A method for detecting formaldehyde based on gold nanoparticles and hybridization chain reaction amplification

Abstract

The present invention discloses a method for detecting formaldehyde based on gold nanoparticles and hybridization chain reaction amplification, comprising the following steps: modifying the Oligo 1 probe sequence on the surface of gold nanoparticles to obtain a gold nanoparticle probe; mixing the gold nanoparticle probe, a sample to be tested, an HP sequence, an H1 sequence, an H2 sequence, and silver ions to undergo a hybridization chain reaction, and quantifying the formaldehyde content in the sample to be tested according to the RGB value of the color of the mixed solution after the reaction; or using a dynamic light scattering method to measure the particle size of the gold nanoparticle probe in the mixed solution after the reaction to quantify the formaldehyde content in the sample to be tested. The detection method of the present invention is sensitive and reliable, does not require a complicated operation process, can achieve on-site detection in a relatively short time, has strong selectivity and high sensitivity, and the detection limit is only 0.02 mg·L ‑1 and 0.01mg·L ‑1 , which has extremely high application value.

Description

Technical Field

[0001] The invention belongs to the field of molecular detection, and in particular relates to a method for detecting formaldehyde based on gold nanoparticles and hybridization chain reaction amplification. Background Art

[0002] In recent years, smartphone RGB colorimetric analysis has been widely used in the analytical detection industry. Its advantages are that the response of the analyte can be read directly and quickly with the naked eye, and the values ​​of the R, G, and B channels can be captured through smartphone applications, so that the concentration information of the analyte can be obtained by analyzing the obtained channel values. Therefore, to a certain extent, the application of smartphones to capture RGB can replace high-cost instrument technology, and the RGB colorimetric analysis can reduce the consumption of reagents and samples, achieving the purpose of simple, portable, visual and rapid analysis. This also makes the smartphone-based RGB colorimetric analysis have great application prospects in analytical detection.

[0003] Dynamic light scattering has been increasingly used in recent years. Dynamic light scattering is a non-invasive way to measure the size of particles, which is usually used as a material characterization method. Dynamic light scattering can be used to analyze the particle size and particle size distribution of nanoparticles, proteins and polymers. However, in related technologies, dynamic light scattering has not been used in conjunction with colorimetric analysis and other methods in actual sample detection, but is more of a characterization method. Therefore, it has greatly limited the further development and development of related detection methods.

[0004] Gold nanoparticles are a type of functional nanoparticle with great potential. Since their discovery, their unique physical and optical properties have attracted widespread attention. The degree of aggregation of gold nanoparticles under different conditions determines its color change. Therefore, based on this feature, they are widely used as probes for diagnosis and sensing.

[0005] Formaldehyde is the simplest aldehyde substance and has long been considered a carcinogenic pollutant that is harmful to the human body. Even continuous exposure to very low concentrations of formaldehyde can cause eye irritation and a variety of diseases, such as allergies, cancer, cerebral infarction, headaches and neurological disorders. However, in real life, adhesives and paints are used in the production of furniture, and adhesives and paints generally contain formaldehyde, which leads to a large amount of formaldehyde remaining after interior decoration. According to the World Health Organization's guidelines on residential indoor air quality, the formaldehyde exposure limit is defined as 0.1mg m -3The International Agency for Research on Cancer has classified formaldehyde as a Class I carcinogen based on its carcinogenicity studies on humans and animals. At present, although there are a variety of formaldehyde detection methods such as high performance liquid chromatography, gas chromatography, fluorescence, and quartz crystal microbalance, these methods all have their own defects, such as limited sensitivity and selectivity, over-reliance on specific instruments, unstable detection substrates, or limitations due to the use of organic solvents or concentrated acids, etc. Therefore, it is necessary and meaningful to develop a new and effective method for detecting formaldehyde in the air. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a formaldehyde detection method based on gold nanoparticles and hybridization chain reaction. The method in the present invention improves the sensitivity of the method based on a specially designed amplification technology (hybridization chain reaction), and can increase the reliability of the method based on dual signal detection (RGB colorimetric detection and dynamic light scattering detection), thereby realizing trace monitoring of formaldehyde.

[0007] In a first aspect, the present invention provides a method for detecting formaldehyde based on gold nanoparticles and hybridization chain reaction amplification, comprising the following steps:

[0008] (1) Modifying the Oligo 1 probe sequence on the surface of gold nanoparticles to obtain gold nanoparticle probes;

[0009] (2) The gold nanoparticle probe, the sample to be tested, the HP sequence, the H1 sequence, the H2 sequence, and the silver ions are mixed to cause a hybridization chain reaction, and the formaldehyde content in the sample to be tested is quantified according to the RGB value of the color of the mixed solution after the reaction; or the particle size of the gold nanoparticle probe in the mixed solution after the reaction is determined by a dynamic light scattering method to quantify the formaldehyde content in the sample to be tested.

[0010] According to the first aspect of the present invention, in some embodiments of the present invention, the Oligo 1 probe sequence is an oligonucleotide sequence.

[0011] In some embodiments of the present invention, the Oligo 1 probe sequence contains 5-1000 thymine sequences and is connected to a linker group at the 5' or 3' end.

[0012] In some embodiments of the present invention, the linking group is a thiol group (SH-C6-).

[0013] In some embodiments of the present invention, the Oligo 1 probe sequence is The 5' end is modified with a thiol group.

[0014] The underlined bold part is the nucleic acid sequence that is complementary to the H1 sequence and the H2 sequence.

[0015] In some embodiments of the present invention, the HP sequence is a nucleotide sequence that can undergo specific recognition enhancement with silver ions and form a hairpin structure, and the non-hairpin HP sequence can open the hairpin H1 sequence to expose its sticky end.

