CN102507952B - Method for detecting protein and metal ions - Google Patents

Abstract

The invention relates to the field of biotechnology, and discloses a method for detecting proteins and metal ions. The method is to provide a probe molecule with a reporter fluorescent group that can specifically bind to a target; The nanomaterial solution of π electrons is mixed, then added to the sample to be tested, and the fluorescence is detected; wherein, the nanomaterial rich in π electrons is selected from co-polymers whose monomers are phenylenediamine, 3,4-ethylenedioxythiophene or styrene Conjugated polymers, silver(I)-4,4'-bipyridine, copper(II)-4,4'-bipyridine, chloroplatinic acid-3,3',5,5'-tetramethylbiphenyl Amines, chloroplatinic acid-p-phenylenediamine or tetrachloroalloy acid-4,4'-bipyridine coordination polymers, porphyrin clusters, mesoporous carbon and nano C ₆₀ . The detection method of the invention has the advantages of low cost, simple operation, short detection time, high sensitivity and wide application range.

Description

A method for detecting proteins and metal ions

technical field

The invention relates to the field of biotechnology, in particular to a method for detecting proteins and metal ions.

Background technique

Protein is an important biomacromolecule in the human body. It is not only very important and essential for maintaining life, but also closely related to the translation of life genetic code, transcription of information, and replication of DNA. The content of protein in body fluids can be used as an important indicator of human nutrition, health and disease diagnosis. For example, the content of urinary protein is one of the basis for diagnosing nephritis and diabetes. The limit is an abnormal value and is a sign of disease. For example, serum protein is an important living substance in the human body, and its content in body fluids can be used as an important indicator of human nutritional health and disease diagnosis. Another example is Alzheimer's disease (AD), which is a group of primary degenerative diseases of the central nervous system with unknown causes. It is still an incurable disease. Quantitative determination of Tau protein in cerebrospinal fluid may become a useful indicator for early diagnosis of AD. Biological assays, such as cerebrospinal fluid Tau protein assay, may help clinicians to achieve the purpose of early diagnosis and prevention of AD. Therefore, the trace detection of specific proteins can realize the early diagnosis of diseases, so as to treat early and prolong the life of patients.

Metal ions play an important role in the fields of environment, biology, and medicine, and their concentration, type, and valence state have an important impact on life activities and the environment. For example, metal ions such as potassium, iron, and zinc are essential elements for living organisms to maintain normal physiological activities. And some heavy metal ions, because they cannot be decomposed in the environment and will gradually accumulate in the upper layer of the biological chain through the food chain, are likely to cause chronic poisoning. According to the "Beijing Youth Daily" report: Since 1974, a total of 58 people died in Longling Village, Huaxian County, Shaanxi Province, 29 people died of cancer, 22 people died of pulmonary heart disease and cerebrovascular disease, and only 1 person died of natural causes. After investigation, the soil in this area is mainly polluted by lead and arsenic, while the flour eaten by local villagers is seriously polluted by four elements: lead, arsenic, zinc, and chromium. The contents of cadmium and lead in beans are 5.3 times and 2.55 times, which is a seriously polluted vegetable. Therefore, the detection of metal ions is of great significance in environmental monitoring and disease diagnosis. At present, atomic luminescence and absorption spectroscopy are mainly used for the detection of metal ions. Although this method has the advantages of high sensitivity and high accuracy, it requires the use of large instruments, and the detection cost is high, which is not conducive to widespread promotion. Therefore, it is necessary to develop a simple, rapid, highly selective, and highly sensitive method for the detection of metal ions.

Contents of the invention

In view of this, the purpose of the present invention is to provide a method for detecting protein and metal ions with simple operation, high sensitivity and strong selectivity for the defects of existing detection methods.

