CN111948183A - Detection object for determining bilirubin in blood plasma and determination method - Google Patents

Abstract

The invention discloses a detection object for measuring bilirubin in blood plasma, which is obtained by loading gold nanoclusters stabilized by bovine serum albumin on filter paper to prepare a gold nanocluster-filter paper sheet stabilized by bovine serum albumin. The detection object is convenient to carry, realizes visual observation of the content of bilirubin in blood plasma, is simple and quick, has no requirement on external conditions of detection, realizes field detection of bilirubin under the condition that detection conditions such as street blood donation are insufficient, and achieves effective control of blood plasma quality.

Description

A kind of detection substance and determination method for determining bilirubin in plasma

【Technical field】

The invention belongs to the technical field of medical detection, and particularly relates to a detection substance and a detection method for measuring bilirubin in plasma.

【Background technique】

Bilirubin is an important routine liver function measurement index. At present, the clinical methods for measuring bilirubin include diazo reagent method, chemical oxidation method, bilirubin oxidase end-point method, etc. The external conditions are required, and it is suitable for testing in the laboratory. Moreover, the detection method takes a long time, and results cannot be obtained immediately; the requirements for specimens are relatively high, and hemolyzed specimens will lead to falsely high detection values, and severe jaundice will lead to falsely low detection values; some reagents are expensive.

At present, there is no effective detection standard for blood donation on the street, only blood type and transaminase are detected. Even if it is a suspicious blood sample on the spot, there is no condition to detect bilirubin in time. After the blood sample was brought back, the bilirubin test was carried out. This increases the scrapping rate and can't remind blood donors in time.

There are three detection methods for neonatal bilirubin value: 1. venous blood biochemical method, 2. microbilirubin method, 3. percutaneous bile measurement method; the first two methods require blood collection, which have certain trauma and are not suitable for bilirubin. Dynamic monitoring of rubin; transdermal bilirubin value within a certain range is similar to serum bilirubin value for reference. But regular correction is required. It cannot be detected immediately after phototherapy, as it is not representative of serum bilirubin levels, which are greatly affected by the skin surface.

[Content of the invention]

The purpose of the present invention is to provide a detection substance and a determination method for measuring bilirubin in plasma, the detection substance is easy to carry, realizes visual observation of bilirubin content in plasma, is simple and fast, and does not require external conditions for detection , In the case of insufficient detection conditions such as street blood donation, on-site detection of bilirubin is realized, and effective control of plasma quality is achieved.

The present invention adopts the following technical scheme: a detection substance for measuring bilirubin in plasma is loaded on filter paper by bovine serum albumin-stabilized gold nanoclusters to prepare bovine serum albumin-stabilized gold nanoclusters- Filter paper, that is.

The above-mentioned preparation method of a test substance for measuring bilirubin in plasma is characterized in that, the filter paper is immersed in the bovine serum albumin-stabilized gold nanocluster solution of the test substance, and after the absorption is saturated, the filter paper is taken out, and placed in the test substance. Dry in the oven, that is.

The concentration of the above-mentioned bovine serum albumin-stabilized gold nanoclusters was 13.44 mg/mL.

The invention also discloses another test substance for measuring bilirubin in plasma, which is fixed in agarose gel by the gold nano-cluster stabilized by bovine serum albumin, encapsulated and solidified by the agarose gel, and cut into Gold nanoclusters - agarose gel sheets, that is, obtained.

The preparation method of the above-mentioned test substance for measuring bilirubin in plasma is as follows: adding bovine serum albumin-stabilized gold nanocluster solution to the melted agarose solution, obtaining a mixed solution, cooling and solidifying, and preparing a bovine serum albumin Serum albumin-stabilized gold nano-cluster-agarose gel is obtained by making the gold nano-cluster-agarose gel into a circular sheet.

In the above mixture, the concentration of the bovine serum albumin-stabilized gold nanoclusters is 13.44 mg/mL.

The above-mentioned method for measuring bilirubin in plasma by a test substance for measuring bilirubin in plasma, the method is as follows:

A standard curve was established between bilirubin concentration and the degree of fluorescence quenching of BSA-stabilized gold nanoclusters, and the linear range of bilirubin concentration was determined.

