the detection module is used to detect the enzyme-like activity color reaction of the renal injury detection reagent in the target urine and the normal urine.

[0058] The analysis module is used to compare the fluorescence intensity and its change rate of the renal injury detection reagent in the target kidney region and the normal kidney region, and / or, the analysis module is used to compare the enzyme-like activity color reaction of the renal injury detection reagent in the target urine and the normal urine.

[0059] In some alternative embodiments, the detection module includes a first detection module, a second detection module, a third detection module, and a fourth detection module.

[0060] Among them. The first detection module is used to detect the fluorescence intensity and its change rate of the renal injury detection reagent in the target kidney region; the second detection module is used to detect the fluorescence intensity and its change rate of the renal injury detection reagent in the normal kidney region; the third detection module is used to detect the enzyme-like activity color reaction of the renal injury detection reagent in the target urine; the fourth detection module is used to detect the enzyme-like activity color reaction of the renal injury detection reagent in the normal urine.

[0061] In some alternative embodiments, the analysis module includes a first analysis module and a second analysis module.

[0062] Among them, the first analysis module is used to compare the fluorescence intensity and its change rate of the renal injury detection reagent in the target kidney region and the normal kidney region; the second analysis module is used to compare the enzyme-like activity color reaction of the renal injury detection reagent in the target urine and the normal urine.

[0063] It should be noted that the fluorescence intensity and its change rate of the renal injury detection reagent in the above-mentioned normal kidney region and the enzyme-like activity color reaction of the renal injury detection reagent in the normal urine can be carried out only once, and the results can be directly used in subsequent applications. For different target kidneys, the fluorescence intensity, its change rate, and the enzyme-like activity color reaction need to be carried out for each target kidney.

[0064] In addition, the present invention also provides a preparation method of the above-mentioned renal injury detection reagent, including the following steps: reducing the gold-sulfur coordination complex formed by the gold source and the thiol compound under alkaline conditions.

[0065] In some alternative embodiments, the preparation of the gold-sulfur coordination complex includes: performing a first vortex stirring on the gold source solution and the thiol compound solution.

[0066] Among them, the concentration of the gold source solution can be 1 mmol / L to 100 mmol / L, such as 1 mmol / L, 5 mmol / L, 10 mmol / L, 20 mmol / L, 50 mmol / L, 80 mmol / L, or 100 mmol / L, etc.; the concentration of the thiol compound solution can be 0.5 mmol / L to 50 mmol / L, such as 0.5 mmol / L, 1 mmol / L, 5 mmol / L, 10 mmol / L, 15 mmol / L, 20 mmol / L, 25 mmol / L, 30 mmol / L, 35 mmol / L, 40 mmol / L, 45 mmol / L, or 50 mmol / L, etc.; the volume ratio of the gold source solution to the thiol compound solution can be 1:1 to 1:10, such as 1:1, 1:2, 1:5, 1:8, or 1:10, etc. The time of the first vortex stirring can be 5 min to 60 min, such as 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, or 60 min, etc.

[0067] In some relatively typical embodiments, the concentration of the gold source solution can be 10 mmol / L, the concentration of the thiol compound solution can be 3 mmol / L; the volume ratio of the gold source solution to the thiol compound solution can be 1:3. The time of the first vortex stirring can be 10 min.

[0068] Exemplarily, the above-mentioned gold source may include chloroauric acid.

[0069] In some alternative embodiments, the reduction reaction includes: subjecting the solution of the gold-sulfur coordination complex to a second vortex stirring with a reducing agent solution under alkaline conditions.

[0070] Among them, the alkaline conditions are provided by an alkaline solution, and the alkaline solution may include a sodium hydroxide solution with a concentration of 0.5 mol / L to 5 mol / L (such as 0.5 mol / L, 1 mol / L, 2 mol / L, 3 mol / L, 4 mol / L or 5 mol / L, etc.). The reducing agent solution may include a sodium borohydride solution with a concentration of 0.05 mol / L to 0.5 mol / L (such as 0.05 mol / L, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L or 0.5 mol / L); the time of the second vortex stirring may be 1 h to 10 h (such as 1 h, 2 h, 5 h, 8 h or 10 h, etc.).

[0071] In some relatively typical embodiments, the alkaline solution may include a sodium hydroxide solution with a concentration of 1 mol / L. The reducing agent solution may include a sodium borohydride solution with a concentration of 0.1 mol / L; the volume ratio of the solution of the gold-sulfur coordination complex to the alkaline solution and the reducing agent solution may be 40:3:1. The time of the second vortex stirring may be 4 h.