For realizing the purpose of the present invention, the present invention adopts following technical scheme:

A method for detecting proteins and metal ions,

Provide a probe molecule with a reporter fluorescent group that can specifically bind to the target;

Mixing the probe molecule with a nanomaterial solution rich in π electrons, then adding the sample to be tested, and detecting the fluorescence;

Wherein, the target is a protein and a metal ion, and the nano-material rich in π electrons is selected from a conjugated polymer whose monomer is phenylenediamine, 3,4-ethylenedioxythiophene or styrene, silver (I )-4,4'-bipyridine, copper (II)-4,4'-bipyridine, chloroplatinic acid-3,3',5,5'-tetramethylbenzidinediamine, chloroplatinic acid-p Phenylenediamine or tetrachloroalloy acid-4,4'-bipyridine coordination polymers, porphyrin clusters, mesoporous carbon and nano C ₆₀ .

With the development of DNA in vitro evolution technology, the cost of screening and synthesis of probe DNA molecular sequences is getting lower and lower, so the detection platform based on DNA molecules has broad application prospects. The specific recognition of DNA molecules and proteins is a research hotspot in recent years, and it is widely used in the identification and detection of specific proteins. For example, Landegren et al. from the Uppsala Biomedical Center in Sweden used DNA as a probe to realize the detection of PDGF protein. High sensitivity and high selectivity detection, the minimum detection concentration is 40×10 ^-21 mol [Fredriksson, S.Gullberg, M.; Jarvius, J.; Olsson, C.; Pietras, K.; Gustafsdottir, SM; Ostman , A.; Landegren, U., Nat. Biotechnol, 2002, 20, 473-477]. Specially screened DNA sequences can form stable complexes with metal ions, showing specificity of action, such as a colorimetric detection method for Hg ²⁺ ions, using the interaction between Hg ²⁺ ions and the T base in the DNA sequence AGR0100, The detection limit was 5OnM [Li, T.; Dong, SJ; Wang, EK, Anal Chem, 2009, 81, 2144-2149]. DNA molecules that have been screened by in vitro evolution can specifically recognize small organic molecules, thereby inducing a change in the conformation of DNA molecules. The selective detection of α, with a detection limit of 20 μM [Wang, Y.; Liu, B., Analyst, 2008, 133, 1593-1598].

The method for detecting proteins and metal ions of the present invention utilizes the π-π interaction to adsorb the probe molecules specifically bound to the target on the surface of nanomaterials rich in π electrons, quench the fluorescence of the probe molecules, and then the probe molecules Mixed with the sample to be tested, the probe molecule is combined with the target, so that the probe molecule is detached from the surface of the nanomaterial, the fluorescence quenching effect disappears, and the detection of the sample to be tested is realized by detecting the fluorescent signal. Moreover, according to the change of the fluorescence intensity, the content of the target object in the sample to be tested can also be determined. The method for detecting proteins and metal ions of the present invention is low in cost, the preparation method of the nano-material rich in π electrons is simple, the price of raw materials is low, and it can be prepared on a large scale; the operation is simple, and only the π electrons rich in The nanomaterials, probe molecules and samples to be tested are mixed, and the fluorescence is detected by using a simple fluorescence detection instrument, without the need for expensive large-scale analysis instruments; the detection performance is excellent, the detection time is short, and the sensitivity is high. It is a simple, fast, and A low-cost, broad-ranging method for the detection of proteins and metal ions.

Description of drawings

Figure 1 shows a schematic diagram of the principle of the detection method of the present invention, wherein the spherical shape only represents nanomaterials rich in π electrons, and does not indicate that the material itself is spherical, and the target contains proteins and metal ions; the folding of the probe DNA represents the occurrence of its conformation changing, but not limited to folding;

Figure 2 shows the results of detection of thrombin protein by poly-m-phenylenediamine nanorods. a is the fluorescence emission spectrum of probe DNA (20nM _TA ); b is the fluorescence emission spectrum of probe DNA (20nM _TA )+target protein (100nM _TB ); c is probe DNA (20nM _TA )+target protein (100nM _TB )+PMPD nanorod fluorescence emission spectrum; d is the fluorescence emission spectrum of probe DNA (20nM _TA )+PMPD nanorod; e is the fluorescence emission spectrum of PMPD nanorod self;

Fig. 3 shows the results of specific selection of poly-m-phenylenediamine nanorods to thrombin; the other two interfering proteins are respectively bovine serum albumin (BSA) and human immunoglobulin (IgG), both at a concentration of 100 nM.

