CN105806842A - A Cu2+ signal amplification detection test strip based on click chemistry - Google Patents

Abstract

The invention discloses a preparation method and application of a Cu ²⁺ signal amplification detection test strip based on click chemistry. The test strip is respectively adhered to the PVC liner by sample pad, colloidal gold pad (detection pad and signal amplification pad), nitrocellulose membrane (sprayed detection line and quality control line) and water-absorbing pad. It is characterized in that the detection pad and the signal amplification pad are respectively sprayed with nano-gold recognition probe 1 and nano-gold signal amplification probe 2, and the nitrocellulose membrane has a detection line coated with streptavidin and a coated There is a control line of ssDNA complementary to Cu ²⁺ -induced click chemistry oligonucleotides. And by this test strip can realize Cu ²⁺ rapid qualitative, semi-quantitative detection by direct observation, without other auxiliary equipment, the test strip provided by the present invention has simple operation, high sensitivity, fast detection speed, low cost It is suitable for screening and on-site monitoring of a large number of samples.

Description

A Cu2+ signal amplification detection test strip based on click chemistry

technical field

The invention relates to a Cu ²⁺ signal amplification detection test strip based on click chemistry, which belongs to the technical field of analytical chemistry.

Background technique

As people pay more attention to environmental protection, the detection of heavy metal ions in the environment has been paid more attention. Copper is an essential trace element for the human body. It is widely distributed in biological tissues, most of which exist in organic complexes, and many are metalloproteins, which play functional roles in the form of enzymes. Excessive intake of copper ions can denature proteins and lose their physiological activity, thus endangering the health of organisms. At the same time, heavy metal copper is a pollutant with strong biological toxicity in the environment. The pollution of copper ions mainly comes from metallurgy, electroplating and chemical industries. Therefore, the development of highly sensitive and rapid detection methods for trace copper ions is gradually becoming a research hotspot. At present, the methods for detecting copper ions mainly include atomic absorption emission spectrometry, atomic fluorescence spectrometry, and electrochemical methods. The above methods can achieve highly sensitive, stable and accurate quantitative detection of copper ions, but they usually require expensive instruments. Long detection time and complex sample preparation process are not conducive to real-time detection on site.

Chromatographic test strips are a friendly, on-line analysis method, which is relatively fast, low in cost, does not require professionals and sophisticated laboratory equipment, and has been widely used in food safety, environmental monitoring, etc. Rapid detection of toxic and hazardous substances in this field. There are also reports on the analysis of Cu ²⁺ immunochromatographic test strips, but due to the low immunogenicity of heavy metal ions and the difficulty in preparing highly efficient antibodies, it greatly increases the difficulty of developing detection methods and limits the use of immunochromatographic test strips. Application and development in the rapid detection of Cu ²⁺ .

Cu ²⁺ catalyzed alkyne-azido cycloaddition reaction (also known as click chemistry) has been applied to the detection of Cu ²⁺ by many researchers in recent years due to its high productivity and high specificity. In the present invention, we use Cu ²⁺ -induced click chemistry combined with rapid chromatography technology to establish a Cu ²⁺ signal amplification detection test strip based on click chemistry to achieve rapid and sensitive on-site detection of Cu ²⁺ .

Contents of the invention

The purpose of the present invention is to overcome the existing deficiencies in Cu ²⁺ detection technology, using Cu ²⁺ induced click chemistry combined with rapid chromatography technology, to establish a Cu ²⁺ signal amplification detection test strip based on click chemistry, to achieve Fast, sensitive on-site detection of Cu ²⁺ . Due to the introduction of ssDNA hybridization signal amplification technology, the sensitivity of the method is greatly improved.

The present invention is realized by the following technical scheme: a kind of Cu ²⁺ signal amplification detection test strip based on click chemistry comprises:

(1) Assembly of test strips: sample pads, colloidal gold pads, nitrocellulose membranes, and water-absorbent pads are adhered to the PVC liner in sequence, and adjacent pads are overlapped and connected at the joints. The colloidal gold pad is composed of a detection pad and a signal amplification pad, and the detection pad contains single-stranded DNA (S1) complementary to the Cu2 ⁺ -induced click chemistry oligonucleotide and single-stranded DNA complementary to the signal amplification probe (S2 ) a double-labeled nano-gold recognition probe 1, the signal amplification pad contains a single-stranded DNA (S3)-labeled nano-gold signal amplification probe 2 complementary to S2. The nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with streptavidin, and the quality control line contains a streptavidin-biotin system coupled with a Cu ²⁺ -induced click Chemical oligonucleotides complementary to single-stranded DNA-C.

