A method for detecting hemoglobin

Abstract

The application discloses a kind of hemoglobin detection methods, comprising: taking 0.1-0.2mmol / L (6,5)-SWCNTs solution, add to the ultrafiltration centrifuge tube with the molecular weight of 100kDa, centrifugal 1-10min, discard filtrate;In the inclusion, add the same volume of SDS solution with filtrate, repeat centrifugal 6-15 times;Again, solution ultrasonic dispersion 0.2-1h, and centrifugal 2-15min;In DPPE-mPEG5000 solution, add the supernatant above, after mixing sufficiently, stand overnight;Again, using the ultrafiltration centrifuge tube with the molecular weight of 10kDa and 100kDa respectively to the mixed solution above sequentially centrifugal filtration, take the supernatant above, add hemoglobin solution to be measured, after mixing uniformly, stand incubation 10S above;With water as blank control, carry out UV-Vis-NIR absorption spectrum and near infrared fluorescence spectrum determination;Hemoglobin standard product is prepared into different concentration stock solution, and standard curve is drawn;The concentration of hemoglobin in sample is calculated according to standard curve.The application is simple in operation, short in time, and low in detection cost.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of hemoglobin detection, in particular to a hemoglobin detection method. BACKGROUND

[0002] Hemoglobin is a kind of iron-containing metalloprotein with heme as a cofactor existing in prokaryotic and eukaryotic cells, which 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 based on microbial cell factories has gradually become a research hotspot. At present, the literature / patents report more on the detection of hemoglobin content in whole blood, serum, plasma and urine. However, the current detection methods are more complicated, or the cost is higher.

[0003] Hemoglobin (Hb) is an important metalloprotein in red blood cells, which is composed of two alpha subunits and two beta subunits, and each subunit contains a peptide chain and a heme. The spherical tetrameric protein formed by the accumulation and folding of four peptide chains is called globin. Heme, also known as ferroprotoporphyrin, is a cyclic molecule composed of four pyrrole groups, with a ferrous ion at the center of the ring. In living organisms, hemoglobin has many functions such as transporting oxygen and carbon dioxide, maintaining blood acid-base balance, and its content is an important indicator for diagnosing anemia. The concentration of Hb in normal adult male blood is 120-165 g / L, that of adult female is 110-150 g / L, and that of children is 120-140 g / L, and lower than this concentration will lead to anemia. According to statistics, about 2 billion people in the world suffer from anemia. In addition, abnormal content of hemoglobin is also related to many other diseases, such as leukemia, obstructive pulmonary emphysema, congenital heart disease and cancer. Therefore, accurate determination of the content of hemoglobin in clinical diagnosis is of great significance for evaluating the health status of the human body. At present, the common methods for detecting hemoglobin include cyanmethemoglobin method, colorimetric method, fluorescence method, liquid chromatography and electrochemical method, but these methods mostly have the defects of complex operation, long time-consuming, need for expensive reagents and instruments, etc. SUMMARY

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

[0005] The technical scheme of the present application is a hemoglobin detection method, comprising the following steps:

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

[0007] (2) Add an equal volume of 0.1-3wt% SDS solution to the retentate, and centrifuge 6-15 times. After centrifugation, sonicate the solution for 0.2-1h and centrifuge at room temperature and centrifugation force of 19000g-23000g for 2-15min.

[0008] (3) Add the supernatant of the above SDS-dispersed (6,5)-SWCNTs to a 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 centrifugal force of 900g-1100g, the above mixed solution was centrifuged and filtered using ultrafiltration centrifuge tubes with molecular weight cutoff of 10kDa and 100kDa respectively to remove excess surfactant and excess DPPE-mPEG5000.

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

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

[0012] (6,5)-SWCNTs stands for single-chiral (6,5) single-walled carbon nanotubes.

[0013] Centrifugal force 400-600g refers to the centrifugal force set during centrifugation as 400-600×g, where g is the acceleration due to gravity.

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

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

[0016] According to a method for detecting hemoglobin according to the present invention, preferably, the centrifugation time in step (1) is 4-6 min.

[0017] According to a method for detecting hemoglobin according to 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 according to 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 according to the present invention, preferably, the centrifugation time in step (4) is 0.2-1h.

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

[0021] According to the method for detecting hemoglobin of the present invention, preferably, step (5) involves static incubation for 10 seconds to 8 minutes. The fluorescence quenching rate is higher during this time period.

[0022] Furthermore, step (5) involves static incubation for 4-6 minutes. A higher fluorescence quenching rate is achieved during static incubation for 4-6 minutes.

[0023] Beneficial effects:

[0024] This invention relates to an innovative method for hemoglobin detection, characterized by its simple operation, rapid detection speed, and low cost. This method eliminates the need for complex pretreatment processes, significantly reducing detection time compared to other methods, requiring only a few minutes. Furthermore, by using near-infrared fluorescence detection through a biological transparent window, background interference from biological tissues is effectively avoided, resulting in excellent stability and reproducibility. The method also features high sensitivity and a low detection limit, accurately capturing even minute changes in hemoglobin concentration, which is of great significance for early diagnosis and treatment monitoring. Attached Figure Description

[0025] Figure 1aThe absorption spectra of DPPE-mPEG5000-(6,5) before and after the addition of Hb are shown. Figure 1b These are the near-infrared fluorescence spectra of DPPE-mPEG5000-(6,5) before and after the addition of Hb.