[0016] In some embodiments of the present invention, the HP sequence is: 5'-TACGCCATCAGCTCCAACCTACCGCAGCAGACTAGC-3'.

[0017] In some embodiments of the present invention, the H1 sequence is a nucleotide sequence that can form a hairpin structure, the sticky end on the H1 sequence can open the hairpin structure of the H2 sequence, exposing the sticky end of the H2 sequence, and there is a fragment in the H1 sequence that is complementary to the Oligo 1 probe sequence.

[0018] In some embodiments of the present invention, the H1 sequence is: 5'-GGAGCTGATGGCGTACATAGTTACGCCATCAAAAAAAAAAAA-3'.

[0019] In some embodiments of the present invention, the H2 sequence is a nucleotide sequence that can form a hairpin structure, the sticky end on the H2 sequence can open the hairpin structure of the H1 sequence, exposing the sticky end of the H1 sequence, and there is a fragment in the H2 sequence that is complementary to the Oligo 1 probe sequence.

[0020] In some embodiments of the present invention, the H2 sequence is: 5'-TACGCCATCAGCTCCGATGGCGTAACTATGAAAAAAAAAAAA-3'.

[0021] In some embodiments of the present invention, the gold nanoparticles can be commercially available gold nanoparticles or prepared by conventional methods in the art, such as sodium citrate reduction method.

[0022] Gold nanoparticles (AuNPs) are an important component of nanomaterials. The inventors found that the dispersion / aggregation state of AuNPs modified with DNA will change significantly, and its absorbance, color and particle size will also change accordingly. Therefore, AuNPs probe can be used as an ideal colorimetric, RGB and dynamic light scattering signal probe to display the amount of formaldehyde.

[0023] In some embodiments of the present invention, the gold nanoparticles are prepared by: adding 2 mL of freshly prepared 38.8 mmol L-1 Sodium citrate solution was quickly added to 20 mL of boiling 1.0 mmol L -1 In chloroauric acid solution. At this time, the reaction solution changes from light yellow to black, then to purple, and finally to wine red. After turning into wine red, continue to heat and reflux, and stir for 20 to 60 minutes. Cool the reaction solution to room temperature (keep stirring), and then filter with a 0.1 to 5.0 μm nylon filter membrane to obtain.

[0024] In some embodiments of the present invention, the silver ion is selected from at least one of silver nitrate and Ag(NH3)2OH.

[0025] In some embodiments of the present invention, the silver ions are selected from silver nitrate.

[0026] In the present invention, the inventors use HCR and DLS to detect formaldehyde, wherein each sequence has a special structural design, such as the need to introduce Ag + The principle of promoting CC base pair mismatch. In the present invention, Ag + Ag can be bound between the N3 nitrogen atoms of cytosine in the CC base pair, and within the pH range of the present application (according to the inventor's research, it is found that the pH range of the detection system in the present invention can be stable at 5 to 9, and there is no situation of being too low or too high). + The presence of α significantly stabilizes the CC mismatch double helix, and the CC base pair mismatch binds Ag + High specificity (Ag + Mainly used to promote the stability of the CGC tribasic body). This characteristic, combined with the probe and hairpin primer design in this application, can be used to change the structure of the HCR initiation chain, so that the initiation chain of the original structure forms a hairpin structure, thereby affecting the occurrence of HCR. The occurrence of HCR directly affects the dispersion of gold nanoparticles, which is then reflected in the visual detection results.

[0027] In some embodiments of the present invention, the specific steps of the detection method are:

[0028] (1) Preparation of gold nanoparticle probes:

[0029] The Oligo 1 probe sequence is treated with TCEP, and then the Oligo 1 probe sequence treated with TCEP is mixed with AuNPs at a molar ratio of Oligo 1 probe sequence: AuNPs of 180 to 220:1 and incubated for 1 to 100 hours, and NaCl with a final concentration of 0.01 to 1.0 M is added for salt aging treatment to obtain a gold nanoparticle probe.

[0030] Au-S, Au-N bonds are used to modify nucleic acid molecules, proteins, antibodies, etc. on the surface of gold nanoparticles to form probes, so that the gold nanoparticles maintain specificity, dispersibility and stability and can specifically detect various substances. In the present invention, the nucleic acid molecule modified on the surface of the gold nanoparticles is single-stranded DNA, which can hybridize with complementary chains, has extremely high efficiency and good specificity, and can easily detect complementary chain DNA or RNA.

[0031] (2) Mix according to the following system:

[0032] Components Dosage <![CDATA[1.0μmol L -1 HP sequence]]> 4μL <![CDATA[10μmol L -1 H1 sequence]]> 2μL <![CDATA[10μmol L -1 H2 sequence]]> 2μL <![CDATA[62μmol L -1 Silver ion solution]]> 4μL Formaldehyde samples 4μL <![CDATA[10mmol L -1 PBS buffer (pH = 7.0)]]> 28μL <![CDATA[33nmol L -1 Gold Nanoparticle Probes]]> 6μL

[0033] Use an imaging device to take a picture of the system after the reaction, identify the RGB value of the picture (R value in this embodiment), and achieve quantitative detection of formaldehyde based on the linear relationship between the RGB value and the formaldehyde concentration (using a formaldehyde standard to determine the standard curve);

[0034] Alternatively, a dynamic light scattering device is used to detect the gold nanoparticles in the system to obtain its dynamic light scattering signal, and quantitative detection of formaldehyde is achieved based on the linear relationship between the dynamic light scattering signal and the formaldehyde concentration (using a formaldehyde standard to determine the standard curve).

[0035] In some embodiments of the present invention, the RGB value signal output may be an R value, a G value, a B value, an RGB total value, an RGB average value, or an RGB ratio (R / G, R / B, or G / B).

