CN110441529A - Formaldehyde detection reagent kit and its detection method - Google Patents

Formaldehyde detection reagent kit and its detection method Download PDF

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CN110441529A
CN110441529A CN201910738174.0A CN201910738174A CN110441529A CN 110441529 A CN110441529 A CN 110441529A CN 201910738174 A CN201910738174 A CN 201910738174A CN 110441529 A CN110441529 A CN 110441529A
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formaldehyde
polypeptide
magnetic
peptide
solution
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陈翠贤
王思捷
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Fuzhou Sijida Biomedical Technology Co Ltd
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
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Abstract

The present invention provides a kind of accurate and reliable, easy economical concentration of formaldehyde new detecting methods and its kit, it is mainly based upon a kind of novel functional polypeptide T1 with efficient formaldehyde binding ability, its sequence is as shown in SEQ ID NO:1, peptide T 1 can prepare magnetic and carry polypeptide via modifying the biotin of connection on terminal phenylalanine with the magnetic bead of coating Streptavidin and combining, and must be added to (NH4) of final concentration of 0.182mol/L in the combination anchor of magnetic load polypeptide and formaldehyde2SO4Solution.Magnetic has absorption maximum after carrying peptide T 1 in conjunction with formaldehyde at 446nm, and the relationship between absorbance x under formaldehyde binding capacity y and 482nm wavelength is y=45.716x-0.7713.

Description

Formaldehyde detection reagent kit and its detection method
Technical field
The present invention relates to the detection kit of harmful substance and its detection method, especially novel formaldehyde detection reagent kit And its detection method.
Background technique
Interior especially occupies the health that indoor air quality is related to everyone.The materials such as finishing, furniture and object The formaldehyde gas of the long-term slow release of product is the important component of room air pollution.Formaldehyde is huge as a kind of pair of mankind's harm Gas, be known as indoor " taking life killer by force ", especially in China, seriously endanger new finishing family, especially children, Yun Fuhe The health of old man is the major incentive of leukemia of children and fetal anomaly.It is published within 2017 the top academic journal in the world Research (A Class of Environmental and Endogenous Toxins Induces on " Cell "BRCA2 Haploinsufficiency and Genome Instability) to show that formaldehyde can degrade be tumor suppressor protein originally BRCA2 albumen, destroy DNA damage repair mechanism, make formaldehyde become induce cancer a high risk factor！In addition, industry is raw Formaldehyde waste liquid caused by production or experimental study is also serious environomental pollution source.
The national standard " Indoor Air Quality standards " (GBT 18883-2002) and live the state that the portion of building formulates that the former Ministry of Public Health formulates The concentration of formaldehyde that indoor security is defined in mark " civil buildings indoor environmental pollution control specification " (GB50325-2010) will be distinguished In 0.10mg/m3With 0.08 mg/m3It is (a kind of architectural engineering) and following.The method for determining formaldehyde listed in national standard has spectrophotometric Method and gas chromatography, wherein phenol reagent method is recommended method.It needs in phenol reagent method using toxic Harmful organic reagent, and operation is troublesome, sensitivity is not high；And gas-chromatography rule needs expensive equipment, sensitive electricity Chemical sensor is expensive, and both of which is more difficult universal.There is also similarly ask in the measurement of formaldehyde waste liquid for these methods Topic.
Removing for indoor formaldehyde, it is recognized that basic measures be long-term ventilation, but it is time-consuming very long, take effect slowly.Plant Purification and activated carbon adsorption and other effects are limited, and will discharge again after activated carbon adsorption saturation.Medical method poor in timeliness, number after spray Hour fails once volatilizing, and often introduces other pollutants.Catalyzed conversion (photocatalyst decomposition) method plays efficiency and needs purple Outside line excitation, can not remove in all directions.In short, being led due to formaldehyde pollution source multiplicity, micro the features such as accumulating, is long-term lasting Many administering methods are caused to have certain limitation.It is not only formaldehyde gas, formaldehyde waste liquid also lacks high-efficiency environment friendly and letter at present Just economic processing method.
There is an urgent need to develop the formaldehyde conjugate of environmental protection and economy and formaldehyde removing and the inspections again of novel high-efficient simple at present Survey method.Applicant's early period has found that specific structural domain has good formaldehyde binding ability in formaldehyde catalysis enzyme system, leads to It crosses computer simulation and obtains efficient formaldehyde combination polypeptide through screening, transformation and optimization, and is more based on newfound functionality Peptide have developed accurate and reliable, easy economy concentration of formaldehyde new detecting method and kit and high-efficiency and economic, environmental protection it is practical Formaldehyde removing new method and kit.
Summary of the invention
The present invention provides two kinds of novel functional polypeptides with efficient formaldehyde binding ability, and open on this basis Corresponding Analysis Methods for Formaldehyde and kit and formaldehyde removing method and kit are sent out.
It is more one of the objects of the present invention is to provide two kinds of functional polypeptide T1 and T2 with efficient formaldehyde binding performance Peptide T1 and T2 sequence are respectively as shown in SEQ ID NO:1 and SEQ ID NO:2, and the C-terminal of peptide T 1 and T2 the last one ammonia Base acid, that is, phenylalanine is modified with biotin, and such polypeptide can be synthesized by the biotech firm of business, it is desirable that purity is not Lower than 95%.
In order to facilitate practical application and recycling, peptide T 1 and T2 can be via modification connections on terminal phenylalanine Biotin prepares magnetic in conjunction with the magnetic bead of coating Streptavidin and carries polypeptide suspension, and (NH4) is added when needing2SO4Solution is allowed into Polypeptide working solution is carried for formaldehyde association reaction working solution, that is, magnetic.The key step that magnetic carries the preparation of polypeptide suspension is as follows:
A. Streptavidin MagneSphere is used, takes 0.1ml Streptavidin to carry out the preparation of magnetic bead according to product description, uses 15ml Centrifuge tube load, using in magnetic bead product carry buffer, finally obtain 10ml work liquid status under magnetic bead liquid；
B. the peptide T 1(or T2 of 10mmol/L are added in the magnetic bead liquid into a.) then polypeptide additive amount is 1 μ to solution 0.1ml( Mol, sufficiently combining rear magnetic to carry polypeptide amount is 1 μm of ol), it keeps mixing well 15min at room temperature；
C. magnetic bead 3 times for being coated with polypeptide are washed by magnet and with the buffer carried in magnetic bead product, remove unbonded object Matter is added 10ml pure water later to get to magnetic and carries polypeptide suspension, and it is stand-by can be placed in preservation for a long time in -20 degree environment；Need into (NH4) is added in row formaldehyde combination Shi Zaixiang reaction system2SO4Solution.
Peptide T 1 and T2 are provided with efficient formaldehyde binding ability, after being prepared into magnetic load polypeptide easy to use, every 1 μm of ol Magnetic carries peptide T 1 and T2 its formaldehyde binding capacity can achieve 87.25mg and 84.69mg respectively, and magnetic carries polypeptide and the combination of formaldehyde is anti- Answer (NH4) that must be added to final concentration of 0.1-0.3mol/L in system2SO4Solution, and (NH4) optimal in system2SO4It is dense Degree is 0.182mol/L.The combination that magnetic carries peptide T 1 and T2 and formaldehyde can be broken by the mixed solution system of NaOH and NaCl It is bad, the wherein final concentration of 0.05-0.15mol/L of NaOH, the final concentration of 0.1-0.3mol/L of NaCl, and NaOH and NaCl Concentration proportioning be 1:2, and optimal NaOH and NaCl final concentration is respectively 0.075mol/L and 0.15mol/L in system.
Magnetic has an absorption maximum at 446nm and 482nm respectively after carrying peptide T 1 and T2 with formaldehyde in conjunction with, and absorbance and Formaldehyde binding capacity is directly proportional, wherein for T1 polypeptide system, absorbance x under formaldehyde binding capacity y and 446nm wavelength it Between relationship be y=45.716x-0.7713, regression coefficient R2=0.9998；For T2 polypeptide system, formaldehyde binding capacity y Relationship between the absorbance x under 482nm wavelength is y=45.763x-0.7826, regression coefficient R2=0.9999。
The second object of the present invention is to provide formaldehyde detection reagent kit and detection method based on new type functional polypeptide. Wherein, the formaldehyde detection reagent kit based on new type functional polypeptide mainly includes following component:
(1) polypeptide freeze dried powder；
(2) it is coated with the magnetic bead of Streptavidin；
(3) (NH4)2SO4Solution；
(4) magnet.
