CN110433650B - Scavenging kit and scavenging method for formaldehyde in air - Google Patents

Scavenging kit and scavenging method for formaldehyde in air Download PDF

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CN110433650B
CN110433650B CN201910738209.0A CN201910738209A CN110433650B CN 110433650 B CN110433650 B CN 110433650B CN 201910738209 A CN201910738209 A CN 201910738209A CN 110433650 B CN110433650 B CN 110433650B
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陈翠贤
王思捷
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Fuzhou Sijida Biomedical Technology Co ltd
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Abstract

The invention provides a novel method for removing formaldehyde in air with high efficiency, economy, environmental protection and practicability and a kit thereof, which are mainly based on a novel functional polypeptide T1 with high-efficiency formaldehyde binding capacity, the sequence of the functional polypeptide T1 is shown as SEQ ID NO:1, the polypeptide T1 can be combined with magnetic beads coated with streptavidin through biotin modified and connected on terminal phenylalanine to prepare magnetic-carried polypeptide, the formaldehyde binding capacity of each 1 mu mol of the magnetic-carried polypeptide T1 can reach 87.25mg, and (NH 4) with the final concentration of 0.182mol/L must be added into a combination reaction system of the magnetic-carried polypeptide and the formaldehyde2SO4The solution, the combination of the magnetic polypeptide T1 and formaldehyde can be destroyed by a mixed solution system of NaOH and NaCl, can be recycled, does not produce secondary pollution, and is safe and environment-friendly.

Description

Scavenging kit and scavenging method for formaldehyde in air
Technical Field
The invention relates to a kit and a method for removing harmful gases in air, in particular to a novel kit and a method for removing formaldehyde in air.
Background
The quality of air in a room, particularly a living room, is a concern to the health of each individual. The formaldehyde gas released slowly for a long time from materials and articles such as decoration, furniture and the like is an important group of indoor air pollutionAnd (4) dividing. Formaldehyde, as a gas with great harm to human, is known as indoor "life-saving killer", especially in China, seriously harms the health of newly-decorated families, especially children, pregnant women and old people, and is an important cause of leukemia and fetal deformity of children. 2017, a study on Cell, the Top academic journal of the world (A Class of Environmental and Environmental toxics industries)BRCA2Haploinsufficiency and Genome availability) show that formaldehyde can degrade BRCA2 protein which is originally tumor suppressor protein, destroy DNA damage repair mechanism and make formaldehyde a high risk factor for inducing cancer! In addition, formaldehyde waste liquid generated in industrial production or experimental research is also a serious environmental pollution source.
The formaldehyde concentration for indoor safety defined in the national standard of indoor air quality Standard (GBT 18883-2002) formulated by the ministry of health and the national standard of civil building indoor environmental pollution control Specification (GB 50325-2010) formulated by the Ministry of construction is 0.10mg/m3And 0.08 mg/m3(class I building engineering) and the following. The formaldehyde determination methods listed in the national standard include spectrophotometry and gas chromatography, of which phenol reagent spectrophotometry is the recommended method. Toxic and harmful organic reagents are needed in the phenol reagent spectrophotometry, the operation is very complicated, and the sensitivity is not high; gas chromatography requires expensive equipment, and sensitive electrochemical sensors are expensive, both of which are difficult to popularize. These methods also have the same problems in the measurement of formaldehyde waste liquid.
For the removal of formaldehyde in rooms, the accepted basic measure is long-term ventilation, but the time is long and the effect is slow. The effects of plant purification, activated carbon adsorption and the like are limited, and the activated carbon is released again after being adsorbed to saturation. The pharmacological method is not time-efficient, fails after several hours of volatilization after spraying, and often introduces other pollutants. The catalytic conversion (photocatalytic decomposition) method requires ultraviolet excitation for its performance and cannot be eliminated in all aspects. In a word, due to the characteristics of various formaldehyde pollution sources, trace accumulation, long-term durability and the like, a plurality of treatment methods have certain limitations. Not only formaldehyde gas, but also formaldehyde waste liquid is lack of a high-efficiency, environment-friendly, simple and economic treatment method at present.
At present, the development of a novel formaldehyde binding substance which is efficient, simple, environment-friendly and economical and a formaldehyde removing and detecting method are urgently needed. The applicant discovers that a specific structural domain has good formaldehyde binding capacity in a formaldehyde catalytic enzyme system in the earlier stage, obtains high-efficiency formaldehyde binding polypeptide through computer simulation and screening, transformation and optimization, and develops a new method and a kit for detecting the concentration of formaldehyde, a new method and a kit for removing formaldehyde, which are accurate, reliable, simple, convenient and economical, high-efficiency, economical, environment-friendly and practical, based on newly discovered functional polypeptide.
Disclosure of Invention
The invention provides two novel functional polypeptides with high-efficiency formaldehyde binding capacity, and develops a corresponding formaldehyde detection method and a corresponding formaldehyde removal kit on the basis of the two novel functional polypeptides.
One of the purposes of the invention is to provide two functional polypeptides T1 and T2 with high-efficiency formaldehyde binding performance, wherein the sequences of the polypeptides T1 and T2 are respectively shown as SEQ ID NO 1 and SEQ ID NO 2, and the final amino acid at the C terminal of the polypeptides T1 and T2, namely phenylalanine, is modified by biotin, so that the polypeptides can be synthesized by commercial biological companies and the purity is required to be not less than 95%.
For practical application and recycling, the polypeptides T1 and T2 can be combined with streptavidin-coated magnetic beads via biotin modified and connected on the terminal phenylalanine to prepare magnetic polypeptide-carrying suspension, and if necessary, the polypeptide-carrying suspension is added (NH 4)2SO4The solution is made into formaldehyde combination reaction working solution, namely magnetic polypeptide-carrying working solution. The preparation method of the magnetic carrier polypeptide suspension mainly comprises the following steps:
a. preparing magnetic beads by using streptavidin magnetic beads and 0.1ml of streptavidin according to a product specification, loading the magnetic beads by using a 15ml centrifugal tube, and finally obtaining magnetic bead liquid in a 10ml working solution state by using a buffer solution carried in the magnetic bead product;
b. adding 0.1ml of 10mmol/L polypeptide T1 (or T2) solution (the addition amount of the polypeptide is 1 mu mol, and the amount of the polypeptide after full combination is 1 mu mol) into the magnetic bead solution in the step a, and keeping the mixture fully and uniformly mixed for 15min at room temperature;
c. washing the magnetic beads coated with the polypeptide for 3 times by using a buffer solution carried by the magnetic bead products by using a magnet, removing unbound substances, and adding 10ml of purified water to obtain a magnetic polypeptide-carrying suspension which can be stored in an environment of-20 ℃ for a long time for later use; adding into the reaction system (NH 4) when the formaldehyde is needed to be combined2SO4And (3) solution.
The polypeptides T1 and T2 both have high-efficiency formaldehyde binding capacity, after the magnetic-carried polypeptides are prepared for convenient use, the formaldehyde binding capacity of each 1 mu mol of the magnetic-carried polypeptides T1 and T2 can reach 87.25mg and 84.69mg respectively, and (NH 4) with the final concentration of 0.1-0.3mol/L must be added into a binding reaction system of the magnetic-carried polypeptides and formaldehyde2SO4Solution, and most preferred in the system (NH 4)2SO4The concentration was 0.182 mol/L. The combination of the magnetic polypeptide T1 and T2 and formaldehyde can be destroyed by a mixed solution system of NaOH and NaCl, wherein the final concentration of NaOH is 0.05-0.15mol/L, the final concentration of NaCl is 0.1-0.3mol/L, the concentration ratio of NaOH to NaCl is 1:2, and the optimal final concentrations of NaOH and NaCl in the system are 0.075mol/L and 0.15mol/L respectively.
The magnetic-carried polypeptides T1 and T2 have maximum absorption at 446nm and 482nm after being combined with formaldehyde respectively, and the absorbance is in direct proportion to the formaldehyde binding capacity, wherein for a T1 polypeptide system, the relationship between the formaldehyde binding capacity y and the absorbance x at the wavelength of 446nm is y =45.716x-0.7713, and the regression coefficient R is2= 0.9998; for the T2 polypeptide system, the relationship between the formaldehyde binding capacity y and the absorbance x at the wavelength of 482nm is y =45.763x-0.7826, and the regression coefficient R2=0.9999。
The invention also aims to provide a formaldehyde detection kit and a formaldehyde detection method based on the novel functional polypeptide. The formaldehyde detection kit based on the novel functional polypeptide mainly comprises the following components:
(1) polypeptide freeze-drying powder;
(2) magnetic beads coated with streptavidin;
（3）（NH4）2SO4a solution;
(4) and a magnet.