Figure 4 shows the results of mesoporous carbon particles detecting thrombin in human serum samples;

Figure 5 shows the detection results of nano-C ₆₀ (nano-C ₆₀ ) on metal ion Hg ²⁺ , the fluorescence emission spectrum of probe DNA ( _PH , 100nM) under different conditions: (a) _PH , (b) _PH + Hg ²⁺ (8 μM), (c) _PH + nano-C60, (d) _PH + nano-C ₆₀ + Hg ²⁺ (8 μM);

Fig. 6 shows the result graph of the selectivity of nano-C ₆₀ to metal ion Hg ²⁺ . Among them, the concentration of Hg ²⁺ is 8 μM, the concentration of other interfering ions in Figure A is 5 μM, and the concentration of other interfering ions in Figure B is 50 μM;

Figure 7 shows the results of nano-C ₆₀ detecting metal ion Hg ²⁺ in lake water. a is the fluorescence emission spectrum of lake water, b is the fluorescence emission spectrum of lake water +30nM Hg ²⁺ .

Detailed ways

The embodiment of the invention discloses a method for detecting protein and metal ions. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method of the present invention has been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the method described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention .

For realizing the purpose of the present invention, the present invention adopts following technical scheme:

Provide a probe molecule with a reporter fluorescent group that can specifically bind to the target;

Mixing the probe molecule with a nanomaterial solution rich in π electrons, then adding the sample to be tested, and detecting the fluorescence;

Wherein, the target is a protein and a metal ion, and the nano-material rich in π electrons is selected from a conjugated polymer whose monomer is phenylenediamine, 3,4-ethylenedioxythiophene or styrene, silver (I )-4,4'-bipyridine, copper (II)-4,4'-bipyridine, chloroplatinic acid-3,3',5,5'-tetramethylbenzidinediamine, chloroplatinic acid-p Phenylenediamine or tetrachloroalloy acid-4,4'-bipyridine coordination polymers, porphyrin clusters, mesoporous carbon and nano C ₆₀ .

The method for detecting proteins and metal ions described in the present invention mixes probe molecules specifically bound to the target with nanomaterials rich in π electrons. Due to the π-π interaction, the probe molecules are adsorbed on the surface of the material, and then the probe molecules The energy transfer between the reporter fluorescent group on the molecule and the nanomaterial rich in π electrons can quench the fluorescence emitted by the fluorescent group. After adding the sample to be tested, the target in the sample to be tested is combined with the probe molecule, so that the probe molecule is detached from the surface of the nanomaterial, the fluorescence quenching effect disappears, and the fluorescence of the probe molecule is restored, and the detection of the sample to be tested is realized by detecting the fluorescence signal. detection. Moreover, according to the change of the fluorescence intensity, the content of the target object in the sample to be tested can also be determined.

The probe molecule of the present invention has a reporter fluorescent group, and the reporter fluorescent group can be labeled at the 3' end or the 5' end of the probe molecule, or can be simultaneously labeled with the reporter fluorescent group at the 3' end and the 5' end, so as to Increase the fluorescence intensity and improve the sensitivity of detection. Probe-labeled reporter fluorophores include, but are not limited to, the following fluorophores: FAM, TET, JOE, VIC, HEX, ROX, TAMRA, CY3, CY3.5, CY5, CY5.5, Oregon Green, CAL Red, Red640, and Texas Red.

The nanometer material rich in π electrons in the present invention is selected from conjugated polymers whose monomers are phenylenediamine, 3,4-ethylenedioxythiophene or styrene. Wherein, the preparation method of the conjugated polymer is to mix the solution of the monomer and the oxidizing agent according to the molar ratio of 1:0.1-20, and leave it for 1min-24h. The monomer is phenylenediamine, 3,4-ethylenedioxythiophene or styrene, and the oxidizing agent is preferably ammonium persulfate or ferric chloride.