Wherein, the polypeptide of component (1) is one of peptide T 1 or T2 i.e. peptide T 1(or peptide T 2), peptide T 1 and polypeptide The sequence of T2 is respectively as shown in SEQ ID NO:1 and SEQ ID NO:2, and the C-terminal of polypeptide the last one amino acid, that is, phenylpropyl alcohol Propylhomoserin is modified with biotin, is synthesized by biotech firm, and purity is not less than 95%, is made into 10mmol/L with preceding plus pure water Solution.
Wherein, component (2) is coated with the magnetic bead of Streptavidin, it is preferable that can choose U.S. Thermo The Streptavidin MagneSphere M-280(Dynabeads of Scientific companyTMM-280 Streptavidin), comprising matched Buffer.
Application method, that is, Analysis Methods for Formaldehyde the key step for carrying the formaldehyde detection reagent kit of polypeptide based on magnetic is as follows:
(1) magnetic must be prepared using kit each component before carrying out formaldehyde and combining carry polypeptide suspension, main preparation process are as follows: a. It takes 0.1ml Streptavidin to carry out the preparation of magnetic bead according to product description, is loaded with the centrifuge tube of 15ml, using magnetic bead product In carry buffer, finally obtain 10ml work liquid status under magnetic bead liquid；B. 10mmol/ is added in the magnetic bead liquid into a. The peptide T 1(or T2 of L) solution 0.1ml, it keeps mixing well 15min at room temperature；C. by magnet and in magnetic bead product from The buffer washing of band has been coated with magnetic bead 3 times of polypeptide, removes unbonded material, pure water 10ml is added later to get magnetic is arrived Polypeptide suspension is carried, can be placed in -20 degree environment and save for a long time for use.This carries being typically prepared process and matching for polypeptide suspension for magnetic Than that can be adjusted according to actual needs.
(2) magnetic is carried polypeptide suspension to be added in 0.1ml formaldehyde sample to be measured, adds (NH4)2SO4Solution is to (NH4)2SO4Final concentration of 0.182mol/L mixes postposition and is combined reaction 10min at room temperature, during which mixes again no less than 2 times；
(3) magnetic is carried into polypeptide using magnet and formaldehyde conjugate is adsorbed on tube wall, reaction system is sucked out；
(4) 1ml pure water is added and mixes elution 1 time, adsorb conjugate again, then clear water eluent is sucked out；
(5) mixing of 1ml pure water is added, takes 3ul magnetic to carry the aqueous solution of polypeptide and formaldehyde conjugate, is existed using microplate reader 446nm(, which is used, uses the wavelength when peptide T 1) or 482nm(use and use the wavelength when peptide T 2) measure absorbance under wavelength；
(6) the absorbance x of measurement is substituted into when formula y=45.716x-0.7713(uses peptide T 1 and uses the formula) or y= 45.763x-0.7826(, which is used, uses the formula when peptide T 2) formaldehyde binding capacity can be calculated, if formaldehyde binding capacity is super It crosses 80mg/ μm of ol magnetic and carries polypeptide, then suitably dilute formalin to be measured and again measurement until being lower than the numerical value, further change It is counted as concentration of formaldehyde or content of formaldehyde.
Wherein, sample to be tested is that solution then directly measures content of formaldehyde therein in Analysis Methods for Formaldehyde step (2)；If to Test sample sheet is air, then utilizes larger bubbles absorption tube and Constant flow sample referring to the sampling operation in GB/T 18204.26-2000 Device carries out the absorption of formaldehyde gas and is dissolved into solution, then the measurement of row content of formaldehyde.
The third object of the present invention be to provide the formaldehyde in air based on new type functional polypeptide scavenger reagent box and Sweep-out method.By probing into and optimizing, the scavenger reagent box of the formaldehyde in air based on new type functional polypeptide mainly includes such as Lower component:
(1) polypeptide freeze dried powder；
(2) it is coated with the magnetic bead of Streptavidin；
(3) (NH4) of 0.182mol/L2SO4Solution；
(4) NaOH solution of 0.15mol/L；
(5) NaCl solution of 0.3mol/L；
(6) 2ml plastic centrifuge tube 5,15ml plastic centrifuge tube 2；
(7) each 10 of plastic suction pipe 1ml and 10ml with a scale；
(8) 2 pieces of magnet；
(9) 15ml volume plastic jar with a scale is no less than 10.
Wherein, it is peptide T 1(or peptide T 2 that the polypeptide of component (1), which is one of peptide T 1 or peptide T 2), by biological public Department's synthesis, the sequence of peptide T 1 and T2 respectively as shown in SEQ ID NO:1 and SEQ ID NO:2, and the C-terminal of polypeptide last A amino acid, that is, phenylalanine is modified with biotin, and purity is not less than 95%, is made into the molten of 10mmol/L with preceding plus water Liquid.
Wherein, component (2) is coated with the magnetic bead of Streptavidin, it is preferable that can choose U.S. Thermo The Streptavidin MagneSphere M-280(Dynabeads of Scientific companyTMM-280 Streptavidin).
Application method, that is, formaldehyde in air sweep-out method of the scavenger reagent box of the formaldehyde in air of polypeptide is carried based on magnetic Key step it is as follows:
(1) it to be first prepared into magnetic before carrying out formaldehyde removing and carries polypeptide suspension, preparation process are as follows: a. uses Streptavidin magnetic Pearl takes 0.1ml Streptavidin to carry out the preparation of magnetic bead according to magnetic bead product description, is loaded, is used with the centrifuge tube of 15ml The buffer carried in magnetic bead product finally obtains the magnetic bead liquid under 10ml work liquid status；B. add in the magnetic bead liquid into a. Enter the peptide T 1(or T2 of 10mmol/L) solution 0.1ml, it keeps mixing well 15min at room temperature；C. by magnet and magnetic is used The buffer washing carried in pearl product has been coated with magnetic bead 3 times of polypeptide, removes unbonded material, pure water is added later 10ml carries polypeptide suspension to get to magnetic, can be placed in -20 degree environment and save for a long time for use.This carries the general of polypeptide suspension for magnetic Preparation process and proportion can be adjusted according to actual needs.
(2) it removes pure water and then (NH4) of 0.182mol/L is added2SO4Solution obtains magnetic and carries polypeptide working solution, 10ml magnetic load polypeptide working solution is drawn to be fitted into plastic jar, at plastic jar formaldehyde volatilization disposed within source, selection yin Cool corner is placed, every 50m2Space need to only place 1 bottle；
(3) water is added once into plastic jar, to supply at working solution to 10ml scale weekly；
(4) after placing 1 month, magnetic load polypeptide is adsorbed on centrifuge tube side wall using magnet, removes centrifuge tube as far as possible with suction pipe In solution, then remove pure water with after pure water washing magnetic bead 2 times；
(5) NaCl solution of the NaOH solution and 5ml 0.3mol/L that are subsequently added into 5ml 0.15mol/L is resuspended magnetic bead, room temperature It is placed in ventilation 5min, recycles for example preceding operation removal NaOH solution of magnet and suction pipe and NaCl solution, and with pure water washing Pure water is removed after magnetic bead 2 times；
(6) (NH4) of the 0.182mol/L of 10ml is added2SO4The magnetic that solution is updated carries polypeptide working solution, and plastics are small Bottle is put in original position again；
(7) step (3)-(6) are repeated until Formaldehyde Concentration of Indoor Air meets national requirements or actual demand.
The fourth object of the present invention is to provide in the waste liquid based on new type functional polypeptide formaldehyde removing kit and clear Except method.By probing into and optimizing, formaldehyde removing kit mainly includes such as the following group in the waste liquid based on new type functional polypeptide Point:
(1) polypeptide freeze dried powder；
(2) it is coated with the magnetic bead of Streptavidin；
(3) (NH4)2SO4Solution；
(4) NaOH solution；
(5) NaCl solution；
(6) 2 pieces of magnet.
Wherein, it is peptide T 1(or peptide T 2 that the polypeptide of component (1), which is one of peptide T 1 or peptide T 2), by biological public Department's synthesis, the sequence of peptide T 1 and T2 respectively as shown in SEQ ID NO:1 and SEQ ID NO:2, and the C-terminal of polypeptide last A amino acid, that is, phenylalanine is modified with biotin, and purity is not less than 95%, is made into the molten of 10mmol/L with preceding plus water Liquid.
Wherein, component (2) is coated with the magnetic bead of Streptavidin, it is preferable that can choose U.S. Thermo The Streptavidin MagneSphere M-280(Dynabeads of Scientific companyTMM-280 Streptavidin), comprising matched Buffer.
Furthermore, it is necessary to provide pH test paper and acid-base accommodation liquid (such as hydrochloric acid and NaOH solution) for oneself.
