CN105241943A - Preparation and applications of electrochemical immunosensor used for detecting circulating tumor cells - Google Patents
Preparation and applications of electrochemical immunosensor used for detecting circulating tumor cells Download PDFInfo
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- CN105241943A CN105241943A CN201510619623.1A CN201510619623A CN105241943A CN 105241943 A CN105241943 A CN 105241943A CN 201510619623 A CN201510619623 A CN 201510619623A CN 105241943 A CN105241943 A CN 105241943A
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Abstract
The invention discloses a sensor used for detecting circulating tumor cells via differential pulse voltammetry. A preparation method of the sensor comprises following steps: vanadium pentoxide nanowires and gold-silver core-shell nanoparticles are prepared via a conventional method; the vanadium pentoxide nanowires and gold-silver core-shell nanoparticles are combined so as to modify aptamers; the surface of an electrode is modified with gold nanoparticles, and then the modified electrode is connected with the vanadium pentoxide nanowire gold-silver core-shell nanoparticle modified thiolation aptamers; the obtained electrochemical sensor is used for detecting circulating tumor cells via combination with an electrochemical workstation; and the electrochemical workstation is used for detecting. The sensor is high in specificity and sensitivity; operation is simple; and detection limit is low.
Description
Technical field
The present invention relates to Cell Measurement Technique field, be a kind of by semiconductor catalysis generation reduction current in particular, alloy strengthens reduction current, thus detects circulating tumor cell number, belongs to the technology of preparing of overdelicate electro-chemical cells immunosensor.
Background technology
In recent years, immunoassay is the clinical means of an important tumor disease in clinical examination.All the time, malignant tumour i.e. cancer are serious threats of human health and life security.Cancer cell, refers to that cell that some have division potential there occurs vicious transformation and Clonal hyperplasia and a kind of neoformation of producing under the effect of carcinogenic factor.And cancer cell is except self growth is out of control, and arround also invading, normal structure even can transfer to other parts of health via the circulation system in body or lymphatic system, thus causes health pathology even dead.So the diagnosis that cancer is early stage and treatment early have great importance.But early stage lower being difficult to of cancer cell content of cancer is found, and the mensuration of the cancer cell of low content becomes the important pursuit of analyst.
Immunoassay mainly measures antigen (or antibody) and haptens as selective chemical reagent to analyze based on antibody (or antigen).Its proposition and development are that one of greatest achievement of bioanalytical chemistry estimates that the whole world will carry out the immunoassay of several hundred million times every year.In immunoassay development fast, apply wide first-elected radio immunoassay (RIA), RIA major advantage is, automation equipment, and easy and simple to handle, highly sensitive, sample preparation is simple; Meanwhile, owing to there is not radioactive materials in clinical sample, thus interference is few.But, RIA needs to carry out hot operation, instrument price is expensive again, this just excites the immune analysis method that scientists makes great efforts to explore nonradioactive labeling, has developed the methods such as enzyme-linked immuno assay (ELISA), luminescence immunoassay (LIA), electrochemical immunoanalytical (ECIA) rapidly.But these immune analysis methods are generally consuming time longer and have the operating process such as loaded down with trivial details application of sample, incubation, washing, and operating personnel are more with contacting of immune substance, may work the mischief to the healthy of personnel of operation.And some finding speed is fast, highly sensitive, the low detection means of detection property needs expensive equipment, can not popularize in developing country.
Summary of the invention
The technical problem to be solved in the present invention there is provided a kind of highly sensitive, detection speed is fast, reagent dosage is few, detects the electrochemical immunosensor of circulating tumor cell.
In order to solve the problems of the technologies described above, the present invention is realized by following measures: a kind of preparations and applicatio method detecting the electrochemical sensor of circulating tumor cell, and it comprises the following steps:
(1) vanadium pentoxide nanowires is prepared according to existing method, gold and silver core-shell nano;
(2) vanadium pentoxide nanowires-gold and silver core-shell nano is combined modification aptamers;
(3) golden nanometer particle is modified in electrode surface, the aptamers that rear connection is Thiolation, specific binding circulating tumor cell, then connect the aptamers of vanadium pentoxide nanowires-gold and silver core-shell nano modification;
(4) the electrochemical sensor combined with electrochemical workstation made is detected circulating tumor cell.
