CN105806842A - Click chemistry-based Cu2+ signal amplification and detection test strip - Google Patents
Click chemistry-based Cu2+ signal amplification and detection test strip Download PDFInfo
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- CN105806842A CN105806842A CN201610372989.8A CN201610372989A CN105806842A CN 105806842 A CN105806842 A CN 105806842A CN 201610372989 A CN201610372989 A CN 201610372989A CN 105806842 A CN105806842 A CN 105806842A
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- 238000012360 testing method Methods 0.000 title claims abstract description 45
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical group [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 title claims abstract description 35
- 230000003321 amplification Effects 0.000 title claims abstract description 26
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 26
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/18—Water
- G01N33/1813—Specific cations in water, e.g. heavy metals
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- Plasma & Fusion (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a production method and application of a click chemistry-based Cu2+ signal amplification and a detection test strip. The test strip is bonded to a PVC (polyvinyl chloride) liner successively through a sample pad, a colloidal gold pad (a detection pad and a signal amplifying pad), a nitrocellulose membrane (a spray detection line and a quality control line) and an absorbent pad. The test strip is characterized by comprising the detection and the signal amplifying pad, a nanogold recognition probe 1 and a nanogold signal amplifying probe 2 are sprayed to the detection pad and the signal amplifying pad respectively, the nitrocellulose membrane has a streptavidin-coated detection line and a quality control line coated with single-stranded DNA complementary with Cu2+ induced click chemistry oligonucleotides. Quick qualitation and semi-quantitative detection for Cu2+ can be achieved through direct observation with the test strip, without other auxiliary instrumentation and equipment, and the test strip provided herein has the advantages such as operation simplicity, high sensitivity, high detection speed and low cost and is suitable for screening and field monitoring of mass samples.
Description
Technical field
The present invention relates to a kind of Cu based on click chemistry2+Signal amplification detection test strips, belongs to technical field of analytical chemistry.
Background technology
Along with people's concern to environmental conservation, in environment, the detection of heavy metal ion is subject to the people's attention.Copper is the trace element of needed by human, is distributed widely in biological tissue, and major part exists with organic double compound, is much metalloprotein, plays function with the form of enzyme.The Excess free enthalpy of copper ion can make protein denaturation, loses physiologically active, thus endangering the health of organism.Heavy metal copper is the pollutant that in environment, a kind of bio-toxicity is stronger simultaneously, and the pollution of copper ion is mainly derived from the industries such as metallurgy, plating chemical industry.Therefore, the exploitation of the high sensitivity method for quick of trace copper ion is increasingly becoming the focus of research.At present for the method for copper ion detection mainly just like Atomic Absorption emission spectrometry, atomic fluorescence spectrometry and electrochemical method etc., it is highly sensitive that above method can realize copper ion, stable and accurate detection by quantitative, but they typically require the instrument of costliness, longer detection time and the Sample Preparation Procedure of complexity, be also unfavorable for on-the-spot detection in real time simultaneously.
Chromatograph test strip is a kind of method of close friend, on-line analysis, the method is relatively fast, expense is low, do not need the laboratory equlpment of professional and precision, the method to be widely used in the quick detection of the poisonous of the multiple fields such as food safety, environmental monitoring and harmful substance.For Cu2+Immuno-chromatographic test paper strip analysis also have been reported that, but owing to heavy metal ion immunogenicity is low, the existence of the problems such as the preparation of Effective Anti body is difficult, considerably increase the development difficulty of detection method, limit immuno-chromatographic test paper strip at Cu2+The application of quick context of detection and development.
Cu2+The alkynes of catalysis-nitrine cycloaddition reaction (being also called click chemistry), due to its high yield ability and high specific, is applied to Cu by vast researcher in recent years2+Detection.In the present invention, we utilize Cu2+The click chemistry of induction, in conjunction with flash chromatography technology, establishes a kind of Cu based on click chemistry2+Signal amplification detection test strips, it is achieved for Cu2+Quick, sensitive Site Detection.
Summary of the invention
It is an object of the invention to overcome existing for Cu2+The deficiency of detection technique, utilizes Cu2+The click chemistry of induction, in conjunction with flash chromatography technology, establishes a kind of Cu based on click chemistry2+Signal amplification detection test strips, it is achieved for Cu2+Quick, sensitive Site Detection.Owing to introducing ssDNA hybridization signal amplifying technique so that the sensitivity of the method is greatly improved.