The coordination polymer of the present invention is silver (I)-4,4'-bipyridine, copper (II)-4,4'-bipyridine, chloroplatinic acid-3,3',5,5'-tetra Methyl benzidine diamine, chloroplatinic acid-p-phenylenediamine or tetrachloroalloy acid-4,4'-bipyridine coordination polymer, composed of 4,4'-bipyridine and tetramethyl bipyridine containing π electrons The phenylenediamine molecule is used as a monomer, and the transition metal ion silver, copper, platinum or gold is used as an initiator to polymerize. The preparation method of the coordination polymer is prepared by mixing the solution of transition metal ions and organic molecules containing π electrons according to the molar ratio of 1:0.1-20, and standing for 1min-24h.

Organic molecular clusters are simple molecular aggregates formed by organic molecules approaching each other driven by hydrophobic and lipophilic interactions in water or a mixed system of water and organic solvents. The porphyrin clusters in the present invention are formed by clustering porphyrin molecules containing π electrons close to each other in a mixed solution of water and an organic solvent. Wherein, the preparation method of the porphyrin cluster is as follows: porphyrin molecules containing π electrons are dissolved in an organic solvent to obtain a solution with a concentration of 1 mmol/L to 0.5 mol/L, and then mixed with water at a volume ratio of 0.001 to 1:1. Mix and place for 1min to 24h to prepare. Wherein, the organic solvent is preferably methanol, ethanol, propanol, ethylene glycol, dimethyl sulfoxide, tetrahydrofuran or N-methylpyrrolidone.

Both the mesoporous carbon and nano- _C60 of the present invention belong to carbon materials, wherein the preparation method of the mesoporous carbon is to introduce the carbon source precursor into the pores of mesoporous silicon oxide by impregnation method, and make the precursor The material is thermally decomposed and deposited in the pores of the template mesoporous material, calcined and carbonized to obtain a carbon-silicon composite, and then the silicon template is removed to obtain mesoporous carbon. Among them, the carbon source precursor is preferably glucose, sucrose acetylene, mesophase pitch, furan methanol, and phenol/formaldehyde resin. The preparation method of the nano- _C60 in the present invention is that the _C60 powder is dissolved in acetone, then mixed with acetonitrile, and the khaki precipitate is collected, and then evenly dispersed in water.

In order to further understand the present invention, the present invention will be described in detail below in conjunction with examples.

Among them, in the embodiments of the present invention, human thrombin protein is used as the target protein ( _TB ) for detection, and a single-stranded DNA sequence modified by a fluorophore (FAM) that has specific recognition ability for human thrombin protein is used as a fluorescent detection protein fluorescent probe (T _A ). In the embodiment, Hg ²⁺ and Ag ⁺ are used as the target metal ions for detection respectively, and the single-stranded DNA sequence rich in T bases modified by fluorophore (FAM) with specific binding ability to Hg ²⁺ is used as the fluorescence detection Hg ²⁺ probe DNA (P _H ), the single-stranded DNA sequence rich in C bases modified by fluorophore (FAM) with specific binding ability to Ag ⁺ is used as the probe DNA for fluorescent detection of Ag ⁺ (P _Ag ). See Table 1 for the sequence of each probe.

Table 1 Related Probe Sequences

Embodiment 1: the preparation of the nanorod of polym-phenylenediamine (PMPD) and detection protein

Material preparation and processing: at room temperature, mix 0.06mL of 0.5M ammonium persulfate aqueous solution with 0.84mL of water, then add 0.1mL of 0.1M m-phenylenediamine aqueous solution and shake, and then a large amount of polym-phenylenediamine can be seen Precipitation of nanospheres of amine occurs. The resulting precipitate was washed with secondary water and centrifuged, and the operation was repeated several times, and then dispersed in water to prepare poly-m-phenylenediamine (PMPD) nanorods, which were stored at 4°C for future use.