(2) Test strip analysis of samples containing Cu ²⁺ : mix alkyne and azide-modified Cu ^{2 +} -induced click chemistry oligonucleotides (DNA-A and DNA-B) with the sample to be tested, Cu 2+ ⁺ Catalyze DNA-A and DNA-B to form a new DNA fragment, which is then applied to test strip analysis. The visual detection limit and quantitative detection limit of Cu ²⁺ in the water sample on the test strip were 5nM and 3.7nM, respectively.

Beneficial effect

(1) A kind of Cu ²⁺ signal amplification detection test strip based on click chemistry provided by the present invention solves the problems of long analysis time, high cost and low specificity in the detection of Cu ²⁺ by using traditional analysis techniques. Qualitative or semi-quantitative analysis of Cu ²⁺ can be performed with naked eyes, and can be used for simple and rapid detection of Cu ²⁺ content in field water samples.

(2) A kind of Cu ²⁺ signal amplification detection test strip based on click chemistry provided by the present invention, due to the introduction of ssDNA hybridization signal amplification technology, the sensitivity of the method is greatly improved, and its minimum detection limit is far less than National minimum standard for Cu ²⁺ in drinking water.

Description of drawings

Fig. 1 is a schematic diagram of the principle of the Cu ²⁺ signal amplification detection test strip based on click chemistry of the present invention.

Figure 2 is the detection results of Cu ²⁺ signal amplification detection test strips based on click chemistry of the present invention for different concentrations of Cu ²⁺ (a) common test strips; (b) signal amplification test strips of the present invention.

Fig. 3 is the specificity test of Cu ²⁺ by the click chemistry-based Cu ²⁺ signal amplification detection test strip of the present invention.

detailed description

The composition of embodiment 1 test strip

A kind of Cu ²⁺ signal amplification detection test strip based on click chemistry provided by the present invention, it is by sample pad, colloidal gold pad, nitrocellulose membrane and water-absorbing pad successively sticking on the PVC backing board, adjacent pads are simultaneously Joins overlap joins. The colloidal gold pad is composed of a detection pad and a signal amplification pad, and the detection pad contains single-stranded DNA (S1) complementary to the Cu2 ⁺ -induced click chemistry oligonucleotide and single-stranded DNA complementary to the signal amplification probe (S2 ) a double-labeled gold nanometer recognition probe 1, and the signal amplification pad contains a gold nanometer signal amplification probe 2 labeled with a single-stranded DNA (S3) complementary to S2. The nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with streptavidin, and the quality control line contains a streptavidin-biotin system coupled with a Cu ²⁺ -induced click Chemical oligonucleotides complementary to single-stranded DNA-C. The sequence composition of each DNA fragment is shown in Table 1.

Table 1 DNA fragment sequence used in the present invention forms

Example 2 Preparation of Nano-gold Recognition Probe 1, Signal Amplification Probe 2 and Quality Control Line Probe

(1) Preparation of nano-gold recognition probe 1, which is characterized by adding 45 μL of 1OD capture strand S1 and 135 μL of 1OD amplification strand S2 to 900 μL of 10-fold concentrated 20nm colloidal gold solution for reaction for 24 hours, aging with 300 mM NaCl for 24 hours, blocking with 0.01% SDS for 4 hours, and centrifuging for 2 hours. Once, suspend with 200 μL buffer (20 mM Na ₃ PO ₄ , 5% BSA, 0.25% Tween-20, and 10% sucrose), and store at 4°C.

(2) Preparation of nano-gold signal amplification probe 2, which is characterized in that 180 μL of 1OD amplification chain S3 is added to 900 μL of 10-fold concentrated 20nm colloidal gold solution for 24 hours, 300 mM NaCl aging for 24 hours, 0.01% SDS for 4 hours, centrifugation twice, and 200 μL Buffer (20mM Na ₃ PO ₄ , 5% BSA, 0.25% Tween-20, and 10% sucrose) was suspended and stored at 4°C.

