Hemoglobin detection method

Abstract

The invention discloses a hemoglobin detection method which comprises the following steps: taking a (6, 5)-SWCNTs solution of 0.1-0.2 mmol / L, adding the (6, 5)-SWCNTs solution into an ultrafiltration centrifuge tube with the molecular weight cut-off of 100 kDa, centrifuging for 1-10 minutes, and discarding the filtrate; adding an SDS solution with the same volume as the filtrate into the retentate, and repeatedly centrifuging for 6-15 times; carrying out ultrasonic dispersion on the solution for 0.2-1 hour, and centrifuging for 2-15 minutes; adding the supernate into a DPPE-mPEG5000 solution, fully mixing, and standing overnight; sequentially carrying out centrifugal filtration on the mixed solution by respectively using ultrafiltration centrifuge tubes with molecular weight cutoff of 10kDa and 100kDa, taking the supernate, adding a to-be-detected hemoglobin solution, uniformly mixing, and standing and incubating for more than 10S; carrying out UV-Vis-NIR absorption spectrum and near infrared fluorescence spectrum determination by taking water as blank control; preparing a hemoglobin standard product into stock solutions with different concentrations, and drawing a standard curve; and calculating the concentration of hemoglobin in the sample according to the standard curve. The method is simple to operate, short in time and low in detection cost.

Description

Technical Field

[0001] The present invention relates to the field of hemoglobin detection, and in particular to a hemoglobin detection method. Background Art

[0002] Hemoglobin is a type of iron-containing metalloprotein with heme as a cofactor that exists in prokaryotic and eukaryotic cells. It has many important functions in the body, such as transporting and storing oxygen, regulating intracellular pH, and regulating physiological metabolism. In recent years, the synthesis of hemoglobin from different sources using microbial cell factories as a platform has gradually become a research hotspot. Currently, most of the reports in the literature / patents are about detecting the content of hemoglobin in whole blood, serum, plasma, and urine. However, the current detection methods are relatively cumbersome or costly.

[0003] Hemoglobin (Hb) is an important metalloprotein in red blood cells, consisting of two α subunits and two β subunits, each of which contains a peptide chain and a heme. The spherical tetrameric protein formed by the stacking and folding of four peptide chains is called globin. Heme, also known as ferrous protoporphyrin, is a ring molecule composed of four pyrrole groups, with a ferrous ion bound to the center of the ring. In organisms, hemoglobin has multiple functions such as transporting oxygen and carbon dioxide and maintaining blood acid-base balance, and its content is an important indicator for diagnosing anemia. The concentration of Hb in the blood of normal adult males is 120-165g / L, 110-150g / L for adult females, and 120-140g / L for children. A concentration lower than this will cause anemia. According to statistics, about 2 billion people suffer from anemia worldwide. In addition, abnormal hemoglobin content is also related to many other diseases, such as leukemia, obstructive pulmonary emphysema, congenital heart disease and cancer. Therefore, accurate determination of hemoglobin content in clinical diagnosis is of great significance for assessing human health status. At present, common methods for hemoglobin detection include cyanide hemoglobin method, colorimetry, fluorescence method, liquid chromatography and electrochemical method, but most of these methods have defects such as complex operation, long time consumption, and the need for expensive reagents and instruments. Summary of the invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide a method for detecting hemoglobin with low cost, convenient operation and high accuracy.

[0005] The technical solution of the present invention is a method for detecting hemoglobin, comprising the following steps:

[0006] (1) Take 0.1-0.2mmol / L (6,5)-SWCNTs solution and add it to an ultrafiltration centrifuge tube with a molecular weight cutoff of 100kDa. Centrifuge at room temperature and a centrifugal force of 400-600g for 1-10min, and discard the filtrate;

[0007] (2) adding an SDS solution with a concentration of 0.1-3 wt% in the same volume as the filtrate to the retentate, and repeating the centrifugation for 6-15 times; after the centrifugation is completed, the solution is ultrasonically dispersed for 0.2-1 h, and centrifuged at room temperature and a centrifugal force of 19000 g-23000 g for 2-15 min;

[0008] (3) Add the supernatant of the SDS-dispersed (6,5)-SWCNTs to the 0.5-2 mg / mL DPPE-mPEG5000 solution at a volume ratio of 1-2:1-5, mix thoroughly and let stand overnight;

[0009] (4) at room temperature and a centrifugal force of 900 g to 1100 g, the mixed solutions were centrifuged using ultrafiltration centrifuge tubes with a molecular weight cutoff of 10 kDa and 100 kDa, respectively, to remove excess surfactant and excess DPPE-mPEG5000;

[0010] (5) Take the above DPPE-mPEG5000-(6,5) supernatant, add hemoglobin (Hb) solution with a final concentration of 0.5-2 μM, mix well and incubate for more than 10 seconds; use water as a blank control and perform UV-Vis-NIR absorption spectroscopy and near-infrared fluorescence spectroscopy.

