A biosensor for detecting heme, hemoglobin and / or L-cysteine ​​and its application

Abstract

The present invention discloses a biosensor for detecting heme, hemoglobin and / or L-cysteine ​​and its application, belonging to the field of biosensing and analysis. The biosensor comprises a hairpin nucleic acid HP, a binding buffer solution, heme, TMB, H2O2 and a HAc-NaAc buffer solution; the sequence of the hairpin nucleic acid HP is shown in SEQ No. 1, and its 5' end is labeled with a quenching group BHQ1, and its 3' end is labeled with a fluorescent group FAM. The present invention can realize the optical detection of heme and hemoglobin in the "turn-on" mode and the rapid detection of L-cysteine. It has higher sensitivity than existing detection methods, and experiments have shown that it is still applicable in complex serum systems. It is expected to provide a simple, rapid and sensitive method for the detection of heme, hemoglobin and L-cysteine ​​in actual clinical samples.

Description

Technical Field

[0001] The present invention belongs to the field of biosensing and analysis, and in particular relates to a biosensor for detecting heme, hemoglobin and / or L-cysteine ​​and its application. Background Art

[0002] Deoxyribozyme (DNAzyme) is a type of nucleic acid with catalytic function discovered by scientists. So far, more than ten types of DNAzyme have been discovered, such as 8-17 and 10-23 DNAzyme with RNA cleavage enzyme activity, Cu-DNAzyme with DNA cleavage enzyme activity, E47 DNAzyme with DNA ligase activity, G-quadruplex / heme DNAzyme with peroxidase activity, and UV1C DNAzyme that can repair thymine dimers. Among them, G-quadruplex / heme DNAzyme is highly favored by researchers due to its simple preparation, stable performance, low price, easy modification and easy storage. G-quadruplex / heme DNAzyme is usually composed of a nucleic acid sequence rich in G bases in the K + 、Na + NH4 + Under the presence of conditions such as α-glucose and β-glucose, G-quadruplex structures are formed, which then bind to heme. The G-rich nucleic acid sequence can be one or two single-stranded DNA sequences containing repeated tandem G sequences. These sequences form cyclic G4 sheets connected by Hoogsteen hydrogen bonds within or between molecules. Each G4 sheet is composed of four Gs, and two or more G4 sheets can further form G-quadruplexes through π-π stacking. In recent years, G-quadruplex / heme DNAzymes have been widely used in fields such as biosensing, bioimaging, and disease diagnosis and treatment.

[0003] Heme is an important iron porphyrin compound and a prosthetic group in hemoglobin, myoglobin, cytochromes, peroxidases, and catalases. It plays a key role in oxygen transport in human blood and maintaining normal cellular function. Rapid, sensitive, and low-cost detection of heme has important practical applications in drug screening, disease diagnosis, and forensic identification. In recent years, the main methods reported for heme detection include fluorescence analysis, colorimetry, chemiluminescence, and electrochemistry. Fluorescence analysis has been widely used due to its high sensitivity, good selectivity, and ease of use. Currently, fluorescence-based heme analysis focuses on two main areas: specific detection based on the fluorescence quenching effect of heme on nanomaterials; and the development of highly sensitive heme detection strategies based on G-rich nucleic acid probes. These methods have good anti-interference properties, but generally have low sensitivity. Furthermore, they require the preparation of fluorescent nanomaterials and characterization of their morphology and properties, which can be cumbersome, expensive, and time-consuming. In comparison, DNA synthesis and labeling technology is very mature. Heme sensing strategies can be easily constructed by using DNA with labeled groups, or with the help of DNA embedded dyes or peroxidase substrates. Therefore, they have obvious advantages in terms of time, cost, sensitivity, etc.