[0036] In some embodiments of the present invention, the dynamic light scattering signal value output may be an average particle size value or an average particle size ratio value.

[0037] According to the inventor's experiments, in the system of the present invention, when the formaldehyde concentration increases from small to large, the R value of the system varies from 80 to 170, and the average particle size varies from 50 to 450.

[0038] In some embodiments of the present invention, the imaging device includes at least one of a smartphone, a still camera, a video camera, a microscope lens, and a webcam.

[0039] In some embodiments of the present invention, the imaging device is a smartphone.

[0040] In some embodiments of the present invention, the specific photographing steps are: fix the mobile phone with a bracket so that the camera is about 10 cm away from the solution, set the built-in camera of the mobile phone to turn off the flash and automatic white balance, set the exposure time to 0.01s, adjust the light source in the shooting parameters to incandescent lamp mode, and take a picture of the sample.

[0041] In some embodiments of the present invention, the RGB value recognition operation is specifically: using a color identifier to read the RGB value of the photo, and realizing quantitative detection of formaldehyde according to the linear relationship between the RGB value and the formaldehyde concentration.

[0042] In some embodiments of the present invention, specific parameters of the dynamic light scattering device are: test temperature is 25° C., equilibrium time is 60 s, measurement angle is 90°, and single measurement time is 60 s.

[0043] In some embodiments of the present invention, the specific detection operation of the dynamic light scattering device is: add 50 μL of the sample to be tested (the system after the reaction) into a 50 μL plastic sample pool, and then perform dynamic light scattering measurement according to the above parameters to obtain a signal value, and realize quantitative detection of formaldehyde based on the linear relationship between the dynamic light scattering signal and the formaldehyde concentration (using the formaldehyde standard to determine the standard curve).

[0044] The detection principle of the method for detecting formaldehyde based on gold nanoparticles and hybridization chain reaction amplification in the present invention is as shown in the accompanying drawings of the specification. Figure 1 As shown, specifically:

[0045] When formaldehyde is not present in the system, the substrate chain HP and silver ions in the system will preferentially form a hairpin structure, so that the substrate chain HP will not further trigger the opening of the hairpin structure in the H1 sequence and the H2 sequence, and thus no hybridization chain reaction will occur. At this time, AuNPs-oligo 1 (gold nanoparticle probe) can only bind to the H1 sequence and the H2 sequence alone, and cannot form an aggregated state, and the corresponding solution color is red.

[0046] When formaldehyde is present in the system, the addition of formaldehyde can quickly consume the silver ions in the system, thereby liberating the substrate chain HP, causing the hairpin structure HP to unwind, and then triggering a hybridization chain reaction between H1 and H2 to form a double-stranded hybridization chain reaction product with a long gap structure. At this time, AuNPs-oligo 1 in the system combines with the hybridization chain reaction product to form an aggregated state, and the corresponding color changes to purple-red. After color recognition through smartphone software, the actual formaldehyde concentration in the system can be calculated based on the linear relationship between the RGB value and the formaldehyde concentration. Alternatively, the particle size can be measured using dynamic light scattering, and the formaldehyde concentration can also be obtained based on the change in particle size.

[0047] Through the hybridization chain reaction, it is very easy to obtain a long single-stranded gapped double-stranded DNA. The single-stranded DNA modified on the surface of the gold nanoparticles can hybridize with the single strand on the gapped double-stranded DNA, causing the gold nanoparticles to aggregate and the color to change from red to purple. Moreover, unlike the polymerase chain reaction, the hybridization chain reaction requires a thermal cycle and a heat-resistant DNA polymerase. It is a simple isothermal enzyme-free reaction process. The hybridization chain reaction is carried out in a constant temperature solution or solid support, and has the advantages of simplicity, strong versatility, high energy efficiency, low cost, high sensitivity and rapid response. However, the formation of a hybridization chain reaction also requires the initiator chain to maintain its original structure. When the original structure of the initiator chain is destroyed, the hybridization chain reaction cannot be triggered.

[0048] Colorimetric analysis combined with dynamic light scattering analysis is one of the core of the present invention. The combination of the two can not only sensitively detect the particle size, distribution and color change of the sample, but the method of the present invention is mainly based on particle size, distribution and color change to reflect the concentration difference. Therefore, it can be well adapted to the method of the present invention to achieve efficient detection of formaldehyde.

[0049] A second aspect of the present invention provides a formaldehyde detection product, which includes an Oligo 1 probe sequence, an HP sequence, an H1 sequence and an H2 sequence.

[0050] In some embodiments of the present invention, the Oligo 1 probe sequence is an oligonucleotide sequence.

[0051] In some embodiments of the present invention, the Oligo 1 probe sequence contains 5-1000 thymine sequences and is connected to a linker group at the 5' or 3' end.

[0052] In some embodiments of the present invention, the linking group is a thiol group (SH-C6-).

[0053] In some embodiments of the present invention, the Oligo 1 probe sequence is The 5' end is modified with a thiol group.

[0054] The underlined bold part is the nucleic acid sequence that is complementary to the H1 sequence and the H2 sequence.

[0055] In some embodiments of the present invention, the HP sequence is a nucleotide sequence that can undergo specific recognition enhancement with silver ions and form a hairpin structure, and the non-hairpin HP sequence can open the hairpin H1 sequence to expose its sticky end.

[0056] In some embodiments of the present invention, the HP sequence is: 5'-TACGCCATCAGCTCCAACCTACCGCAGCAGACTAGC-3'.

[0057] In some embodiments of the present invention, the H1 sequence is a nucleotide sequence that can form a hairpin structure, the sticky end on the H1 sequence can open the hairpin structure of the H2 sequence, exposing the sticky end of the H2 sequence, and there is a fragment in the H1 sequence that is complementary to the Oligo 1 probe sequence.