Vanadium pentoxide nanowires of the present invention-gold and silver core-shell nano combines the preparations and applicatio modifying aptamers and electrochemical sensor and comprises the following steps:
(1) preparation of vanadium pentoxide nanowires: it is characterized in that water miscible vanadic sulfate at room temperature to mix with oxygenant potassium bromate, magnetic stirring reaction 30min, acidity is reduced afterwards by nitric acid, mixture aqueous solution is reacted 24h with 180 DEG C in autoclave, take out autoclave and be cooled to room temperature, intermediate water repeatedly rinses to obtain thick yellow precipitate, will be deposited in dry for standby in 80 DEG C of baking ovens;
(2) preparation of golden nanometer particle storing solution: it is characterized in that the gold chloride of 1.25mL10mM to add 42.65mL intermediate water, be placed in three mouthfuls of round-bottomed flasks of 100mL capacity, 30min is heated when magnetic stirs, after add the sodium citrate of 2.5mL1%, in 10min, solution first becomes pewter from yellow, finally becomes soft pink colour, and solution is through centrifugal and secondary washing, after be scattered in again in intermediate water, obtain golden nanometer particle storing solution;
(3) preparation of gold and silver core-shell nano: it is characterized in that 7mL(2) the golden nanometer particle storing solution prepared mixes with 0.65mL0.1% polyvinylpyrrolidone, magnetic stirs 20min, after add the beta-schardinger dextrin-of 1.5mL0.05mM and the halfcystine of 1.5mL0.05mM, magnetic stir a few minutes mixing.Add sodium hydroxide solution hatching 10min and adjust pH to 10, by mixed solution 85 ° of C water-baths.Rear magnetic continuously stirs 30min, and dropwise add the liquor argenti nitratis ophthalmicus of 3mL0.1mM, in dropping process, solution becomes glassy yellow from pink colour gradually, obtains gold and silver core-shell nano;
(4) preparation of vanadium pentoxide nanowires-gold and silver core-shell nano: it is characterized in that vanadium pentoxide nanowires prepared by (1) being got 10mg is dissolved in 1mL intermediate water, add the chlorination hexadiene dimethyl amine solution of 1%, ultrasonic 30min, centrifugal washing 4 times, after add the gold and silver core-shell nano 0.5mL (3) prepared, magnetic stirs 1h, and pelleting centrifugation washing is repeatedly constant to color, finally obtains vanadium pentoxide nanowires-gold and silver core-shell nano;
(5) aptamers that vanadium pentoxide nanowires-gold and silver core-shell nano is modified is prepared: the pH to 9 that it is characterized in that first being adjusted by the NaOH of 0.1M vanadium pentoxide nanowires-gold and silver core-shell nano, after add 5 μMs of Thiolation aptamers of 50 μ L, mixed solution magnetic under 4 ° of C conditions stirs 12h, wash by phosphate buffered solution afterwards, discard supernatant liquor, will be precipitated and dissolved in containing 1mMCa
2+, 1mMMn
2+with in the incubation buffer of 0.1% bovine serum albumin(BSA), 4 ° of C transfer use of purchasing;
(6) modification of glass-carbon electrode: it is characterized in that the gold chloride cleaned glass-carbon electrode being inserted 2mL1mM, electro-deposition 40s under-0.2V voltage, golden nanometer particle in modification, phosphate buffered solution is rinsed, and dries for subsequent use;
(7) glass-carbon electrode that (6) have been modified is connected Thiolation aptamers, it is characterized in that encapsulating site by 6-sulfydryl-1-hexanol, then circulating tumor cell is caught, connect the aptamers that vanadium pentoxide nanowires-gold and silver core-shell nano is modified, electrode inserts and comprises in the 0.01M phosphate buffered solution of 25 μMs of thionines, detects reduction current obtain result by differential pulse voltammetry.
The phosphate buffered solution that the wash solution used in the present invention is pH7.4.
Circulating tumor cell of the present invention is breast cancer cell (MCF-7 and T47D).
Beneficial effect of the present invention:
(1) the present invention utilizes differential pulse voltammetry to measure, and operation is simple fast, and reaction and result complete and record automatically by instrument, avoid the impact of subjective factor, and ensure good repeatability;
(2) utilize biologically active to stablize, vanadium pentoxide that catalytic property is good as catalyzer, and vanadium pentoxide can under hydrogen peroxide existent condition catalysis small molecule dyes, produce reduction current;
(3) gold and silver core-shell nano has high conductivity, is exaggerated reduction current simultaneously, plays amplifying signal effect.