The master of formaldehyde removing method in application method, that is, waste liquid of formaldehyde removing kit in the waste liquid of polypeptide is carried based on magnetic Want that steps are as follows:
(1) magnetic must be prepared using reagent constituents before carrying out formaldehyde removing and carries polypeptide suspension, preparation process are as follows: a. takes 0.1ml Streptavidin carries out the preparation of magnetic bead according to product description, is loaded with the centrifuge tube of 15ml, using in magnetic bead product Included buffer finally obtains the magnetic bead liquid under 10ml work liquid status；B. 10mmol/L is added in the magnetic bead liquid into a. Peptide T 1(or T2) solution 0.1ml, at room temperature keep mix well 15min；C. by magnet and in magnetic bead product from The buffer washing of band has been coated with magnetic bead 3 times of polypeptide, removes unbonded material, pure water 10ml is added later to get magnetic is arrived Polypeptide suspension is carried, can be placed in -20 degree environment and save for a long time for use.This carries being typically prepared process and matching for polypeptide suspension for magnetic Than that can be adjusted according to actual needs.
(2) pH in waste liquid is adjusted to 7.0 first with acid-base accommodation liquid and pH test paper, then prepared magnetic is carried into polypeptide Suspension is added in formaldehyde waste liquid, is subsequently added into (NH4)2SO4Solution is to (NH4)2SO4Final concentration of 0.182mol/L mixes postposition It is sufficiently reacted at room temperature；
(3) it recycles magnet to adsorb magnetic load polypeptide, pours out processed formaldehyde waste liquid, then with pure water washing magnetic bead 2 times After remove pure water；
(4) polypeptide being carried then according to every 1 μm of ol magnetic, the NaOH solution of 5ml 0.15mol/L and the NaCl of 5ml 0.3mol/L is added Solution is resuspended magnetic bead, and room temperature is placed in ventilation 5min, recycles for example preceding operation removal NaOH solution of magnet and NaCl solution, and Pure water is removed with after pure water washing magnetic bead 2 times；
(5) by treated in (4), magnetic load polypeptide is rejoined in the formaldehyde waste liquid poured out in (3), and pH is adjusted to 7.0 again After add (NH4)2SO4Solution is to (NH4)2SO4Final concentration of 0.182mol/L, mixing postposition carry out sufficiently anti-at room temperature It answers；
(6) step (3)-(5) are repeated until concentration of formaldehyde meets national requirements or actual demand in waste liquid.
The present invention provides two kinds of functional polypeptides with efficient formaldehyde binding ability, and formaldehyde binding ability is strong, and And can efficiently combine, it recycles, secondary pollution, safety and environmental protection will not be generated；Concentration of formaldehyde based on polypeptide exploitation Detection kit and detection method, have the characteristics that accurate and reliable, easy economy and high-efficiency environment friendly, formaldehyde removing kit and clear Except method has the characteristics that high-efficiency and economic, environmental protection are practical.
Detailed description of the invention
Fig. 1 is the full wavelength scanner map of the formaldehyde conjugate sample of peptide T 1 in embodiment 4.
Fig. 2 is the full wavelength scanner map of the formaldehyde conjugate sample of peptide T 2 in embodiment 4.
Fig. 3 is blank control group full wavelength scanner map in embodiment 4.
Fig. 4 is the formaldehyde binding capacity and absorbance graph of relation that magnetic carries 1 system of peptide T in embodiment 5.
Fig. 5 is the formaldehyde binding capacity and absorbance graph of relation that magnetic carries 2 system of peptide T in embodiment 5.
Specific embodiment
Below in conjunction with specific embodiment, the present invention is further explained.
1 target magnetic of embodiment carries polypeptide and has formaldehyde binding performance
If target polypeptides (i.e. T1 or T2) have formaldehyde binding performance, in a certain specific formaldehyde systems (such as formalin) The target polypeptides of certain content are added, the two is made to contact with each other, after sufficiently combining a period of time under suitable conditions, then will The target polypeptides for combining formaldehyde remove out the formaldehyde systems, inevitable by the concentration of formaldehyde being measured by sampling in the then formaldehyde systems It can decline, it is possible thereby to verify and probe into the formaldehyde binding performance of target polypeptides, detailed process is as follows.
The peptide T 1 and T2 of Shanghai Tao Pu Biotechnology Co., Ltd synthesis sequence as shown in SEQ ID NO:1 and 2 are entrusted, And the last one amino acid phenylalanine (the amino acid single letter code is F) of its C-terminal of both polypeptides uses biotin (Biotin, Sigma-Aldrich) is modified, and the purity of synthesis polypeptide is not less than 95%.By the polypeptide of synthesis Freeze-dried powder is configured to the peptide T 1 and T2 solution (peptide T 1 of biotin modification and mole of T2 of 10mmol/L with pure water respectively Quality is respectively 6578.34g/mol and 6024.81g/mol).The combination of biotin and Streptavidin is in current nature Known strongest noncovalent interaction.In order to which performance study and practical application are convenient, peptide T 1 and T2 are passed through into its end The biotin modification of phenylpropyl alcohol amino acid acts on the magnetic bead of coating Streptavidin, is prepared into magnetic and carries polypeptide.Using the U.S. The Streptavidin MagneSphere M-280(DynabeadsTM M-280 Streptavidin of Thermo Scientific company, Invitrogen), method relies on biotin (modification group on peptide T 1 and T2 end amino acid) and strepto- to specifications Strong affinity interaction power between Avidin (being coated on M-280 magnetic bead surfaces), the magnetic bead of preparation coating polypeptide, and utilize the coating The functional magnetic bead of polypeptide probes into the formaldehyde binding performance of polypeptide.Specific steps are as follows:
(1) 0.1ml magnetic bead is taken to carry out the preparation of M-280 magnetic bead according to method in Streptavidin MagneSphere M-280 product description, It is loaded with the centrifuge tube of 15ml, buffer finally obtains 10ml work using the B&W Buffer carried in magnetic bead product Magnetic bead liquid under liquid status；
(2) into the magnetic bead liquid in (1) be added 10mmol/L polypeptide solution 0.1ml(then polypeptide additive amount is 1 μm of ol, fill Point rear magnetic is combined to carry polypeptide amount is 1 μm of ol), at the same be arranged blank control group (blank control group does not add peptide solution, but with The deionized water of 0.1ml substitutes), it is all made of vertical rotary blending instrument and keeps mixing well 15min at room temperature；
(3) it is washed by magnet or magnetic frame (Shanghai Sheng Gong bioengineering Co., Ltd) and with B&W Buffer and has been coated with polypeptide Magnetic bead 3 times, remove unbonded material, later be added 10.1ml 0.2mol/L(NH4)2SO4(1ml is then added in solution in next step After formalin, (NH4)2SO4Ultimate density be 0.182mol/L, probed into this as optimal final concentration), then sample sets obtain Magnetic carries polypeptide suspension, and what blank control group obtained is the coated suspension containing magnetic beads of no polypeptide；
(4) (mass fraction is the formaldehyde of 35-40% to the pure formalin of magnetic load polypeptide suspension addition 1ml analysis into (3) immediately Aqueous solution is commonly called as formalin, is purchased from Shanghai Yuan Mu Biotechnology Co., Ltd), it is placed in and is combined reaction 30min at room temperature (period shakes up 2 times)；
(5) when reaction proceeds to 10min and 30min in (4), magnetic bead is adsorbed on to the wall of 15ml centrifuge tube with magnetic frame On, then with liquid-transfering gun the reaction solution in 10ul centrifuge tube is drawn, using electrochemical sensor method (referring to national standard GBT18204.2- 7.5 part in 2014) its concentration of formaldehyde is measured, use Britain to be based on the PPM- of electrochemical sensor principle (PPM-Tech) 400 ST methylene oxide detecting instruments are detected (parallel determination is averaged three times, similarly hereinafter) to specifications, and the results are shown in Table 1.