The present invention is achieved through the following technical solutions: a kind of Cu based on click chemistry2+Signal amplification detection test strips includes:
(1) assembling of test strips: sample pad, colloidal gold pad, nitrocellulose membrane and adsorptive pads stick on PVC liner plate successively, adjacent each pad is overlapping in junction simultaneously connects.Colloidal gold pad is amplified pad by detecting pad and signal and is formed, and described detecting pad contains and Cu2+The nanometer gold identification probe 1 of the single stranded DNA (S1) that the click chemistry oligonucleotide of induction is complementary and single stranded DNA (S2) double labeling with the complementation of signal amplifying probe, described signal amplifies the pad nano-gold signal amplifying probe 2 containing single stranded DNA (S3) labelling complementary with S2.Nitrocellulose membrane is provided with detection line and nature controlling line, and described detection line is coated Streptavidin, and described nature controlling line contains energy and the Cu of Streptavidin-biotin system coupling2+Single stranded DNA-the C that the click chemistry oligonucleotide of induction is complementary.
(2) containing Cu2+The test strips analysis of sample: the Cu that alkynes and nitrine are modified2+Click chemistry oligonucleotide (DNA-A and DNA-B) of induction mixes with testing sample, Cu2+Catalytic dna-A, DNA-B form DNA fragmentation one section new, are then applied to test strips analysis.To water sample kind Cu in test strips2+Visual detection limit and detection by quantitative limit respectively 5nM and 3.7nM.
Beneficial effect
(1) a kind of Cu based on click chemistry provided by the invention2+Signal amplification detection test strips, solves and utilizes conventional analytical techniques for Cu2+The analysis time that detection exists is long, cost height, and the problems such as specificity is low utilize naked eyes can carry out Cu2+Qualitative or semi-quantitative analysis, can be used for on-the-spot water sample Cu2+The simple and quick detection of content.
(2) a kind of Cu based on click chemistry provided by the invention2+Signal amplification detection test strips, owing to introducing ssDNA hybridization signal amplifying technique so that the sensitivity of the method is greatly improved, its lowest detectable limit is far smaller than country for Cu in drinking water2+Minimum content standard.
Accompanying drawing explanation
Fig. 1 is the Cu based on click chemistry of the present invention2+Signal amplification detection test strips principle schematic.
Fig. 2 is the Cu based on click chemistry of the present invention2+Signal amplification detection test strips is for variable concentrations Cu2+The common test strips of testing result (a)；B () signal of the present invention amplifies test strips.
Fig. 3 is the Cu based on click chemistry of the present invention2+Signal amplification detection test strips is to Cu2+Specific assay.
Detailed description of the invention
The composition of embodiment 1 test strips
A kind of Cu based on click chemistry provided by the invention2+Signal amplification detection test strips, it is to be sticked on PVC liner plate successively by sample pad, colloidal gold pad, nitrocellulose membrane and adsorptive pads, and adjacent each pad is overlapping in junction simultaneously connects.Colloidal gold pad is amplified pad by detecting pad and signal and is formed, and described detecting pad contains and Cu2+The nanometer gold identification probe 1 of the single stranded DNA (S1) that the click chemistry oligonucleotide of induction is complementary and single stranded DNA (S2) double labeling with the complementation of signal amplifying probe, described signal amplifies the pad nano-gold signal amplifying probe 2 containing single stranded DNA (S3) labelling complementary with S2.Nitrocellulose membrane is provided with detection line and nature controlling line, and described detection line is coated Streptavidin, and described nature controlling line contains energy and the Cu of Streptavidin-biotin system coupling2+Single stranded DNA-the C that the click chemistry oligonucleotide of induction is complementary.Wherein each sequence dna fragment composition is as shown in table 1.
Sequence dna fragment composition used in table 1 present invention
The preparation of embodiment 2 nanometer gold identification probe 1 and signal amplifying probe 2 and nature controlling line probe
(1) preparation of nanometer gold identification probe 1, its feature includes 45 μ L1OD and catches chain S1 and 135 μ L1OD amplifier chain S2 and join in the 900 μ L10 times 20nm colloidal gold solution concentrated reaction 24h, the aging 24h of 300mMNaCl, 0.01%SDS closes 4h, centrifugal 2 times, with 200 μ L buffer (20mMNa3PO4, 5%BSA, 0.25%Tween-20, and10%sucrose) suspend, 4 DEG C of preservations.