[0072] Continuing from the above, by first adding an alkaline solution and then sodium borohydride to the gold-sulfur coordination complex, the gold ions in chloroauric acid are reduced to gold atoms, and the gold atoms gradually form gold clusters through the interaction between gold-gold. Mercapto-β-cyclodextrin forms a coordination bond with the surface of the gold cluster through the sulfur atom of the mercapto group, stabilizing the gold cluster, preventing excessive aggregation between gold atoms, and helping to control the size and dispersibility of the gold cluster.

[0073] Furthermore, the reaction mixture obtained from the reduction reaction is dialyzed to remove excess gold source, mercapto-β-cyclodextrin, alkaline substances and reducing agent.

[0074] In some alternative embodiments, the number of dialysis times may be 3 times exemplarily, and each time may be 2 h. Substances with a molecular weight cut-off of 3000 Da are retained during the dialysis process.

[0075] After dialysis, the dialysis product is concentrated (such as ultrafiltration concentration), and the concentration of the obtained gold cluster nanoparticles after concentration may be 7 mg / mL.

[0076] The features and properties of the present invention will be further described in detail below in conjunction with examples.

[0077] The following mercapto-β-cyclodextrin, glutathione, and 3-mercaptopropionic acid (S30728) were all purchased from commercially available products, specifically from products S29877, S52684, and S30728 of Shanghai Yuanye Bio-Technology Co., Ltd.

[0078] Example 1

[0079] This example provides a renal injury detection reagent (mercapto-β-cyclodextrin capped gold clusters, abbreviated as "CD-GC"), and its preparation method is as follows: Take 1 mL of chloroauric acid aqueous solution with a concentration of 10 mmol / L and 3 mL of mercapto-β-cyclodextrin aqueous solution with a concentration of 3 mmol / L, and perform the first vortex stirring for 10 min; after the reaction is sufficient, add 0.3 mL of sodium hydroxide aqueous solution with a concentration of 1 mol / L and 0.1 mL of sodium borohydride aqueous solution with a concentration of 0.1 mmol / L in sequence, and perform the second vortex stirring for 4 h. After the stirring ends, dialyze 3 times with a dialysis bag with a molecular weight cut-off of 3000 Da, 2 h each time. After dialysis, use an ultrafiltration tube with a cut-off of 3000 Da to ultrafilter and concentrate the gold clusters, and take half of the concentrated gold clusters and freeze-dry them to determine the concentration (7 mg / mL).

[0080] Example 2

[0081] This example provides a renal injury detection reagent (mercapto-β-cyclodextrin capped gold clusters, abbreviated as "CD-GC"), and its preparation method is as follows: Take 1 mL of chloroauric acid aqueous solution with a concentration of 1 mmol / L and 3 mL of mercapto-β-cyclodextrin aqueous solution with a concentration of 0.5 mmol / L, and perform the first vortex stirring for 5 min; after the reaction is sufficient, add 0.3 mL of sodium hydroxide aqueous solution with a concentration of 0.5 mol / L and 0.1 mL of sodium borohydride aqueous solution with a concentration of 0.05 mmol / L in sequence, and perform the second vortex stirring for 1 h. After the stirring ends, dialyze 3 times with a dialysis bag with a molecular weight cut-off of 3000 Da, 2 h each time. After dialysis, use an ultrafiltration tube with a cut-off of 3000 Da to ultrafilter and concentrate the gold clusters, and take half of the concentrated gold clusters and freeze-dry them to determine the concentration (7 mg / mL).

[0082] Example 3

[0083] This example provides a renal injury detection reagent (mercapto-β-cyclodextrin capped gold clusters, abbreviated as "CD-GC"), and its preparation method is as follows: Take 1 mL of chloroauric acid aqueous solution with a concentration of 100 mmol / L and 3 mL of mercapto-β-cyclodextrin aqueous solution with a concentration of 50 mmol / L, and perform the first vortex stirring for 60 min; after the reaction is sufficient, add 0.3 mL of sodium hydroxide aqueous solution with a concentration of 5 mol / L and 0.1 mL of sodium borohydride aqueous solution with a concentration of 0.5 mmol / L in sequence, and perform the second vortex stirring for 10 h. After the stirring is completed, dialyze 3 times with a 3000 Da molecular weight dialysis bag, 2 h each time. After dialysis, use an ultrafiltration tube with a cut-off molecular weight of 3000 Da to ultrafilter and concentrate the gold clusters. Take half of the concentrated gold clusters and lyophilize them to determine the concentration (7 mg / mL).