Wherein, the polypeptide of the component (1) is one of the polypeptide T1 or T2, namely the polypeptide T1 (or the polypeptide T2), the sequences of the polypeptide T1 and the polypeptide T2 are respectively shown as SEQ ID NO:1 and SEQ ID NO:2, the last amino acid at the C end of the polypeptide, namely phenylalanine, is modified by biotin, the modified polypeptide is synthesized by biological companies, the purity is not less than 95 percent, and purified water is added before the modified polypeptide is used to prepare a solution of 10 mmol/L.
The component (2) is streptavidin-coated magnetic beads, preferably streptavidin magnetic beads M-280 (Dynabeads) from Thermo Scientific, USATMM-280 Streptavidin), including a compatible buffer.
The application method of the formaldehyde detection kit based on the magnetic-carried polypeptide, namely the formaldehyde detection method, mainly comprises the following steps:
(1) before formaldehyde combination, each component of the kit must be utilized to prepare magnetic-carrier polypeptide suspension, and the main preparation process is as follows: a. preparing magnetic beads by taking 0.1ml of streptavidin according to a product specification, loading the streptavidin by using a 15ml centrifuge tube, and finally obtaining magnetic bead liquid in a 10ml working solution state by using a buffer solution carried in a magnetic bead product; b. adding 0.1ml of 10mmol/L polypeptide T1 (or T2) solution into the magnetic bead solution in the step a, and keeping the mixture fully and uniformly mixed for 15min at room temperature; c. washing the magnetic beads coated with the polypeptide for 3 times by using a buffer solution carried by the magnetic bead products by using a magnet, removing unbound substances, and adding 10ml of purified water to obtain a magnetic polypeptide-carrying suspension which can be stored in an environment of-20 ℃ for a long time for later use. The preparation process and the proportion of the magnetic carrier polypeptide suspension can be adjusted according to actual requirements.
(2) Adding the magnetic polypeptide suspension into 0.1ml formaldehyde sample to be detected, and then adding (NH 4)2SO4Solution to (NH 4)2SO4The final concentration is 0.182mol/L, the mixture is uniformly mixed and then is placed at room temperature for bonding reaction for 10min, and the mixture is uniformly mixed for not less than 2 times;
(3) adsorbing the magnetic-carrying polypeptide and formaldehyde conjugate on the tube wall by using a magnet, and sucking out a reaction system;
(4) adding 1ml of purified water, uniformly mixing, eluting for 1 time, re-adsorbing the conjugate, and sucking out clear water eluent;
(5) adding 1ml of purified water, mixing, collecting 3ul of aqueous solution of magnetic carrier polypeptide and formaldehyde conjugate, and measuring absorbance at 446nm (using the wavelength of polypeptide T1) or 482nm (using the wavelength of polypeptide T2) with enzyme-labeling instrument;
(6) and substituting the measured absorbance x into a formula y =45.716x-0.7713 (the formula is adopted when the polypeptide T1 is used) or y =45.763x-0.7826 (the formula is adopted when the polypeptide T2 is used), so that the formaldehyde binding capacity can be calculated, and if the formaldehyde binding capacity exceeds 80 mg/mu mol of the magnetic-loaded polypeptide, properly diluting the formaldehyde solution to be measured, measuring the formaldehyde solution again until the formaldehyde binding capacity is lower than the value, and further converting the formaldehyde concentration or the formaldehyde content.
Wherein, the formaldehyde content in the sample to be detected in the step (2) of the formaldehyde detection method is directly measured if the sample is a solution; if the sample to be detected is air, absorbing and dissolving formaldehyde gas into solution by using a large-scale bubble absorption tube and a constant-current sampler according to the sampling operation in GB/T18204.26-2000, and then determining the content of formaldehyde.
The invention also aims to provide a novel functional polypeptide-based kit and a novel functional polypeptide-based method for removing formaldehyde in the air. Through exploration and optimization, the novel functional polypeptide-based air formaldehyde removal kit mainly comprises the following components:
(1) polypeptide freeze-drying powder;
(2) magnetic beads coated with streptavidin;
(3) 0.182mol/L (NH 4)2SO4A solution;
(4) 0.15mol/L NaOH solution;
(5) 0.3mol/L NaCl solution;
(6) 5 plastic centrifuge tubes of 2ml, 2 plastic centrifuge tubes of 15 ml;
(7) 10 plastic straws with scales of 1ml and 10ml respectively;
(8) 2 magnets;
(9) the volume of 15ml scaled plastic vials is not less than 10.
Wherein, the polypeptide of the component (1) is one of polypeptide T1 or polypeptide T2, namely polypeptide T1 (or polypeptide T2), which is synthesized by biological companies, the sequences of the polypeptides T1 and T2 are respectively shown as SEQ ID NO:1 and SEQ ID NO:2, and the last amino acid at the C end of the polypeptide, namely phenylalanine, is modified by biotin, the purity is not lower than 95 percent, and water is added before use to prepare 10mmol/L solution.
The component (2) is streptavidin-coated magnetic beads, preferably streptavidin magnetic beads M-280 (Dynabeads) from Thermo Scientific, USATM M-280 Streptavidin）。
The use method of the kit for removing formaldehyde in air based on the magnetic-carried polypeptide, namely the method for removing formaldehyde in air, mainly comprises the following steps:
(1) the preparation method comprises the following steps of preparing magnetic-carrier polypeptide suspension before formaldehyde removal, wherein the preparation process comprises the following steps: a. preparing magnetic beads by using streptavidin magnetic beads and 0.1ml of streptavidin according to the specification of the magnetic bead products, loading the magnetic beads by using a 15ml centrifugal tube, and finally obtaining magnetic bead liquid in a 10ml working solution state by using a buffer solution carried in the magnetic bead products; b. adding 0.1ml of 10mmol/L polypeptide T1 (or T2) solution into the magnetic bead solution in the step a, and keeping the mixture fully and uniformly mixed for 15min at room temperature; c. washing the magnetic beads coated with the polypeptide for 3 times by using a buffer solution carried by the magnetic bead products by using a magnet, removing unbound substances, and adding 10ml of purified water to obtain a magnetic polypeptide-carrying suspension which can be stored in an environment of-20 ℃ for a long time for later use. The preparation process and the proportion of the magnetic carrier polypeptide suspension can be adjusted according to actual requirements.
(2) After removing the purified water, 0.182mol/L (NH 4) is added2SO4Obtaining magnetic polypeptide working solution, sucking 10ml of magnetic polypeptide working solution, placing into a plastic small bottle, placing the plastic small bottle at indoor formaldehyde volatilization source, and placing at every 50m2The space only needs to be provided with 1 bottle;
(3) adding water into a plastic bottle once a week, and replenishing the working solution to a scale of 10 ml;
(4) after the magnetic beads are placed for 1 month, adsorbing the magnetic-carrying polypeptide on the side wall of the centrifugal tube by using a magnet, removing the solution in the centrifugal tube as much as possible by using a suction tube, washing the magnetic beads by using purified water for 2 times, and removing the purified water;
(5) then adding 5ml of 0.15mol/L NaOH solution and 5ml of 0.3mol/L NaCl solution to resuspend the magnetic beads, placing the mixture in a ventilated place for 5min at room temperature, removing the NaOH solution and the NaCl solution by using a magnet and a straw in the previous operation, washing the magnetic beads for 2 times by using purified water, and removing the purified water;
(6) 10ml of 0.182mol/L (NH 4) were added2SO4The solution is used for obtaining updated magnetic polypeptide-carrying working solution, and the plastic small bottle is put in place again;
(7) and (5) repeating the steps (3) to (6) until the concentration of the formaldehyde in the indoor air meets the national requirement or the actual requirement.
The fourth purpose of the invention is to provide a novel functional polypeptide-based formaldehyde removal kit and a removal method from waste liquid. Through exploration and optimization, the formaldehyde removal kit in the waste liquid based on the novel functional polypeptide mainly comprises the following components:
(1) polypeptide freeze-drying powder;
(2) magnetic beads coated with streptavidin;
（3）（NH4）2SO4a solution;
(4) NaOH solution;
(5) NaCl solution;
(6) and 2 magnets.
Wherein, the polypeptide of the component (1) is one of polypeptide T1 or polypeptide T2, namely polypeptide T1 (or polypeptide T2), which is synthesized by biological companies, the sequences of the polypeptides T1 and T2 are respectively shown as SEQ ID NO:1 and SEQ ID NO:2, and the last amino acid at the C end of the polypeptide, namely phenylalanine, is modified by biotin, the purity is not lower than 95 percent, and water is added before use to prepare 10mmol/L solution.
The component (2) is streptavidin-coated magnetic beads, preferably streptavidin magnetic beads M-280 (Dynabeads) from Thermo Scientific, USATMM-280 Streptavidin), including a compatible buffer.
In addition, self-contained pH paper and acid-base adjusting solutions (e.g., hydrochloric acid and NaOH solutions) are required.
