CN109856210A - A kind of electrochemical luminescence biosensor and preparation method thereof detecting early stage of lung cancer marker NAP-2 albumen - Google Patents
A kind of electrochemical luminescence biosensor and preparation method thereof detecting early stage of lung cancer marker NAP-2 albumen Download PDFInfo
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Abstract
The present invention provides a kind of electrochemical luminescence biosensor and preparation method thereof for detecting early stage of lung cancer marker NAP-2 albumen, comprising: step 1, prepares p-aminobenzoic acid modified electrode；Step 2, preparation NAP-2 protein-specific identifies probe: TW and J1-Ru being added in DNA buffer and be mixed, heating is hybridized to TW/J1-Ru, then cools to room temperature and continues hybridization and obtain TW/J1-Ru probe；Step 3, p-aminobenzoic acid modified electrode in step 1 is immersed in the Tris-HCl buffer containing NHS and EDC and is activated, the 4-ABA modified electrode activated, which immerses in TW/J1-Ru probe solution obtained in step 2, to be modified, then is closed, and electrochemical luminescence biosensor is obtained.The present invention can detect immediately early stage of lung cancer marker NAP-2 albumen, high sensitivity, high specificity and cheap and simple.
Description
Technical field
The invention belongs to lung cancer detection fields, are related to a kind of electrochemistry hair for detecting early stage of lung cancer marker NAP-2 albumen
Optical biosensor and preparation method thereof.
Background technique
Lung cancer is one of most common cancer mortality reason in the whole world, has more than 120 ten thousand people to die of lung cancer every year.Lung cancer is in early stage
Lack typical symptom, most patient is late just diagnosed with lung cancer and is difficult to cure.It is reported that lung cancer is in early stage
5 years survival rates after being diagnosed treatment are 70% to 90%, and 5 years survival rates of Patients with Advanced Lung Cancer are only 16%.
Currently, the technology for detecting lung cancer mainly includes traditional X-ray technology, lung CT, draws through bronchus ultrasound
Needle biopsy, positron emission computerized tomography (PET), bronchial perfusate oncology marker etc. are led, but these technologies are all
It is unable to satisfy the requirement of lung cancer early detection.For example, X-ray technology is difficult to detect 10mm lump below, false lung cancer is negative
Rate is very high, and also has certain radiation injury；Though the sensitivity of lung CT detection is higher than X-ray technology, specific
Poor and higher cost is not easy to popularize in general population；It is thin that pneumothorax, cancer are easily caused through bronchus ultrasonic guidance needle biopsy
Born of the same parents send out plantation, local hemorrhage, if there is focus of infection may also lead to other site infections.It is able to detect examining for micron order tumour
Disconnected test (such as CT scan or bronchoscopy) is expensive, it is difficult to which that realization carries out large-scale screening in general population.
Therefore, high sensitivity, high specificity and simple, cheap method, on-site test early stage of lung cancer biological marker are researched and developed
Object, in lung cancer High risk group screening patients with lung cancer be of great significance.
Summary of the invention
Aiming at the problems existing in the prior art, the present invention provides a kind of detection early stage of lung cancer marker NAP-2 albumen
Electrochemical luminescence biosensor and preparation method thereof can detect immediately early stage of lung cancer marker NAP-2 albumen, sensitive
Degree height, high specificity and cheap and simple, can carry out screening lung cancer in general population.
The present invention is to be achieved through the following technical solutions:
A kind of preparation method for the electrochemical luminescence biosensor detecting early stage of lung cancer marker NAP-2 albumen, including
Following steps:
Step 1, working electrode is placed in the p-aminobenzoic acid solution with diazonium cations, passes through cyclic voltammetry scan
P-aminobenzoic acid modified electrode is prepared in method；
Step 2, preparation NAP-2 protein-specific identifies probe:
TW and J1-Ru is added in DNA buffer and is mixed, heating is hybridized to TW/J1-Ru, then cools to room temperature continuation
Hybridization obtains NAP-2 protein-specific identification probe, is named as TW/J1-Ru probe；
Step 3, the assembling of electrochemical luminescence sensor；
Step 3.1, p-aminobenzoic acid modified electrode in step 1 is immersed in the buffering of the tris-HCl containing NHS and EDC
It is activated in liquid, the 4-ABA modified electrode activated；
Step 3.2, the 4-ABA modified electrode after activation is immersed in TW/J1-Ru probe solution obtained in step 2
Modification, obtains TW/J1-Ru/4-ABA modified electrode；
Step 3.3, the DNA buffer containing bovine serum albumin is dripped into step 3.2 gained TW/J1-Ru/4-ABA modification
Electrode surface is closed, and electrochemical luminescence biosensor is obtained.