[0084] Comparative Example 1

[0085] This comparative example provides a glutathione capped gold cluster (abbreviated as "GSH-GC"), and its preparation method is as follows: Take 2 mL of chloroauric acid aqueous solution with a concentration of 1.5 mmol / L and 10 mL of glutathione aqueous solution with a concentration of 15 mmol / L, and perform the first vortex stirring for 10 min; after the reaction is sufficient, add 2 mL of sodium borohydride aqueous solution with a concentration of 6 mg / mL and allow the reaction to proceed for 24 h. In order to obtain pure GSH-GC, dialyze the product in water and purify it using a 3 kDa ultrafiltration centrifugal filter at 4000 rpm, and repeat each process three times.

[0086] Comparative Example 2

[0087] This comparative example provides a 3-mercaptopropionic acid capped gold cluster (abbreviated as "MPA-GC"), and its preparation method is as follows: Mix 1 mL of chloroauric acid aqueous solution with a concentration of 5 mmol / L and 1.6 mL of 3-mercaptopropionic acid aqueous solution with a concentration of 7.5 mmol / L and stir for 5 min. Subsequently, add 0.3 mL of NaOH aqueous solution with a concentration of 1 mol / L and 0.1 mL of sodium borohydride aqueous solution with a concentration of 4.3 mg / mL in sequence, and react for 6 h under dark conditions. In order to obtain pure MPA-GC, dialyze the product in water and purify it using 3 kDa and 10 kDa ultrafiltration filters at 4000 rpm, and repeat each process three times.

[0088] Test Example 1

[0089] Perform cytotoxicity and photostability tests on the gold cluster materials prepared in Example 1 and Comparative Examples 1-2.

[0090] (1) The cytotoxicity test method is as follows: The gold cluster materials prepared in Example 1 and Comparative Examples 1-2 were added to the cell culture medium, and the gold cluster concentrations were 1500 μg / mL, 750 μg / mL, 350 μg / mL, 175 μg / mL, and 87.5 μg / mL, respectively. Each gold cluster material was cultured with HEK-293T cells at the above different concentrations in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and incubated in a humidified culture environment at 37 °C and 5% CO2 in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin for 24 h, and then the CCK-8 experiment was carried out. The CCK-8 experiment method is as follows: After the cells were incubated in different concentrations of gold cluster materials for 24 h, the cells were washed three times with PBS, and then CCK-8 reagent was added and incubated for about 30 minutes. Finally, the percentage of cell viability was calculated by measuring the absorbance at 450 nm. At the same time, PBS was used as a control, and the results are as Figure 1 shown. Figure 1 In " Figure 1 ", "ns" means p > 0.05; "****" means p < 0.0001, the same below.

[0091] It can be seen from Figure 1 that: No obvious cytotoxicity was found in the CD-GC provided in Example 1 and GSH-G provided in Comparative Example 1 at both low and high concentrations. The MPA-GC provided in Comparative Example 2 had relatively low toxicity at low concentrations, but the cytotoxicity increased with the increase of concentration.

[0092] (2) The photostability test method is as follows: The gold cluster materials prepared in Example 1 and Comparative Examples 1-2 were irradiated with an 808 nm laser with a power of 200 W / cm 2 for 2 h. During this period, the fluorescence brightness changes of the three gold clusters were identified using a second near-infrared imaging system at different time intervals (0 min, 30 min, 60 min, and 120 min), and the results are as Figure 2 shown.

[0093] It can be seen from Figure 2 that: The CD-GC provided in Example 1 and the MPA-GC provided in Comparative Example 2 have good photostability, while the GSH-GC provided in Comparative Example 1 has poor photostability.

[0094] Combined with Figure 1 and Figure 2 : The gold cluster nanomaterial provided in Example 1 of the present invention can have both good photostability and biocompatibility (low cytotoxicity).