The use method of the kit for removing formaldehyde from the waste liquid based on the magnetic-carried polypeptide, namely the method for removing formaldehyde from the waste liquid, mainly comprises the following steps:
(1) the magnetic carrier polypeptide suspension is prepared by using kit components before formaldehyde removal, and the preparation process comprises the following steps: a. preparing magnetic beads by taking 0.1ml of streptavidin according to a product specification, loading the streptavidin by using a 15ml centrifuge tube, and finally obtaining magnetic bead liquid in a 10ml working solution state by using a buffer solution carried in a magnetic bead product; b. adding 0.1ml of 10mmol/L polypeptide T1 (or T2) solution into the magnetic bead solution in the step a, and keeping the mixture fully and uniformly mixed for 15min at room temperature; c. washing the magnetic beads coated with the polypeptide for 3 times by using a buffer solution carried by the magnetic bead products by using a magnet, removing unbound substances, and adding 10ml of purified water to obtain a magnetic polypeptide-carrying suspension which can be stored in an environment of-20 ℃ for a long time for later use. The preparation process and the proportion of the magnetic carrier polypeptide suspension can be adjusted according to actual requirements.
(2) Firstly, pH in the waste liquor is adjusted to 7.0 by utilizing acid-base regulating solution and pH test paper, then the prepared magnetic-carried polypeptide suspension is added into the formaldehyde waste liquor, and then (NH 4)2SO4Solution to (NH 4)2SO4The final concentration is 0.182mol/L, and the mixture is evenly mixed and placed at room temperature for full reaction;
(3) adsorbing the magnetic-carried polypeptide by using a magnet, pouring out the treated formaldehyde waste liquid, washing the magnetic beads by using purified water for 2 times, and then removing purified water;
(4) then, 5ml of 0.15mol/L NaOH solution and 5ml of 0.3mol/L NaCl solution are added into every 1 mu mol of magnetic polypeptide to resuspend the magnetic beads, the magnetic beads are placed in a ventilated place for 5min at room temperature, the NaOH solution and the NaCl solution are removed by using a magnet according to the previous operation, the magnetic beads are washed by purified water for 2 times, and then the purified water is removed;
(5) adding the magnetic polypeptide treated in the step (4) into the formaldehyde waste liquid poured out in the step (3), adjusting the pH value to 7.0 again, and then adding (NH 4)2SO4Solution to (NH 4)2SO4The final concentration is 0.182mol/L, and the mixture is evenly mixed and placed at room temperature for full reaction;
(6) and (5) repeating the steps (3) to (5) until the concentration of the formaldehyde in the waste liquid meets the national requirement or the actual requirement.
The invention provides two functional polypeptides with high-efficiency formaldehyde binding capacity, which have strong formaldehyde binding capacity, can be efficiently bound and repeatedly utilized, do not produce secondary pollution, and are safe and environment-friendly; the formaldehyde concentration detection kit and the detection method developed based on the polypeptide have the characteristics of accuracy, reliability, simplicity, convenience, economy, high efficiency and environmental protection, and the formaldehyde removal kit and the removal method have the characteristics of high efficiency, economy, environmental protection and practicability.
Drawings
FIG. 1 is a full wavelength scan of a sample of the formaldehyde conjugate of the polypeptide T1 from example 4.
FIG. 2 is a full wavelength scan of a sample of the formaldehyde conjugate of the polypeptide T2 from example 4.
FIG. 3 is the full wavelength scan pattern of the blank control group in example 4.
FIG. 4 is a graph showing the relationship between the amount of bound formaldehyde and the absorbance of the system of example 5 in which the polypeptide T1 is magnetically supported.
FIG. 5 is a graph showing the relationship between the amount of bound formaldehyde and the absorbance of the system of example 5 in which the polypeptide T2 is magnetically supported.
Detailed Description
The invention is further illustrated below with reference to specific examples.
Example 1 the target magnetically-supported polypeptide has Formaldehyde binding Properties
If the target polypeptide (i.e. T1 or T2) has formaldehyde binding property, a certain amount of the target polypeptide is added into a specific formaldehyde system (e.g. aqueous formaldehyde solution), and the two are contacted with each other, and after the target polypeptide combined with formaldehyde is sufficiently bound for a period of time under appropriate conditions, the target polypeptide combined with formaldehyde is removed from the formaldehyde system, and the concentration of formaldehyde in the formaldehyde system is inevitably reduced by sampling and determining, so that the formaldehyde binding property of the target polypeptide can be verified and explored.
The polypeptides T1 and T2 with the sequences shown in SEQ ID NO 1 and 2 were synthesized by Shanghai Tanpu Biotech limited, and the final amino acid phenylalanine (the amino acid one letter code is F) at the C-terminal of both polypeptides was modified with Biotin (Biotin, Sigma-Aldrich, USA), and the purity of the synthesized polypeptide was not less than 95%. And respectively preparing the synthesized polypeptide freeze-dried powder into 10mmol/L polypeptide T1 and T2 solutions (the molar masses of the biotin-modified polypeptides T1 and T2 are 6578.34g/mol and 6024.81g/mol respectively) by using purified water. Binding of biotin to streptavidin is the strongest non-covalent interaction now known in nature. For the convenience of performance research and practical application, the polypeptides T1 and T2 are acted on magnetic beads coated with streptavidin through biotin modification of styrene-acrylic acid at the tail ends of the polypeptides to prepare magnetic-carried polypeptides. The polypeptide-coated magnetic beads were prepared using Streptavidin magnetic beads M-280 (dynabeads tm M-280 Streptavidin, Invitrogen) from Thermo Scientific, usa, according to the method described in the specification, relying on strong affinity between biotin (modifying groups on the amino acids at the ends of polypeptides T1 and T2) and Streptavidin (coated on the surface of M-280 magnetic beads), and the formaldehyde binding properties of the polypeptides were investigated using the polypeptide-coated functional magnetic beads. The method comprises the following specific steps:
(1) preparing M-280 magnetic beads by taking 0.1ml of magnetic beads according to a method in a specification of a streptavidin magnetic bead M-280 product, loading the magnetic beads by using a 15ml centrifuge tube, wherein a Buffer solution is B & W Buffer carried in the magnetic bead product, and finally obtaining magnetic bead solution in a 10ml working solution state;
(2) adding 0.1ml of 10mmol/L polypeptide solution into the magnetic bead solution in the step (1) (the adding amount of the polypeptide is 1 mu mol, and the amount of the polypeptide is 1 mu mol after the polypeptide is fully combined), and meanwhile, setting a blank control group (the blank control group is not added with the polypeptide solution, but is replaced by 0.1ml deionized water), and fully and uniformly mixing the mixture at room temperature for 15min by adopting a vertical rotation mixing instrument;
(3) by means of magnets or magnetic frames (Shanghai Bioengineering Co., Ltd.) and using B&The W Buffer washed the polypeptide-coated magnetic beads 3 times to remove unbound material, and then 10.1ml of 0.2mol/L (NH 4) was added2SO4Solution (after addition of 1ml formaldehyde solution in the next step, (NH 4)2SO4The final concentration of (a) is 0.182mol/L, probedThe optimal final concentration is obtained), the sample group obtains magnetic polypeptide suspension, and the blank control group obtains magnetic bead suspension without polypeptide coating;
(4) immediately adding 1ml of analytically pure formalin (35-40% by mass of aqueous formaldehyde solution commonly known as formalin from Shanghai Yuanmu Biotech Co., Ltd.) into the magnetic polypeptide-loaded suspension in (3), and performing binding reaction at room temperature for 30min (shaking up for 2 times);
(5) when the reaction in (4) is carried out for 10min and 30min, magnetic beads are adsorbed on the wall of a 15ml centrifuge tube by using a magnetic frame, 10ul of reaction liquid in the centrifuge tube is then absorbed by using a pipette gun, the formaldehyde concentration of the reaction liquid is measured by using an electrochemical sensor method (see part 7.5 in national standard GBT 18204.2-2014), a PPM-400 ST formaldehyde detector based on the principle of electrochemical sensors (PPM-Tech) in British is used, and the detection is carried out according to the instruction (three-time parallel measurement and average value taking, the same below), and the results are shown in Table 1.