[0004] Hemoglobin is the main component of red blood cells in the blood and is responsible for the transport of oxygen and carbon dioxide within the cells. Abnormal hemoglobin levels in the human body can lead to serious diseases such as thalassemia, leukemia, cardiovascular disease, and diabetes. Currently, the reported methods for hemoglobin detection mainly include electrochemistry, surface plasmon resonance, and fluorescence. Among them, some methods require the hydrolysis of hemoglobin with trypsin to expose its heme subunits, and then detect hemoglobin by detecting heme; some methods achieve detection based on the quenching effect of hemoglobin on fluorescence. This "turn-off" mode of optical detection has many interfering factors and is not conducive to the analysis of target substances in complex systems. Therefore, there is still a need to develop new methods that are simple, rapid, and sensitive for the direct detection of hemoglobin.

[0005] Cysteine ​​is an essential amino acid containing a sulfhydryl group in the body. It is an important amino acid that constitutes the functional part of proteins and enzymes, and plays an important role in protein synthesis and cell reduction. Abnormal levels of L-cysteine ​​in cells are associated with a variety of diseases, such as skin damage, brain damage, liver damage, and Alzheimer's disease. At present, the main methods for detecting L-cysteine ​​are fluorescence, colorimetry, electrochemistry, and electrochemiluminescence. Among them, the new type of L-cysteine ​​detection based on simulating peroxidase activity has received special attention. Researchers have reported the use of nitrogen, boron, and sulfur co-doped carbon dots, iron nanoparticles @ Co3O4 hollow nanocages, FeMnO3 nanoparticles filled with polypyrrole nanotubes, Mo 6+There are methods for detecting L-cysteine ​​by mimicking peroxidase by doping Co3O4 nanotubes. However, these methods are generally limited by disadvantages such as complex operation and high cost. Summary of the Invention

[0006] Technical problem solved: In response to the above technical problems, the present invention provides a biosensor for detecting heme, hemoglobin and / or L-cysteine ​​and its application, which can realize the "turn-on" mode optical detection of heme and hemoglobin and the rapid detection of L-cysteine. Compared with the existing detection methods, it has higher sensitivity, and experiments have shown that it is still applicable to complex serum systems. It is expected to provide a simple, rapid and sensitive method for the detection of heme, hemoglobin and L-cysteine ​​in actual clinical samples.

[0007] Technical solution: A biosensor for detecting heme, hemoglobin and / or L-cysteine, comprising a hairpin nucleic acid HP, a binding buffer solution, heme, TMB, H2O2 and a HAc-NaAc buffer solution; the sequence of the hairpin nucleic acid HP is shown in SEQ No. 1, and its 5' end is labeled with a quenching group BHQ1 and its 3' end is labeled with a fluorescent group FAM.

[0008] Preferably, the binding buffer solution comprises: 0.55 mM MgCl2, 0.137 M NaCl, 0.0027 M KCl, 0.01 M Na2HPO4, 0.0018 M KH2PO4, and has a pH of 7.4.

[0009] Preferably, the hairpin nucleic acid HP is prepared from a Tris-HCl buffer solution comprising 10 mM Tris-HCl and having a pH of 8.0.

[0010] Preferably, the HAc-NaAc buffer solution comprises 0.2 M NaAc, 0.3 M HAc, and has a pH of 4.2.

[0011] A method for detecting heme or hemoglobin comprises the following steps:

[0012] (1) Adding the hairpin nucleic acid HP to the binding buffer solution to form a nucleic acid probe solution;

[0013] (2) Add the test solution to the nucleic acid probe solution for reaction;

[0014] (3) After the reaction is completed, fluorescence spectroscopy is performed with an excitation wavelength of 480 nm, and the concentration of hemoglobin or hemoglobin in the test solution is calculated based on the test results.

[0015] Preferably, the concentration of the hairpin nucleic acid HP in the nucleic acid probe solution is 20 nM.

[0016] Preferably, when the test solution does not contain serum, the detection concentration range of heme or hemoglobin is 0-400 nM; when the test solution contains serum, the detection concentration range of heme or hemoglobin is 20-400 nM.