(2) preparation of nano-gold signal amplifying probe 2, its feature includes 180 μ L1OD amplifier chain S3 and joins aging 24h, the 0.01%SDS closing 4h of reaction 24h, 300mMNaCl in the 900 μ L10 times 20nm colloidal gold solution concentrated, centrifugal 2 times, with 200 μ L buffer (20mMNa3PO4, 5%BSA, 0.25%Tween-20, and10%sucrose) suspend, 4 DEG C of preservations.
(3) preparation of nature controlling line probe, its feature includes 20 μ L1OD biotinylated DNA-C and 200 μ L2mg/mL Streptavidin hybrid reaction 1h, centrifugal 1 time, suspends with 500 μ L0.01MPBS buffer, 4 DEG C of preservations.
The process of embodiment 3 sample pad
Sample pad is soaked in commercial membrane closure liquid (Invitrogen), uniform wet, takes out standby after 37 DEG C of baking 90min.
Embodiment 4 detecting pad and signal amplify the spraying of pad
Utilizing XYZ3060 spray film instrument that the nanometer gold identification probe 1 prepared and nano-gold signal amplifying probe 2 are respectively adopted gas blowout method to be sprayed on detecting pad and signal amplification pad, spray speed is 10 μ L/cm.Take out after being placed in 42 DEG C of drying 60min, dry environment saves backup (humidity < 20%).
Embodiment 5 detects the spraying of line and nature controlling line
2mg/mL-Streptavidin and 2mg/mL nature controlling line probe are respectively adopted a spray and are sprayed at detection line and nature controlling line, and it is spaced apart 5mm, and detection line and nature controlling line width are 2mm, and spray speed is 2 μ L/cm.Take out after being placed in 37 DEG C of drying 60min, dry environment saves backup (humidity < 20%).
The assembling of embodiment 6 test strips
Sample pad, detecting pad and signal being amplified pad, nitrocellulose membrane and adsorptive pads and sticks on PVC liner plate successively, adjacent each pad is overlapping in junction simultaneously connects, and between each pad, the length of lap is 2mm.Utilizing CM4000 cutting instrument that the test strips assembled is cut into the wide little bar of 4mm, be placed in special plastic clip, dry 4 DEG C save backup.
Embodiment 7 is to Cu2+Specific assay
It is respectively compared Mg2+, Hg2+, pb2+, Cd2+, Ca2+, Zn2+, Fe3+, Na+8 heavy metal species ions are for a kind of Cu based on click chemistry provided by the invention2+The signal response of signal amplification detection test strips.Wherein Cu2+Concentration is 100nM, and all the other ion concentrations are 1000nM.Even if Fig. 3 shows that the concentration of other 8 metal ion species is Cu2+10 times, only Cu2+Under condition, the T line of this test strips demonstrates redness, it was shown that a kind of Cu based on click chemistry provided by the invention2+Signal amplification detection test strips is for Cu2+Having very high specificity, cross reaction is less than 0.01%.
Embodiment 8 drinking water and river actual sample Cu2+Detection
River sample first passes through 0.45 μM of biofilter in advance and removes impurity.Due to Cu in the actual water sample product that adopt2+Content be respectively less than the lowest detectable limit of the present invention.Utilize standard addition method, by certain density Cu2+Join in actual water sample product, after join containing 0.1 μM of DNA-A, 0.1 μM of DNA-B, 800 μMs of sodium ascorbates, 0.01MTris-HCl (the 0.2MNaCl of 1mMTHPTA, pH7.0) buffer reacts 30min, take 20 μ L mixed liquors and add in 80 μ L4 × SSC buffer, drop to sample pad, react 10min, it is determined that result.
Result judges: negative (-): T line, without colour developing C line colour developing, represents Cu in sample2+Content is lower than detection limit.
Positive (+): T line C line all develops the color, and represents Cu in sample2+Content is equal to or higher than detection limit
Invalid: C line does not develop the color, represent the deterioration failure of incorrect operating procedure or test strips.
Claims (9)
1. the Cu based on click chemistry2+Signal amplification detection test strips, it is characterised in that comprise the Cu with alkynes and nitrine modification2+Click chemistry oligonucleotide (DNA-A and DNA-B) of induction.
2. a kind of Cu based on click chemistry described in claim 12+Signal amplification detection test strips, this test strips consists of sample pad, colloidal gold pad, nitrocellulose membrane and adsorptive pads and sticks on PVC liner plate successively, and adjacent each pad is overlapping in junction simultaneously connects.