TABLE 1 concentration of Formaldehyde in the reaction solution of the sample group and blank control group (unit: mg/ml)
Figure 352514DEST_PATH_IMAGE001
It was determined that the concentration of formaldehyde added to the reaction system, i.e., the original formaldehyde concentration, was 34.0mg/ml (total reaction solution volume was 11.1 ml), and therefore the amount of formaldehyde added was 377.4mg by 1ml of formalin, which was an amount sufficient to cause very serious persistent indoor formaldehyde contamination, and was suitable as a model amount. As can be seen from table 1, the relative percentage deviations (the difference between the sample concentration value and the original concentration value is a percentage of the original concentration value) of the formaldehyde concentration of the reaction solution of the magnetic beads without the polypeptides coating, i.e., the blank control group, as compared with the original formaldehyde concentration are 0.24% (reaction for 10 min) and 0.29% (reaction for 30 min), which are almost negligible, indicating that the magnetic beads without the polypeptides coating do not have significant formaldehyde absorption and binding performance. However, the formaldehyde concentration of the magnetic beads coated with the polypeptide, i.e., the reaction solution of the sample group, is significantly reduced compared with the original formaldehyde concentration, even by more than 15%. Wherein, for the magnetic bead sample coated with the polypeptide T1, the formaldehyde concentration reduction percentage is respectively 5.29% (reaction for 10 min) and 18.59% (reaction for 30 min); for the magnetic bead sample coated with the polypeptide T2, the percentage decrease in formaldehyde concentration was 6.91% (reaction 10 min) and 21.06% (reaction 30 min), respectively. Therefore, the magnetic beads coated with the polypeptide (T1 or T2) have obvious formaldehyde absorption and binding performance, and the formaldehyde absorption and binding amount of the magnetic beads is preliminarily shown to increase along with the prolonging of the reaction time.
Example 2 saturated binding Capacity of magnetically-loaded Polypeptides to Formaldehyde
As can be seen from example 1, the target polypeptide has good formaldehyde binding ability, but the formaldehyde binding amount per unit of the target polypeptide must be further studied to provide reference and basis for subsequent applications. Under specific systems and conditions, the formaldehyde binding capacity of the unit target polypeptide increases along with the prolonging of the reaction time until the saturated binding capacity is reached and maintained, so that the saturated binding degree of the target polypeptide to formaldehyde can be explored by utilizing the characteristic, and the specific process is as follows.
The investigation of example 2 was conducted with reference to example 1 (a blank control was also set), except that the reaction time of step (4) in example 1 was only 30min, the reaction time of example 2 was extended to 24h, and the formaldehyde concentration in the reaction solution was measured and averaged three times at 10 reaction time points of 30s, i.e., 0.5min, 1min, 10min, 30min, 1h, 3h, 5h, 10h, 15h and 24h, respectively, and the results were as shown in table 2 (the measured formaldehyde concentration in the reaction system of example 2, i.e., the original formaldehyde concentration, was 33.9 mg/ml).
TABLE 2 Formaldehyde concentration in the reaction solution at different reaction times and its percentage reduction
Figure 885127DEST_PATH_IMAGE002
As can be seen from Table 2, in the case of the samples of the group of polypeptide T1, when the reaction time reached 5 hours or more, the concentration of formaldehyde in the reaction solution remained unchanged at 26.04mg/ml, in which case the polypeptide T1 coated on the magnetic beads had reached the saturation binding of formaldehyde. Therefore, the saturation binding amount of the polypeptide T1 to formaldehyde per micromole is 87.25mg (the total volume of the reaction solution is 11.1 ml), and the reaction time is not less than 5 h. For the samples of the group of polypeptide T2, when the reaction time reached 3h and more, the concentration of formaldehyde in the reaction solution remained unchanged at 26.27mg/ml, in which case the polypeptide T2 coated on the magnetic beads had reached the saturation binding of formaldehyde. Therefore, the saturated binding capacity of the peptide T2 to formaldehyde per micromole is 84.69mg (the total volume of the reaction solution is 11.1 ml), and the reaction time is not less than 3 h.
Example 3 recovery of binding Capacity of magnetically-loaded polypeptide and Formaldehyde
After the target polypeptide is saturated and combined with formaldehyde, in order to realize cyclic and cyclic utilization of the target polypeptide for practical application, the condition of removing combination after formaldehyde combination is searched for, so that the formaldehyde combination capacity is restored again, and the specific process is as follows.
For the magnetic polypeptide-carrying system reacted for 24 hours in example 2 (at this time, the polypeptide has reached the saturation binding state of formaldehyde), the magnetic frame is adsorbed on the side wall of the centrifuge tube, the reaction solution in the centrifuge tube is removed as much as possible by using a pipette, and the magnetic beads are washed with purified water for 2 times and then purified water is removed. 5ml of 0.15mol/L NaOH solution and 5ml of 0.3mol/L NaCl solution (the final concentrations of NaOH and NaCl are 0.075mol/L and 0.15mol/L, respectively, and the optimum concentration and ratio are obtained by research) are added to resuspend the magnetic beads, the mixture is placed at room temperature for 5min (the period is shaken for 1 time, and the step is carried out in a fume hood or a ventilation place to prevent formaldehyde release from causing indoor pollution), then the NaOH solution and the NaCl solution are removed by using a magnetic frame and a pipette gun in the previous operation, and purified water is removed after the magnetic beads are washed 2 times by using purified water. The applicant researches and shows that, as the added NaOH can change the configuration of the polypeptide and the pH value of the system, the added NaCl can change the ion concentration of the system, and the combination of the original polypeptide and formaldehyde is damaged, so that the release of the formaldehyde combined with the target polypeptide is realized, and the polypeptide is renewed in terms of formaldehyde combination. Next, with respect to the renewed polypeptide (renewed polypeptide), the study of the recombination of formaldehyde was conducted with reference to the process steps in examples 1 and 2 in order to achieve the recycling as follows.
Next, 10.1ml of 0.2mol/L (NH 4) was added to the renewed magnetic beads2SO4The solution obtained magnetic bead suspension, then added with 1ml of analytically pure formalin, placed at room temperature for 24h for binding reaction (shaking for 2-3 times), and then measured by PPM-400 ST formaldehyde detector for formaldehyde concentration in the reaction solution after 15h and 24h for secondary binding reaction, and converted into formaldehyde binding amount of polypeptide, and the results are shown in the following table 3.
TABLE 3 Experimental data for the ability of polypeptides to recombine Formaldehyde
Figure 84027DEST_PATH_IMAGE003
The concentration of formaldehyde added to the reaction system in this time, i.e., the original formaldehyde concentration, was measured to be 34.1mg/ml (the total volume of the reaction solution was 11.1 ml). In example 2, it was determined that the binding of formaldehyde was saturated in the original polypeptide system at 15h and 24h, and the saturated formaldehyde binding amounts were 87.25mg (polypeptide T1) and 84.69mg (polypeptide T2), respectively. For the renewed polypeptide, 24h and 15h were separated by 9h, however, the concentration of formaldehyde in the reaction solution was the same, and it was confirmed that the saturated binding state of formaldehyde was also achieved. The calculated saturated formaldehyde binding capacity of the renewed polypeptide system (Table 3) was 80.03mg (renewed polypeptide T1) and 79.03mg (renewed polypeptide T2), respectively.
If the percentage of the saturated binding capacity of formaldehyde of the updated polypeptide to that of the original polypeptide is referred to as the recovery rate based on the saturated binding capacity of formaldehyde of the original polypeptide, the recovery rates of the updated polypeptides T1 and T2 are 91.7% and 93.3%, respectively. Continuing to perform experiments such as secondary updating and tertiary updating, and finding that when the updating is repeated 11 times (from the second updating, the interval time between every two updating is 10 days, and the total time lasts 100 days), the recovery rate of the polypeptide T1 still remains 50.83%; when the renewal was repeated 11 times (from the second renewal, the interval between two renewals was 10 days, and the total time was 100 days), the recovery rate of the polypeptide T2 was 53.11%. Therefore, the magnetic-carrying polypeptides T1 and T2 have good formaldehyde repeated binding capacity and can be recycled.
Example 4 spectral absorption Properties of magnetically Supported Polypeptides and Formaldehyde conjugates
The polypeptide can generate maximum absorption (usually between 210-220 nm) under ultraviolet, however, under the condition of combination or complexation with other substances, the spectral absorption performance of the polypeptide is likely to generate certain change, and detection methods based on spectral characteristics and the like have the characteristics of simplicity, rapidness, economy, practicability and the like, so the spectral absorption characteristics of the target polypeptide and the formaldehyde conjugate are researched so as to facilitate subsequent related applications, and the specific research process is as follows.
The magnetic polypeptide reaction solution samples (including blank control group, polypeptide T1 sample group and polypeptide T2 sample group) which are reacted for 24 hours in example 2 and reach saturated formaldehyde combination are reserved, and full-wavelength scanning of the full waveband is carried out so as to find out the maximum absorption wavelength of the polypeptide and formaldehyde combination. The Multiskan GO full-wavelength microplate reader of Thermo Fisher company in USA is adopted, the sample loading amount is 3ul, full-wavelength scanning is carried out at room temperature in 1nm stepping amount within the spectral band of 200nm-1000nm according to the steps of the operation instruction, and the result is shown in figures 1-3.