Preferably, step 1 includes the following steps:
Step 1.1, first p-aminobenzoic acid is dissolved in HCl solution, adds NaNO2, ice-water bath and logical condition of nitrogen gas
Under be stirred to react 5-20min, obtain the p-aminobenzoic acid solution with diazonium cations；P-aminobenzoic acid and NaNO2Rub
You are than being 1:(0.8-1.2)；
Step 1.2, the p-aminobenzoic acid solution with diazonium cations prepared using step 1.1 is electrolyte, with work
Electrode, the three-electrode system formed to electrode and reference electrode carry out cyclic voltammetry scan, obtain p-aminobenzoic acid modification electricity
Pole.
Preferably, in step 1, working electrode is one in glass-carbon electrode, graphite electrode, ITO electrode and noble metal electrode
Kind.
Preferably, in step 2, the molar ratio of TW and J1-Ru are 1:(1.1-1.5).
Preferably, in step 2,
TW:5 '-NH2-(CH2)6-O-TCT CCA GCG TCG AGG TTT TCT GGT ATT ATA CCA GCC GTT
ACG CAA TCC TTC TCG AGC TAA A-3'；
J1-Ru:Ru-HN-(CH2)6-O-5'-TTT AGC TCG AGA CGA CGC TGG AGA-3'。
Preferably, in step 2, first heating hybridization 2-10min under the conditions of 60-90 DEG C, it is miscellaneous to then cool to room temperature continuation
Hand over 10-15h.
Preferably, in step 3.1, activation time 0.5-2h.
Preferably, in step 3.2, the concentration of TW/J1-Ru probe solution is 0.01-2 μM, and the modification time is 0.5-2h.
Preferably, in step 3.3, bovine serum albumen solution mass concentration is 0.05%-1%, off-period 30-
60min。
The electrochemical luminescence biology for the detection early stage of lung cancer marker NAP-2 albumen that the preparation method is prepared passes
Sensor.
Compared with prior art, the invention has the following beneficial technical effects:
P-aminobenzoic acid (4-ABA) of the present invention can generate positively charged diazonium cations after diazotising, when with diazonium
When p-aminobenzoic acid solution after change is that electrolyte carries out cyclic voltammetry scan, the p-aminobenzoic acid with diazonium cations
By covalence graft is to electrode surface in a manner of electrochemical reduction, so that electrode surface be made successfully to modify one layer of p-aminophenyl first
Acid molecule layer；Prepared NAP-2 albumen probe TW/J1-Ru is made of two single stranded DNAs in the present invention, wherein one single-stranded
(TW) 5 ' ends are marked with amino, and 5 ' ends of another chain (J1), which are marked with, can produce swashing for electrochemical luminescence (ECL) signal
Send out object-ruthenium bipyridine complex.When the carboxyl on the p-aminobenzoic acid of electrode surface is through 1- (3- dimethylamino-propyl) -3-
It, can be with the ammonia that is marked on probe after ethyl-carbodiimide hydrochloride (EDC) and N-N- HOSu NHS (NHS) activation
Classical amidation process occurs for base, so that probe covalent modification obtains TW/J1-Ru/4-ABA modified electrode to electrode surface.