[0017] A method for detecting L-cysteine ​​comprises the following steps:

[0018] (1) Mixing the hairpin nucleic acid HP with the hemoglobin solution for reaction;

[0019] (2) adding the test solution to the solution obtained in step (1) to carry out a reaction;

[0020] (3) Add TMB, H2O2 and HAc-NaAc buffer solution to the solution obtained in step (2) to carry out reaction;

[0021] (4) The solution obtained in step (3) is subjected to UV-visible absorption spectrum measurement, the absorption value at 652 nm is determined, and the concentration of L-cysteine ​​in the test solution is calculated based on the absorption value.

[0022] Preferably, in the solution obtained after the reaction in step (3), the concentration of the hairpin nucleic acid HP is 16 nM, the concentration of heme is 320 nM, the concentration of TMB is 2 mM, and the concentration of H2O2 is 12.5 mM.

[0023] Preferably, the detection concentration range of L-cysteine ​​is 0~45 μM.

[0024] Beneficial effects: In response to the shortcomings of current detection methods such as complex operation, high cost, and low sensitivity, as well as the deficiencies of the "turn-off" detection method, the present invention constructs a multifunctional biosensor with a fluorescent "turn-on" mode. This method can improve the shortcomings of low "turn-off" specificity, improve detection accuracy, and has higher sensitivity than current detection methods. It can still detect hemoglobin and hemoglobin in complex serum systems.

[0025] In the research of the present invention, "turn-on" mode fluorescence detection was used as the main analytical means, based on a novel G-base-rich hairpin nucleic acid probe and a G-quadruplex / heme DNAzyme. The 5' and 3' ends of the nucleic acid probe were labeled with a quencher group BHQ1 and a fluorescent group FAM as fluorescent probes, respectively. This achieved rapid, simple and highly sensitive analysis of heme and hemoglobin in complex systems. At the same time, the peroxidase activity of the novel G-quadruplex / heme DNAzyme was utilized to achieve the detection and analysis of L-cysteine, providing valuable theoretical and experimental basis for future development in this field.

[0026] The present invention labels a guanine (G)-rich hairpin nucleic acid probe (HP) with a quencher group (BHQ1) at its 5' end and a fluorescent group (FAM) at its 3' end. In a buffered solution, HP forms a hairpin structure. Due to the close proximity between FAM and BHQ1, efficient fluorescence resonance energy transfer (FRET) occurs, quenching FAM's fluorescence and placing the signal in a "turn-off" state. In the presence of heme or hemoglobin, heme binds to G-rich DNA sequences to form a stable G-quadruplex, increasing the distance between FAM and BHQ1. This significantly reduces FRET efficiency, allowing FAM's fluorescence to recover and placing the signal in a "turn-on" state. Furthermore, because the G-quadruplex / heme DNAzyme exhibits peroxidase-mimicking activity, it catalyzes the redox reaction between the substrate 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2, generating a blue product with an absorption peak at 652 nm. When L-cysteine ​​is present in the system, the color of the system changes from blue to colorless, and the absorption peak intensity significantly decreases, due to L-cysteine's inhibition of peroxidase activity. Therefore, by monitoring the fluorescence intensity, color, or UV-visible absorption intensity of the system, qualitative and quantitative detection of heme, heme proteins, and L-cysteine ​​can be achieved simultaneously. The method of the present invention is not only simple, rapid, and low-cost, but also highly sensitive, selective, and practical, demonstrating excellent detection performance even in complex biological serum. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG1 is a diagram showing the working principle of the biosensor for detecting hemoglobin and L-cysteine ​​using the present invention;

[0028] FIG2 is a specific analysis diagram of hemoglobin detection using the biosensor of the present invention;

[0029] FIG3 is a sensitivity analysis graph of hemoglobin detection in a buffer solution using the biosensor of the present invention; FIG3 is a trend graph showing the change in fluorescence intensity versus hemoglobin concentration in the buffer solution; FIG3 is a linear relationship graph showing the change in fluorescence intensity versus hemoglobin concentration within the concentration range of 20 to 400 nM; and FIG3 is a linear relationship graph showing the change in fluorescence intensity versus hemoglobin concentration within the concentration range of 0 to 2 nM.