Concentration of formaldehyde (unit: mg/ml) in 1 sample sets of table and blank control group reaction solution
After measured, it is that 34.0mg/ml(reaction solution is overall that concentration caused by formaldehyde, that is, original concentration of formaldehyde is added in reaction system Product is 11.1ml), therefore formaldehyde additive amount caused by added 1ml formalin is 377.4mg, which is enough to cause non- Normal serious duration Formaldehyde Pollution of Indoor Air, is suitable as model value.In conjunction with table 1 it is found that not being coated with the magnetic bead of polypeptide i.e. The concentration of formaldehyde of its reaction solution of blank control group is compared with original concentration of formaldehyde, percentage deviation (sample concentration value and original Difference between beginning concentration value accounts for the percentage of original concentration value) be respectively 0.24%(reaction 10min) and 0.29%(reaction 30min), it almost can be ignored, show that the magnetic bead for not being coated with polypeptide does not have apparent formaldehyde absorption binding performance.So And the magnetic bead i.e. concentration of formaldehyde of its reaction solution of sample sets for being coated with polypeptide is decreased significantly compared with original concentration of formaldehyde, very To more than 15%.Wherein, for the Magnetic bead sample of coating peptide T 1, concentration of formaldehyde decline percentage is respectively 5.29%(reaction 10min) and 18.59%(reacts 30min)；For being coated with the Magnetic bead sample of peptide T 2, concentration of formaldehyde decline percentage is respectively 6.91%(reacts 10min) and 21.06%(reaction 30min).It is indicated above that the magnetic bead of coating polypeptide (T1 or T2) is provided with obviously Formaldehyde absorption binding performance, and tentatively show that its formaldehyde absorption binding capacity increases with the extension of reaction time.
The saturation binding capacity of 2 magnetic of embodiment load polypeptide PARA FORMALDEHYDE PRILLS(91,95)
By embodiment 1 it is found that target polypeptides have good formaldehyde binding ability, however it must further probe into unit target The formaldehyde binding capacity of polypeptide, to provide reference and foundation for subsequent application.Specific system and under the conditions of, unit target polypeptides Formaldehyde binding capacity will be increased up with the extension of reaction time and reach and its is maintained to be saturated binding capacity, therefore can use This feature probes into the saturation conjugation of target polypeptides PARA FORMALDEHYDE PRILLS(91,95), and detailed process is as follows.
Carry out probing into and (being equally also provided with blank control group) for embodiment 2 referring to embodiment 1, the difference is that in embodiment 1 The step of (4) reaction time only arrive 30min, 2 reaction time of embodiment extends to for 24 hours, and 30s, that is, 0.5min, 1min, The separately sampled first measured in reaction solution three times of 10min, 30min, 1h, 3h, 5h, 10h, 15h and for 24 hours this 10 reaction time points Aldehyde concentration is simultaneously averaged, and the results are shown in Table 2, and (concentration of formaldehyde caused by after measured, adding in 2 reaction system of embodiment is i.e. former The concentration of formaldehyde of beginning is 33.9mg/ml).
Concentration of formaldehyde and its percentage range of decrease under the 2 differential responses time of table in reaction solution
As shown in Table 2, for 1 group of sample of peptide T, when reacted between when reaching 5h or more, the concentration of formaldehyde in reaction solution is protected Holding does not become 26.04mg/ml, and the saturation that coated peptide T 1 has had reached formaldehyde on magnetic bead in the case of this combines.Therefore per micro- It is 11.1ml that polypeptide T1, which is 87.25mg(reaction solution total volume for the saturation binding capacity of formaldehyde), the reaction time is not low In 5h.For 2 groups of samples of peptide T, when reacted between when reaching 3h or more, the concentration of formaldehyde in reaction solution remain unchanged for 26.27mg/ml, the saturation that coated peptide T 2 has had reached formaldehyde on magnetic bead in the case of this combine.Therefore every micromole's polypeptide It is 11.1ml that T2, which is 84.69mg(reaction solution total volume for the saturation binding capacity of formaldehyde), the reaction time is not less than 3h.
The recovery of 3 magnetic of embodiment load polypeptide and formaldehyde binding ability
After target polypeptides PARA FORMALDEHYDE PRILLS(91,95) realizes that saturation combines, in order to realize that moving in circles for target polypeptides utilizes so as to applied to real Border, it is necessary to seek the condition for releasing combination after its formaldehyde combines, make it restore formaldehyde binding ability again, detailed process is as follows.
Carrying polypeptide system for the magnetic after reacting for 24 hours in embodiment 2, (polypeptide has reached the saturation bonding state of formaldehyde at this time ), it is adsorbed on centrifuge tube side wall using magnetic frame, removes the reaction solution in centrifuge tube as far as possible with liquid-transfering gun, then with pure Water purification removes pure water after washing magnetic bead 2 times.The NaCl of NaOH solution and 5ml 0.3mol/L that 5ml 0.15mol/L is added is molten (final concentration of NaOH and NaCl is respectively 0.075mol/L and 0.15mol/L to liquid at this time, is probed into this as optium concentration and matched Than) magnetic bead is resuspended, it is rocked during being placed in 5min(at room temperature 1 time, which in draught cupboard or ventilation carries out that formaldehyde is prevented to release Put and cause indoor pollution), recycle for example preceding operation of magnetic frame and liquid-transfering gun to remove NaOH solution and NaCl solution, and use pure water Pure water is removed after washing magnetic bead 2 times.Applicant probes into the configuration and body for showing that the NaOH due to addition can change polypeptide The pH value of system, the NaCl of addition can change the ion concentration of system, these will lead to the combination quilt of original polypeptide and formaldehyde It destroys, so far realizes the release of the combined formaldehyde of target polypeptides, the polypeptide for formaldehyde combination at this time has realized update.It connects For updated polypeptide (more novel polypeptide), referring to method and step in Examples 1 and 2, carry out formaldehyde in conjunction with probing into, with Phase realizes the utilization that moves in circles, and detailed process is as follows.
Then, the 0.2mol/L(NH4 of 10.1ml is added into the magnetic bead updated)2SO4Solution obtains suspension containing magnetic beads, later 1ml is added and analyzes pure formalin, is placed in and is combined for 24 hours at room temperature reaction (period rocks 2-3 times), then with PPM-400 ST Methylene oxide detecting instrument measure its recombine reaction 15h and for 24 hours when reaction solution in concentration of formaldehyde, and be converted into the formaldehyde knot of polypeptide Resultant, as a result as shown in table 3 below.
3 polypeptide of table is in conjunction with formaldehyde capacity experimental data
After measured, it is 34.1mg/ml(reaction solution that concentration caused by formaldehyde, that is, original concentration of formaldehyde is this time added in reaction system Total volume is 11.1ml).Detected in embodiment 2 original polypeptide system association reaction 15h and for 24 hours when PARA FORMALDEHYDE PRILLS(91,95) combination reached To being saturated, and being saturated formaldehyde binding capacity is respectively 87.25mg(peptide T 1) and 84.69mg(peptide T 2).For more novel polypeptide, For 24 hours with 15h interphase every 9h, however concentration of formaldehyde is the same in reaction solution, it was demonstrated that also has reached the saturation bonding state of formaldehyde. It is computed, the saturation formaldehyde binding capacity (table 3) of more novel polypeptide system is respectively that 80.03mg(updates peptide T 1) and 79.03mg(is more Novel polypeptide T2).
If the formaldehyde saturation binding capacity of more novel polypeptide accounts for original polypeptide on the basis of the formaldehyde of original polypeptide saturation binding capacity Formaldehyde saturation binding capacity percentage be known as response rate, then the response rate for updating peptide T 1 and T2 is respectively 91.7% He 93.3%.Continue secondary update, the three times experiment such as update, and when discovery updates 11 times repeatedly (update is started for the second time, and every two The interval time of secondary update is 10 days, continues 100 days total time), the response rate of peptide T 1 still has 50.83%；It updates 11 times repeatedly When (second update start, be 10 days per the interval time updated twice, continue 100 days total time), the response rate of peptide T 2 Still have 53.11%.It is indicated above magnetic load peptide T 1 and T2 all has good formaldehyde binding ability repeatedly, can be recycled.
The spectral absorption performance of 4 magnetic of embodiment load polypeptide and formaldehyde conjugate
Absorption maximum (usually between 210-220nm) can occur under ultraviolet for polypeptide, however in the combination of itself and other materials Or in the case where complexing, spectral absorption performance will be likely to that certain change, and the detection based on spectral characteristic occurs It is quickly and economical and practical that method etc. has the characteristics that simplicity, therefore probes into the spectral absorption characteristics of target polypeptides and formaldehyde conjugate In order to subsequent related application, specific probe process is as follows.
It leaves and takes the reaching magnetic that saturation formaldehyde combines after reacting for 24 hours in embodiment 2 and carries polypeptides reactive liquid sample (including blank 2 sample sets of control group, 1 sample sets of peptide T and peptide T), full wave full wavelength scanner is carried out, to find out polypeptide and formaldehyde The maximum absorption wavelength of conjugate.Using the Multiskan GO all-wave length microplate reader of Thermo Fisher company, the U.S., Applied sample amount is 3ul, is carried out at room temperature in 200nm-1000nm spectral band with 1nm stepping-in amount according to operational manual step complete Length scanning, as a result as shown in Figs. 1-3.