As can be seen from FIGS. 1 and 2, the maximum absorption wavelengths of the formaldehyde conjugate samples of the polypeptides T1 and T2 were 446nm and 482nm, respectively, which are much higher than the peptide bond absorption peak (at 220 nm). For the blank control group (reaction system without polypeptide), which only contains magnetic beads and a reaction solution system, and does not contain polypeptide, therefore, no conjugate of polypeptide and formaldehyde is generated, and as can be seen from the full-wavelength scanning spectrum (FIG. 3), no significant absorption occurs at 446nm and 482 nm. From this, it was found that the absorption at the wavelength of 446nm was the characteristic absorption peak of the polypeptide T1 and the formaldehyde conjugate, and the absorption at the wavelength of 482nm was the characteristic absorption peak of the polypeptide T2 and the formaldehyde conjugate.
Example 5 absorption of magnetically-loaded polypeptide Formaldehyde conjugates at maximum wavelength as a function of Formaldehyde binding Capacity
On the basis of obtaining the maximum absorption wavelength of the target polypeptide and the formaldehyde conjugate in example 4, the relationship between the absorption of the target polypeptide and the formaldehyde conjugate at the maximum wavelength and the formaldehyde binding amount can be further researched, so that a simple and convenient detection method of the formaldehyde binding amount (or the formaldehyde content) can be developed by means of the characteristic, and the specific process is as follows.
Based on example 2, while the remaining formaldehyde concentration in the system was measured at 10 selected reaction time points (30 s, i.e., 0.5min, 1min, 10min, 30min, 1h, 3h, 5h, 10h, 15h, 24 h), the absorbance (averaged over 3 measurements) was measured at the maximum absorption wavelength (446 nm for the T1 polypeptide system and 482nm for the T2 polypeptide system) according to the method of example 4, and the results are shown in Table 4 below. The concentration of formaldehyde in the reaction solution at each reaction time point was measured in example 2 (see table 2 for details), from which the amount of formaldehyde bound at each reaction time point was calculated, see table 4 for details. The absorbance of the samples at 5h was substantially consistent for the polypeptide T1 system and at 3h for the polypeptide T2 system, consistent with the observation that formaldehyde saturation binding was achieved in example 2.
TABLE 4 Formaldehyde binding amount and absorbance in the reaction solution at different reaction times
Figure 752906DEST_PATH_IMAGE004
A scatter diagram of the formaldehyde binding amount and the absorbance corresponding to each reaction time point is listed on Excel (only the time point of first reaching saturated binding is taken), a linear relationship between the two is found, and the results are shown in FIGS. 4-5 by linear fitting. For the T1 polypeptide system, the relationship between the formaldehyde binding capacity y and the absorbance x at the wavelength of 446nm is y =45.716x-0.7713, and the regression coefficient R2=0.9998 demonstrates that the linear equation well reflects the linear relationship between the formaldehyde binding capacity and the absorbance of the magnetically supported polypeptide T1; for the T2 polypeptide system, the relationship between the formaldehyde binding capacity y and the absorbance x at the wavelength of 482nm is y =45.763x-0.7826, and the regression coefficient R2=0.9999 demonstrates that the linear equation well reflects the linear relationship between formaldehyde binding amount and absorbance of the magnetically supported polypeptide T2.
Polypeptide of interest and summary of its properties
From the previous research and the results of examples 1-5, it can be known that the functional polypeptides T1 and T2 with high formaldehyde binding performance have the sequences shown in SEQ ID NO 1 and SEQ ID NO 2, respectively, and the last amino acid at the C terminal of the polypeptides T1 and T2, namely phenylalanine, is modified by biotin; the polypeptides T1 and T2 can be synthesized by commercial biological companies, and the purity is required to be not less than 95%; the polypeptides T1 and T2 can be combined with magnetic beads coated with streptavidin through biotin modified and connected on terminal phenylalanine to prepare magnetic-carried polypeptides, so that the magnetic-carried polypeptides can be applied to practical application more effectively, simply and economically. Through comparative analysis, the saturated formaldehyde binding amount of the unit polypeptide T1 is higher than that of the unit polypeptide T2, however, the binding rate of the polypeptide T2 and formaldehyde is faster than that of the polypeptide T1, and the two have respective advantages.
The polypeptides T1 and T2 both have high formaldehyde binding capacity, the formaldehyde binding capacity of each 1 mu mol of the magnetic-carried polypeptides T1 and T2 can reach 87.25mg and 84.69mg respectively, and (NH 4) with the final concentration of 0.1-0.3mol/L must be added into a binding reaction system of the magnetic-carried polypeptides and formaldehyde2SO4Solution, and most preferred in the system (NH 4)2SO4The concentration was 0.182 mol/L. The combination of the magnetic polypeptide T1 and T2 and formaldehyde can be destroyed by a mixed solution system of NaOH and NaCl, wherein the final concentration of NaOH is 0.05-0.15mol/L, the final concentration of NaCl is 0.1-0.3mol/L, the concentration ratio of NaOH to NaCl is 1:2, and the optimal final concentrations of NaOH and NaCl in the system are 0.075mol/L and 0.15mol/L respectively. Furthermore, the formaldehyde-bound magnetic-carried polypeptides T1 and T2 still have high formaldehyde recombination capability after releasing the bound formaldehyde, and the binding force can still reach more than 50% of the first binding force after the binding, releasing and recombining are repeated for 10 times.
The magnetic-carried polypeptides T1 and T2 have maximum absorption at 446nm and 482nm after being combined with formaldehyde respectively, and the absorbance is in direct proportion to the formaldehyde binding capacity, wherein for a T1 polypeptide system, the relationship between the formaldehyde binding capacity y and the absorbance x at the wavelength of 446nm is y =45.716x-0.7713, and the regression coefficient R is2= 0.9998; for the T2 polypeptide system, the relationship between the formaldehyde binding capacity y and the absorbance x at the wavelength of 482nm is y =45.763x-0.7826, and the regression coefficient R2=0.9999。
Example 6 Polypeptides T1 and T2 having high Formaldehyde binding Properties
The functional polypeptides T1 and T2 with high formaldehyde binding performance have the sequences shown in SEQ ID NO 1 and SEQ ID NO 2, respectively, and the final amino acid at the C terminal of the polypeptides T1 and T2, namely phenylalanine, is modified by biotin, so that the polypeptides can be synthesized by commercial biological companies and the purity is required to be not less than 95%.
For practical application and recycling, the polypeptides T1 and T2 can be combined with magnetic beads coated with streptavidin via biotin modified and connected with terminal phenylalanine to prepare magnetic-carried polypeptides, and the magnetic-carried polypeptides are added when in use (NH 4)2SO4The solution is made into formaldehyde combination reaction working solution, namely magnetic polypeptide-carrying working solution. The main steps for preparing the magnetic carrier polypeptide suspension are as follows (which is only a general example of the preparation of the magnetic carrier polypeptide and can be scaled up or down or adjusted according to actual needs):
a. preparing magnetic beads by using streptavidin magnetic beads and 0.1ml of streptavidin according to a product specification, loading the magnetic beads by using a 15ml centrifugal tube, and finally obtaining magnetic bead liquid in a 10ml working solution state by using a buffer solution carried in the magnetic bead product;
b. adding 10mmol/L of polypeptide T1 (or T2) solution 0.1ml (the addition amount of the polypeptide is 1 mu mol, and the amount of the polypeptide after full combination is 1 mu mol) into the magnetic bead solution in the step a, and keeping the mixture fully and uniformly mixed for 15min at room temperature;
c. washing the magnetic beads coated with the polypeptide for 3 times by using a buffer solution carried by the magnetic bead products by using a magnet, removing unbound substances, and adding 10ml of purified water to obtain a magnetic polypeptide-carrying suspension which can be stored in an environment of-20 ℃ for a long time for later use; adding (NH 4) into the reaction system when formaldehyde is combined2SO4And (3) solution.
The polypeptides T1 and T2 both have high formaldehyde binding capacity, the formaldehyde binding capacity of each 1 mu mol of the magnetic-carried polypeptides T1 and T2 can reach 87.25mg and 84.69mg respectively, and (NH 4) with the final concentration of 0.1-0.3mol/L must be added into a binding reaction system of the magnetic-carried polypeptides and formaldehyde2SO4Solution, and most preferred in the system (NH 4)2SO4The concentration was 0.182 mol/L. The magnetic carrier polypeptide T1 and T2 can be combined with formaldehyde by NaOH and NaClThe mixed solution system is destroyed, wherein the final concentration of NaOH is 0.05-0.15mol/L, the final concentration of NaCl is 0.1-0.3mol/L, the concentration ratio of NaOH to NaCl is 1:2, and the optimal final concentrations of NaOH and NaCl in the system are 0.075mol/L and 0.15mol/L respectively.