[0095] Test Example 2

[0096] (1) The gold cluster nanomaterials provided in Example 1 were used for the detection of prerenal acute kidney injury. For the prerenal acute kidney injury model: an ischemia-reperfusion acute kidney injury model was adopted. C57BL / 6J mice aged 10 - 12 weeks were used. Under anesthesia, the renal artery was clamped with a surgical clamp for 30 min and then reperfusion blood flow was restored. For early prerenal acute kidney injury, the reperfusion time was 2 h, and for late prerenal acute kidney injury, the reperfusion time was extended to 24 h.

[0097] A. Detection of early prerenal acute kidney injury: For the detection of early renal acute injury, normal mice (control group) and mice with early ischemia-reperfusion acute kidney injury model (early prerenal acute kidney injury group) were injected with gold cluster nanomaterials at a concentration of 7 mg / mL via the tail vein. Subsequently, near-infrared second-region imaging was immediately performed on the mice, and the urine of the mice was collected for the chromogenic reaction of catalase-like substances.

[0098] B. Detection of late prerenal acute kidney injury: For the detection of late renal acute injury, normal mice and mice with late ischemia-reperfusion acute kidney injury model (late prerenal acute kidney injury group) were injected with gold cluster nanomaterials at a concentration of 7 mg / mL via the tail vein. Subsequently, near-infrared second-region imaging was immediately performed on the mice, and the urine of the mice was collected for the chromogenic reaction of catalase-like substances.

[0099] The imaging conditions for the above near-infrared second-region imaging were a long-pass filter of 1000 nm, an exposure time of 500 ms - 1000 ms, an excitation laser of 808 nm, and a power of 200 mW / cm 2 . The urine of the mice collected was the urine 1 h after injecting the gold cluster nanomaterials. The detection results are as shown in Figure 3 and Figure 4 .

[0100] From Figure 3 the near-infrared second-region imaging results, it can be seen that: the real-time fluorescence intensity of the renal region of mice with early prerenal acute kidney injury is significantly lower than that of normal mice; the real-time fluorescence intensity of the renal region of mice with late prerenal acute kidney injury is significantly lower than that of normal mice.

[0101] From Figure 4 the chromogenic reaction results, it can be seen that: the chromogenic reaction of the urine of mice with early prerenal acute kidney injury is significantly weaker than that of the urine collected from normal mice; the chromogenic reaction of the urine of mice with late prerenal acute kidney injury is significantly weaker than that of the urine collected from normal mice.

[0102] (2) The serum creatinine and serum urea nitrogen of the normal mice (control group), mice with early prerenal acute kidney injury (early group), and mice with late prerenal acute kidney injury (late group) in (1) above were detected by currently commonly used detection methods (serum creatinine combined with serum urea nitrogen detection). The results are as shown in Figure 5as shown in (a) and (b) in

[0103] It can be seen from Figure 5 that this detection method cannot detect early pre-renal acute kidney injury.

[0104] Combined with Figures 3 to 5 , it shows that the gold cluster nanomaterials provided in Example 1 of the present invention can detect acute kidney injury in real time, rapidly and accurately at the early stage of pre-renal acute kidney injury, while the currently commonly used detection methods (serum creatinine and serum urea nitrogen) cannot detect early pre-renal acute kidney injury.

[0105] The reason for the above differences is as follows: Ischemic reperfusion injury acute kidney injury belongs to pre-renal acute kidney injury, and its main pathological characteristics are a sharp decrease in renal blood flow and a decrease in glomerular filtration rate. And at the early stage of acute kidney injury, the renal blood flow and glomerular filtration rate of the kidney have changed, but the metabolic wastes in the blood such as creatinine and urea nitrogen have not had time to accumulate, so serum creatinine and urea nitrogen cannot be used for detection. And the gold cluster nanomaterials are mainly transported to the kidney through the blood and excreted through the urine in the form of the gold cluster nanomaterial prototype. Therefore, when pre-renal acute kidney injury occurs, the renal blood flow is damaged, and the gold cluster nanoparticles flowing through the kidney in the blood decrease, so the result of weakened fluorescence intensity in the renal area appears. The present invention intuitively monitors the metabolic process of the gold cluster nanomaterials in the kidney through near-infrared second-region fluorescence imaging, thereby reflecting the renal blood flow and glomerular filtration rate through the change of fluorescence intensity, and screening and judging early acute kidney injury by comparing with the fluorescence intensity change in the renal area of normal mice. In addition, the gold cluster nanomaterials used in the present invention have catalase-like activity, so the gold cluster nanomaterial prototype in the urine after passing through the kidney also has catalase-like activity. By collecting the urine and using 3,3',5,5'-tetramethylbenzidine color reaction, the amount of gold cluster nanomaterials metabolized through the urine per unit time can be intuitively reflected, and this method can also effectively reflect the glomerular filtration rate.