[0030] FIG4 is a graph showing the sensitivity of the biosensor of the present invention for detecting hemoglobin in a 10% serum system; FIG4 is a graph showing the trend of fluorescence intensity versus hemoglobin concentration in 10% serum; FIG4 is a graph showing the linear relationship between fluorescence intensity and hemoglobin concentration within the concentration range of 20 to 400 nM; ...

[0031] FIG5 is a specific analysis diagram of hemoglobin detection using the biosensor of the present invention;

[0032] FIG6 is a sensitivity analysis graph of the biosensor of the present invention for detecting hemoglobin in a buffer solution; FIG6 is a graph showing the trend of fluorescence intensity versus hemoglobin concentration; FIG6 is a graph showing the linear relationship between fluorescence intensity and hemoglobin concentration in the range of 20 to 400 nM; and FIG6 is a graph showing the linear relationship between fluorescence intensity and hemoglobin concentration in the range of 0 to 2 nM.

[0033] FIG7 is a graph showing the sensitivity of the biosensor of the present invention for detecting hemoglobin in a 10% serum system; FIG7 is a graph showing the trend of fluorescence intensity versus hemoglobin concentration in 10% serum; FIG7 is a graph showing the linear relationship between fluorescence intensity and hemoglobin concentration in the concentration range of 20 to 400 nM; FIG7 is a graph showing the sensitivity of the biosensor of the present invention for detecting hemoglobin in a 10% serum system; FIG7 is a graph showing the trend of fluorescence intensity versus hemoglobin concentration in the 10% serum system; FIG7 is a graph showing the linear relationship between fluorescence intensity and hemoglobin concentration in the concentration range of 20 to 400 nM;

[0034] Figure 8 is a sensitivity analysis graph for detecting L-cysteine ​​using the biosensor of the present invention; Figure A shows the UV-visible absorption spectra of the reaction between different concentrations of L-Cys and G-quadruplex / heme DNAzyme; Figure B shows the relationship between L-Cys concentration and absorbance (λ = 652 nm) (the inset shows the linear relationship in the range of 0 to 45 μM). DETAILED DESCRIPTION

[0035] The present invention establishes a multifunctional biosensor based on a hairpin nucleic acid probe. Leveraging the ability of heme to bind to G-rich nucleic acid sequences, the 5' and 3' ends of the nucleic acid probe are labeled with a quencher group, BHQ1, and a fluorescent group, FAM, respectively. In the absence of the target substance, the probe forms a hairpin structure, quenching fluorescence. In the presence of the target substance, the hairpin structure transforms into a G-quadruplex structure, restoring the fluorescence signal. By utilizing the fluorescence signal changes before and after the nucleic acid probe binds to the target molecule, rapid, simple, and highly sensitive analysis of heme and hemoglobin can be achieved. Due to the peroxidase activity of the G-quadruplex / heme DNAzyme, colorimetric analysis of L-cysteine ​​can be achieved by monitoring the color change of the substrate, TMB, upon oxidation. This detection method exhibits high selectivity and practicality, and its performance for heme detection surpasses that of reported methods. Furthermore, heme-containing proteins can be directly detected without prior hydrolysis to release the heme, shortening detection time and reducing costs.

[0036] The present invention is further described below with reference to the accompanying drawings and examples.

[0037] The present invention can be better understood by referring to the following examples. However, it is readily understood by those skilled in the art that the specific material ratios, process conditions, and results described in the examples are only for illustrating the present invention and should not and will not limit the technical solutions in the claims.

[0038] Fluorescence detection was performed using a Shimadzu RF-6000 fluorescence spectrophotometer (Shimadzu Corporation, Japan). Fluorescence spectra were measured using a xenon lamp with an excitation wavelength of 480 nm, a scan range of 490–650 nm, and 5 nm slit widths for both excitation and emission. Measurements were performed using a quartz cuvette with a maximum volume of 600 µL, and a total sample volume of 400 µL. All samples were measured at room temperature.