A series of standard pictures of bilirubin concentration versus fluorescence quenching of bovine serum albumin-stabilized gold nanoclusters were obtained over the linear range of bilirubin concentration.

Take 100 μL of plasma to be tested, drop it on BSA-stabilized gold nanoclusters-filter paper sheet or BSA-stabilized gold nanoclusters-agarose gel sheet, and place it at room temperature to react for 40min, and observe the wavelength of 365nm. The fluorescence quenching degree of the gold nanoclusters was compared with the standard picture, and the concentration of bilirubin in the plasma to be detected was obtained semi-quantitatively.

The specific process of obtaining a series of standard pictures above is as follows: within the linear range of bilirubin concentration, take 100 μL of a plurality of standard solutions of bilirubin with different concentrations, and drop a plurality of the standard solutions on a plurality of gold nanometers correspondingly. On the cluster-filter paper or gold nanoclusters-agarose gel sheet, put it at room temperature for 40min reaction, observe the fluorescence quenching of gold nanoclusters under 365nm wavelength ultraviolet lamp, and take pictures respectively to obtain the bilirubin linear range. A series of standard pictures of fluorescence quenching of gold nanoclusters.

The invention also discloses a method for measuring bilirubin in plasma, the method is as follows:

Step 1: Establish a standard curve between the concentration of bilirubin and the degree of fluorescence quenching of BSA-stabilized gold nanoclusters, and obtain a standard curve equation;

Step 2: Take the plasma to be detected, add bovine serum albumin-stabilized gold nanoclusters to the plasma to be detected, measure the fluorescence intensity of the plasma at this time as I', and substitute the I' into the In the standard curve equation, the concentration C' of bilirubin in the plasma to be detected is obtained.

The specific process of the above step 1 is as follows: the blank solution and multiple bilirubin standard solutions with different concentrations c are respectively added to the corresponding multiple bovine serum albumin-stabilized gold nanoclusters, respectively, and the fluorescence intensity is measured. The blank solution is: Deionized water, the fluorescence intensity of the blank solution is I ₀ , and the fluorescence intensity of the bilirubin standard solution is I, with (I ₀ -I)/I ₀ as the ordinate, and the bilirubin concentration as the abscissa to establish a standard curve to obtain The standard curve equation is (I ₀ -I)/I ₀ =0.98c-6.10.

The beneficial effects of the present invention are as follows: 1. Visual observation can be achieved by using the detection substance, and the content of bilirubin in plasma can be obtained semi-quantitatively, which is simple and fast, and has no requirements on the external conditions of detection, and is not sufficient for street blood donation and other detection conditions. Under the circumstance, on-site detection of bilirubin is realized, effective control of plasma quality is achieved, and the quality of clinical plasma is guaranteed. 2. The amount of plasma required is small, and it will not cause trauma to the skin of patients, especially newborns. 3. Using bovine serum albumin-stabilized gold nanoclusters to detect bilirubin in plasma, the method is reliable and the detection process is simple. 4 The test object is easy to carry.

【Description of drawings】

Fig. 1 is the reaction temperature optimization diagram of bilirubin and bovine serum albumin-stabilized gold nanoclusters;

Fig. 2 is the reaction time optimization diagram of bilirubin and bovine serum albumin-stabilized gold nanoclusters;

Fig. 3 is the reaction pH optimization diagram of bilirubin and bovine serum albumin-stabilized gold nanoclusters;

Figure 4 is a standard curve graph of bilirubin concentration and the degree of fluorescence quenching of BSA-stabilized gold nanoclusters;

Figure 5 is the detection chart of the control group and the sample group.

【Detailed ways】

The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

The embodiment of the present invention discloses a test substance for measuring bilirubin in plasma. The bovine serum albumin-stabilized gold nanoclusters are loaded on filter paper to prepare bovine serum albumin-stabilized gold nanoclusters-filter paper. piece, that's it.

The preparation method of the above-mentioned test substance for measuring bilirubin in plasma is as follows: soak the filter paper in the bovine serum albumin-stabilized gold nanocluster solution of the test substance, take out the filter paper after the absorption is saturated, and place it in an oven. Dry and get it. The concentration of the above-mentioned bovine serum albumin-stabilized gold nanoclusters was 13.44 mg/mL. The above-mentioned filter paper is in strip shape.