Accompanying drawing explanation
Figure 1 shows that process flow diagram of the present invention.
A is depicted as golden nanometer particle; B is depicted as aptamers; C is depicted as circulating tumor cell; D is depicted as vanadium pentoxide nanowires; E is depicted as gold and silver core-shell nano.
Embodiment
Detect a preparations and applicatio method for the electrochemical sensor of circulating tumor cell, it comprises the following steps:
(1) vanadium pentoxide nanowires is prepared according to existing method, gold and silver core-shell nano;
(2) vanadium pentoxide nanowires-gold and silver core-shell nano is combined modification aptamers;
(3) golden nanometer particle is modified in electrode surface, the aptamers that rear connection is Thiolation, specific binding circulating tumor cell, then connect the aptamers of vanadium pentoxide nanowires-gold and silver core-shell nano modification;
(4) the electrochemical sensor combined with electrochemical workstation made is detected circulating tumor cell.
Vanadium pentoxide nanowires of the present invention-gold and silver core-shell nano combines the preparations and applicatio modifying aptamers and electrochemical sensor and comprises the following steps:
(1) preparation of vanadium pentoxide nanowires: it is characterized in that water miscible vanadic sulfate at room temperature to mix with oxygenant potassium bromate, magnetic stirring reaction 30min, acidity is reduced afterwards by nitric acid, mixture aqueous solution is reacted 24h with 180 DEG C in autoclave, take out autoclave and be cooled to room temperature, intermediate water repeatedly rinses to obtain thick yellow precipitate, will be deposited in dry for standby in 80 DEG C of baking ovens;
(2) preparation of golden nanometer particle storing solution: it is characterized in that the gold chloride of 1.25mL10mM to add 42.65mL intermediate water, be placed in three mouthfuls of round-bottomed flasks of 100mL capacity, 30min is heated when magnetic stirs, after add the sodium citrate of 2.5mL1%, in 10min, solution first becomes pewter from yellow, finally becomes soft pink colour, and solution is through centrifugal and secondary washing, after be scattered in again in intermediate water, obtain golden nanometer particle storing solution;
(3) preparation of gold and silver core-shell nano: it is characterized in that 7mL(2) the golden nanometer particle storing solution prepared mixes with 0.65mL0.1% polyvinylpyrrolidone, magnetic stirs 20min, after add the beta-schardinger dextrin-of 1.5mL0.05mM and the halfcystine of 1.5mL0.05mM, magnetic stir a few minutes mixing.Add sodium hydroxide solution hatching 10min and adjust pH to 10, by mixed solution 85 ° of C water-baths.Rear magnetic continuously stirs 30min, and dropwise add the liquor argenti nitratis ophthalmicus of 3mL0.1mM, in dropping process, solution becomes glassy yellow from pink colour gradually, obtains gold and silver core-shell nano;
(4) preparation of vanadium pentoxide nanowires-gold and silver core-shell nano: it is characterized in that vanadium pentoxide nanowires prepared by (1) being got 10mg is dissolved in 1mL intermediate water, add the chlorination hexadiene dimethyl amine solution of 1%, ultrasonic 30min, centrifugal washing 4 times, after add the gold and silver core-shell nano 0.5mL (3) prepared, magnetic stirs 1h, and pelleting centrifugation washing is repeatedly constant to color, finally obtains vanadium pentoxide nanowires-gold and silver core-shell nano;
(5) aptamers that vanadium pentoxide nanowires-gold and silver core-shell nano is modified is prepared: the pH to 9 that it is characterized in that first being adjusted by the NaOH of 0.1M vanadium pentoxide nanowires-gold and silver core-shell nano, after add 5 μMs of Thiolation aptamers of 50 μ L, mixed solution magnetic under 4 ° of C conditions stirs 12h, wash by phosphate buffered solution afterwards, discard supernatant liquor, will be precipitated and dissolved in containing 1mMCa
2+, 1mMMn
2+with in the incubation buffer of 0.1% bovine serum albumin(BSA), 4 ° of C transfer use of purchasing;
(6) modification of glass-carbon electrode: it is characterized in that the gold chloride cleaned glass-carbon electrode being inserted 2mL1mM, electro-deposition 40s under-0.2V voltage, golden nanometer particle in modification, phosphate buffered solution is rinsed, and dries for subsequent use;
(7) glass-carbon electrode that (6) have been modified is connected Thiolation aptamers, site is encapsulated by 6-sulfydryl-1-hexanol, then circulating tumor cell is caught, connect the aptamers that vanadium pentoxide nanowires-gold and silver core-shell nano is modified, electrode inserts and comprises in the 0.01M phosphate buffered solution of 25 μMs of thionines, detects reduction current obtain result by differential pulse voltammetry.