[0058] In some embodiments of the present invention, the H1 sequence is: 5'-GGAGCTGATGGCGTACATAGTTACGCCATCAAAAAAAAAAAA-3'.

[0059] In some embodiments of the present invention, the H2 sequence is a nucleotide sequence that can form a hairpin structure, the sticky end on the H2 sequence can open the hairpin structure of the H1 sequence, exposing the sticky end of the H1 sequence, and there is a fragment in the H2 sequence that is complementary to the Oligo 1 probe sequence.

[0060] In some embodiments of the present invention, the H2 sequence is: 5'-TACGCCATCAGCTCCGATGGCGTAACTATGAAAAAAAAAAAA-3'.

[0061] In some embodiments of the present invention, the formaldehyde detection product also includes gold nanoparticles (AuNPs) and silver ions.

[0062] In some embodiments of the present invention, the gold nanoparticles can be commercially available gold nanoparticles or prepared by conventional methods in the art, such as sodium citrate reduction method.

[0063] In some embodiments of the present invention, the silver ion is selected from at least one of silver nitrate and Ag(NH3)2OH.

[0064] In some embodiments of the present invention, the silver ions are selected from silver nitrate.

[0065] In some embodiments of the present invention, the product includes a detection reagent, a detection kit, and a detection chip.

[0066] A third aspect of the present invention provides a formaldehyde detection device, which includes a detection module, an RGB colorimetric device and / or a dynamic light scattering device.

[0067] In some embodiments of the present invention, the detection module includes an Oligo 1 probe sequence, an HP sequence, an H1 sequence, and an H2 sequence, and the detection module is used to perform detection of the detection sample and output a post-detection product.

[0068] In some embodiments of the present invention, the Oligo 1 probe sequence is an oligonucleotide sequence.

[0069] In some embodiments of the present invention, the Oligo 1 probe sequence contains 5-1000 thymine sequences and is connected to a linker group at the 5' or 3' end.

[0070] In some embodiments of the present invention, the linking group is a thiol group (SH-C6-).

[0071] In some embodiments of the present invention, the Oligo 1 probe sequence is The 5' end is modified with a thiol group.

[0072] The underlined bold part is the nucleic acid sequence that is complementary to the H1 sequence and the H2 sequence.

[0073] In some embodiments of the present invention, the HP sequence is a nucleotide sequence that can undergo specific recognition enhancement with silver ions and form a hairpin structure, and the non-hairpin HP sequence can open the hairpin H1 sequence to expose its sticky end.

[0074] In some embodiments of the present invention, the HP sequence is: 5'-TACGCCATCAGCTCCAACCTACCGCAGCAGACTAGC-3'.

[0075] In some embodiments of the present invention, the H1 sequence is a nucleotide sequence that can form a hairpin structure, the sticky end on the H1 sequence can open the hairpin structure of the H2 sequence, exposing the sticky end of the H2 sequence, and there is a fragment in the H1 sequence that is complementary to the Oligo 1 probe sequence.

[0076] In some embodiments of the present invention, the H1 sequence is: 5'-GGAGCTGATGGCGTACATAGTTACGCCATCAAAAAAAAAAAA-3'.

[0077] In some embodiments of the present invention, the H2 sequence is a nucleotide sequence that can form a hairpin structure, the sticky end on the H2 sequence can open the hairpin structure of the H1 sequence, exposing the sticky end of the H1 sequence, and there is a fragment in the H2 sequence that is complementary to the Oligo 1 probe sequence.

[0078] In some embodiments of the present invention, the H2 sequence is: 5'-TACGCCATCAGCTCCGATGGCGTAACTATGAAAAAAAAAAAA-3'.

[0079] In some embodiments of the present invention, the detection module further includes gold nanoparticles (AuNPs) and silver ions.

[0080] In some embodiments of the present invention, the gold nanoparticles can be commercially available gold nanoparticles or prepared by conventional methods in the art, such as sodium citrate reduction method.

[0081] In some embodiments of the present invention, the silver ion is selected from at least one of silver nitrate and Ag(NH3)2OH.

[0082] In some embodiments of the present invention, the silver ions are selected from silver nitrate.

[0083] In some embodiments of the present invention, the RGB colorimetric device and / or the dynamic light scattering device performs formaldehyde quantification based on the post-detection product.

[0084] In some embodiments of the present invention, the RGB colorimetric device obtains RGB values ​​based on the recorded solution color, and calculates the formaldehyde concentration according to the RGB values.

[0085] In some embodiments of the present invention, the dynamic light scattering device is used to detect the dynamic light scattering signal in the solution, thereby obtaining the average particle size of the gold nanoparticles and calculating the formaldehyde concentration.

[0086] In some embodiments of the present invention, the formaldehyde detection device further comprises an imaging module according to the actual detection method used. The imaging module is used to record the color of the solution.

[0087] In some embodiments of the present invention, the color recognition function is implemented based on an RGB color detector.

[0088] In some embodiments of the present invention, the RGB color detector is composed of a mobile phone containing a charge coupled device image sensor CCD (or complementary metal oxide semiconductor CMOS), or other detectors containing RGB three-primary color sensors.

[0089] The fourth aspect of the present invention provides the use of the formaldehyde detection product described in the second aspect of the present invention or the formaldehyde detection device described in the third aspect of the present invention in the detection of formaldehyde residues in food and environment.

[0090] In some preferred embodiments of the present invention, the food comprises fresh fruits and vegetables.

[0091] In some preferred embodiments of the present invention, the food is green vegetables.

[0092] In some preferred embodiments of the present invention, the environment includes a working environment and a living environment that are easily polluted by formaldehyde, including but not limited to home decoration and outdoor painting.