As seen from Figure 1, Figure 2, the maximum absorption wavelength of the formaldehyde conjugate sample of peptide T 1 and T2 be respectively 446nm, 482nm is much higher than its peptide bond absorption peak (at 220nm).For blank control group (reaction system without polypeptide), containing only magnetic Therefore pearl and reaction solution system there will not be the conjugate of polypeptide and formaldehyde without polypeptide, in conjunction with its full wavelength scanner map (figure 3) it is found that being absorbed without obvious at 446nm and 482nm.It follows that being absorbed as peptide T 1 and formaldehyde at 446nm wavelength The characteristic absorption peak of conjugate, the characteristic absorption peak for being absorbed as peptide T 2 and formaldehyde conjugate at 482nm wavelength.
5 magnetic of embodiment carries the relationship of absorption and formaldehyde binding capacity of the polypeptide formaldehyde conjugate under maximum wavelength
On the basis of embodiment 4 obtains target polypeptides and formaldehyde conjugate maximum absorption wavelength, mesh can be further probed into The relationship for marking the absorption and formaldehyde binding capacity of polypeptide and formaldehyde conjugate under maximum wavelength, thus by the feature development formaldehyde The convenient detection method of binding capacity (or content of formaldehyde), detailed process is as follows.
Based on embodiment 2, selected 10 reaction time points (30s, that is, 0.5min, 1min, 10min, 30min, 1h, 3h, 5h, 10h, 15h, for 24 hours) while residual formaldehyde concentration in measurement system, response sample is left and taken referring to side in embodiment 4 Method (T1 polypeptide system is 446nm, and T2 polypeptide system is 482nm) under maximum absorption wavelength measures its absorbance and (measures 3 times and take It is average), as a result as shown in table 4 below.The concentration of formaldehyde in reaction solution at each reaction time point is surveyed by embodiment 2 (is detailed in table 2), it is possible thereby to calculate the formaldehyde binding capacity of each reaction time point, see Table 4 for details.For 1 system of peptide T, 5h rises each Time point sample absorbance is almost the same, and for 2 system of peptide T, 3h various time points sample absorbances are almost the same, This is consistent with the conclusion that formaldehyde saturation is combined that reaches observed in embodiment 2.
Formaldehyde binding capacity and absorbance under the 4 differential responses time of table in reaction solution
The scatter plot that the corresponding formaldehyde binding capacity of each reaction time point and absorbance are listed on Excel (is only got and is reached at first At the time of saturation combines), both discoveries are in a linear relationship, carry out linear fit, as a result as illustrated in figures 4-5.For T1 polypeptide body For system, the relationship between absorbance x under formaldehyde binding capacity y and 446nm wavelength is y=45.716x-0.7713, returns system Number R2=0.9998 proves the line that the linear equation has been well reflected between the formaldehyde binding capacity and absorbance of magnetic load peptide T 1 Sexual intercourse；For T2 polypeptide system, the relationship between absorbance x under formaldehyde binding capacity y and 482nm wavelength be y= 45.763x-0.7826 regression coefficient R2=0.9999 proves that the linear equation has been well reflected the formaldehyde that magnetic carries peptide T 2 Linear relationship between binding capacity and absorbance.
Target polypeptides and its characteristic brief summary
By early-stage study and embodiment 1-5 result it is found that functional polypeptide T1 and T2 with efficient formaldehyde binding performance, sequence Column are respectively as shown in SEQ ID NO:1 and SEQ ID NO:2, and the C-terminal of peptide T 1 and T2 the last one amino acid, that is, phenylpropyl alcohol Propylhomoserin is modified with biotin；Peptide T 1 and T2 can be synthesized by commercialized biotech firm, and purity is required to be not less than 95%；Peptide T 1 and T2 can be via the magnetic bead knots of the biotin and coating Streptavidin of modifying connection on terminal phenylalanine Conjunction prepares magnetic and carries polypeptide, practical more effectively, easily and economically to be applied to.It is found by comparative analysis, unit peptide T 1 Saturation formaldehyde binding capacity be higher than peptide T 2, however the association rate of peptide T 2 and formaldehyde is faster than peptide T 1, and the two respectively has respectively Advantage.
Peptide T 1 and T2 are provided with efficient formaldehyde binding ability, and every 1 μm of ol magnetic carries peptide T 1 and T2 its formaldehyde binding capacity Can achieve 87.25mg and 84.69mg respectively, magnetic carry must be added in the combination anchor of polypeptide and formaldehyde it is final concentration of (NH4) of 0.1-0.3mol/L2SO4Solution, and (NH4) optimal in system2SO4Concentration is 0.182mol/L.Magnetic carries peptide T 1 Combination with T2 and formaldehyde can be destroyed by the mixed solution system of NaOH and NaCl, and wherein NaOH's is final concentration of The final concentration of 0.1-0.3mol/L of 0.05-0.15mol/L, NaCl, and the concentration proportioning of NaOH and NaCl is 1:2, and body Optimal NaOH and NaCl final concentration is respectively 0.075mol/L and 0.15mol/L in system.Further, it is carried in conjunction with the magnetic of formaldehyde Peptide T 1 and T2 still have very high formaldehyde in conjunction with ability, in conjunction with release in conjunction with repeatedly 10 after discharging combined formaldehyde Binding force still can achieve 50% or more of binding force for the first time after secondary.
Magnetic has an absorption maximum at 446nm and 482nm respectively after carrying peptide T 1 and T2 with formaldehyde in conjunction with, and absorbance and Formaldehyde binding capacity is directly proportional, wherein for T1 polypeptide system, absorbance x under formaldehyde binding capacity y and 446nm wavelength it Between relationship be y=45.716x-0.7713, regression coefficient R2=0.9998；For T2 polypeptide system, formaldehyde binding capacity y Relationship between the absorbance x under 482nm wavelength is y=45.763x-0.7826, regression coefficient R2=0.9999。
Embodiment 6 has the peptide T 1 and T2 of efficient formaldehyde binding performance
Functional polypeptide T1 and T2 with efficient formaldehyde binding performance, sequence is respectively such as SEQ ID NO:1 and SEQ ID Shown in NO:2, and the C-terminal of peptide T 1 and T2 the last one amino acid, that is, phenylalanine is modified with biotin, such Polypeptide can be synthesized by the biotech firm of business, it is desirable that purity is not less than 95%.
In order to facilitate practical application and recycling, peptide T 1 and T2 can be via modification connections on terminal phenylalanine Biotin is combined with the magnetic bead of coating Streptavidin to be prepared magnetic and carries polypeptide, addition (NH4) when use2SO4Solution makes first Aldehyde association reaction working solution, that is, magnetic carries polypeptide working solution.The key step that magnetic carries the preparation of polypeptide suspension is following (here only as magnetic The general example that polypeptide is prepared is carried, can be scaled up according to actual needs or be reduced or adjust):
A. Streptavidin MagneSphere is used, takes 0.1ml Streptavidin to carry out the preparation of magnetic bead according to product description, uses 15ml Centrifuge tube load, using in magnetic bead product carry buffer, finally obtain 10ml work liquid status under magnetic bead liquid；
B. the peptide T 1(or T2 of 10mmol/L are added in the magnetic bead liquid into a.) then polypeptide additive amount is 1 μ to solution 0.1ml( Mol, sufficiently combining rear magnetic to carry polypeptide amount is 1 μm of ol), it keeps mixing well 15min at room temperature；
C. magnetic bead 3 times for being coated with polypeptide are washed by magnet and with the buffer carried in magnetic bead product, remove unbonded object Matter is added 10ml pure water later to get to magnetic and carries polypeptide suspension, and it is stand-by can be placed in preservation for a long time in -20 degree environment；Carry out first (NH4) is added in aldehyde combination Shi Zaixiang reaction system2SO4Solution.
Peptide T 1 and T2 are provided with efficient formaldehyde binding ability, and every 1 μm of ol magnetic carries peptide T 1 and T2 its formaldehyde binding capacity Can achieve 87.25mg and 84.69mg respectively, magnetic carry must be added in the combination anchor of polypeptide and formaldehyde it is final concentration of (NH4) of 0.1-0.3mol/L2SO4Solution, and (NH4) optimal in system2SO4Concentration is 0.182mol/L.Magnetic carries peptide T 1 Combination with T2 and formaldehyde can be destroyed by the mixed solution system of NaOH and NaCl, and wherein NaOH's is final concentration of The final concentration of 0.1-0.3mol/L of 0.05-0.15mol/L, NaCl, and the concentration proportioning of NaOH and NaCl is 1:2, and body Optimal NaOH and NaCl final concentration is respectively 0.075mol/L and 0.15mol/L in system.