The magnetic-carried polypeptides T1 and T2 have maximum absorption at 446nm and 482nm after being combined with formaldehyde respectively, and the absorbance is in direct proportion to the formaldehyde binding capacity, wherein for a T1 polypeptide system, the relationship between the formaldehyde binding capacity y and the absorbance x at the wavelength of 446nm is y =45.716x-0.7713, and the regression coefficient R is2= 0.9998; for the T2 polypeptide system, the relationship between the formaldehyde binding capacity y and the absorbance x at the wavelength of 482nm is y =45.763x-0.7826, and the regression coefficient R2=0.9999。
Example 7 Formaldehyde detection kit and detection method based on novel polypeptide T1
Through exploration and optimization, the formaldehyde detection kit based on the novel functional polypeptide T1 mainly comprises the following components:
(1) the polypeptide T1 freeze-dried powder has a sequence shown in SEQ ID NO. 1;
(2) magnetic beads coated with streptavidin;
（3）（NH4）2SO4a solution;
(4) and a magnet.
Wherein, the component (1), namely the polypeptide T1 is synthesized by biological companies, the last amino acid at the C end of the polypeptide T1, namely phenylalanine, is modified by biotin, the purity is not lower than 95 percent, and purified water is added before use to prepare a solution of 10 mmol/L.
The component (2) is streptavidin-coated magnetic beads, preferably streptavidin magnetic beads M-280 (Dynabeads) from Thermo Scientific, USATMM-280 Streptavidin), including a compatible buffer.
The application method of the formaldehyde detection kit based on the magnetic-carried polypeptide T1, namely the formaldehyde detection method, mainly comprises the following steps:
(1) the components of the kit must be used to prepare a suspension of magnetically-loaded polypeptide T1 prior to formaldehyde conjugation, as described in example 6;
(2) adding the magnetic polypeptide T1 suspension into 0.1ml formaldehyde sample to be detected, and then adding (NH 4)2SO4Solution to (NH 4)2SO4The final concentration is 0.182mol/L, the mixture is uniformly mixed and then is placed at room temperature for bonding reaction for 10min, and the mixture is uniformly mixed for not less than 2 times;
(3) adsorbing the magnetic-carrying polypeptide and formaldehyde conjugate on the tube wall by using a magnet, and sucking out a reaction system;
(4) adding 1ml of purified water, uniformly mixing, eluting for 1 time, re-adsorbing the conjugate, and sucking out clear water eluent;
(5) adding 1ml of purified water, mixing, taking 3ul of aqueous solution of the magnetic carrier polypeptide and formaldehyde conjugate, and measuring absorbance at 446nm by using an enzyme-labeling instrument;
(6) and substituting the measured absorbance x into a formula y =45.716x-0.7713 to calculate the formaldehyde binding capacity, and if the formaldehyde binding capacity exceeds 80 mg/mu mol of the magnetic-carried polypeptide, properly diluting the formaldehyde solution to be measured, measuring again until the formaldehyde binding capacity is lower than the value, and further converting into the formaldehyde concentration or the formaldehyde content.
Wherein, the formaldehyde content in the sample to be detected in the step (2) of the formaldehyde detection method is directly measured if the sample is a solution; if the sample to be detected is air, absorbing and dissolving formaldehyde gas into solution by using a large-scale bubble absorption tube and a constant-current sampler according to the sampling operation in GB/T18204.26-2000, and then determining the content of formaldehyde.
Example 8 Formaldehyde detection kit and detection method based on novel polypeptide T2
Through exploration and optimization, the formaldehyde detection kit based on the novel functional polypeptide T2 mainly comprises the following components:
(1) the polypeptide T2 freeze-dried powder has a sequence shown in SEQ ID NO. 2;
(2) magnetic beads coated with streptavidin;
（3）（NH4）2SO4a solution;
(4) and a magnet.
Wherein, the component (1), namely the polypeptide T2 is synthesized by biological companies, the last amino acid at the C end of the polypeptide T2, namely phenylalanine, is modified by biotin, the purity is not lower than 95 percent, and purified water is added before use to prepare a solution of 10 mmol/L.
Wherein the component (2), i.e., the magnetic beads, are streptavidin-coated magnetic beads, preferably streptavidin magnetic beads M-280 (Dynabeads) from Thermo Scientific Co., U.S.A.TMM-280 Streptavidin), including a compatible buffer.
The application method of the formaldehyde detection kit based on the magnetic-carried polypeptide T2, namely the formaldehyde detection method, mainly comprises the following steps:
(1) the components of the kit must be used to prepare a suspension of magnetically-loaded polypeptide T2 prior to formaldehyde conjugation, as described in example 6;
(2) adding the magnetic polypeptide T2 suspension into 0.1ml formaldehyde sample to be detected, and then adding (NH 4)2SO4Solution to (NH 4)2SO4The final concentration is 0.182mol/L, the mixture is uniformly mixed and then is placed at room temperature for bonding reaction for 10min, and the mixture is uniformly mixed for not less than 2 times;
(3) adsorbing the magnetic-carrying polypeptide and formaldehyde conjugate on the tube wall by using a magnet, and sucking out a reaction system;
(4) adding 1ml of purified water, uniformly mixing, eluting for 1 time, re-adsorbing the conjugate, and sucking out clear water eluent;
(5) adding 1ml of purified water, mixing, taking 3ul of aqueous solution of the magnetic carrier polypeptide and formaldehyde conjugate, and measuring absorbance at 482nm wavelength by using an enzyme-labeling instrument;
(6) and substituting the measured absorbance x into a formula y =45.763x-0.7826 to calculate the formaldehyde binding capacity, and if the formaldehyde binding capacity exceeds 80 mg/mu mol of the magnetic-carried polypeptide, properly diluting the formaldehyde solution to be measured, measuring again until the formaldehyde binding capacity is lower than the value, and further converting into the formaldehyde concentration or the formaldehyde content.
Wherein, the formaldehyde content in the sample to be detected in the step (2) of the formaldehyde detection method is directly measured if the sample is a solution; if the sample to be detected is air, absorbing and dissolving formaldehyde gas into solution by using a large-scale bubble absorption tube and a constant-current sampler according to the sampling operation in GB/T18204.26-2000, and then determining the content of formaldehyde.
Example 9 kit and method for removing Formaldehyde from air based on novel functional Polypeptides
Through exploration and optimization, the novel functional polypeptide-based kit for removing formaldehyde from air mainly comprises the following components:
(1) polypeptide freeze-drying powder;
(2) magnetic beads coated with streptavidin;
(3) 0.182mol/L (NH 4)2SO4A solution;
(4) 0.15mol/L NaOH solution;
(5) 0.3mol/L NaCl solution;
(6) 5 plastic centrifuge tubes of 2ml, 2 plastic centrifuge tubes of 15 ml;
(7) 10 plastic straws with scales of 1ml and 10ml respectively;
(8) 2 magnets;
(9) the volume of 15ml scaled plastic vials is not less than 10.
Wherein, the polypeptide of the component (1) is one of polypeptide T1 or polypeptide T2, namely polypeptide T1 (or polypeptide T2), which is synthesized by biological companies, the sequences of the polypeptides T1 and T2 are respectively shown as SEQ ID NO:1 and SEQ ID NO:2, and the last amino acid at the C end of the polypeptide, namely phenylalanine, is modified by biotin, the purity is not lower than 95 percent, and water is added before use to prepare 10mmol/L solution.
The component (2) is streptavidin-coated magnetic beads, preferably streptavidin magnetic beads M-280 (Dynabeads) from Thermo Scientific, USATMM-280 Streptavidin), including a compatible buffer.
The use method of the kit for removing formaldehyde in air based on the magnetic-carried polypeptide, namely the method for removing formaldehyde in air, mainly comprises the following steps:
(1) the preparation process is as shown in example 6, wherein the magnetic carrier polypeptide suspension is prepared before formaldehyde removal;
(2) after removing the purified water from the suspension of magnetically-supported polypeptide, 0.182mol/L (NH 4) was added2SO4Obtaining magnetic carrier in solutionThe peptide working solution is prepared by sucking 10ml of magnetic polypeptide-carrying working solution, placing into a plastic small bottle, placing the plastic small bottle at the indoor formaldehyde volatilization source, and placing at every 50m2The space only needs to be provided with 1 bottle;
(3) adding water into a plastic bottle once a week, and replenishing the working solution to a scale of 10 ml;
(4) after the magnetic beads are placed for 1 month, adsorbing the magnetic-carrying polypeptide on the side wall of the centrifugal tube by using a magnet, removing the solution in the centrifugal tube as much as possible by using a suction tube, washing the magnetic beads by using purified water for 2 times, and removing the purified water;
(5) then adding 5ml of 0.15mol/L NaOH solution and 5ml of 0.3mol/L NaCl solution to resuspend the magnetic beads, placing the mixture in a ventilated place for 5min at room temperature, removing the NaOH solution and the NaCl solution by using a magnet and a straw in the previous operation, washing the magnetic beads for 2 times by using purified water, and removing the purified water;
(6) 10ml of 0.182mol/L (NH 4) are added2SO4The solution is used for obtaining updated magnetic polypeptide-carrying working solution, and the plastic small bottle is put in place again;
(7) and (5) repeating the steps (3) to (6) until the concentration of the formaldehyde in the indoor air meets the national requirement or the actual requirement.