After bovine serum albumin (BSA) closing, it is possible to reduce the non-specific adsorption of electrode surface obtains BSA/TW/J1-Ru/4-ABA
Modified electrode.The TW/J1-Ru probe of NAP-2 protein-specific identification is a DNA three-dimensional connected structure, the TW chain in probe
It is adapted to body region containing the NAP-2 albumen that the aglucon phyletic evolution technology (SELEX) through index concentration filters out, so NAP-2
The combination of specificity can occur with the probe for albumen.When there are NAP-2 albumen, due in NAP-2 albumen and TW/J1-Ru
Adaptation body region occur specificity combination, causing TW/J1-Ru conformation to change causes Ru compound far from electrode table
Face, ECL signal strength weakening.The concentration of NAP-2 albumen is higher in solution to be measured, and ECL signal strength is weaker, establishes NAP-2
Relationship between the concentration and ECL signal strength of albumen can obtain in sample to be tested according to the ECL signal strength detected
The concentration of NAP-2 albumen, here it is electrochemical luminescence biosensor quantitative detection lung cancer marker NAP-2 albumen of the present invention
Mechanism, as shown in Figure 1.The present invention has the advantages that (1) high sensitivity, the present invention is using Electrochemiluminescence technology as signal
The way of output, the technology itself have high sensitivity, and the electrochemical luminescence biosensor in the present invention can quantitative detection
0.008pM NAP-2 albumen.(2) selectivity is high, using NAP-2 albumen and its aptamers specific recognition, induces TW/J1-Ru
Conformation changes；It is demonstrated experimentally that common MMP-14, CD44, PEX-14, chymotrypsin, albumin (Alb), flesh are red
Albumen (Mb) is noiseless to detecting.(3)Ru(bpy)3 2+System is with good stability, higher ECL quantum yield and life
Object compatibility, by Ru (bpy)3 2+It is fixed on electrode surface, can not only reduce the usage amount of expensive reagent, but also is also enhanced
ECL signal strength and simplify experimentation.(4) signal stabilization, TW/J1-Ru probe are modified by way of covalent linkage
Electrode surface avoids traditional Au-S self assembly when current potential is+0.9V, the fracture of Au-S key.(5) low in cost, required examination
Dosage is few.
Detailed description of the invention
Fig. 1 is the electrochemical luminescence biosensor of present invention detection early stage of lung cancer marker for detecting NAP-2 albumen
Schematic diagram.
Fig. 2 is to modify the cyclic voltammogram of 4-ABA molecular layer in glassy carbon electrode surface.
(a) is the corresponding electrochemical luminescence signals of various concentration NAP-2 albumen in Fig. 3, (b) is luminous intensity values and NAP-
The linear relationship of 2 protein concentrations.
Fig. 4 is the selective result figure that electrochemical luminescence biosensor detects NAP-2 albumen.
Fig. 5 is the stability result figure that electrochemical luminescence biosensor detects NAP-2 albumen.
Specific embodiment
Below with reference to specific embodiment, the present invention is described in further detail, it is described be explanation of the invention and
It is not to limit.
A kind of preparation method for the electrochemical luminescence biosensor detecting early stage of lung cancer marker, early stage lung detected
Carcinoma marker is NAP-2 albumen, and specific preparation process is as follows:
(1) preparation of p-aminobenzoic acid (4-ABA) modified electrode
A. first 4-ABA is dissolved in 0.5M HCl solution, adds NaNO2.Under dark condition, the system is in ice-water bath
5-20min is reacted under conditions of middle stirring and logical nitrogen, obtains the p-aminobenzoic acid solution with diazonium cations.P-aminophenyl
Formic acid and NaNO2Molar ratio be 1:(0.8-1.2).
B. by the working electrode that diameter is 3mm successively use 1.0 μM, 0.3 μM, at 0.05 μM of aluminum oxide powder polishing
Reason, it is then successively clean spare with ethyl alcohol and deionized water ultrasonic cleaning again.With in above-mentioned steps a with diazonium cations to ammonia
Yl benzoic acid solution is electrolyte, is to electrode and Ag/AgCl as reference electrode (saturation KCl) using working electrode, platinum electrode
The three-electrode system of composition carries out cyclic voltammetry scan, and potential range is+0.5V~+2.0V, with 20-100mVS-1Sweep speed
Scan round 2-10 circle.Diazotizing p-aminobenzoic acid is set to be grafted to working electrode surface by way of electrochemical reduction,
Obtain 4-ABA modified electrode.Working electrode can be in glass-carbon electrode (GCE), graphite electrode, ITO electrode and noble metal electrode
Any one.