[0106] Test Example 3

[0107] The gold cluster nanomaterials provided in Example 1 were used for the detection of renal acute kidney injury. For the renal acute kidney injury model: a cisplatin-induced acute kidney injury model was used, and C57BL / 6J mice aged 10 - 12 weeks were intraperitoneally injected with 20 mg / kg of cisplatin. The early renal acute kidney injury was 12 h after injection, and the late acute kidney injury was 48 h.

[0108] A. Detection of early renal acute kidney injury: For the detection of early renal acute injury, normal mice (control group) and mice with early cisplatin-induced acute kidney injury model (early renal acute kidney injury group) were injected with gold cluster nanomaterials at a concentration of 7 mg / mL via the tail vein. Subsequently, near-infrared II imaging was immediately performed on the mice, and mouse urine was collected for catalase-like chromogenic reaction.

[0109] B. Detection of late renal acute kidney injury: For the detection of late renal acute injury, normal mice (control group) and mice with late cisplatin-induced acute kidney injury model (late renal acute kidney injury group) were injected with gold cluster nanomaterials at a concentration of 7 mg / mL via the tail vein. Subsequently, near-infrared II imaging was immediately performed on the mice, and mouse urine was collected for catalase-like chromogenic reaction.

[0110] The imaging conditions for the above near-infrared II imaging were a long-pass filter of 1000 nm, an exposure time of 500 ms to 1000 ms, an excitation laser of 808 nm, and a power of 200 mW / cm 2 . The mouse urine collected was the urine 1 h after injection of the nanogold cluster material. The detection results are as Figure 6 and Figure 7 shown.

[0111] From Figure 6 the near-infrared II imaging results, it can be seen that the real-time renal region fluorescence intensity of mice with early renal acute kidney injury is significantly higher than that of normal mice; the real-time renal region fluorescence intensity of mice with late renal acute kidney injury is significantly higher than that of normal mice.

[0112] From Figure 7 the chromogenic reaction results, it can be seen that the chromogenic reaction of urine from mice with early renal acute kidney injury is significantly weaker than that of urine collected from normal mice; the chromogenic reaction of urine from mice with late renal acute kidney injury is significantly weaker than that of urine collected from normal mice.

[0113] Combined with Figures 6 to 7 , it shows that the gold cluster nanomaterials provided in Example 1 of the present invention can be used for real-time, rapid, and accurate detection in the early and late stages of renal acute kidney injury.

[0114] Test Example 4

[0115] This test example is different from the early prerenal acute kidney injury model in Test Example 2. In this test example, the bilateral renal arteries were clamped unilaterally during modeling, and the subsequent modeling operations were the same as those in Test Example 2. After injecting the gold cluster nanomaterials provided in Example 1 into the model mice, near-infrared II imaging was immediately performed on the mice, and mouse urine was collected for catalase-like chromogenic reaction. The detection conditions were also the same as those in Test Example 2, and the detection results are as Figure 8 and Figure 9 shown.

[0116] From Figure 8 the near-infrared second near-infrared imaging results, it can be seen that: the fluorescence intensity in the renal area on the side of pre-renal acute kidney injury is significantly reduced, while the fluorescence intensity of the contralateral normal kidney increases. From Figure 9 the color reaction results, it can be seen that: there is no significant difference in the urine color reaction results, and the reason is that the contralateral kidney can metabolize normally.

[0117] Test Example 5

[0118] In this test example, the economic benefits of the product were calculated. Taking the gold cluster nanomaterial provided in Example 1 as an example, its reaction yield was about 90%. The price of chloroauric acid trihydrate was 835.52 yuan / g, and the price of β-cyclodextrin-capped mercapto compound was 1027.98 yuan / g. If only considering the amount of gold clusters used in the urine color reaction, the amount used for a single mouse was about 0.1 mg of gold cluster nanomaterial, and the total price was about 0.15 yuan. Through specific surface area conversion, the amount used for an adult male with a body weight of 60 kg was expanded by about 2000 times, that is, the cost of gold cluster nanomaterial for a single person was about 150 yuan, indicating that the detection cost of the metal nanomaterial provided by the present invention is low and has strong promotion and practicality.