[0093] The beneficial effects of the present invention are:

[0094] 1. The detection method of the present invention is sensitive, reliable, does not require complicated operation procedures, can achieve on-site detection in a short time, and its cost and efficiency far exceed other traditional methods.

[0095] 2. The detection method of the present invention requires less sample and has good selectivity. It can effectively eliminate the interference of other structural analogs such as acetaldehyde, methanol, ethanol, acetone, ether, acetic acid, ammonia and toluene. It can accurately detect the content of formaldehyde in the linear range, and the detection limit is 0.02 mg·L -1 and 0.01 mg·L -1 , with extremely high detection sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figure 1 This is a detection principle diagram of a method for detecting formaldehyde based on gold nanoparticles and hybridization chain reaction amplification in an embodiment of the present invention.

[0097] Figure 2 1 is the selective detection result of the formaldehyde detection method in the embodiment of the present invention, and the samples are: formaldehyde (HCHO), water (control, Blank), acetaldehyde (CH3CHO), methanol (CH3OH), ethanol (CH3CH2OH), acetone (CH3COCH3), ether (C2H5OC2H5), acetic acid (CH3COOH), ammonia water (NH3·H2O) and toluene (C6H5CH3).

[0098] Figure 3 is the standard curve of the formaldehyde standard solution obtained by the formaldehyde RGB colorimetric detection method in the embodiment of the present invention, wherein A is 0 mg L -1 ~45mg L -1 Curve in the range, B is 0 mg L -1 ~4mg L -1 Curve within the range.

[0099] Figure 4 It is a standard curve of formaldehyde standard solution obtained by the formaldehyde dynamic light detection method in the embodiment of the present invention.

[0100] Figure 5Photo (A) and UV-visible absorption spectrum (B) to verify the accuracy of the formaldehyde detection method in the embodiment of the present invention, where a is the formaldehyde concentration of 0 mg L -1 Figure 2, b is the formaldehyde concentration of 4.0 mg L -1 .

[0101] Figure 6 The particle size distribution diagram of the dynamic light scattering method for verifying the accuracy of the formaldehyde detection method in the embodiment of the present invention, wherein A is the formaldehyde concentration of 0 mg L -1 Figure 1, B is the formaldehyde concentration of 4.0 mg L -1 .

[0102] Figure 7 The particle size distribution diagram of the transmission electron microscope to verify the accuracy of the formaldehyde detection method in the embodiment of the present invention, A is the formaldehyde concentration of 0mg L -1 Figure 1, B is the formaldehyde concentration of 4.0 mg L -1 . DETAILED DESCRIPTION

[0103] In order to make the invention purpose, technical solution and technical effect of the present invention clearer, the present invention is further described in detail below in conjunction with specific implementation methods. It should be understood that the specific implementation methods described in this specification are only for explaining the present invention, not for limiting the present invention.

[0104] Unless otherwise specified, the experimental materials and reagents used are consumables and reagents that can be obtained from conventional commercial channels.

[0105] A method for detecting formaldehyde based on gold nanoparticles and hybridization chain reaction amplification

[0106] In this embodiment, the method for detecting formaldehyde based on gold nanoparticles and hybridization chain reaction amplification includes the following steps:

[0107] (1) Preparation of gold nanoparticles:

[0108] In this embodiment, gold nanoparticles are prepared according to the adjusted sodium citrate reduction method, and the specific preparation method is as follows:

[0109] 2 mL of freshly prepared 38.8 mmol L -1 Sodium citrate solution was quickly added to 20 mL of boiling 1.0 mmol L -1 In chloroauric acid solution. At this time, the reaction solution changes from light yellow to black, then to purple, and finally to wine red. After turning into wine red, continue to heat and reflux, and stir for 40 minutes. Cool the reaction solution to room temperature. Filter with a 3μm nylon filter membrane to obtain gold nanoparticles, and store at 4℃ for later use.

[0110] (2) Preparation of gold nanoparticle probes:

[0111] Take the gold nanoparticles prepared in step (1) and modify the Oligo 1 probe sequence on the surface to obtain the gold nanoparticle probe. Of course, commercially available gold nanoparticles can also be directly used to replace the gold nanoparticles prepared in step (1).

[0112] The specific steps are:

[0113] a. Construction of Oligo 1 probe sequence:

[0114] The Oligo 1 probe sequence is an oligonucleotide sequence, and its construction rule is: containing 5-1000 thymine sequences, and the Oligo 1 probe sequence has a complementary pairing relationship with the H1 sequence and the H2 sequence, and the complementary sequence part is: 5'-TTTTTTTTTTTT-3' (SEQ ID NO. 1). The 5' or 3' end of the Oligo 1 probe sequence is connected to a thiol group.

[0115] In this example, the Oligo 1 probe sequence is: Among them, the underlined bold part is the complementary sequence part.

[0116] b Modification of gold nanoparticles (AuNPs):

[0117] The Oligo 1 probe shown in SEQ ID NO.2 was treated with TCEP (i.e., TCEP-HCl, full name Tris(2-carboxyethyl)phosphine hydrochloride, Chinese name Tris(2-carboxyethyl)phosphine hydrochloride). Then, the Oligo 1 probe treated with TCEP was mixed with AuNPs at a molar ratio of 200:1 for Oligo 1 probe: AuNPs, and incubated at room temperature for 50 hours. Then, during the incubation process, NaCl was added in small amounts multiple times at irregular intervals for salt aging treatment, and the final concentration of NaCl was controlled to be 0.0.5 mol L -1 After the incubation, the mixed solution was centrifuged at 15000 rpm for 40 minutes to fully remove the free Oligo 1 probe. The centrifugation operation was repeated 3 times. The oily precipitate obtained was the gold nanoparticles modified with the Oligo 1 probe (named as gold nanoparticle probe). The obtained gold nanoparticle probe was dissolved in 10 mmol L -1 PBS buffer (pH = 7.4, NaCl = 0.1 mol L -1 ), and stored at 4℃ for later use.