(2) preparation of NAP-2 protein-specific identification probe
TW and J1-Ru is in DNA buffer (25mM KCl, 1mM MgCl2, 50mM Tris-HCl (pH 8.0)) medium body
Product mixing, heats 2-10 minutes in 60-90 DEG C of water-bath and is hybridized to TW/J1-Ru, be then slowly cooled to room temperature and continue to hybridize
10-15h obtains NAP-2 protein-specific identification probe, is named as TW/J1-Ru probe.The molar ratio of TW and J1-Ru is 1:
(1.1-1.5).Related DNA sequence dna is as follows:
TW:5 '-NH2-(CH2)6-O-TCT CCA GCG TCG AGG TTT TCT GGT ATT ATA CCA GCC GTT
ACG CAA TCC TTC TCG AGC TAA A-3'
J1-Ru:Ru-HN-(CH2)6- O-5'-TTT AGC TCG AGA CGA CGC TGG AGA-3'(3) electrochemistry hair
The assembling of optical sensor
A. it is slow 4-ABA modified electrode in step (1) to be immersed in the 50mM Tris-HCl containing 8mM NHS and 5mM EDC
0.5-2h is activated in fliud flushing (pH=8.0), is then eluted with 50mM Tris-HCl buffer (pH=8.0), the 4- activated
ABA modified electrode.
B. the 4-ABA modified electrode after activation is immersed in TW/J1-Ru probe solution obtained in step (2) and is modified
Then 0.5-2h uses DNA buffer solution (25mM KCl, 1mM MgCl2, 50mM Tris-HCl (pH=8.0)) rinse remove inhale
Attached substance obtains TW/J1-Ru/4-ABA modified electrode.Wherein, the concentration of TW/J1-Ru probe solution is 0.01-2 μM, example
It can be such as any concentration in 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 2 μM.
C. the DNA buffer (pH=8.0) that 10-30 μ L contains 0.05%-1% bovine serum albumin (BSA) is dripped into step b
Gained TW/J1-Ru/4-ABA modified electrode surface-closed 30-60min, to reduce any non-specific adsorption of electrode surface,
After with 50mM Tris-HCl buffer (pH=8.0) rinse electrode surface to get to detectable lung cancer marker (NAP-
2) electrochemical luminescence biosensor.The concentration of Tris-HCl buffer solution can be any value in 0.01-0.1M, pH value
It can be any value in 7-8.
The application method of the electrochemical luminescence biosensor of above-mentioned detection early stage of lung cancer marker, specifically includes following step
It is rapid:
(1) using the electrochemical luminescence biosensor of the detection lung cancer marker as working electrode, Ag/AgCl electrode
For reference electrode (saturation KCl), platinum electrode is to construct three-electrode system to electrode；
(2) in electrochemical luminescence test fluid, electrochemical luminescence intensity I is tested0, the electrochemical method of use: circulation volt
An Fa；Scanning range: 0.2V-1.35V；Sweep speed: 0.1VS-1；
(3) working electrode is immersed to DNA buffer solution (the 25mM KCl, 1mM MgCl of known NAP-2 protein concentration2、
50mM Tris-HCl (pH=8.0)) in, after 20-30min, the substance for removing absorption is rinsed with DNA buffer solution, in electrochemistry
In the test fluid that shines, test obtains the corresponding electrochemical luminescence intensity of the NAP-2 protein concentration；It repeats the step and obtains multiple groups not
The corresponding electrochemical luminescence intensity data with NAP-2 protein concentration；NAP-2 protein concentration range is 0.01pM-1.0pM, and is pressed
It is tested according to the ascending sequence of NAP-2 protein concentration；
(4) the corresponding electrochemical luminescence intensity data of multiple groups difference NAP-2 protein concentration obtained according to step (3), mould
It is quasi- to obtain the matched curve between NAP-2 protein concentration and electrochemical luminescence intensity；
(5) working electrode is immersed into DNA buffer solution (25mM KCl, 1mM MgCl of the 30 μ L containing sample to be tested2、50mM
Tris-HCl (pH 8.0)) in, after 30min, the substance for removing absorption is rinsed with DNA buffer solution (pH=8.0), in electrochemistry
In the test fluid that shines, electrochemical luminescence intensity is tested, sample to be tested is calculated according to electrochemical luminescence intensity and matched curve
The concentration of middle NAP-2 albumen.