In the embodiment of the present invention, another test substance used for the determination of bilirubin in plasma is immobilized in agarose gel by bovine serum albumin-stabilized gold nanoclusters, and then encapsulated and solidified by agarose gel. It is gold nanocluster-agarose gel sheet, that is, it is obtained. The cut solidified agarose gel was a circular sheet.

The preparation method of the above-mentioned test substance for measuring bilirubin in plasma is as follows: adding a bovine serum albumin-stabilized gold nanocluster solution to the melted agarose solution, obtaining a mixed solution, cooling and solidifying, and preparing a bovine serum albumin Serum albumin-stabilized gold nanoclusters-agarose gel, which is obtained by making bovine serum albumin-stabilized gold nanoclusters into circular sheets. The amount of agarose solution added determines the hardness of the circular sheet, and the amount of agarose solution added can be adjusted according to the hardness of the circular sheet.

In the above mixture, the concentration of bovine serum albumin-stabilized gold nanoclusters was 13.44 mg/mL.

The invention also discloses the above-mentioned method for measuring bilirubin in plasma by using the above-mentioned test substance for measuring bilirubin in plasma. The standard curve between the extinction degrees was used to determine the linear range of bilirubin concentration.

A series of standard pictures of bilirubin concentration versus fluorescence quenching of bovine serum albumin-stabilized gold nanoclusters were obtained over the linear range of bilirubin concentration.

Take 100 μL of plasma to be tested, drop it on BSA-stabilized gold nanoclusters-filter paper sheet or BSA-stabilized gold nanoclusters-agarose gel sheet, and place it at room temperature to react for 40min, and observe the wavelength of 365nm. The fluorescence quenching degree of the gold nanoclusters was compared with the standard picture, and the concentration of bilirubin in the plasma to be detected was obtained semi-quantitatively.

The specific process of obtaining a series of standard pictures above is as follows: within the linear range of bilirubin concentration, take 100 μL of a plurality of standard solutions of bilirubin with different concentrations, and drop a plurality of the standard solutions on a plurality of gold nanometers correspondingly. On the cluster-filter paper or gold nanoclusters-agarose gel sheet, put it at room temperature for 40min reaction, observe the fluorescence quenching of gold nanoclusters under 365nm wavelength ultraviolet lamp, and take pictures respectively to obtain the bilirubin linear range. A series of standard pictures of fluorescence quenching of gold nanoclusters.

The preparation process of the above-mentioned bovine serum albumin-stabilized gold nanoclusters is as follows: prepare a bovine serum albumin solution with a concentration of 50 mg/mL. Add 5 mL of bovine serum albumin solution to 5 mL of chloroauric acid solution under vigorous stirring. The concentration of chloroauric acid solution is 10 mmoL/L. After 2 minutes of reaction at 37 °C, add 0.5 mL of 1moL/L sodium hydroxide solution to adjust the reaction. The pH was about 12, and the reaction was continuously stirred at 37 °C for 12 h. The color of the reaction solution changes from light yellow to brown, that is, fluorescence detection, and can emit red fluorescence clearly visible to the naked eye under the excitation of 365 nm. Then, the prepared bovine serum albumin-stabilized gold nanoclusters were stored at 4° C. until use. The glassware used in the reaction process was soaked and washed with Wang.

The invention also discloses a method for measuring bilirubin in plasma, the method is as follows:

Step 1: Establish a standard curve between the concentration of bilirubin and the degree of fluorescence quenching of the bovine serum albumin-stabilized gold nanoclusters, and obtain a standard curve equation.

Step 2: Take the plasma to be detected, add bovine serum albumin-stabilized gold nanoclusters to the plasma to be detected, measure the fluorescence intensity of the plasma at this time as I', and substitute the I' into the In the standard curve equation, the concentration C' of bilirubin in the plasma to be detected is obtained.

The specific process of the above step 1 is as follows: the blank solution and multiple bilirubin standard solutions with different concentrations c are respectively added to the corresponding multiple bovine serum albumin-stabilized gold nanoclusters, respectively, and the fluorescence intensity is measured. The blank solution is: Deionized water, the fluorescence intensity of the blank solution is I ₀ , and the fluorescence intensity of the bilirubin standard solution is I, with (I ₀ -I)/I ₀ as the ordinate, and the bilirubin concentration as the abscissa to establish a standard curve to obtain The standard curve equation is (I ₀ -I)/I ₀ =0.98c-6.10.