[0118] (3) Sample testing:

[0119] Mix according to the detection system shown in Table 1 (a total of 50 μL of the detection system).

[0120] Table 1 Detection system

[0121] Components Dosage <![CDATA[1.0μmol L -1 HP sequence]]> 4μL <![CDATA[10μmol L -1 H1 sequence]]> 2μL <![CDATA[10μmol L -1 H2 sequence]]> 2μL <![CDATA[62μmol L -1 Silver ion solution]]> 4μL Formaldehyde samples 4μL <![CDATA[10mmol L -1 PBS buffer (pH = 7.0)]]> 28μL <![CDATA[33nmol L -1 Gold Nanoparticle Probes]]> 6μL

[0122] Among them, the HP sequence is a nucleotide sequence that can undergo specific recognition and enhancement with silver ions and form a hairpin structure, and when HP is not in a hairpin structure, it can open the H1 sequence of the hairpin structure and expose the sticky end of the H1 sequence.

[0123] In this example, the HP sequence is: 5'-TACGCCATCAGCTCCAACCTACCGCAGCAGACTAGC-3' (SEQ ID NO. 3).

[0124] The H1 sequence is a nucleotide sequence that can form a hairpin structure, and the sticky end of the H1 sequence can open the hairpin structure of the H2 sequence and expose the sticky end of the H2 sequence, and some sequence fragments in the H1 sequence can complementarily pair with the Oligo 1 probe sequence.

[0125] In this example, the H1 sequence is: 5'-GGAGCTGATGGCGTACATAGTTACGCCATCAAAAAA AAAAAA-3' (SEQ ID NO. 4).

[0126] The H2 sequence is a nucleotide sequence that can form a hairpin structure, and the sticky end of the H2 sequence can open the H1 sequence with a hairpin structure and expose the sticky end of the H1 sequence, and some sequence fragments in the H2 sequence can complementarily pair with the Oligo 1 probe sequence.

[0127] In this example, the H2 sequence is: 5'-TACGCCATCAGCTCCGATGGCGTAACTATGAAAAAAA AAAAA-3' (SEQ ID NO. 5).

[0128] In this embodiment, the silver ion solution is a silver nitrate solution. Of course, those skilled in the art can also use other silver ion solutions that do not interfere with the test results to replace it.

[0129] The reaction procedure of the detection system is: let the reaction stand at room temperature for 4 hours.

[0130] (4) Output test results:

[0131] In the formaldehyde method of the present invention, the detection result is mainly obtained based on the color change and the particle size change.

[0132] The specific steps are:

[0133] An imaging device is used to take a picture of the system after the reaction, and the RGB value (R value in this embodiment) of the picture is identified. Quantitative detection of formaldehyde is achieved based on the linear relationship between the RGB value and the formaldehyde concentration (using a formaldehyde standard to determine the standard curve).

[0134] Alternatively, a dynamic light scattering device is used to detect the gold nanoparticles in the system to obtain its dynamic light scattering signal, and quantitative detection of formaldehyde is achieved based on the linear relationship between the dynamic light scattering signal and the formaldehyde concentration (using a formaldehyde standard to determine the standard curve).

[0135] In this embodiment, the imaging device uses a mobile phone camera, and the specific steps of taking pictures are: fix the mobile phone with a bracket, so that the camera is about 10 cm away from the solution, set the built-in camera of the mobile phone to turn off the flash and automatic white balance, set the exposure time to 0.01s, adjust the light source in the shooting parameters to the incandescent lamp mode, and take pictures of the sample. The RGB value recognition operation is specifically: read the RGB value of the photo with a color identifier, and realize the quantitative detection of formaldehyde according to the linear relationship between the RGB value and the formaldehyde concentration.

[0136] In this embodiment, the specific parameters of the dynamic light scattering device are: test temperature is 25°C, equilibrium time is 60s, measurement angle is 90°, and single measurement time is 60s. The specific detection operation is: add 50μL of the sample to be tested (the system after reaction) into a 50μL plastic sample pool, and then perform dynamic light scattering measurement according to the above parameters to obtain the signal value, and realize quantitative detection of formaldehyde according to the linear relationship between the dynamic light scattering signal and the formaldehyde concentration (using the formaldehyde standard to determine the standard curve).

[0137] Detection effect of formaldehyde detection method based on gold nanoparticles and hybridization chain reaction amplification

[0138] (1) Quantitative detection effect (selectivity):

[0139] In order to explore the detection selectivity of the above-mentioned formaldehyde detection method based on gold nanoparticles and hybridization chain reaction amplification, the inventors used different compounds as detection samples (all with a concentration of 10.0 mg L -1 ) for testing.

[0140] In this embodiment, the test samples are specifically formaldehyde (HCHO), water (control, Blank), acetaldehyde (CH3CHO), methanol (CH3OH), ethanol (CH3CH2OH), acetone (CH3COCH3), ether (C2H5OC2H5), acetic acid (CH3COOH), ammonia (NH3·H2O) and toluene (C6H5CH3).

[0141] The detection method is the same as that in the above embodiment.

[0142] Test results such as Figure 2 shown.

[0143] Depend on Figure 2 A It can be seen that the detection method in the embodiment of the present invention will have different responses to different interferents, but only has a selective response to formaldehyde. Specifically, the R value generated by the detection method in the embodiment of the present invention for interferents with similar structures to formaldehyde, such as acetaldehyde, methanol, ethanol, acetone, ether, acetic acid, ammonia and toluene, is similar to the RGB value of the control sample (water) (no significance), indicating that the detection method in the embodiment of the present invention will not produce a selective response to these structural analogs, and only the RGB value of formaldehyde drops sharply (with significance), indicating that the detection method in the above embodiment only has a selective response to formaldehyde.