Detection of protein: the single-stranded DNA sequence modified by fluorophore (FAM) with specific recognition ability for human thrombin protein is used as the fluorescent probe (T _A ) for detection of protein, and human thrombin protein is used as the target protein for detection (T _B ). Detection method: first, PMPD nanorods are added to a solution of fluorescent probes (50nM _TA ), and single-stranded DNA probes are adsorbed to the surface of PMPD nanorods, causing fluorescence quenching. After a certain reaction time (60min), the fluorescence quenching reached equilibrium (98%). When human thrombin protein (100nM _TB ) was added, the fluorescence quenching was inhibited due to the presence of the protein, and only 50% of the quenching occurred (60min to reach equilibrium). The test results are shown in Figure 2. The results show that the detection limit of the detection method based on PMPD nanorods can reach 100pM. After adding other proteins (100nM bovine serum albumin BSA or human immunoglobulin 100nM IgG) to the solution of fluorescent probe (50nM _TA ), then add PMPD nanorods, the results are shown in FIG. 3 . It can be seen from Fig. 3 that the fluorescence quenching is not suppressed, and the ratio of the fluorescence intensity when BSA and IgG exist to the fluorescence intensity when _TB exists is 10.6% and 8.6%, so the detection method based on PMPD nanorods has no effect on human thrombin The protein has a high specific detection ability.

Example 2: Preparation of nano-C ₆₀ and detection of protein and metal ions

Material preparation and processing: Dissolve 2.5 mg of C ₆₀ in 2 mL of acetone, then add 12 mL of acetonitrile dropwise while stirring, and then a large amount of khaki precipitate appears. Wash the precipitate with acetonitrile and centrifuge, repeat the operation several times, and then sonicate for 30 minutes to uniformly disperse the obtained Nano-C ₆₀ in 8 mL of water for later use.

Detection of protein: the single-stranded DNA sequence modified by fluorophore (FAM) with specific recognition ability for human thrombin protein is used as the fluorescent probe (T _A ) for fluorescent detection of protein, and human thrombin protein is used as the target protein for detection (T _B ). First, nano-C ₆₀ is added to the solution of fluorescent probe (50nMT _A ), the single-stranded DNA probe will be adsorbed to the surface of nano-C ₆₀ , causing fluorescence quenching. After a certain reaction time (60min), the fluorescence quenching reached equilibrium (86%). Adding human thrombin protein (100nM _TB ) and adding nano-C ₆₀ , the quenching of fluorescence was inhibited due to the presence of protein, and only 60% quenching occurred (60min to reach equilibrium). The detection limit of the detection method based on nano-C ₆₀ can reach 1nM. When adding other proteins (100nM bovine serum albumin BSA or human immunoglobulin 100nM IgG) to the fluorescent probe (50nM _TA ) solution, then adding nano-C ₆₀ , the fluorescence quenching was not inhibited, and BSA The ratio of the fluorescence intensity in the presence of IgG to the fluorescence intensity in the presence of _TB is 27.3% and 29.3%, so the detection method based on nano-C ₆₀ has a high specificity detection ability for human thrombin protein.

Detection of metal ions (Hg ²⁺ , Ag ⁺ ): The single-stranded DNA sequence rich in T bases modified by fluorophore (FAM) and having specific binding ability to Hg ²⁺ is used as a fluorescent probe for fluorescent detection (P _H ), with Hg ²⁺ as the target metal ion detected. First, nano-C ₆₀ is added to the solution of fluorescent probe (50nM _PH ), the single-stranded DNA probe will be adsorbed to the surface of nano-C ₆₀ , causing 88% fluorescence quenching. In the presence of Hg ²⁺ , due to the formation of folded structure caused by T-Hg ²⁺ -T, the quenching of fluorescence is suppressed, and the fluorescence intensity at this time is 6 times that of the absence of Hg ²⁺ . Through the above process, the detection limit of the method for detecting Hg ²⁺ ions based on nano-C ₆₀ can be as low as 500pM. The results are shown in Figures 5 and 6. When other metal ions are added, the detection method also has high selectivity, and this method can be detected in the actual sample lake water, the results are shown in Figure 7. Furthermore, this method can be applied to Ag ⁺ detection with a detection limit reaching 1 nM.