[0011] In step (1), a 100 kDa ultrafiltration centrifuge tube is used to replace the mixed surfactant (DOC / SC / SDS) coated on the surface of (6,5)-SWCNTs with the subsequent SDS solution by ultrafiltration.

[0012] (6,5)-SWCNTs is the full name of single-chirality (6,5) single-walled carbon nanotubes.

[0013] The centrifugal force of 400-600g means that the centrifugal force set during centrifugation is 400-600×g, where g is the acceleration due to gravity.

[0014] In step (4), the excess surfactant refers to free SDS. In this step, a 10 kDa ultrafiltration tube is first used to remove the surfactant, and then a 100 kDa ultrafiltration tube is used to remove the DPPE-mPEG5000 that is not bound to SWCNTs. The supernatant obtained is the DPPE-mPEG5000 dispersed (6,5)-SWCNTs. Steps (1) to (4) are the preparation of the probe.

[0015] In step (5), the static incubation here means that before the spectral measurement, the probe (i.e., DPPE-mPEG5000-(6,5)) is allowed to fully react with the detection substance (i.e., hemoglobin). The sample to be tested is the hemoglobin mentioned above. It is found that the fluorescence of the probe is quenched immediately (about 10 seconds) after the addition of hemoglobin. The final concentration of the hemoglobin (Hb) solution is 0.5-2μM, which means that the unknown sample needs to be controlled within this concentration range.

[0016] According to a hemoglobin detection method of the present invention, preferably, the centrifugation time in step (1) is 4-6 minutes.

[0017] According to the hemoglobin detection method of the present invention, preferably, the concentration of the SDS solution in step (2) is 0.8-2.2%.

[0018] According to a method for detecting hemoglobin of the present invention, preferably, the concentration of the DPPE-mPEG5000 solution in step (3) is 1-1.5 mg / mL.

[0019] According to a method for detecting hemoglobin of the present invention, preferably, the centrifugation time in step (4) is 0.2-1h.

[0020] According to a method for detecting hemoglobin of the present invention, preferably, in step (5), the volume ratio of the DPPE-mPEG5000-(6,5) supernatant to the hemoglobin solution is 3-5:1.

[0021] According to a hemoglobin detection method of the present invention, preferably, the static incubation in step (5) is 10S-8min. The fluorescence quenching rate during this period is relatively high.

[0022] Furthermore, in step (5), the static incubation is performed for 4-6 minutes. When the static incubation is performed for 4-6 minutes, the fluorescence quenching rate is higher.

[0023] Beneficial effects:

[0024] The present invention relates to an innovative hemoglobin detection method, which is characterized by simple operation, fast detection speed and low detection cost. The method does not require a complicated pretreatment process and significantly shortens the detection time compared with other methods, which only takes a few minutes. In addition, by performing near-infrared fluorescence detection on a biological transparent window, the background interference of biological tissues is effectively avoided, and it has good stability and reproducibility. The method also has the characteristics of high sensitivity and low detection limit. Even trace changes in hemoglobin concentration can be accurately captured, which is of great significance for early diagnosis and treatment monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1ais the absorption spectrum of DPPE-mPEG5000-(6,5) before and after adding Hb; Figure 1b This is the near-infrared fluorescence spectrum of DPPE-mPEG5000-(6,5) before and after adding Hb.

[0026] Figure 2 It is the near-infrared fluorescence spectrum of DPPE-mPEG5000-(6,5) after adding different concentrations of Hb (0-2000nM).