[0144] Depend on Figure 2 B shows that only the particle size of the system with formaldehyde as the detection sample is significantly larger than that of the system with other compounds as the detection sample, and the particle size of the system with other compounds as the detection sample is almost the same as that of the blank sample (water) system, indicating that only the system with formaldehyde as the detection sample can affect the change of particle size. The above results show that the detection method of the present invention has good selectivity.

[0145] (2) Detection sensitivity:

[0146] In order to explore the detection sensitivity of the above-mentioned formaldehyde detection method based on gold nanoparticles and hybridization chain reaction, the inventors used formaldehyde standard solutions of different concentrations for testing.

[0147] The detection method is the same as that in the above embodiment.

[0148] Test results such as Figure 3 and Figure 4 shown.

[0149] Figure 3 Based on different concentrations (0.05 mg L -1 , 0.10mg L -1 , 0.25mg L -1 , 0.50mg L -1 , 1.0mg L -1 , 2.0mg L -1 , 3.0mg L -1 , 3.5mg L -1 , 4.0mg L -1 ) and the RGB values ​​obtained by detection to construct a standard curve, and the constructed standard curve is:

[0150] R = 167.8-20.05C;

[0151] Among them, the linear correlation coefficient R = 0.9958, R is the RGB value obtained by detection; C is the concentration of formaldehyde. The linear range is 0.05~4.0mg·L -1 , the detection limit is 0.02mg·L -1 .

[0152] Figure 4 Based on different concentrations (0.02 mg L -1 , 0.05mg L -1 , 0.13mg L -1 , 0.25mg L -1 , 0.50mgL -1 , 1.0mg L -1 , 2.0mg L -1 , 3.0mg L -1 , 3.5mg L -1 , 4.0mg L -1 ) and the dynamic light scattering signal obtained by detection to construct a standard curve, and the constructed standard curve is:

[0153] D=60.39+100.73C

[0154] Among them, the linear correlation coefficient R = 0.9959, D is the dynamic light scattering signal obtained by detection; C is the concentration of formaldehyde. The linear range is 0.02~4.0mg·L -1 , the detection limit is 0.01 mg·L -1 .

[0155] It can be found that based on the above formaldehyde detection method, it is possible to achieve 0.02-4.0 mg·L -1 High-precision detection of formaldehyde samples within a wide concentration range, with a detection limit of 0.01 mg·L -1 .

[0156] Practical application of formaldehyde detection method based on gold nanoparticles and hybridization chain reaction amplification

[0157] In order to explore the actual use effect of the formaldehyde detection method based on gold nanoparticles and hybridization chain reaction, the inventors obtained three air samples from the Chemical Materials Building of the East Campus of Sun Yat-sen University and used the formaldehyde detection method in the above embodiment for detection.

[0158] The detection method is the same as that in the above embodiment.

[0159] Among them, the obtained air sample needs to be pre-treated. The specific pre-treatment operation is: pump the air sample into a gas bag, and then pump the gas in the gas bag into formaldehyde absorption liquid (water, the amount is 1mL), which can be used for formaldehyde detection method in the present invention.

[0160] The spike recovery test was performed on the pre-treated samples according to the addition amount shown in Table 2.

[0161] The results are shown in Table 2.

[0162] Table 2 Actual sample spike recovery test results (n = 3)

[0163]

[0164] It can be found that by using dynamic light scattering to detect the absorption liquid of indoor air, three concentrations of standard formaldehyde solutions shown in Table 2 were added to the absorption liquid with known formaldehyde content (three parallel samples were made for each sample), and the spiked recovery rates of the two air samples were 85.7% to 106.8% respectively, and the relative standard deviation was 2.3% to 6.0%. These results show that the method of the present invention has good accuracy and repeatability, and it is confirmed that the method is feasible to determine the formaldehyde concentration in indoor air.

[0165] The formaldehyde concentration in the air can be further calculated based on the formaldehyde content of the absorption liquid. The specific idea is: first, the formaldehyde mass in the absorption liquid can be calculated by the formaldehyde content of the absorption liquid, and then the formaldehyde mass can be divided by the sampling volume under standard conditions to obtain the formaldehyde concentration in the air. Among them, the sampling volume of 1L is converted into the sampling volume under standard conditions, which is 0.93L.

[0166] Taking the data in Table 2 as an example, the formaldehyde concentration in the absorption liquid of sample 2 is 0.04 mg L -1 , it can be calculated that the formaldehyde concentration in the air is 0.04 mg m -3 This concentration is lower than the indoor formaldehyde concentration limit specified by the national standard (0.1mg m -3 ), so it can be determined that the collected air samples meet the indoor air standards.

[0167] Comparison of formaldehyde detection method based on gold nanoparticles and hybridization chain reaction amplification with conventional detection methods

[0168] In order to verify the reliability of the detection effect of the formaldehyde detection method based on gold nanoparticles and hybridization chain reaction amplification, the inventors also used conventional measurement methods (ultraviolet-visible absorption spectroscopy, dynamic light scattering measurement, transmission electron microscopy) for auxiliary testing.

[0169] The concentration of the test sample was set to 0 mg L -1 and 4.0 mg L -1 of formaldehyde standards.

[0170] The detection steps of the formaldehyde detection method based on gold nanoparticles and hybridization chain reaction amplification are the same as those in the above embodiment.

[0171] In this embodiment, the UV-visible absorption spectrum is obtained based on the gold nanoparticle solution as the solvent. In this system, the solution without formaldehyde is red, and the color of the solution changes to purple after adding formaldehyde (such as Figure 5 ). The detection steps of the UV-visible absorption spectrum are: scanning the absorbance of the system under the conditions of a slit width of 1.0 nm and a wavelength range of 750 nm-450 nm.