[0039] UV absorption measurements were performed using a Shimadzu UV-3600 UV-Vis spectrophotometer (Shimadzu Corporation, Japan). UV absorption spectra were measured at 652 nm over a scanning range of 500–800 nm, with slit widths of 3 nm for both excitation and emission. Measurements were performed using a quartz cuvette with a maximum volume of 700 µL and a total sample volume of 500 µL. All samples were measured at room temperature.

[0040] The oligonucleotides used in the examples of the present invention were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China). The hairpin nucleic acid probe HP sequence used was

[0041] HP: 5'-TACAGGTTCTGGGGGGTGGGTGGGGAACCTGTT-3', (SEQ No. 1)

[0042] Hemin was purchased from Shanghai Angyi Biotechnology Co., Ltd.; protoporphyrin IX (PPIX) was purchased from Sigma-Aldrich, USA; PD-L1 protein was purchased from Beijing Sino Biological; fetal bovine serum (Gibco) was purchased from KeyGen Biotechnology Co., Ltd. (Nanjing, China); L-serine, L-glutamic acid, L-cysteine, and bovine serum albumin were purchased from Shanghai Huixing Biochemical; lysozyme and hemoglobin were purchased from Shanghai Sangon Biotechnology Co., Ltd.; TMB (3,3',5,5'-tetramethylbenzidine) and H2O2 were purchased from Aladdin Reagent (Shanghai) Co., Ltd.

[0043] All buffers were prepared using Milli-Q water (18.2 MΩ·cm). The hairpin nucleic acid HP used in the experiment was labeled with the quencher BHQ1 and the fluorophore FAM at its 5' and 3' ends, respectively, and was prepared and diluted in Tris-HCl buffer (10 mM Tris-HCl, pH 8.0). Binding buffer (0.55 mM MgCl2, 0.137 M NaCl, 0.0027 M MKCl, 0.01 M Na2HPO4, 0.0018 M KH2PO4, pH 7.4) was used for the reaction between HP and heme and hemoglobin and subsequent fluorescence detection. HAc-NaAc buffer (0.2 M NaAc, 0.3 M HAc, pH 4.2) was used for the reaction between HP and the G-quadruplex / heme DNAzyme and L-cysteine.

[0044] Example 1: Specificity analysis of hemoglobin detection

[0045] To 36 μL of binding buffer, 4 μL of 2 μM hairpin nucleic acid HP was added to form nucleic acid probe solution I. The test solutions were then added to nucleic acid probe solution I and allowed to react at 37°C for 30 minutes. The binding buffer was then added to a final volume of 400 μL, and fluorescence spectra were measured. The test solutions consisted of 20 μL of 8 μM heme solution, 20 μL of 100 μM protoporphyrin IX, 20 μL of 100 μM L-serine, 20 μL of 100 μM L-glutamic acid, and 20 μL of 100 μM L-cysteine.

[0046] like Figure 2As shown in the figure, the fluorescence intensity of the system containing heme (400 nM) was 4 times that of the blank group, while the fluorescence intensities of the systems containing PPIX, L-Ser, L-Glu and L-Cys were 1.9, 1.5, 1.4 and 1.3 times that of the blank group, respectively. The results showed that the method had good specificity.

[0047] Example 2: Sensitivity Analysis of Hemoglobin Detection in Buffer Solution

[0048] For the 0-400 nM sensitivity analysis, the test solution consisted of 20 μL of heme solutions at concentrations of 0, 0.04, 0.4, 2, 4, 6, and 8 μM. Fluorescence spectra were measured after the reaction. For the 0-2 nM sensitivity analysis, the test solution consisted of 20 μL of heme solutions at concentrations of 0, 0.01, 0.02, 0.03, and 0.04 μM. Other conditions were the same as those used in the specificity study.

[0049] like Figure 3 As shown in the figure, with the increase of hemoglobin concentration, the fluorescence intensity of the system gradually increases. The trend diagram between hemoglobin concentration and system fluorescence intensity shows that there is a good linear relationship in the range of 20~400 nM. The regression equation is y= 0.11x + 139.31, and the linear correlation coefficient R 2 =0.95399. Further, the low concentration range (0, 0.5, 1, 1.5, 2 nM) of hemoglobin was analyzed, and a good linear relationship was also found in the range of 0~2 nM. The regression equation was y = 21.1x + 52.71, R 2 = 0.97098. Based on the 3σ principle, the sensitivity of this method for detecting hemoglobin is estimated to be 282 pM.