(3) Preparation of quality control line probes, which include 20 μL 1OD biotinylated DNA-C and 200 μL 2mg/mL streptavidin mixed reaction for 1 hour, centrifuged once, suspended in 500 μL 0.01MPBS buffer, and stored at 4°C .

The processing of embodiment 3 sample pads

The sample pad was soaked in commercial membrane sealing solution (Invitrogen), evenly wetted, baked at 37°C for 90min, and then taken out for use.

The spraying of embodiment 4 detection pad and signal amplification pad

The prepared nano-gold recognition probe 1 and nano-gold signal amplification probe 2 were sprayed on the detection pad and the signal amplification pad by air spraying with a spray rate of 10 μL/cm by XYZ3060 film spraying apparatus. Dry at 42°C for 60 minutes, take it out, and store it in a dry environment (humidity <20%) for later use.

The spraying of embodiment 5 detection line and quality control line

2mg/mL ^- streptavidin and 2mg/mL quality control line probes were sprayed on the test line and the quality control line respectively by dot spraying method, the interval was 5mm, the width of the test line and the quality control line was 2mm, and the spraying speed was 2 μL/cm. Dry at 37°C for 60 minutes, take it out, and store it in a dry environment (humidity <20%) for later use.

The assembly of embodiment 6 test strips

Adhere the sample pad, detection pad, signal amplification pad, nitrocellulose membrane, and water-absorbent pad to the PVC liner in sequence, and at the same time, adjacent pads are overlapped and connected at the joint, and the length of the overlapping part between each pad is 2mm. Use CM4000 strip cutter to cut the assembled test strips into 4mm wide strips, place them in special plastic cards, dry them at 4°C and store them for later use.

Embodiment 7 is to the specificity test of Cu ²⁺

Compared Mg ²⁺ , Hg ²⁺ , pb ²⁺ , Cd ²⁺ , Ca ²⁺ , Zn ²⁺ , Fe ³⁺ , Na ⁺ 8 kinds of heavy metal ions for Cu ^{2 +} Signal amplification detects the signal response of the test strip. The concentration of Cu ²⁺ is 100nM, and the concentration of other ions is 1000nM. Figure 3 shows that even if the concentration of other 8 kinds of metal ions is 10 times that of Cu ²⁺ , only under Cu ²⁺ condition, the T line of the test strip shows red color, indicating that a kind of Cu 2+ based ^on click chemistry provided by the present invention ⁺ Signal amplification test strips have very high specificity for Cu ²⁺ , with less than 0.01% cross-reactivity.

The detection of embodiment 8 drinking water and river water actual sample Cu ²⁺

River water samples were pre-passed through a 0.45 μM bacterial filter to remove impurities. Because the content of Cu ²⁺ in the actual water sample adopted is all less than the minimum detection limit of the present invention. Using the standard addition method, a certain concentration of Cu ²⁺ was added to the actual water sample, and then added to 0.01M Tris-HCl (0.2M NaCl, pH 7.0 ) buffer for 30 minutes, take 20 μL of the mixture and add it to 80 μL of 4×SSC buffer, add it dropwise to the sample pad, react for 10 minutes, and determine the result.

Result judgment: Negative (-): No color development on T line and color development on C line, indicating that the Cu ²⁺ content in the sample is lower than the detection limit.

Positive (+): Both the T line and the C line are colored, indicating that the Cu ²⁺ content in the sample is equal to or higher than the detection limit

Invalid: The C line does not show color, indicating incorrect operation steps or the test strip has deteriorated and failed.

Claims (9)