Example 10 removal of Formaldehyde from waste streams based on novel functional Polypeptides
Through exploration and optimization, the formaldehyde removal kit in the waste liquid based on the novel functional polypeptide mainly comprises the following components:
(1) polypeptide freeze-drying powder;
(2) magnetic beads coated with streptavidin;
（3）（NH4）2SO4a solution;
(4) NaOH solution;
(5) NaCl solution;
(6) and 2 magnets.
Wherein, the polypeptide of the component (1) is one of polypeptide T1 or polypeptide T2, namely polypeptide T1 (or polypeptide T2), which is synthesized by biological companies, the sequences of the polypeptides T1 and T2 are respectively shown as SEQ ID NO:1 and SEQ ID NO:2, and the last amino acid at the C end of the polypeptide, namely phenylalanine, is modified by biotin, the purity is not lower than 95 percent, and water is added before use to prepare 10mmol/L solution.
The component (2) is streptavidin-coated magnetic beads, preferably streptavidin magnetic beads M-280 (Dynabeads) from Thermo Scientific, USATMM-280 Streptavidin), including a compatible buffer.
In addition, self-contained pH paper and acid-base adjusting solutions (e.g., hydrochloric acid and NaOH solutions) are required.
The use method of the kit for removing formaldehyde from the waste liquid based on the magnetic-carried polypeptide, namely the method for removing formaldehyde from the waste liquid, mainly comprises the following steps:
(1) the components of the kit must be used to prepare a magnetically-loaded polypeptide suspension prior to formaldehyde removal, as described in example 6;
(2) firstly, pH in the waste liquor is adjusted to 7.0 by utilizing acid-base regulating solution and pH test paper, then the prepared magnetic-carried polypeptide suspension is added into the formaldehyde waste liquor, and then (NH 4)2SO4Solution to (NH 4)2SO4The final concentration is 0.182mol/L, and the mixture is evenly mixed and placed at room temperature for full reaction;
(3) adsorbing the magnetic-carried polypeptide by using a magnet, pouring out the treated formaldehyde waste liquid, washing the magnetic beads by using purified water for 2 times, and then removing purified water;
(4) then, 5ml of 0.15mol/L NaOH solution and 5ml of 0.3mol/L NaCl solution are added into every 1 mu mol of magnetic polypeptide to resuspend the magnetic beads, the magnetic beads are placed in a ventilated place for 5min at room temperature, the NaOH solution and the NaCl solution are removed by using a magnet according to the previous operation, the magnetic beads are washed by purified water for 2 times, and then the purified water is removed;
(5) adding the magnetic polypeptide treated in the step (4) into the formaldehyde waste liquid poured out in the step (3), adjusting the pH value to 7.0 again, and then adding (NH 4)2SO4Solution to (NH 4)2SO4The final concentration is 0.182mol/L, and the mixture is evenly mixed and placed at room temperature for full reaction;
(6) and (5) repeating the steps (3) to (5) until the concentration of the formaldehyde in the waste liquid meets the national requirement or the actual requirement.
Example 11 Formaldehyde scavenging and detection experiments in shop air
With a commercial body (decorated by a developer) completed in 2018 and 4 months as an object, 6 sets of SOHO shops (# 1.1-1.3 and # 2.1-2.3) are selected for removing and detecting formaldehyde. The formaldehyde detection was performed by selecting the kit and method of examples 7 (# 1.1-1.3) and 8 (# 2.1-2.3), and taking the average value of three measurements while using a PPM-400 ST formaldehyde detector as a detection reference. The reagent kit and method in example 9 were selected for formaldehyde removal, and the reference for activated carbon removal and the commercial formaldehyde scavenger based on photocatalyst was also selected, and the specific data are shown in table 5 below.
#1.1, #1.2 and #1.3 are 21m2Shops on different floors of the same house type, #2.1, #2.2 and #2.3 are 30m2In the same house type of shops with different floors, all 6 places are 1, and the experiment of the embodiment is started immediately after finishing decoration. The formaldehyde removal period is 3 months, and only 1 bottle of magnetic polypeptide-carrying removal solution is placed at each time by adopting the method; the dosage of the active carbon is 0.1kg/m2Placed in the room at 5, and changed once a month; the dosage of the photocatalyst scavenger is 0.5 bottle/10 m2Spray heavily on formaldehyde-rich sites and again every month. The formaldehyde removing method is not supplemented with additional ventilation during the formaldehyde treatment, and the door and the window are closed for 12 hours before the concentration is measured.
TABLE 5 New shop air formaldehyde detection and removal experimental data mg/m3
Figure DEST_PATH_IMAGE005
Note: the relative percentage deviation is the absolute value of the difference between the formaldehyde concentration detected by the method of the invention and the concentration detected by the formaldehyde meter, which is the percentage of the concentration detected by the formaldehyde meter.
As can be seen from Table 5, for #1-6, the formaldehyde concentration measured by the method of the present invention and the formaldehyde concentration measured by the formaldehyde detector were measured by any cleaning method, whether before or after the formaldehyde was cleanedThe relative percentage deviation between the two is less than 5 percent, and the average is only 2.205 percent. Therefore, the formaldehyde detection kit and the detection method are accurate and reliable, achieve the accuracy of a specialized formaldehyde detector, and have the advantages of simplicity, economy, high efficiency, environmental protection and the like. In addition, the concentration of the formaldehyde eliminated by the formaldehyde eliminating method based on the magnetic-carried polypeptide is less than 0.1 mg/m3(see the numerical values of #1.1 and 2.1), which meet the national regulations for the safe concentration of formaldehyde in buildings. The formaldehyde removing method adopting the active carbon and the catalyst has the formaldehyde concentration still higher than 0.1 mg/m after removal in the same time3(see values other than #1.1 and 2.1), the formaldehyde concentration is 6.1 times to 18.6 times the safe range. Therefore, the formaldehyde removal kit and the removal method based on the magnetic-carried polypeptide are proved to be efficient, and have the advantages of simplicity, convenience, economy, environmental protection and the like.
Example 12 Formaldehyde removal and detection experiments in air in commercial Room
With a commodity house (self-decoration) delivered in 2018 and 9 months as an object, 2 sets of house-type houses (# 1 and # 2) are selected for formaldehyde removal and detection. The formaldehyde detection was performed by selecting the kit and method of examples 7 (# 1) and 8 (# 2), and measuring three times with a PPM-400 ST formaldehyde detector as a detection reference. The reagent kit and method in example 9 were selected for formaldehyde removal, and the reference for activated carbon removal and the commercial formaldehyde scavenger based on photocatalyst was also selected, and the specific data are shown in table 6 below.
#1 and #2 are 120m2The commodity rooms on different floors of the same house type are 3 bedrooms (# 1.1-1.3 and #2.1-2.3, and #1.1 and #2.1 are 12m2#1.2 and 2.2 are 15m2#1.3 and 2.3 are 20m2) The experiment of this example was started after finishing the finishing. The formaldehyde removal period is 3 months, and only 1 bottle of magnetic polypeptide-carrying removal solution is placed in each room by adopting the method; the dosage of the active carbon is 0.1kg/m2Placed in each room at 5, and replaced once a month; the dosage of the photocatalyst scavenger is 0.5 bottle/10 m2Spraying in the place rich in formaldehydeAnd sprayed again once every month. The adopted formaldehyde removing method is used for normal ventilation during formaldehyde treatment, and the door and the window are closed for 12 hours before concentration measurement.
TABLE 6 detection and elimination experimental data mg/m for formaldehyde in newly-installed commercial room air3
Figure 667641DEST_PATH_IMAGE006
Note: the relative percentage deviation is the absolute value of the difference between the formaldehyde concentration detected by the method of the invention and the concentration detected by the formaldehyde meter, which is the percentage of the concentration detected by the formaldehyde meter.
As can be seen from Table 6, for #1.1-1.3 and #2.1-2.3, the relative percent deviation between the formaldehyde concentration measured by the method of the present invention and the formaldehyde concentration measured by the formaldehyde meter was less than 5%, and on average only 2.76%, regardless of whether the cleaning method was used before or after the formaldehyde was cleaned. Therefore, the formaldehyde detection kit and the detection method are accurate and reliable, achieve the accuracy of a specialized formaldehyde detector, and have the advantages of simplicity, economy, high efficiency, environmental protection and the like. In addition, the concentration of the formaldehyde eliminated by the formaldehyde eliminating method based on the magnetic-carried polypeptide is less than 0.08 mg/m3(see the numerical values of #1.3 and # 2.3), and meets the national regulation on the safe concentration of formaldehyde in the living room. The formaldehyde removing method adopting the active carbon and the catalyst has the formaldehyde concentration still higher than 0.1 mg/m after removal in the same time3(see values other than #1.3 and 2.3), the formaldehyde concentration is 2.1 times to 3.1 times the safe range. Therefore, the formaldehyde removal kit and the removal method based on the magnetic-carried polypeptide are proved to be efficient, and have the advantages of simplicity, convenience, economy, environmental protection and the like.