[0026] Figure 2 The near-infrared fluorescence spectra of DPPE-mPEG5000-(6,5) after adding different concentrations of Hb (0-2000nM) are shown.

[0027] Figure 3 The standard curve is plotted with Hb concentration on the x-axis and fluorescence intensity change rate (F0-F / F0) on the y-axis, within the range of Hb concentration from 0.05 to 100 nM. Detailed Implementation

[0028] I. Probe Preparation:

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

[0030] Take 300 μL of a 1 mg / mL DPPE-mPEG5000 solution, add 300 μL of 1% SDS-(6,5), mix thoroughly, and let stand overnight. Centrifuge the above mixture at 25℃ and 1000g using ultrafiltration centrifuge tubes with molecular weight cutoffs of 10 kDa and 100 kDa, respectively, to remove excess surfactant and DPPE-mPEG5000. Centrifugation time is 0.5 h. After centrifugation, collect the supernatant to obtain DPPE-mPEG5000-dispersed (6,5)-SWCNTs, denoted as DPPE-mPEG5000-(6,5).

[0031] II. Hemoglobin Detection

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

[0033] like Figure 1a and Figure 1bAs shown, after the addition of Hb, the absorbance of (6,5)-SWCNTs decreased slightly, while the fluorescence intensity was almost completely quenched. Furthermore, the characteristic absorption peak wavelength of (6,5)-SWCNTs showed a significant redshift of 8 nm, while the maximum fluorescence emission wavelength only redshifted by 1 nm. The simultaneous decrease in absorbance and fluorescence intensity indicates that the quenching of DPPE-mPEG5000-(6,5) fluorescence by Hb is a static quenching. This may be because the combination of DPPE-mPEG5000 and (6,5)-SWCNTs forms a ground-state complex that is unfavorable for luminescence.

[0034] III. Test Results

[0035] Accurately weigh hemoglobin standards to prepare stock solutions of different concentrations. Add 20 μL of each stock solution to an 80 μL LDPPE-mPEG5000-(6,5) probe to achieve final hemoglobin concentrations of 0.05, 0.2, 0.5, 1, 2, 5, 10, 25, 50, 75, and 100 nM. Vortex mix and then measure the near-infrared fluorescence spectrum. Each sample was measured in triplicate. A standard curve was plotted with purified water as a blank control (F0), hemoglobin concentration (C) on the x-axis, and the fluorescence intensity change rate of the probe (F0-F / F0) on the y-axis.

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

[0037] Example 1

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

[0039] Example 2

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

[0041] Example 3

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

Claims

1. A method for detecting hemoglobin, characterized in that: Includes the following steps: (1) Take 0.1-0.2 mmol / 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 centrifugation force of 400-600g for 1-10 min and discard the filtrate. (2) Add an SDS solution with a concentration of 0.1-3wt% to the retentate in the same volume as the above filtrate, and repeat centrifugation 6-15 times; after centrifugation, sonicate the solution for 0.2-1h, and centrifuge at room temperature and centrifugation force of 19000g-23000g for 2-15min. (3) Add the supernatant of the above SDS-dispersed (6,5)-SWCNTs to a 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 centrifugal force of 900g-1100g, the above mixed solution was centrifuged and filtered sequentially using ultrafiltration centrifuge tubes with molecular weight cutoff of 10kDa and 100kDa respectively to remove excess surfactant and excess DPPE-mPEG5000; first use 10kDa ultrafiltration tube to remove surfactant, and then use 100kDa ultrafiltration tube to remove DPPE-mPEG5000 that has not been bound to SWCNTs, and the supernatant obtained is (6,5)-SWCNTs dispersed by DPPE-mPEG5000; steps (1)-(4) are the preparation of probes; (5) Take the supernatant of the above DPPE-mPEG5000-(6,5)-SWCNTs, add hemoglobin (Hb) solution with a final concentration of 0.5-2μM, mix well and let stand for more than 10 seconds; use water as a blank control, perform UV-Vis-NIR absorption spectroscopy and near-infrared fluorescence spectroscopy; accurately weigh the hemoglobin standard to prepare stock solutions of different concentrations, plot the standard curve with the concentration of hemoglobin (C) as the abscissa and the fluorescence intensity change rate of the probe (F0-F / F0) as the ordinate; calculate the concentration of hemoglobin in the sample according to the standard curve.

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

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

1.

7. The method for detecting hemoglobin according to claim 1, characterized in that: Step (5) involves static incubation for 10 seconds to 8 minutes.

8. The method for detecting hemoglobin according to claim 7, characterized in that: Step (5) involves static incubation for 4-6 minutes.

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