Magnetic has an absorption maximum at 446nm and 482nm respectively after carrying peptide T 1 and T2 with formaldehyde in conjunction with, and absorbance and Formaldehyde binding capacity is directly proportional, wherein for T1 polypeptide system, absorbance x under formaldehyde binding capacity y and 446nm wavelength it Between relationship be y=45.716x-0.7713, regression coefficient R2=0.9998；For T2 polypeptide system, formaldehyde binding capacity y Relationship between the absorbance x under 482nm wavelength is y=45.763x-0.7826, regression coefficient R2=0.9999。
Formaldehyde detection reagent kit and detection method of the embodiment 7 based on novel polypeptide T1
By probing into and optimizing, the formaldehyde detection reagent kit based on new type functional peptide T 1 mainly includes following component:
(1) 1 freeze dried powder of peptide T, sequence is as shown in SEQ ID NO:1；
(2) it is coated with the magnetic bead of Streptavidin；
(3) (NH4)2SO4Solution；
(4) magnet.
Wherein, component (1) i.e. peptide T 1 is synthesized by biotech firm, and the C-terminal of peptide T 1 the last one amino acid, that is, benzene Alanine is modified with biotin, and purity is not less than 95%, and the solution of 10mmol/L is made into preceding plus pure water.
Wherein, component (2) is coated with the magnetic bead of Streptavidin, it is preferable that can choose U.S. Thermo The Streptavidin MagneSphere M-280(Dynabeads of Scientific companyTMM-280 Streptavidin), comprising matched Buffer.
Application method, that is, Analysis Methods for Formaldehyde the key step for carrying the formaldehyde detection reagent kit of peptide T 1 based on magnetic is as follows:
(1) magnetic must being prepared using kit each component before carrying out formaldehyde and combining and carrying 1 suspension of peptide T, preparation process is referring to reality Apply example 6；
(2) magnetic is carried 1 suspension of peptide T to be added in 0.1ml formaldehyde sample to be measured, adds (NH4)2SO4Solution is to (NH4)2SO4 Final concentration of 0.182mol/L mixes postposition and is combined reaction 10min at room temperature, during which mixes again no less than 2 times；
(3) magnetic is carried into polypeptide using magnet and formaldehyde conjugate is adsorbed on tube wall, reaction system is sucked out；
(4) 1ml pure water is added and mixes elution 1 time, adsorb conjugate again, then clear water eluent is sucked out；
(5) mixing of 1ml pure water is added, takes 3ul magnetic to carry the aqueous solution of polypeptide and formaldehyde conjugate, is existed using microplate reader Absorbance is measured under 446nm wavelength；
(6) formaldehyde binding capacity can be calculated by the absorbance x of measurement being substituted into formula y=45.716x-0.7713, if formaldehyde Binding capacity is more than that 80mg/ μm of ol magnetic carries polypeptide, then suitably dilutes formalin to be measured and again measurement until being lower than the numerical value, then Further it is converted into concentration of formaldehyde or content of formaldehyde.
Wherein, sample to be tested is that solution then directly measures content of formaldehyde therein in Analysis Methods for Formaldehyde step (2)；If to Test sample sheet is air, then utilizes larger bubbles absorption tube and Constant flow sample referring to the sampling operation in GB/T 18204.26-2000 Device carries out the absorption of formaldehyde gas and is dissolved into solution, then the measurement of row content of formaldehyde.
Formaldehyde detection reagent kit and detection method of the embodiment 8 based on novel polypeptide T2
By probing into and optimizing, the formaldehyde detection reagent kit based on new type functional peptide T 2 mainly includes following component:
(1) 2 freeze dried powder of peptide T, sequence is as shown in SEQ ID NO:2；
(2) it is coated with the magnetic bead of Streptavidin；
(3) (NH4)2SO4Solution；
(4) magnet.
Wherein, component (1) i.e. peptide T 2 is synthesized by biotech firm, and the C-terminal of peptide T 2 the last one amino acid, that is, benzene Alanine is modified with biotin, and purity is not less than 95%, and the solution of 10mmol/L is made into preceding plus pure water.
Wherein, component (2) i.e. magnetic bead is the magnetic bead for being coated with Streptavidin, it is preferable that can choose U.S. Thermo The Streptavidin MagneSphere M-280(Dynabeads of Scientific companyTMM-280 Streptavidin), comprising matched Buffer.
Application method, that is, Analysis Methods for Formaldehyde the key step for carrying the formaldehyde detection reagent kit of peptide T 2 based on magnetic is as follows:
(1) magnetic must being prepared using kit each component before carrying out formaldehyde and combining and carrying 2 suspension of peptide T, preparation process is referring to reality Apply example 6；
(2) magnetic is carried 2 suspension of peptide T to be added in 0.1ml formaldehyde sample to be measured, adds (NH4)2SO4Solution is to (NH4)2SO4 Final concentration of 0.182mol/L mixes postposition and is combined reaction 10min at room temperature, during which mixes again no less than 2 times；
(3) magnetic is carried into polypeptide using magnet and formaldehyde conjugate is adsorbed on tube wall, reaction system is sucked out；
(4) 1ml pure water is added and mixes elution 1 time, adsorb conjugate again, then clear water eluent is sucked out；
(5) mixing of 1ml pure water is added, takes 3ul magnetic to carry the aqueous solution of polypeptide and formaldehyde conjugate, is existed using microplate reader Absorbance is measured under 482nm wavelength；
(6) formaldehyde binding capacity can be calculated by the absorbance x of measurement being substituted into formula y=45.763x-0.7826, if formaldehyde Binding capacity is more than that 80mg/ μm of ol magnetic carries polypeptide, then suitably dilutes formalin to be measured and again measurement until being lower than the numerical value, then Further it is converted into concentration of formaldehyde or content of formaldehyde.
Wherein, sample to be tested is that solution then directly measures content of formaldehyde therein in Analysis Methods for Formaldehyde step (2)；If to Test sample sheet is air, then utilizes larger bubbles absorption tube and Constant flow sample referring to the sampling operation in GB/T 18204.26-2000 Device carries out the absorption of formaldehyde gas and is dissolved into solution, then the measurement of row content of formaldehyde.
Formaldehyde in air scavenger reagent box and sweep-out method of the embodiment 9 based on new type functional polypeptide
By probing into and optimizing, the formaldehyde in air scavenger reagent box based on new type functional polypeptide mainly includes following component:
(1) polypeptide freeze dried powder；
(2) it is coated with the magnetic bead of Streptavidin；
(3) (NH4) of 0.182mol/L2SO4Solution；
(4) NaOH solution of 0.15mol/L；
(5) NaCl solution of 0.3mol/L；
(6) 2ml plastic centrifuge tube 5,15ml plastic centrifuge tube 2；
(7) each 10 of plastic suction pipe 1ml and 10ml with a scale；
(8) 2 pieces of magnet；
(9) 15ml volume plastic jar with a scale is no less than 10.
Wherein, it is peptide T 1(or peptide T 2 that the polypeptide of component (1), which is one of peptide T 1 or peptide T 2), by biological public Department's synthesis, the sequence of peptide T 1 and T2 respectively as shown in SEQ ID NO:1 and SEQ ID NO:2, and the C-terminal of polypeptide last A amino acid, that is, phenylalanine is modified with biotin, and purity is not less than 95%, is made into the molten of 10mmol/L with preceding plus water Liquid.
Wherein, component (2) is coated with the magnetic bead of Streptavidin, it is preferable that can choose U.S. Thermo The Streptavidin MagneSphere M-280(Dynabeads of Scientific companyTMM-280 Streptavidin), comprising matched Buffer.
Application method, that is, formaldehyde in air sweep-out method master of the formaldehyde in air scavenger reagent box of polypeptide is carried based on magnetic Want that steps are as follows:
(1) it to be first prepared into magnetic before carrying out formaldehyde removing and carries polypeptide suspension, preparation process is referring to embodiment 6；
(2) magnetic is removed to carry the pure water in polypeptide suspension and then (NH4) of 0.182mol/L is added2SO4Solution obtains magnetic Polypeptide working solution is carried, 10ml magnetic load polypeptide working solution is drawn and is fitted into plastic jar, plastic jar formaldehyde disposed within is volatilized At source, shady and cool corner is selected to place, every 50m2Space need to only place 1 bottle；
(3) water is added once into plastic jar, to supply at working solution to 10ml scale weekly；
(4) after placing 1 month, magnetic load polypeptide is adsorbed on centrifuge tube side wall using magnet, removes centrifuge tube as far as possible with suction pipe In solution, then remove pure water with after pure water washing magnetic bead 2 times；
(5) NaCl solution of the NaOH solution and 5ml 0.3mol/L that are subsequently added into 5ml 0.15mol/L is resuspended magnetic bead, room temperature It is placed in ventilation 5min, recycles for example preceding operation removal NaOH solution of magnet and suction pipe and NaCl solution, and with pure water washing Pure water is removed after magnetic bead 2 times；
(6) (NH4) of 10ml 0.182mol/L is added2SO4The magnetic that solution is updated carries polypeptide working solution, and plastics are small Bottle is put in original position again；
(7) step (3)-(6) are repeated until Formaldehyde Concentration of Indoor Air meets national requirements or actual demand.