When the bilirubin concentration was 8.55-85.52 μmoL/L, the fluorescence quenching degree of gold nanoclusters ((I ₀ -I)/I ₀ ) showed a good linear relationship with the bilirubin concentration, and the linear relationship was (I ₀ -I)/I ₀ =0.9837c-6.10, the correlation coefficient r=0.9985.

In the present invention, bovine serum albumin-stabilized gold nanocluster solution and bilirubin solution are prepared, and experiments are carried out to obtain pH and detection time in the process of hemoglobin detection. as follows:

Add 100 μL of bilirubin solution to 50 μL of bovine serum albumin-stabilized gold nanocluster solution, vortex to mix, and add PBS buffer solution to make up to 500 μL. The concentration of bilirubin solution was 214 μmoL/L and then verified as follows:

1. Verified the effect of pH change of phosphate buffer solution on the degree of bilirubin quenching the fluorescence of gold nanoclusters: The selected pH is shown in Figure 3. At different pH, the fluorescence intensity of the solution was measured, and the pH The change has little effect on the test results, and the pH value close to human blood is selected as the final test pH value.

2. Influence of incubation temperature and incubation time on the detection results: The samples were processed at different incubation temperatures to perform fluorescence detection. The results are shown in Figure 1. It can be seen from the figure that when the temperature reaches 37 °C, the degree of fluorescence quenching is the largest. At the same time, the effect of incubation time on the degree of quenching was also verified. As shown in Figure 2, when the incubation time reached 30 min, the degree of fluorescence quenching reached the maximum. It can be seen that when the method of the present invention is used to measure bilirubin in plasma, the time required for the detection is short.

In order to verify the feasibility of the method adopted in the present invention, the following experiments are carried out:

Five normal plasmas were selected, the control group was BSA-stabilized gold nanoclusters + plasma + PBS buffer, and the sample group was BSA-stabilized gold nanoclusters + plasma + bilirubin standard solution.

Control group: add 100 μL of the above 5 normal plasmas to 50 μL of BSA-stabilized gold nanoclusters, vortex and mix, add pbs buffer solution (pH 7.4) to make up to 500 μL, vortex and mix, and then place at 37 The reaction was incubated in a dry thermostat for 40 min, and the fluorescence was measured. As shown in Figure 5.

Sample group: Add 100 μL of the above 5 normal plasmas to 50 μL of bovine serum albumin-stabilized gold nanoclusters. After vortex mixing, add 100 μL of bilirubin standard solution with a final concentration of 171 μmoL/L, and then add PBS buffer. The solution (pH 7.4) was adjusted to 500 μL, vortexed and mixed, and then placed in a dry thermostat at 37° C. for 40 min to incubate the reaction, and the fluorescence was measured. As shown in Figure 5.

It can be seen from Figure 5 that when bilirubin is not added, other components in the plasma will not be detected, indicating that there will be no interference from other components when using the fluorescence detection method to detect bilirubin in the plasma.

In order to verify the accuracy of the method in the present invention, a sample addition and recovery experiment was carried out, the original concentration of bilirubin was selected as 50 μmoL/L, three samples were selected, and 5 μmoL/L, 10 μmoL/L and 5 μmoL/L were added to the samples. 15 μmoL/L of bilirubin, the theoretical concentrations of bilirubin finally obtained are 55 μmoL/L, 60 μmoL/L and 65 μmoL/L, respectively, and then the method for measuring bilirubin in plasma in the present invention is used to determine, to obtain bilirubin. The measured concentrations of erythrocytes were 53.43 μmoL/L, 61.75 μmoL/L and 65.82 μmoL/L, respectively. The recovery rates are shown in Table 1, the recovery rates are 96% to 103%, the RSDs are 2.27, 1.51 and 1.34, respectively, and the recovery rates are about 100%, indicating that the method of the present invention is used to determine the concentration of bilirubin in plasma. High accuracy.