[0027] Figure 3 The standard curve is drawn with Hb concentration as the horizontal axis and fluorescence intensity change rate (F0-F / F0) as the vertical axis within the Hb concentration range of 0.05-100 nM. DETAILED DESCRIPTION

[0028] 1. Preparation of probe:

[0029] Take 400 μL of the diluted (6,5)-SWCNTs solution, add it to an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa, and centrifuge it at 25°C and 500g for 5 minutes. Discard the filtrate, add 400 μL of 1% SDS to the retentate to maintain a constant volume of the solution, and repeat the centrifugation 10 times. After the centrifugation is completed, the solution is ultrasonically dispersed for 0.5 hours and centrifuged at 25°C and 21000g for 10 minutes. Take the supernatant to obtain 1% SDS-dispersed (6,5)-SWCNTs, which is recorded as 1% SDS-(6,5).

[0030] Take 300 μL of 1 mg / mL DPPE-mPEG5000 solution, add 300 μL of 1% SDS-(6,5), mix thoroughly and let stand overnight. At 25°C and 1000g, use ultrafiltration centrifuge tubes with a molecular weight cutoff of 10 kDa and 100 kDa to centrifuge the mixed solution to remove excess surfactant and DPPE-mPEG5000. The centrifugation time is 0.5 h. After centrifugation, take out the supernatant to obtain DPPE-mPEG5000 dispersed (6,5)-SWCNTs, recorded as DPPE-mPEG5000-(6,5).

[0031] 2. Hemoglobin test

[0032] Take 80 μL of DPPE-mPEG5000-(6,5), add 20 μL of hemoglobin (Hb) solution with a final concentration of 1 μM, mix well and incubate for a period of time. Use water as a blank control to perform UV-Vis-NIR absorption spectroscopy and near-infrared fluorescence spectroscopy.

[0033] like Figure 1a and Figure 1bAs shown in the figure, after adding Hb, the absorbance of (6,5)-SWCNTs decreased slightly, while the fluorescence intensity was almost completely quenched. In addition, the characteristic absorption peak wavelength of (6,5)-SWCNTs was significantly red-shifted by 8nm, while the maximum fluorescence emission wavelength was only red-shifted by 1nm. The simultaneous decrease in absorbance and fluorescence intensity indicates that the quenching of DPPE-mPEG5000-(6,5) fluorescence by Hb is static quenching, which may be due to the combination of DPPE-mPEG5000 and (6,5)-SWCNTs to form a ground state complex that is not conducive to luminescence.

[0034] 3. Test results

[0035] Accurately weigh the hemoglobin standard to prepare stock solutions of different concentrations, take 20 μL of each and add it to 80 μL of DPPE-mPEG5000-(6,5) probe to make the final concentrations of hemoglobin 0.05, 0.2, 0.5, 1, 2, 5, 10, 25, 50, 75, 100 nM, respectively. Vortex mix and measure the near-infrared fluorescence spectrum. Each sample is measured in parallel 3 times. Use pure water as the blank control (F0), the concentration of hemoglobin (C) as the horizontal axis, and the fluorescence intensity change rate of the probe (F0-F / F0) as the vertical axis to draw a standard curve.

[0036] from Figure 2 and Figure 3 It can be seen that in the range of Hb concentration of 0-100nM, as the Hb concentration increases, the fluorescence intensity of DPPE-mPEG5000-(6,5) at the maximum emission wavelength gradually decreases. The standard curve is drawn with Hb concentration (CHb) as the horizontal axis and the fluorescence intensity change rate of (6,5)-SWCNTs (F0-F / F0) as the vertical axis. As shown in the right figure, in the range of 0.05-100nM, the fitted equation is y=0.12422x 0.3884 , R 2 =0.996. The detection limit (LOD) of hemoglobin was 0.01 nM, and the quantification limit (LOQ) was 0.034 nM.

[0037] Example 1

[0038] Accurately weigh the hemoglobin standard to prepare a stock solution with a concentration of 1 μM. Take 200 μL of artificial serum, add 1000 μL of methanol, vortex mix, and centrifuge at 10000 rpm for 30 minutes. After centrifugation, take the supernatant and add the above stock solution to obtain a spiked serum sample with a hemoglobin concentration of 125 nM. Then take 20 μL and add it to 80 μL of DPPE-mPEG5000-(6,5) probe to make the final concentration of hemoglobin 25.0 nM. Vortex mix, let it stand for 5 minutes, and measure the near-infrared fluorescence spectrum. With the addition of blank serum as a control, the concentration of hemoglobin in the sample is calculated to be 24.2 nM according to the standard curve.