embodiment 1(breast cancer cell, MCF-7)
(1) select easily to measure with the MCF-7 of lymphatic system transfer; :
(2) preparation of vanadium pentoxide nanowires: water miscible vanadic sulfate is at room temperature mixed with oxygenant potassium bromate, magnetic stirring reaction 30min, acidity is reduced afterwards by nitric acid, mixture aqueous solution is reacted 24h with 180 DEG C in autoclave, take out autoclave and be cooled to room temperature, intermediate water repeatedly rinses to obtain thick yellow precipitate, will be deposited in dry for standby in 80 DEG C of baking ovens;
(3) gold chloride of the preparation of golden nanometer particle storing solution: 1.25mL10mM adds 42.65mL intermediate water, be placed in three mouthfuls of round-bottomed flasks of 100mL capacity, 30min is heated when magnetic stirs, after add the sodium citrate of 2.5mL1%, in 10min, solution first becomes pewter from yellow, finally becomes soft pink colour, and solution is through centrifugal and secondary washing, after be scattered in again in intermediate water, obtain golden nanometer particle storing solution;
(4) preparation of gold and silver core-shell nano: by 7mL(2) the golden nanometer particle storing solution prepared mixes with 0.65mL0.1% polyvinylpyrrolidone, magnetic stirs 20min, after add the beta-schardinger dextrin-of 1.5mL0.05mM and the halfcystine of 1.5mL0.05mM, magnetic stir a few minutes mixing.Add sodium hydroxide solution hatching 10min and adjust pH to 10, by mixed solution 85 ° of C water-baths.Rear magnetic continuously stirs 30min, and dropwise add the liquor argenti nitratis ophthalmicus of 3mL0.1mM, in dropping process, solution becomes glassy yellow from pink colour gradually, obtains gold and silver core-shell nano;
(5) preparation of vanadium pentoxide nanowires-gold and silver core-shell nano: vanadium pentoxide nanowires prepared by (1) is got 10mg and be dissolved in 1mL intermediate water, add the chlorination hexadiene dimethyl amine solution of 1%, ultrasonic 30min, centrifugal washing 4 times, after add the gold and silver core-shell nano 0.5mL (3) prepared, magnetic stirs 1h, and pelleting centrifugation washing is repeatedly constant to color, finally obtains vanadium pentoxide nanowires-gold and silver core-shell nano;
(6) aptamers that vanadium pentoxide nanowires-gold and silver core-shell nano is modified is prepared: the pH to 9 first being adjusted vanadium pentoxide nanowires-gold and silver core-shell nano by the NaOH of 0.1M, after add the Thiolation SYL3C aptamers of 5 μMs of 50 μ L, mixed solution magnetic under 4 ° of C conditions stirs 12h, wash by phosphate buffered solution afterwards, discard supernatant liquor, will be precipitated and dissolved in containing 1mMCa
2+, 1mMMn
2+with in the incubation buffer of 0.1% bovine serum albumin(BSA), 4 ° of C transfer use of purchasing;
(7) modification of glass-carbon electrode: cleaned glass-carbon electrode inserts the gold chloride of 2mL1mM, electro-deposition 40s under-0.2V voltage, golden nanometer particle in modification, phosphate buffered solution is rinsed, and dries for subsequent use;
(8) glass-carbon electrode that (7) have been modified is connected Thiolation aptamers, site is encapsulated by 6-sulfydryl-1-hexanol, then MCF-7 cell is caught, connect the SYL3C aptamers that vanadium pentoxide nanowires-gold and silver core-shell nano is modified, electrode inserts and comprises in the 0.01M phosphate buffered solution of 25 μMs of thionines, detects reduction current obtain result by differential pulse voltammetry.