Example 13 removal and detection experiment of Formaldehyde in Formaldehyde waste liquid
Two formaldehyde waste liquids #1 and #2 from the laboratory, 1L each, were sampled and their formaldehyde concentrations were determined according to the kits and methods of examples 7 and 8, and the mean values were determined three times with a PPM-400 ST formaldehyde detector as the detection reference. Adding magnetic polypeptide according to the measured formaldehyde content in the sample, wherein the working fluid volume of the magnetic polypeptide added per 1g of formaldehyde in the sample is 10 ml. Formaldehyde removal was performed according to the formaldehyde removal kit and method in waste liquid of example 10, in which polypeptide T1 was renewed every 5h (according to the method in example 10, not replaced), polypeptide T2 was renewed every 3h (as described for polypeptide T1), and the concentration of formaldehyde in the sample was monitored before every renewal of the magnetically loaded polypeptide; in addition, parallel samples (# 1.1 and #1.2 and #2.1 and # 2.2) were taken as a scavenging control with activated carbon, 25g of activated carbon was added to each sample containing 1g of formaldehyde and the activated carbon was replaced every 3h, and the experimental data are shown in table 7.
As can be seen from Table 7, for #1 and #2, the relative percent deviation between the formaldehyde concentration measured by the method of the present invention and the formaldehyde concentration measured by the formaldehyde meter was less than 5%, and on average only 1.09%, regardless of the cleaning method used, either before or after the formaldehyde was cleaned. Therefore, the formaldehyde detection method is accurate and reliable, and achieves the accuracy of a specialized formaldehyde detector. As for the waste liquid #1 (the initial formaldehyde concentration is 1.33 g/L), the waste liquid #1 is removed by the polypeptide T1 (the waste liquid # is updated for 2 times, the saturated binding time of the polypeptide T1, namely 5h is selected for each updating interval in order to save time, the total time is 15h, the effect is better when the updating interval is selected for 24 h), the formaldehyde concentration in the waste liquid #1 after removal is reduced to 0.04g/L, the reduction ratio is up to 96.99%, and the good removal effect is achieved. For the waste liquid #2 (the initial formaldehyde concentration is 2.53 g/L), the waste liquid #2 is removed by the polypeptide T2 (the waste liquid # is updated for 4 times, the saturated combination time of the polypeptide T2, namely 3h, is selected for saving time at each updating interval, the total time is 12h, the effect is better when the updating interval is selected for 24 h), the formaldehyde concentration in the waste liquid #2 after removal is reduced to 0.07g/L, the reduction ratio is as high as 97.23%, and the good removing effect is achieved. In conclusion, the kit and the method for removing formaldehyde from waste liquid based on the polypeptides T1 and T2 can remove the waste liquid (more than 1.0 g/L) seriously polluted by formaldehyde in a short time (within 1 d), and the removal rate is more than 95%. Under the same conditions, a large amount of activated carbon is consumed, and the clearance rate is only about 30 percent. Therefore, the formaldehyde removing kit and the removing method in the waste liquid provided by the invention are efficient, and have the advantages of simplicity, convenience, economy, environmental protection and the like.
TABLE 7 removal of Formaldehyde from Formaldehyde effluent and test data g/L
Figure DEST_PATH_IMAGE007
Sequence listing
<110> biomedical science and technology Co., Ltd of Tetrajida, Fuzhou
<120> removing kit for formaldehyde in air and removing method thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 54
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Phe Glu Gln Arg His Gly Arg Val Arg Val Lys Thr Arg Gln Gly Ala
1 5 10 15
Arg Val Trp Gln His Phe Ile Val Val Glu Trp Arg Val Gly Ile Thr
20 25 30
Leu Phe Ser Asp Ser Tyr Leu Arg Asp Cys Val Asn Asp Asn Asp Thr
35 40 45
Met Lys Asn Gln Glu Phe
50
<210> 2
<211> 47
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Phe Glu Gln Arg His Gly Arg Val Arg Val Lys Thr Arg Arg Val Trp
1 5 10 15
Gly Gln His Phe Ile Val Glu Trp Arg Val Ser Asp Cys Val Asn Ser
20 25 30
Tyr Leu Arg Asp Asp Asn Cys Ala Met Lys Asn Thr Gln Glu Phe
35 40 45

Claims (3)

1. A kit for removing formaldehyde in air is characterized in that the kit mainly comprises the following components: (1) the polypeptide T1 freeze-dried powder has a polypeptide T1 sequence shown in SEQ ID NO 1, the last amino acid at the C end of the polypeptide, namely phenylalanine, is modified by biotin, is synthesized by biological companies, the purity is not lower than 95%, and water is added before use to prepare a solution of 10 mmol/L; (2) magnetic beads coated with streptavidin; (3) 0.182mol/L (NH 4)2SO4A solution; (4) 0.15mol/L NaOH solution; (5) 0.3mol/L NaCl solution; (6) 5 plastic centrifuge tubes of 2ml, 2 plastic centrifuge tubes of 15 ml; (7) 10 plastic straws with scales of 1ml and 10ml respectively; (8) 2 magnets; (9) the volume of 15ml scaled plastic vials is not less than 10.
2. The kit for removing formaldehyde from the air as claimed in claim 1, wherein the component (2) of the kit, i.e., streptavidin-coated magnetic beads, is selected from streptavidin magnetic beads Dynabeads available from Thermo Scientific, usaTMM-280 Streptavidin, comprising a compatible buffer.
3. The method for using the kit for removing formaldehyde from the air as claimed in claim 1 or 2, which is a method for removing formaldehyde from the air, is characterized in that the method for removing formaldehyde from the air comprises the following main steps:
(1) the preparation method comprises the following steps of preparing magnetic-carrier polypeptide suspension before formaldehyde detection, wherein the preparation process comprises the following steps: a. preparing magnetic beads by using streptavidin magnetic beads and 0.1ml of streptavidin according to a product specification, loading the magnetic beads by using a 15ml centrifugal tube, and finally obtaining magnetic bead liquid in a 10ml working solution state by using a buffer solution carried in the magnetic bead product; b. adding 0.1ml of 10mmol/L polypeptide T1 solution into the magnetic bead solution in the step a, and keeping the mixture fully and uniformly mixed for 15min at room temperature; c. washing the magnetic beads coated with the polypeptide for 3 times by using a buffer solution carried by the magnetic bead products by using a magnet, removing unbound substances, and adding 10ml of purified water to obtain a magnetic polypeptide-carrying suspension which can be stored in an environment of-20 ℃ for a long time for later use;
(2) after removing the purified water, 0.182mol/L (NH 4) is added2SO4Obtaining magnetic polypeptide working solution, sucking 10ml of magnetic polypeptide working solution, placing into a plastic small bottle, placing the plastic small bottle at indoor formaldehyde volatilization source, and placing at every 50m2The space only needs to be provided with 1 bottle;
(3) adding water into a plastic bottle once a week, and replenishing the working solution to a scale of 10 ml;
(4) after the magnetic beads are placed for 1 month, adsorbing the magnetic-carrying polypeptide on the side wall of the centrifugal tube by using a magnet, removing the solution in the centrifugal tube as much as possible by using a suction tube, washing the magnetic beads by using purified water for 2 times, and removing the purified water;
(5) then adding 5ml of 0.15mol/L NaOH solution and 5ml of 0.3mol/L NaCl solution to resuspend the magnetic beads, placing the mixture in a ventilated place for 5min at room temperature, removing the NaOH solution and the NaCl solution by using a magnet and a straw in the previous operation, washing the magnetic beads for 2 times by using purified water, and removing the purified water;
(6) 10ml of 0.182mol/L (NH 4) were added2SO4The solution is used for obtaining updated magnetic polypeptide-carrying working solution, and the plastic small bottle is put in place again;
(7) and (5) repeating the steps (3) to (6) until the concentration of the formaldehyde in the indoor air meets the national requirement or the actual requirement.
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Citations (2)

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Publication number Priority date Publication date Assignee Title
CN108130355A (en) * 2018-02-11 2018-06-08 华南理工大学 A kind of collagen polypeptide rich in glycine and formaldehyde scavenger prepared therefrom
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Publication number Priority date Publication date Assignee Title
CN108130355A (en) * 2018-02-11 2018-06-08 华南理工大学 A kind of collagen polypeptide rich in glycine and formaldehyde scavenger prepared therefrom
CN109647322A (en) * 2019-01-31 2019-04-19 厦门理工学院 A kind of biological safe type formaldehyde scavenger and its preparation method and application

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