1. a kind of Cu ²⁺ signal amplification detection test strip based on click chemistry, it is characterized in that comprising have alkyne and azide modification Cu ²⁺ induce click chemistry oligonucleotide (DNA-A and DNA-B) . 2. a kind of Cu2 ⁺ signal amplification detection test strip based on click chemistry as claimed in claim 1, this test strip is made up of sample pad, colloidal gold pad, nitrocellulose membrane and water-absorbing pad are adhered on the PVC backing plate successively , while adjacent pads overlap and connect at the junction. 3. a kind of Cu2 ⁺ signal amplification detection test strip based on click chemistry according to claim 2, is characterized in that described colloidal gold pad is made up of detection pad and signal amplification pad, and described detection pad contains and Cu ^{2 +} Induced click chemical oligonucleotide complementary single-stranded DNA (S1) and double-labeled nano-gold recognition probe 1 complementary to signal amplification probe (S2), the signal amplification pad contains and S2 Complementary single-stranded DNA (S3)-labeled nanogold signal amplification probe 2. 4. a kind of Cu2 ⁺ signal amplification detection test strip based on click chemistry according to claim 2, is characterized in that described nitrocellulose membrane is provided with detection line and quality control line, and described detection line is coated with streptavidin Avidin, the quality control line contains streptavidin-biotin system coupled single-stranded DNA-C that can be complementary to Cu ²⁺ induced click chemistry oligonucleotides. 5. A kind of Cu ²⁺ signal amplification detection test strip based on click chemistry as claimed in claim 2, characterized in that the length of the overlapping portion between the connected pads is 2mm. 6. A kind of Cu ²⁺ signal amplification detection test strip based on click chemistry according to claim 2, characterized in that the nano-gold particle diameter in the nano-gold recognition probe 1 and the nano-gold signal amplification probe 2 is 20nm. 7. a kind of preparation method based on the Cu of click chemistry ²⁺ signal amplification detection test strip, it is characterized in that comprising the steps: (1) Preparation of nano-gold recognition probe 1, which is characterized by adding 45 μL of 1OD capture strand S1 and 135 μL of 1OD amplification strand S2 to 900 μL of 10-fold concentrated 20nm colloidal gold solution for reaction for 24 hours, aging with 300 mM NaCl for 24 hours, blocking with 0.01% SDS for 4 hours, and centrifuging for 2 hours. Once, suspend with 200 μL buffer (20 mM Na ₃ PO ₄ , 5% BSA, 0.25% Tween-20, and 10% sucrose), and store at 4°C. (2) Preparation of nano-gold signal amplification probe 2, which is characterized in that 180 μL of 1OD amplification chain S3 is added to 900 μL of 10-fold concentrated 20nm colloidal gold solution for 24 hours, 300 mM NaCl aging for 24 hours, 0.01% SDS for 4 hours, centrifugation twice, and 200 μL Buffer (20mM Na ₃ PO ₄ , 5% BSA, 0.25% Tween-20, and 10% sucrose) was suspended and stored at 4°C. (3) Preparation of quality control line probes, which include 20 μL 1OD biotinylated DNA-C and 200 μL 2mg/mL streptavidin mixed reaction for 1 hour, centrifuged once, suspended in 500 μL 0.01MPBS buffer, and stored at 4°C . (4) the spraying of detection pad and signal amplification pad, it is characterized in that the nano-gold recognition probe 1 and the nano-gold signal amplification probe 2 prepared are sprayed on the detection pad and the signal amplification pad by air spraying method respectively, and the spray speed is 10 μL/cm. Dry at 42°C for 60 minutes, take it out, and store it in a dry environment (humidity <20%) for later use. (5) The spraying of test line and quality control line, its feature comprises 2mg/mL ^- streptavidin and 2mg/mL quality control line probe adopts spot spraying method to spray on test line and quality control line respectively, and its interval is 5mm, the width of test line and quality control line is 2mm, and the spray rate is 2μL/cm. Dry at 37°C for 60 minutes, take it out, and store it in a dry environment (humidity <20%) (6) The treatment of the sample pad, which is characterized in that the sample pad is soaked in a commercial membrane blocking solution (Invitrogen), evenly wetted, baked at 37° C. for 90 minutes, and then taken out for use. 8. The application of a Cu ²⁺ signal amplification detection test strip based on click chemistry, its features include 0.1 μM DNA-A, 0.1 μM DNA-B, 800 μM sodium ascorbate, 1mM M H T PTA is added with different concentrations of Cu ²⁺ to react for 30 minutes, take 20 μL The mixed solution was added to 80 μL 4×SSC buffer solution, added dropwise to the sample pad, reacted for 10 minutes, and observed the color of the detection line. 9. the application of a kind of Cu2 ⁺ signal amplification detection test strip based on click chemistry according to claim 8, characterized in that the buffer solution used by the click chemistry reaction induced by the Cu2 ⁺ is 0.01M Tris-HCl containing 0.2 MNaCl, pH 7.0.