Detect circulating tumor cell MCF-7, the range of linearity of this electrochemical immunosensor is 50cells mL
-1~ 1 × 10
7cells mL
-1, detect and be limited to 31cells mL
-1.
embodiment 2(breast cancer cell, T47D)
(1) select easily to measure with the T47D of lymphatic system transfer;
(2) preparation of vanadium pentoxide nanowires: water miscible vanadic sulfate is at room temperature mixed with oxygenant potassium bromate, magnetic stirring reaction 30min, acidity is reduced afterwards by nitric acid, mixture aqueous solution is reacted 24h with 180 DEG C in autoclave, take out autoclave and be cooled to room temperature, intermediate water repeatedly rinses to obtain thick yellow precipitate, will be deposited in dry for standby in 80 DEG C of baking ovens;
(3) gold chloride of the preparation of golden nanometer particle storing solution: 1.25mL10mM adds 42.65mL intermediate water, be placed in three mouthfuls of round-bottomed flasks of 100mL capacity, 30min is heated when magnetic stirs, after add the sodium citrate of 2.5mL1%, in 10min, solution first becomes pewter from yellow, finally becomes soft pink colour, and solution is through centrifugal and secondary washing, after be scattered in again in intermediate water, obtain golden nanometer particle storing solution;
(4) preparation of gold and silver core-shell nano: by 7mL(2) the golden nanometer particle storing solution prepared mixes with 0.65mL0.1% polyvinylpyrrolidone, magnetic stirs 20min, after add the beta-schardinger dextrin-of 1.5mL0.05mM and the halfcystine of 1.5mL0.05mM, magnetic stir a few minutes mixing.Add sodium hydroxide solution hatching 10min and adjust pH to 10, by mixed solution 85 ° of C water-baths.Rear magnetic continuously stirs 30min, and dropwise add the liquor argenti nitratis ophthalmicus of 3mL0.1mM, in dropping process, solution becomes glassy yellow from pink colour gradually, obtains gold and silver core-shell nano;
(5) preparation of vanadium pentoxide nanowires-gold and silver core-shell nano: vanadium pentoxide nanowires prepared by (1) is got 10mg and be dissolved in 1mL intermediate water, add the chlorination hexadiene dimethyl amine solution of 1%, ultrasonic 30min, centrifugal washing 4 times, after add the gold and silver core-shell nano 0.5mL (3) prepared, magnetic stirs 1h, and pelleting centrifugation washing is repeatedly constant to color, finally obtains vanadium pentoxide nanowires-gold and silver core-shell nano;
(6) aptamers that vanadium pentoxide nanowires-gold and silver core-shell nano is modified is prepared: the pH to 9 first being adjusted vanadium pentoxide nanowires-gold and silver core-shell nano by the NaOH of 0.1M, after add the Thiolation SYL3C aptamers of 5 μMs of 50 μ L, mixed solution magnetic under 4 ° of C conditions stirs 12h, wash by phosphate buffered solution afterwards, discard supernatant liquor, will be precipitated and dissolved in containing 1mMCa
2+, 1mMMn
2+with in the incubation buffer of 0.1% bovine serum albumin(BSA), 4 ° of C transfer use of purchasing;
(7) modification of glass-carbon electrode: cleaned glass-carbon electrode inserts the gold chloride of 2mL1mM, electro-deposition 40s under-0.2V voltage, golden nanometer particle in modification, phosphate buffered solution is rinsed, and dries for subsequent use;
(8) glass-carbon electrode that (7) have been modified is connected Thiolation aptamers, site is encapsulated by 6-sulfydryl-1-hexanol (MCH), then T47D cell is caught, connect the SYL3C aptamers that vanadium pentoxide nanowires-gold and silver core-shell nano is modified, electrode inserts and comprises in the 0.01M phosphate buffered solution of 25 μMs of thionines, detects reduction current obtain result by differential pulse voltammetry.
Detect cancer cell T47D, the range of linearity of this electrochemical immunosensor is 50cells mL
-1~ 1 × 10
7cells mL
-1, detect and be limited to 40cells mL
-1.