[0050] Example 3: Sensitivity analysis of hemoglobin detection in 10% serum

[0051] To 36 μL of nucleic acid probe solution I, 4 μL of serum was added to form nucleic acid probe solution II (serum concentration was 10%). Test solutions were then added to each 40 μL of nucleic acid probe solution II: 20 μL of heme solutions at concentrations of 0, 0.04, 0.4, 2, 4, 6, and 8 μM. The mixture was reacted at 37°C for 30 minutes. The appropriate amount of binding buffer was then added to the final volume of 400 μL for fluorescence spectroscopy measurement. Other conditions were the same as those used in the specificity study.

[0052] like Figure 4As shown in the figure, with the increase of hemoglobin concentration, the fluorescence intensity of the system gradually increases. The trend diagram between hemoglobin concentration and system fluorescence intensity shows that there is a good linear relationship in the range of 20~400 nM. The regression equation is y=0.16x+205.73, and the linear correlation coefficient R 2 = 0.98151. Based on the 3σ principle, the sensitivity of this method for detecting hemoglobin is estimated to be 40 nM.

[0053] Example 4: Specificity Analysis of Hemoglobin Detection

[0054] To 36 μL of binding buffer, 4 μL of 2 μM hairpin nucleic acid HP was added to form nucleic acid probe solution I. The test solutions were then added to nucleic acid probe solution I and allowed to react at 37°C for 30 minutes. The appropriate amount of binding buffer was then added to the final volume of 400 μL for fluorescence spectroscopy measurement. The test solutions were hemoglobin, PD-L1 protein, bovine serum albumin, and lysozyme, each with a 20 μL volume and an 8 μM concentration.

[0055] like Figure 5 As shown, the fluorescence intensity of the system containing Hb is 140, while the fluorescence intensities of the systems containing BSA, Lys, and PD-L1 protein are 52, 30, and 60, respectively, which are not much different from the blank. The results show that using HP as a recognition probe and detecting the fluorescence signal of the system can significantly distinguish between proteins containing heme and those without heme.

[0056] Example 5: Sensitivity Analysis of Hemoglobin Detection in Buffer Solution

[0057] For sensitivity testing in the 0-400 nM concentration range, the test solution consisted of 20 μL of hemoglobin solutions at concentrations of 0, 0.04, 0.4, 2, 4, 6, and 8 μM. For sensitivity testing in the 0-2 nM concentration range, the test solution consisted of 20 μL of hemoglobin solutions at concentrations of 0, 0.01, 0.02, 0.03, and 0.04 μM. Other conditions were the same as those used in the specificity study.

[0058] like Figure 6 As shown in the figure, with the increase of Hb concentration, the fluorescence intensity of the system gradually increased. In the range of Hb concentration of 20~400 nM, there is a good linear relationship between the fluorescence intensity and Hb concentration. The regression equation is y = 0.14x + 88.38, and the linear correlation coefficient R 2 = 0.98771. The results of the analysis of Hb in the low concentration range showed that it also had a good linear relationship in the range of 0~2 nM, with the regression equation being y = 17.64x+50.88, R 2= 0.99215. According to the 3σ principle, the sensitivity of this method for hemoglobin detection is estimated to be 108 pM.

[0059] Example 6: Sensitivity analysis of hemoglobin detection in 10% serum

[0060] The test solution was 20 μL of hemoglobin solution with concentrations of 0, 0.04, 0.4, 2, 4, 6, and 8 μM. Other conditions were the same as in Example 3.