3. a kind of Cu based on click chemistry described in claim 22+Signal amplification detection test strips, it is characterised in that described colloidal gold pad is amplified pad by detecting pad and signal and formed, and described detecting pad contains and Cu2+The nanometer gold identification probe 1 of the single stranded DNA (S1) that the click chemistry oligonucleotide of induction is complementary and single stranded DNA (S2) double labeling with the complementation of signal amplifying probe, described signal amplifies the pad nano-gold signal amplifying probe 2 containing single stranded DNA (S3) labelling complementary with S2.
4. a kind of Cu based on click chemistry described in claim 22+Signal amplification detection test strips, it is characterised in that described nitrocellulose membrane is provided with detection line and nature controlling line, and described detection line is coated Streptavidin, and described nature controlling line contains energy and the Cu of Streptavidin-biotin system coupling2+Single stranded DNA-the C that the click chemistry oligonucleotide of induction is complementary.
5. a kind of Cu based on click chemistry described in claim 22+Signal amplification detection test strips, it is characterised in that between connected each pad, the length of lap is 2mm.
6. a kind of Cu based on click chemistry described in claim 22+Signal amplification detection test strips, it is characterised in that in nanometer gold identification probe 1 and nano-gold signal amplifying probe 2, nanometer gold particle diameter is 20nm.
7. the Cu based on click chemistry2+The preparation method of signal amplification detection test strips, it is characterised in that comprise the steps:
(1) preparation of nanometer gold identification probe 1, its feature includes 45 μ L1OD and catches chain S1 and 135 μ L1OD amplifier chain S2 and join in the 900 μ L10 times 20nm colloidal gold solution concentrated reaction 24h, the aging 24h of 300mMNaCl, 0.01%SDS closes 4h, centrifugal 2 times, with 200 μ L buffer (20mMNa3PO4, 5%BSA, 0.25%Tween-20, and10%sucrose) suspend, 4 DEG C of preservations.
(2) preparation of nano-gold signal amplifying probe 2, its feature includes 180 μ L1OD amplifier chain S3 and joins aging 24h, the 0.01%SDS closing 4h of reaction 24h, 300mMNaCl in the 900 μ L10 times 20nm colloidal gold solution concentrated, centrifugal 2 times, with 200 μ L buffer (20mMNa3PO4, 5%BSA, 0.25%Tween-20, and10%sucrose) suspend, 4 DEG C of preservations.
(3) preparation of nature controlling line probe, its feature includes 20 μ L1OD biotinylated DNA-C and 200 μ L2mg/mL Streptavidin hybrid reaction 1h, centrifugal 1 time, suspends with 500 μ L0.01MPBS buffer, 4 DEG C of preservations.
(4) detecting pad and signal amplify the spraying of pad, and its feature includes that the nanometer gold identification probe 1 prepared and nano-gold signal amplifying probe 2 are respectively adopted gas blowout method and is sprayed on detecting pad and signal amplification pad, and spray speed is 10 μ L/cm.Take out after being placed in 42 DEG C of drying 60min, dry environment saves backup (humidity < 20%).
(5) spraying of detection line and nature controlling line, its feature includes 2mg/mL-Streptavidin and 2mg/mL nature controlling line probe are respectively adopted a spray and are sprayed at detection line and nature controlling line, and it is spaced apart 5mm, and detection line and nature controlling line width are 2mm, and spray speed is 2 μ L/cm.Take out after being placed in 37 DEG C of drying 60min, dry environment saves backup (humidity < 20%)
(6) process of sample pad, its feature includes being soaked in sample pad in commercial membrane closure liquid (Invitrogen), uniform wet, takes out standby after 37 DEG C of baking 90min.
8. the Cu based on click chemistry2+The application of signal amplification detection test strips, its feature includes 0.1 μM of DNA-A, 0.1 μM of DNA-B, 800 μMs of sodium ascorbates, and 1mMTHPTA adds the Cu of variable concentrations2+Reaction 30min, takes 20 μ L mixed liquors and adds in 80 μ L4 × SSC buffer, drops to sample pad, reacts 10min, observes detection line color.
9. a kind of Cu based on click chemistry described in claim 82+The application of signal amplification detection test strips, it is characterised in that described Cu2+The buffer that the click chemistry reaction of induction uses is that 0.01MTris-HCl is containing 0.2MNaCl, pH7.0.
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