[0172] The results are as follows Figure 5 shown.

[0173] It can be found that it contains 0mg L -1 The absorption peak of the formaldehyde standard system is at 520nm, containing 4.0mg L -1 The absorption peak corresponding to the formaldehyde standard system is at 600nm, and the absorption peak shift changes significantly. The data can be used to make a standard curve of A600 / A520 and concentration.

[0174] The inventors also found that after using the gold nanoparticles and hybrid chain reaction amplification detection method for formaldehyde in the above embodiment, the test result is consistent with the given concentration of the test sample. The method is verified by RGB colorimetry, and it is found that its color change is the same as the solution color change law in the ultraviolet visible absorption spectroscopy, and its color change trend is consistent with the relationship between the formaldehyde concentration and the RGB value change. Therefore, it can be considered that the test result is accurate. Moreover, based on the test results of the ultraviolet visible absorption spectrum, the absorbance change trend of the gold nanoparticles in the test solution is consistent with the relationship between the formaldehyde concentration and the absorbance change of the gold nanoparticles. Therefore, it can be considered that the test result is accurate.

[0175] The detection steps of the dynamic light scattering method are: the solvent is selected as H2O, the detection cell is selected as SM50, and the hydrated particles of the test system are tested. The detection parameters are the same as those in the above embodiment.

[0176] The results are as follows Figure 6 shown.

[0177] It can be found that it contains 0mg L -1 The particle size of the formaldehyde standard system is 61nm, containing 4.0mg L -1 The particle size corresponding to the formaldehyde standard system is 461nm, and the particle size changes significantly, so a standard curve of particle size and concentration can be drawn. The gold nanoparticle size distribution diagram obtained by dynamic light scattering measurement method is consistent with the theoretical estimated distribution of formaldehyde concentration and gold nanoparticle size changes. Therefore, the results obtained by dynamic light scattering measurement method can also show that the detection results of the above-mentioned formaldehyde detection method based on gold nanoparticles and hybrid chain reaction amplification are accurate.

[0178] The detection system in the method for detecting formaldehyde based on gold nanoparticles and hybridization chain reaction amplification in the above embodiment was characterized by transmission electron microscopy in the presence or absence of formaldehyde. It was found that in the absence of formaldehyde, the substances (particles) in the system were dispersed, while when 4.0 mg L -1 After the formaldehyde standard, the mixture becomes aggregated ( Figure 7 ). The gold nanoparticle size diagram obtained by transmission electron microscopy image characterization is consistent with the theoretical estimated distribution of the change in formaldehyde concentration and gold nanoparticle size. Therefore, the results obtained by transmission electron microscopy can also illustrate that the detection results of the above-mentioned formaldehyde detection method based on gold nanoparticles and hybridization chain reaction amplification are accurate.

[0179] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for detecting formaldehyde based on gold nanoparticles and hybridization chain reaction amplification, comprising the following steps: (1) Modifying the Oligo 1 probe sequence on the surface of gold nanoparticles to obtain gold nanoparticle probes; (2) mixing the gold nanoparticle probe, the sample to be tested, the HP sequence, the H1 sequence, the H2 sequence, and the silver ions to generate a hybridization chain reaction, and quantifying the formaldehyde content in the sample to be tested according to the RGB value of the color of the mixed solution after the reaction; or using a dynamic light scattering method to measure the particle size of the gold nanoparticle probe in the mixed solution after the reaction to quantify the formaldehyde content in the sample to be tested; The HP sequence is a nucleotide sequence that can specifically recognize and enhance silver ions and form a hairpin structure, and the non-hairpin HP sequence can open the hairpin H1 sequence to expose its sticky end; The H1 sequence is a nucleotide sequence that can form a hairpin structure, the sticky end of the H1 sequence can open the hairpin structure of the H2 sequence, so that the sticky end of the H2 sequence is exposed, and there is a fragment in the H1 sequence that is complementary to the Oligo 1 probe sequence; The H2 sequence is a nucleotide sequence that can form a hairpin structure, and the sticky end of the H2 sequence can open the hairpin structure of the H1 sequence, so that the sticky end of the H1 sequence is exposed, and there is a fragment in the H2 sequence that is complementary to the Oligo 1 probe sequence; The Oligo 1 probe sequence is 5'-TTTTTTTTTTTTTTTTTTTTTT-3', and the 5' end is modified with a thiol group; The HP sequence is: 5'-TACGCCATCAGCTCCAACCTACCGCAGCAGACTAGC-3'; The H1 sequence is: 5'-GGAGCTGATGGCGTACATAGTTACGCCATCAAAAAAAAAAAA-3'; The H2 sequence is: 5'-TACGCCATCAGCTCCGATGGCGTAACTATGAAAAAAAAAAAA-3'.

2. A formaldehyde detection product, characterized in that: The formaldehyde detection product includes the Oligo 1 probe sequence, HP sequence, H1 sequence and H2 sequence as defined in claim 1.

3. The formaldehyde detection product according to claim 2, characterized in that: The products include detection reagents, detection kits, and detection chips.

4. A formaldehyde detection device, characterized in that: The formaldehyde detection device comprises a detection module, an RGB colorimetric device and / or a dynamic light scattering device; Wherein, the detection module includes the Oligo 1 probe sequence, HP sequence, H1 sequence and H2 sequence defined in claim 1, and the detection module is used to perform detection of the detection sample and output the detection product; The RGB colorimetric device and / or the dynamic light scattering device performs formaldehyde quantification based on the post-detection product.

5. Use of the formaldehyde detection product according to claim 2 or 3 or the formaldehyde detection device according to claim 4 in the detection of formaldehyde residues in food and environment.

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