Claims (6)
1. detect a preparation method for the electrochemical sensor of circulating tumor cell, it comprises the following steps:
First, prepare vanadium pentoxide nanowires according to existing method, gold and silver core-shell nano;
Then, vanadium pentoxide nanowires-gold and silver core-shell nano is combined and modifies aptamers, the pH to 9 of vanadium pentoxide nanowires-gold and silver core-shell nano is first adjusted by the NaOH of 0.1M, after add 5 μMs of Thiolation aptamers of 50 μ L, mixed solution magnetic under 4 ° of C conditions stirs 12h, afterwards with phosphate buffered solution washing, discard supernatant liquor, will be precipitated and dissolved in containing 1mMCa
2+, 1mMMn
2+with in the incubation buffer of 0.1% bovine serum albumin(BSA), 4 ° of C transfer use of purchasing;
Finally, cleaned glass-carbon electrode is inserted the gold chloride of 2mL1mM, electro-deposition 40s under-0.2V voltage, golden nanometer particle in modification, phosphate buffered solution is rinsed, and dries for subsequent use, the aptamers that rear connection is Thiolation, encapsulate site, then specific binding circulating tumor cell by 6-sulfydryl-1-hexanol, then connect the aptamers of vanadium pentoxide nanowires-gold and silver core-shell nano modification.
2. the application process of the sensor is: detected circulating tumor cell by the electrochemical sensor combined with electrochemical workstation made.
3. according to claim 1, the preparation feature of vanadium pentoxide nanowires is, water miscible vanadic sulfate is at room temperature mixed with oxygenant potassium bromate, magnetic stirring reaction 30min, reduce acidity by nitric acid afterwards, mixture aqueous solution is reacted 24h in 180 DEG C in autoclave, take out autoclave and be cooled to room temperature, intermediate water repeatedly rinses to obtain thick yellow precipitate, is deposited in dry for standby in 80 DEG C of baking ovens.
4. according to claim 1, the preparation feature of golden nanometer particle storing solution is, the gold chloride of 1.25mL10mM is added 42.65mL intermediate water, is placed in three mouthfuls of round-bottomed flasks of 100mL capacity, heats 30min when magnetic stirs, after add the sodium citrate of 2.5mL1%, in 10min, solution first becomes pewter from yellow, finally becomes soft pink colour, and solution is through centrifugal and secondary washing, after be scattered in again in intermediate water, obtain golden nanometer particle storing solution.
5. according to claim 1, the preparation feature of gold and silver core-shell nano is, the golden nanometer particle storing solution prepared by 7mL stirs 20min with 0.65mL0.1% polyvinylpyrrolidone mixing magnetic, after add the beta-schardinger dextrin-of 1.5mL0.05mM and the halfcystine of 1.5mL0.05mM, magnetic stir a few minutes mixing; Add sodium hydroxide solution hatching 10min and adjust pH to 10, by mixed solution 85 ° of C water-baths; Rear magnetic continuously stirs 30min, and dropwise add the liquor argenti nitratis ophthalmicus of 3mL0.1mM, in dropping process, solution becomes glassy yellow from pink colour gradually, obtains gold and silver core-shell nano.
6. according to claim 1, the preparation feature of vanadium pentoxide nanowires-gold and silver core-shell nano is, the vanadium pentoxide nanowires of preparation being got 10mg is dissolved in 1mL intermediate water, add the chlorination hexadiene dimethyl amine solution of 1%, ultrasonic 30min, centrifugal washing 4 times, after add the gold and silver core-shell nano 0.5mL of preparation, magnetic stirs 1h, and pelleting centrifugation washing is repeatedly constant to color, finally obtains vanadium pentoxide nanowires-gold and silver core-shell nano.
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| CN108344783A (en) * | 2018-01-23 | 2018-07-31 | 东南大学 | A kind of electro-chemical cells sensor and its preparation method and application |
| CN109696465A (en) * | 2019-01-04 | 2019-04-30 | 中国药科大学 | A kind of circulating tumor cell detection method based on plasma-reinforced electrochemical reaction |
| CN109709174A (en) * | 2019-02-18 | 2019-05-03 | 武汉大学 | The enzyme type electrochemical sensor and the preparation method and application thereof of single core-shell nano line |
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| CN109709174A (en) * | 2019-02-18 | 2019-05-03 | 武汉大学 | The enzyme type electrochemical sensor and the preparation method and application thereof of single core-shell nano line |
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| CN105241943B (en) | 2017-12-12 |
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