[0039] Example 2

[0040] Accurately weigh the hemoglobin standard to prepare a stock solution with a concentration of 1 μM. Take 200 μL of artificial serum, add 1000 μL of methanol, vortex to mix, and centrifuge at 10000 rpm for 30 minutes. After centrifugation, take the supernatant and add the above stock solution to obtain a spiked serum sample with a hemoglobin concentration of 250 nM. Then take 20 μL and add it to 80 μL of DPPE-mPEG5000-(6,5) probe to make the final concentration of hemoglobin 50.0 nM. Vortex to mix, let it stand for 5 minutes, and measure the near-infrared fluorescence spectrum. With the addition of blank serum as a control, the concentration of hemoglobin in the sample is calculated to be 51.5 nM according to the standard curve.

[0041] Example 3

[0042] Accurately weigh the hemoglobin standard to prepare a stock solution with a concentration of 1 μM. Take 200 μL of artificial serum, add 1000 μL of methanol, vortex mix, and centrifuge at 10000 rpm for 30 minutes. After centrifugation, take the supernatant and add the above stock solution to obtain a spiked serum sample with a hemoglobin concentration of 375 nM. Then take 20 μL and add it to 80 μL of DPPE-mPEG5000-(6,5) probe to make the final concentration of hemoglobin 75.0 nM. Vortex mix, let it stand for 5 minutes, and measure the near-infrared fluorescence spectrum. With the addition of blank serum as a control, the concentration of hemoglobin in the sample is calculated to be 70.6 nM according to the standard curve.

Claims

1. A method for detecting hemoglobin, characterized in that: The steps include: (1) Take 0.1-0.2mmol / L (6,5)-SWCNTs solution and add it to an ultrafiltration centrifuge tube with a molecular weight cutoff of 100kDa. Centrifuge at room temperature and a centrifugal force of 400-600g for 1-10min, and discard the filtrate; (2) adding an SDS solution with a concentration of 0.1-3 wt% in the same volume as the filtrate to the retentate, and repeating the centrifugation for 6-15 times; after the centrifugation is completed, the solution is ultrasonically dispersed for 0.2-1 h, and centrifuged at room temperature and a centrifugal force of 19000 g-23000 g for 2-15 min; (3) Add the supernatant of the SDS-dispersed (6,5)-SWCNTs to the 0.5-2 mg / mL DPPE-mPEG5000 solution at a volume ratio of 1-2:1-5, mix thoroughly and let stand overnight; (4) at room temperature and a centrifugal force of 900 g to 1100 g, the mixed solutions were centrifuged using ultrafiltration centrifuge tubes with a molecular weight cutoff of 10 kDa and 100 kDa, respectively, to remove excess surfactant and excess DPPE-mPEG5000; (5) Take the above DPPE-mPEG5000-(6,5) supernatant, add hemoglobin (Hb) solution with a final concentration of 0.5-2 μM, mix well and incubate for more than 10 seconds; use water as a blank control to perform UV-Vis-NIR absorption spectrum and near-infrared fluorescence spectrum measurement; accurately weigh the hemoglobin standard to prepare stock solutions of different concentrations, and draw a standard curve with the hemoglobin concentration (C) as the horizontal axis and the fluorescence intensity change rate of the probe (F0-F / F0) as the vertical axis; calculate the hemoglobin concentration in the sample according to the standard curve.

2. A method for detecting hemoglobin according to claim 1, characterized in that: The centrifugation time in step (1) is 4-6 minutes.

3. A method for detecting hemoglobin according to claim 1, characterized in that: The concentration of the SDS solution in step (2) is 0.8-2.2%.

4. A method for detecting hemoglobin according to claim 1, characterized in that: The concentration of the DPPE-mPEG5000 solution in step (3) is 1-1.5 mg / mL.

5. The method for detecting hemoglobin according to claim 1, characterized in that: The centrifugation time in step (4) is 0.2-1h.

6. A method for detecting hemoglobin according to claim 1, characterized in that: In step (5), the volume ratio of the DPPE-mPEG5000-(6,5) supernatant to the hemoglobin solution is 3-5:

1.

7. A method for detecting hemoglobin according to claim 1, characterized in that: The step (5) is to incubate for 10 seconds to 8 minutes.

8. A method for detecting hemoglobin according to claim 7, characterized in that: The step (5) is to incubate for 4-6 minutes.

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