[0061] like Figure 7 As shown in the figure, with the increase of Hb concentration, the fluorescence intensity of the system gradually increased. In the range of Hb concentration of 20~400 nM, there is a good linear relationship between fluorescence intensity and Hb concentration. The regression equation is y = 0.33x + 157.00, and the linear correlation coefficient R 2 = 0.99424. According to the 3σ principle, the sensitivity of this method for hemoglobin detection is estimated to be 13 nM.

[0062] Example 7: Sensitivity Analysis of L-Cysteine ​​Detection

[0063] 4 μL of 2 μM hairpin nucleic acid HP and 20 μL of 8 μmol / L heme solution were mixed and reacted at 37°C for 30 minutes. Binding buffer was then added to a final volume of 100 μL. Then, 100 μL of L-cysteine ​​solution at concentrations of 0, 18, 36, 72, 180, 720, and 1440 μM was added. After incubation at room temperature for 2 minutes, 100 μL of 10 mM TMB, 100 μL of 50 mM H2O2, and 100 μL of HAc-NaAc buffer (pH = 4.2) were added, respectively. After reacting at 37°C for 20 minutes, UV-visible absorption spectroscopy was performed.

[0064] like Figure 8 As shown in the figure, the absorption value of the system at 652 nm gradually decreases. When the concentration is greater than 180 μM, the absorption peak disappears and the corresponding solution color is colorless. At this time, the DNAzyme activity is completely inhibited. The relationship curve between L-cysteine ​​concentration and 652 nm UV absorption value shows a good linear relationship in the range of 0-45 μM. The regression equation is y=0.33-0.00336x, and the correlation coefficient R 2 =0.99185. According to the 3σ principle, the detection limit was estimated to be 3.9 μM.

[0065] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. Sequence Listing <110> Nanjing University of Posts and Telecommunications <120> A biosensor for detecting heme, hemoglobin and / or L-cysteine ​​and its application <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 33 <212> DNA <213> Artificial Sequence <400> 1 tacaggttct gggggtggg tggggaacct gtt 33

Claims

1. A biosensor for detecting hemoglobin and / or hemoglobin, characterized in that: The invention comprises a hairpin nucleic acid HP and a binding buffer solution; the sequence of the hairpin nucleic acid HP is shown in SEQ No. 1, the 5' end of the hairpin nucleic acid HP is labeled with a quenching group BHQ1, and the 3' end of the hairpin nucleic acid HP is labeled with a fluorescent group FAM; in the absence of target substances heme and / or hemoglobin, the hairpin nucleic acid HP forms a hairpin structure and the fluorescence is quenched; in the presence of the target substance, the hairpin structure is converted into a G-quadruplex structure and the fluorescence signal is restored.

2. The biosensor for detecting hemoglobin and / or hemoglobin according to claim 1, characterized in that: The binding buffer solution includes: 0.55 mM MgCl2, 0.137 M NaCl, 0.0027 M KCl, 0.01 M Na2HPO4, 0.0018 M KH2PO4, and has a pH of 7.

4.

3. The biosensor for detecting hemoglobin and / or hemoglobin according to claim 1, characterized in that: The hairpin nucleic acid HP is prepared from a Tris-HCl buffer solution, which includes 10 mM Tris-HCl and has a pH of 8.

0.

4. A method for detecting hemoglobin or hemoglobin using the biosensor according to claim 1, characterized in that: The steps are as follows: (1) Adding the hairpin nucleic acid HP to the binding buffer solution to form a nucleic acid probe solution; (2) Add the test solution to the nucleic acid probe solution for reaction; (3) After the reaction is completed, fluorescence spectroscopy is performed with an excitation wavelength of 480 nm, and the concentration of hemoglobin or hemoglobin in the test solution is calculated based on the test results.

5. The method for detecting hemoglobin or hemoglobin according to claim 4, characterized in that: The concentration of the hairpin nucleic acid HP in the nucleic acid probe solution is 20 nM.

6. The method for detecting hemoglobin or hemoglobin according to claim 4, characterized in that: When the test solution does not contain serum, the detection concentration range of heme or hemoglobin is 0~400 nM; when the test solution contains serum, the detection concentration range of heme or hemoglobin is 20~400 nM.

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