[0119] In summary, the kidney injury detection reagent provided by the present invention can realize the strategy of early, rapid, effective, real-time and low-cost detection of acute kidney injury, and reveal the subtle changes in the early stage of acute kidney injury.

[0120] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A reagent for detecting kidney injury, characterized in that, The renal injury detection reagent includes gold cluster nanomaterials, and the gold cluster nanomaterials are gold cluster nanoparticles capped with mercapto-β-cyclodextrin; The particle size of the gold cluster nanomaterials is 1.9 nm to 2.11 nm; The gold cluster nanomaterials are used for near-infrared second-region fluorescence imaging in the kidney area and / or for enzyme-like activity color reaction in urine.

2. The renal injury detection reagent according to claim 1, characterized in that, The gold cluster nanomaterials are used for the combined detection of near-infrared second-region fluorescence imaging and enzyme-like activity color reaction in the kidney area and urine.

3. The kidney injury detection reagent according to claim 1 or 2, characterized in that The near-infrared second-zone fluorescence imaging includes: using a long-pass filter above 1000nm, an exposure time of 500ms to 1000ms, an excitation laser of 808nm, and a power of 200mW / cm 2 .

4. The renal injury detection reagent according to claim 1 or 2, characterized in that, The color reagent used for the enzyme-like activity color reaction includes 3,3',5,5'-tetramethylbenzidine.

5. A kidney injury detection device, characterized in that, The renal injury detection device includes a detection module and an analysis module; The detection module is used to detect the fluorescence intensity and its change rate of the renal injury detection reagent in the target kidney area and normal kidney; and / or, the detection module is used to detect the enzyme-like activity color reaction of the renal injury detection reagent in the target urine and normal urine; The analysis module is used to compare the fluorescence intensity and its change rate of the renal injury detection reagent in the target kidney area and normal kidney area, and / or, the analysis module is used to compare the enzyme-like activity color reaction of the renal injury detection reagent in the target urine and normal urine; The renal injury detection reagent is the renal injury detection reagent according to any one of claims 1 to 4.

6. The renal injury detection device according to claim 5, wherein, The detection module includes a first detection module, a second detection module, a third detection module and a fourth detection module; The first detection module is used to detect the fluorescence intensity and its change rate of the renal injury detection reagent in the target kidney area; the second detection module is used to detect the fluorescence intensity and its change rate of the renal injury detection reagent in the normal kidney area; The third detection module is used to detect the enzyme-like activity color reaction of the renal injury detection reagent in the target urine; the fourth detection module is used to detect the enzyme-like activity color reaction of the renal injury detection reagent in the normal urine.

7. The kidney injury detection device according to claim 5, wherein, The analysis module includes a first analysis module and a second analysis module; The first analysis module is used to compare the fluorescence intensity and its change rate of the renal injury detection reagent in the target kidney area and normal kidney area; The second analysis module is used to compare the enzyme-like activity color reaction of the renal injury detection reagent in the target urine and normal urine.

8. A method for preparing a renal injury detection reagent according to any one of claims 1 to 4, characterized in that, It includes the following steps: Performing a reduction reaction on the gold-sulfur coordination complex formed by gold source and mercapto under alkaline conditions; Preferably, it further includes: dialyzing the reaction mixture obtained from the reduction reaction.

9. The preparation method according to claim 8, wherein The preparation of the gold-sulfur coordination complex includes: performing a first vortex stirring on the gold source solution and the mercapto-β-cyclodextrin solution; Wherein, the concentration of the gold source solution is 1 mmol / L to 100 mmol / L, the concentration of the mercapto-β-cyclodextrin solution is 0.5 mmol / L to 50 mmol / L; the molar ratio of the gold source solution to the mercapto-β-cyclodextrin solution is 1:1 to 1:10; the time of the first vortex stirring is 5 min to 60 min; Preferably, the gold source is chloroauric acid.

10. The preparation method according to claim 8, characterized in that, The reduction reaction includes: subjecting the solution of the gold-sulfur coordination complex to a second vortex stirring with a reducing agent solution under alkaline conditions; Wherein, the alkaline conditions are provided by an alkaline solution; the alkaline solution includes a sodium hydroxide solution with a concentration of 0.5 mol / L to 5 mol / L; the reducing agent solution includes a sodium borohydride solution with a concentration of 0.05 mol / L to 0.5 mol / L; the time of the second vortex stirring is 1 h to 10 h.

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