Table 1 Recovery of bilirubin

Claims (10)

1. a detection substance for measuring bilirubin in plasma, is characterized in that, by the gold nano-cluster of bovine serum albumin-stabilized load on filter paper, the gold nano-cluster-filter paper of bovine serum albumin-stabilized is obtained piece, that's it. 2. the preparation method of a kind of detection substance for measuring bilirubin in blood plasma according to claim 1, is characterized in that, filter paper is soaked in the gold nanocluster solution of detection substance bovine serum albumin stable, wait for After the absorption is saturated, take out the filter paper and place it in an oven to dry. 3 . The method for preparing a test substance for measuring bilirubin in plasma according to claim 2 , wherein the concentration of the bovine serum albumin-stabilized gold nanoclusters is 13.44 mg/mL. 4 . 4. a detection substance for measuring bilirubin in plasma, characterized in that, the gold nano-clusters stabilized by bovine serum albumin are fixed in agarose gel, and are packaged and solidified by agarose gel, and are cut into Gold nanoclusters - agarose gel sheets, that is, obtained. 5. a kind of preparation method for measuring the detection substance of bilirubin in blood plasma according to claim 4, is characterized in that, adds the gold nano-cluster solution stabilized by bovine serum albumin in the melted agarose solution, The mixed solution was obtained, cooled and solidified to prepare a bovine serum albumin-stabilized gold nanocluster-agarose gel, and the bovine serum albumin-stabilized gold nanocluster-agarose gel was made into a circular sheet , that is. 6. The preparation method of a detection substance for measuring bilirubin in plasma according to claim 5, wherein in the mixed solution, the concentration of the bovine serum albumin-stabilized gold nanoclusters was 13.44 mg/mL. 7. a kind of method for measuring bilirubin in blood plasma according to a kind of detection substance for measuring bilirubin in blood plasma according to claim 1 and 4, is characterized in that, this method is as follows: A standard curve was established between the concentration of bilirubin and the degree of fluorescence quenching of BSA-stabilized gold nanoclusters, and the linear range of the concentration of bilirubin was determined; A series of standard pictures of bilirubin concentration and fluorescence quenching of BSA-stabilized gold nanoclusters were obtained in the linear range of bilirubin concentration; Take 100 μL of plasma to be tested, drop it on BSA-stabilized gold nanoclusters-filter paper sheet or BSA-stabilized gold nanoclusters-agarose gel sheet, and place it at room temperature to react for 40min, and observe the wavelength of 365nm. The fluorescence quenching degree of the gold nanoclusters was compared with the standard picture, and the concentration of bilirubin in the plasma to be detected was obtained semi-quantitatively. 8. a kind of method for measuring bilirubin in plasma according to claim 7, is characterized in that, the concrete process that draws a series of standard pictures is as follows: in bilirubin concentration Within the linear range of , take 100 μL each of multiple bilirubin standard solutions of different concentrations, and drop the multiple standard solutions on multiple gold nanocluster-filter paper sheets or gold nanoclusters-agarose gel sheets correspondingly, After 40 minutes of reaction at room temperature, the fluorescence quenching of gold nanoclusters under 365nm wavelength ultraviolet light was observed and photographed respectively, and a series of standard pictures of fluorescence quenching of gold nanoclusters in the linear range of bilirubin were obtained. 9. a method for measuring bilirubin in plasma, is characterized in that, the method is as follows: Step 1: Establish a standard curve between the concentration of bilirubin and the degree of fluorescence quenching of the BSA-stabilized gold nanoclusters, and obtain a standard curve equation; Step 2: Take the plasma to be detected, add bovine serum albumin-stabilized gold nanoclusters to the plasma to be detected, measure the fluorescence intensity of the plasma at this time as I ^' , and substitute the I ^' into the In the standard curve equation, the concentration C' of bilirubin in the plasma to be detected is obtained. 10. a kind of method for measuring bilirubin in blood plasma according to claim 9, is characterized in that, the concrete process of described step 1 is as follows: by blank solution and the different bilirubin standard solutions of a plurality of concentrations c, respectively. Add the corresponding multiple bovine serum albumin-stabilized gold nanoclusters to measure the fluorescence intensity respectively, the blank solution is deionized water, the fluorescence intensity of the blank solution is I ₀ , and the fluorescence intensity of the bilirubin standard solution is I, with (I ₀ -I)/I ₀ is the ordinate, and the bilirubin concentration is the abscissa to establish a standard curve, and the equation of the standard curve is (I ₀ -I)/I ₀ =0.98c-6.10.

Images