Formaldehyde removing kit and sweep-out method in waste liquid of the embodiment 10 based on new type functional polypeptide
By probing into and optimizing, formaldehyde removing kit mainly includes following component in the waste liquid based on new type functional polypeptide:
(1) polypeptide freeze dried powder；
(2) it is coated with the magnetic bead of Streptavidin；
(3) (NH4)2SO4Solution；
(4) NaOH solution；
(5) NaCl solution；
(6) 2 pieces of magnet.
Wherein, it is peptide T 1(or peptide T 2 that the polypeptide of component (1), which is one of peptide T 1 or peptide T 2), by biological public Department's synthesis, the sequence of peptide T 1 and T2 respectively as shown in SEQ ID NO:1 and SEQ ID NO:2, and the C-terminal of polypeptide last A amino acid, that is, phenylalanine is modified with biotin, and purity is not less than 95%, is made into the molten of 10mmol/L with preceding plus water Liquid.
Wherein, component (2) is coated with the magnetic bead of Streptavidin, it is preferable that can choose U.S. Thermo The Streptavidin MagneSphere M-280(Dynabeads of Scientific companyTMM-280 Streptavidin), comprising matched Buffer.
Furthermore, it is necessary to provide pH test paper and acid-base accommodation liquid (such as hydrochloric acid and NaOH solution) for oneself.
The master of formaldehyde removing method in application method, that is, waste liquid of formaldehyde removing kit in the waste liquid of polypeptide is carried based on magnetic Want that steps are as follows:
(1) magnetic must be prepared using reagent constituents before carrying out formaldehyde removing and carries polypeptide suspension, preparation process is referring to embodiment 6；
(2) pH in waste liquid is adjusted to 7.0 first with acid-base accommodation liquid and pH test paper, then prepared magnetic is carried into polypeptide suspension It is added in formaldehyde waste liquid, is subsequently added into (NH4)2SO4Solution is to (NH4)2SO4Final concentration of 0.182mol/L, mixing are placed on room Temperature is lower sufficiently to be reacted；
(3) it recycles magnet to adsorb magnetic load polypeptide, pours out processed formaldehyde waste liquid, then with pure water washing magnetic bead 2 times After remove pure water；
(4) polypeptide being carried then according to every 1 μm of ol magnetic, the NaOH solution of 5ml 0.15mol/L and the NaCl of 5ml 0.3mol/L is added Solution is resuspended magnetic bead, and room temperature is placed in ventilation 5min, recycles for example preceding operation removal NaOH solution of magnet and NaCl solution, and Pure water is removed with after pure water washing magnetic bead 2 times；
(5) by treated in (4), magnetic load polypeptide is rejoined in the formaldehyde waste liquid poured out in (3), and pH is adjusted to 7.0 again After add (NH4)2SO4Solution is to (NH4)2SO4Final concentration of 0.182mol/L, mixing postposition carry out sufficiently anti-at room temperature It answers；
(6) step (3)-(5) are repeated until concentration of formaldehyde meets national requirements or actual demand in waste liquid.
Formaldehyde removing and test experience in 11 retail shop's air of embodiment
The business (included developer's finishing) being completed in April, 2018 is object, selects wherein 6 sets of SOHO retail shop (#1.1- 1.3 and #2.1-2.3) carry out formaldehyde removing and detection.Formaldehyde examination selection is embodiment 7(#1.1-1.3) and 8(#2.1- 2.3) kit and method in, in addition simultaneously with PPM-400 ST methylene oxide detecting instrument to detect reference, measurement is averaged three times Value.What formaldehyde removing selected is the kit and method in embodiment 9, in addition simultaneously for active carbon sweep-out method and on the market A using photocatalyst as the formaldehyde scavenger of principle is to remove reference, and specific data are as shown in table 5 below.
#1.1, #1.2 and #1.3 are 21m2With the retail shop of house type difference floor, #2.1, #2.2 and #2.3 are 30m2Same house type The retail shop of different floors is that the present embodiment experiment is immediately begun to after the completion of finishing between 1 at all 6.The removing of formaldehyde schedules to last 3 months, 1 bottle of magnetic was only placed using the method for the present invention every and carries polypeptide removing liquid；Use activated carbon dosage for 0.1kg/m2, point It is placed in the room at 5, and every month, replacement was primary；Use its dosage of photocatalyst scavenger for 0.5 bottle/10m2/ time, in richness It sprays, and is sprayed again every month once containing emphasis at formaldehyde.During used formaldehyde removing method improvement formaldehyde not It is aided with additional ventilation, airtight door window 12h before concentration mensuration.
The detection of 5 new clothes retail shop air formaldehyde of table and removing experimental data mg/m3
Note: difference between the concentration that percentage deviation is detected this refers to the method for the present invention concentration of formaldehyde detected and formaldehyde instrument Absolute value account for the percentage of formaldehyde instrument detectable concentration.
As can be known from Table 5, for #1-6, either before formaldehyde cleaning or after clearing up, either with which kind of method for cleaning, The percentage deviation between concentration of formaldehyde that the concentration of formaldehyde and methylene oxide detecting instrument that the method for the present invention measures measure is respectively less than 5%, Averagely there was only 2.205%.Therefore, it is considered that formaldehyde detection reagent kit and detection method of the invention is accurately and reliably, to have reached special The accuracy of industry methylene oxide detecting instrument, and have many advantages, such as easy economy and high-efficiency environment friendly.In addition, using the present invention is based on magnetic Concentration of formaldehyde after carrying the formaldehyde removing method removing of polypeptide is respectively less than 0.1 mg/m3(referring to the numerical value of #1.1 and 2.1), meets Regulation of the country to formaldehyde safe concentration in building.And the formaldehyde removing method of active carbon and catalyst is used, when identical In, its concentration of formaldehyde is still much higher than 0.1 mg/m after removing3(referring to the numerical value in addition to #1.1 and 2.1), concentration of formaldehyde It is 6.1 times -18.6 times of safe range.Hence it is demonstrated that the present invention is based on formaldehyde removing kit and removing sides that magnetic carries polypeptide Method is efficient, and has many advantages, such as simplicity economical and environmentally friendly.
Formaldehyde removing and test experience in 12 the commercial house air of embodiment
The commercial house (voluntarily fitting up) delivered in September, 2018 is object, and selection wherein carries out for 2 sets with house type house (#1 and #2) The removing and detection of formaldehyde.Formaldehyde examination selection be embodiment 7(#1) and 8(#2) in kit and method, in addition simultaneously It is detection reference with PPM-400 ST methylene oxide detecting instrument, measurement is averaged three times.Formaldehyde removing selection is in embodiment 9 Kit and method, in addition simultaneously using for active carbon sweep-out method and a using photocatalyst as the formaldehyde scavenger of principle on the market To remove reference, specific data are as shown in table 6 below.
#1 and #2 is 120m2It is all 3 bedrooms (#1.1-1.3 and #2.1-2.3, # with the commercial house of house type difference floor 1.1 and 2.1 be 12m2, #1.2 and 2.2 is 15m2, #1.3 and 2.3 is 20m2), it is real to start the present embodiment after the completion of finishing It tests.The removing of formaldehyde 3 months by a definite date, 1 bottle of magnetic was only placed using every room of the method for the present invention and carries polypeptide removing liquid；Using work Property charcoal dosage be 0.1kg/m2, divide at 5 and be placed in every room, and every month, replacement was primary；Using photocatalyst scavenger Its dosage is 0.5 bottle/10m2/ time, it is sprayed rich in emphasis at formaldehyde, and spray again every month once.Used first Aldehyde sweep-out method is normally divulged information during administering formaldehyde, airtight door window 12h before concentration mensuration.
The detection of 6 new clothes the commercial house air formaldehyde of table and removing experimental data mg/m3
Note: difference between the concentration that percentage deviation is detected this refers to the method for the present invention concentration of formaldehyde detected and formaldehyde instrument Absolute value account for the percentage of formaldehyde instrument detectable concentration.