Example 3: Preparation of mesoporous silicon and detection of proteins and metal ions

Material preparation and treatment: Add 1 g of mesoporous silicon (MS) to 5 g of water, then add 1.25 g of glucose and 0.14 g of 98% H ₂ SO ₄ , stir at room temperature for 5 h, and let the resulting mixture react at 100°C and 160°C for 6 h respectively . Afterwards, the reaction mixture was added to ₅ g of water, followed by 0.75 g of sugar and 0.09 g of 98% _H2SO4 . After stirring for 12 h, the reaction mixture was dried at 160 °C for 6 h. Finally, calcining at 800 °C for 4 h under the protection of N ₂ to complete the carbonization process. The obtained carbon-silicon composite was washed with 1M NaOH (water/ethanol mixture) for 12 h to remove the silicon template, and then dried at room temperature to obtain mesoporous carbon (MC) particles.

Detection of protein: the single-stranded DNA sequence modified by fluorophore (FAM) with specific recognition ability for human thrombin protein is used as the fluorescent probe (T _A ) for fluorescent detection of protein, and human thrombin protein is used as the target protein for detection (T _B ). Firstly, MC is added to the solution of fluorescent probe (50nM _TA ), and the single-stranded DNA probe will be adsorbed to the surface of MC, causing fluorescence quenching. After a certain reaction time (60min), the fluorescence quenching reached equilibrium (97%). When human thrombin protein (100nM _TB ) was added, and then MC was added, the fluorescence quenching was inhibited due to the presence of the protein, and only 38% of the quenching occurred (reached equilibrium in 60 minutes). The results are shown in FIG. 4 . The results showed that the detection limit of the MC-based assay could be as low as 250pM. When other proteins (100nM bovine serum albumin BSA or human immunoglobulin 100nM IgG) were added to the solution of fluorescent probe (50nM _TA ) and then MC was added, the fluorescence quenching was not inhibited, and BSA and IgG existed The ratio of the fluorescence intensity in the presence of _TB to the fluorescence intensity in the presence of TB was 6.2% and 7.4%, so this MC-based detection method has a high specificity detection ability for human thrombin protein.

Detection of metal ions: The single-stranded DNA sequence rich in C bases modified by fluorophore (FAM) and having specific binding ability to Ag ⁺ is used as the fluorescent probe (P _Ag ) for fluorescent detection, and Ag ⁺ is used as the detection target metal ions. Firstly, MC was added to the solution of fluorescent probe (50nM P _Ag ), and the single-stranded DNA probe would be adsorbed to the surface of MC, causing 84% fluorescence quenching. However, in the presence of Ag ⁺ , due to the formation of folded structures caused by C-Ag ⁺ -C, the quenching of fluorescence is suppressed, and the fluorescence intensity at this time is 4 times that of the absence of Ag ⁺ . The results showed that the detection limit of MC-based detection method can be as low as 500pM. When other metal ions are added, the detection method also has high selectivity, and the method can be detected in actual sample lake water. In addition, this method can be used for the detection of Hg ²⁺ , and the detection limit can reach 10nM.

The descriptions of the above embodiments are only used to help understand the method and core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, some improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims (1)

1. A method for detecting protein or metal ion, characterized in that, Provide a probe molecule with a reporter fluorescent group that can specifically bind to the target; Mixing the probe molecule with a nanomaterial solution rich in π electrons, then adding the sample to be tested, and detecting the fluorescence; Wherein, the target is a protein or a metal ion, and the nanometer material monomer rich in π electrons is copper (II)-4,4'-bipyridine.

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