As can be known from Table 6, for #1.1-1.3 and #2.1-2.3, either before formaldehyde cleaning or after clearing up, either The percentage between concentration of formaldehyde that the concentration of formaldehyde measured with which kind of method for cleaning, the method for the present invention and formaldehyde instrument measure is inclined Difference is respectively less than 5%, averagely only 2.76%.Therefore, it is considered that formaldehyde detection reagent kit and detection method of the invention is accurate and reliable , reach the accuracy of specialized methylene oxide detecting instrument, and have many advantages, such as easy economy and high-efficiency environment friendly.In addition, using Concentration of formaldehyde after removing the present invention is based on the formaldehyde removing method that magnetic carries polypeptide is respectively less than 0.08 mg/m3(referring to #1.3 and # 2.3 numerical value), meet country to the regulation for occupying indoor formaldehyde safe concentration.And use the formaldehyde removing side of active carbon and catalyst Method, interior at the same time, its concentration of formaldehyde is still much higher than 0.1 mg/m after removing3(referring to the number in addition to #1.3 and 2.3 Value), concentration of formaldehyde is 2.1 times -3.1 times of safe range.Hence it is demonstrated that the present invention is based on the formaldehyde removings that magnetic carries polypeptide Kit and sweep-out method are efficient, and have many advantages, such as simplicity economical and environmentally friendly.
The removing and test experience of formaldehyde in 13 formaldehyde waste liquid of embodiment
Two kinds of formaldehyde waste liquid #1 and #2, the every part of sample 1L for taking laboratory, according in embodiment 7 and 8 kit and method survey Its fixed concentration of formaldehyde, in addition simultaneously with PPM-400 ST methylene oxide detecting instrument to detect reference, measurement is averaged three times.According to survey Content of formaldehyde in fixed sample is added magnetic and carries polypeptide, and the magnetic load polypeptide work liquid measure of the often addition of formaldehyde containing 1g is in sample 10ml.According in 10 waste liquid of embodiment formaldehyde removing kit and method carry out the removing of formaldehyde, wherein the every 5h of peptide T 1 is more New primary (according to method in embodiment 10, non-replaceable), the every 3h of peptide T 2 update primary (homopolypeptide T1 explanation), and every time more New magnetic carries the concentration of monitor sample formaldehyde before polypeptide；In addition, taking parallel sample (#1.1 and #1.2 and #2.1 and #2.2) with work Property charcoal compare as removing, often the active carbon of 25g is added in formaldehyde containing 1g in sample, and replaces an active carbon every 3h, real It is as shown in table 7 to test data.
As can be known from Table 7, for #1 and #2, either before formaldehyde cleaning or after clearing up, either with which kind of cleaning side The percentage deviation between concentration of formaldehyde that method, the concentration of formaldehyde that the method for the present invention measures and formaldehyde instrument measure is respectively less than 5%, Averagely there was only 1.09%.Therefore, it is considered that Analysis Methods for Formaldehyde of the invention be it is accurate and reliable, reached specialized formaldehyde examination The accuracy of instrument.It is 1.33g/L for waste liquid #1(initial amount concentration), it removes and (updates 2 times, when in order to save through peptide T 1 Between every time update be spaced selection be peptide T 1 saturation binding time i.e. 5h, amount to 15h；Updating interval selection, effect is more for 24 hours It is good), the concentration of formaldehyde after removing in waste liquid #1 has decreased to 0.04g/L, and down ratio is up to 96.99%, has good removing Effect.For waste liquid #2(initial amount concentration be 2.53g/L), through peptide T 2 remove (update 4 times, in order to save time every time That update is spaced selection is the saturation binding time i.e. 3h of peptide T 2, amounts to 12h；Update interval selection better effect for 24 hours), clearly Except the concentration of formaldehyde in rear waste liquid #2 has decreased to 0.07g/L, down ratio is up to 97.23%, has good elimination effect. In general, the scavenger reagent box and sweep-out method for formaldehyde in waste liquid provided by the invention based on peptide T 1 and T2, can The waste liquid of serious formaldehyde pollution (being greater than 1.0g/L) to be purged in the short time (in 1d), clearance rate reaches 95% or more. And under equal conditions, big carbon content active is consumed, clearance rate but only has 30% or so.Therefore in waste liquid provided by the invention Formaldehyde removing kit and sweep-out method are efficient, and have many advantages, such as simplicity economical and environmentally friendly.
The removing of formaldehyde and test experience data g/L in 7 formaldehyde waste liquid of table
Sequence table
<110>Foochow four is recorded up to biomedical Science and Technology Ltd.
<120>formaldehyde detection reagent kit and its detection method
<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 (4)

1. a kind of novel formaldehyde detection reagent kit, it is characterised in that the Main Components of kit include: 1 freeze-dried powder of (1) peptide T Agent, the sequence of peptide T 1 is as shown in SEQ ID NO:1, and the C-terminal of polypeptide the last one amino acid, that is, phenylalanine biology Element is modified, and is synthesized by biotech firm, and purity is not less than 95%, and the solution of 10mmol/L is made into preceding plus pure water；(2) it wraps There is the magnetic bead of Streptavidin；(3) (NH4)2SO4Solution；(4) magnet.
2. formaldehyde detection reagent kit according to claim 1, it is characterised in that the component (2) of kit is coated with strepto- That the magnetic bead of Avidin selects is the Streptavidin MagneSphere Dynabeads of Thermo Scientific company, the U.S.TM M-280 Streptavidin includes matched buffer.
3. application method, that is, Analysis Methods for Formaldehyde of formaldehyde detection reagent kit as stated in claim 1 or 2, it is characterised in that The key step of Analysis Methods for Formaldehyde is as follows:
(1) magnetic must be prepared using kit each component before carrying out formaldehyde and combining carry polypeptide suspension, main preparation process are as follows: a. It takes 0.1ml Streptavidin to carry out the preparation of magnetic bead according to product description, is loaded with the centrifuge tube of 15ml, using magnetic bead product In carry buffer, finally obtain 10ml work liquid status under magnetic bead liquid；B. 10mmol/ is added in the magnetic bead liquid into a. The 1 solution 0.1ml of peptide T of L keeps mixing well 15min at room temperature；C. by magnet and slow with what is carried in magnetic bead product Fliud flushing washing has been coated with magnetic bead 3 times of polypeptide, removes unbonded material, pure water 10ml is added later to get to magnetic and carries polypeptide Suspension can be placed in -20 degree environment and save for a long time for use；
(2) magnetic is carried polypeptide suspension to be added in 0.1ml formaldehyde sample to be measured, adds (NH4)2SO4Solution is to (NH4)2SO4It is dense eventually Degree is 0.182mol/L, mixes postposition and is combined reaction 10min at room temperature, during which mixes again no less than 2 times；
(3) magnetic is carried into polypeptide using magnet and formaldehyde conjugate is adsorbed on tube wall, reaction system is sucked out；
(4) 1ml pure water is added and mixes elution 1 time, adsorb conjugate again, then clear water eluent is sucked out；
(5) mixing of 1ml pure water is added, takes 3ul magnetic to carry the aqueous solution of polypeptide and formaldehyde conjugate, is existed using microplate reader Absorbance is measured under 446nm wavelength；
(6) formaldehyde binding capacity can be calculated by the absorbance x of measurement being substituted into formula y=45.716x-0.7713, if formaldehyde Binding capacity is more than 80mg/ μm of ol, then suitably dilutes formalin to be measured and again measurement until being lower than the numerical value, further change It is counted as concentration of formaldehyde or content of formaldehyde.
4. Analysis Methods for Formaldehyde according to claim 3, it is characterised in that test sample in Analysis Methods for Formaldehyde step (2) This is that solution then directly measures content of formaldehyde therein；If sample to be tested is air, referring in GB/T 18204.26-2000 Sampling operation carry out the absorption of formaldehyde gas using larger bubbles absorption tube and constant flow sampler and be dissolved into solution, then row first Aldehyde measurement.
CN201910738174.0A 2019-08-12 2019-08-12 Formaldehyde detection reagent kit and its detection method Pending CN110441529A (en)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
US20020119507A1 (en) * 2000-12-05 2002-08-29 Takahide Kishimoto Determination method of biological component and reagent kit used therefor
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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Publication number Priority date Publication date Assignee Title
US20020119507A1 (en) * 2000-12-05 2002-08-29 Takahide Kishimoto Determination method of biological component and reagent kit used therefor
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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吴明松 等: "室内空气中甲醛去除及评价方法研究进展", 《二氧化氯研究与应用进展——二〇一五二氧化氯与水处理技术研讨会暨二氧化氯专业委员会十周年纪念年会论文集》 *
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