The electrochemical luminescence test fluid is DNA buffer solution (25mM KCl, 1mM of tripropyl amine (TPA) containing 50mM (TPA)
MgCl2, 50mM Tris-HCl (pH=8.0)).
The present invention can detecte early stage of lung cancer marker using the electrochemical luminescence biosensor, step letter when test
It is single, after electrochemical luminescence biosensor assembles, in conjunction with lung cancer marker NAP-2 protein solution to be measured after 20-30min
Can step detection, be advantageously implemented in general population instant screening patients with lung cancer.
It is as follows to prepare embodiment.
Embodiment 1
A kind of preparation method for the electrochemical luminescence biosensor detecting early stage of lung cancer marker, sensor detection
Lung cancer marker is NAP-2 albumen, and specific preparation process is as follows:
(1) preparation of p-aminobenzoic acid (4-ABA) modified electrode
A. 25mL 0.5M HCl solution is first configured, then 0.0343g 4-ABA is added (in 25mL solution into the solution
Concentration is 10mM), in the case where ice bath stirring and logical nitrogen, add 0.0138g NaNO2(concentration is in 25mL solution
8mM).Under dark condition, which stirs in ice-water bath and reacts 20min under conditions of leading to nitrogen, obtains containing diazonium sun
The p-aminobenzoic acid solution of ion.
B. by the glass-carbon electrode that diameter is 3mm successively use 1.0 μM, 0.3 μM, at 0.05 μM of aluminum oxide powder polishing
Reason, it is then successively clean spare with ethyl alcohol and deionized water ultrasonic cleaning again.With p-aminophenyl first diazotizing in above-mentioned steps a
Acid solution is electrolyte, and using glass-carbon electrode as working electrode, platinum filament is to electrode, and Ag/AgCl is reference electrode (saturation KCl) group
At three-electrode system carry out cyclic voltammetry scan, potential range be+0.5V~+2.0V, with 20mV.S-1Sweep fast scan round
8 circles, scanning result figure are as shown in Figure 2.Diazotizing p-aminobenzoic acid covalent modification by way of electrochemical reduction is set to exist
Glassy carbon electrode surface obtains 4-ABA/GCE.
(2) preparation of NAP-2 protein-specific identification probe
With DNA buffer solution (25mM KCl, 1mM MgCl2, 50mM Tris-HCl (pH=8.0)) be respectively configured 1.0 μM
TW100uL and 1.5 μM of J1-Ru100uL, the DNA solution configured is mixed in equal volume.5 points are heated in 80 DEG C of water-baths
Clock is hybridized to TW/J1-Ru, continues hybridization 12h after being slowly cooled to room temperature and obtains NAP-2 protein-specific identification probe, the spy
Needle is named as TW/J1-Ru.Related DNA sequence dna is as follows:
TW:5 '-NH2-(CH2)6-O-TCT CCA GCG TCG AGG TTT TCT GGT ATT ATA CCA GCC GTT
ACG CAA TCC TTC TCG AGC TAA A-3'
J1:Ru-HN-(CH2)6-O-5'-TTT AGC TCG AGA CGA CGC TGG AGA-3'
(3) assembling of electrochemical luminescence sensor
A. the glass-carbon electrode modified of 4-ABA in step (1) is immersed in the 50mM that 30 μ L contain 8mM NHS and 5mM EDC
0.5h is activated in Tris-HCl buffer (pH=8.0), is then eluted, is obtained with 50mM Tris-HCl buffer (pH=8.0)
The 4-ABA/GCE of activation.
B. the 4-ABA/GCE after activation is immersed in TW/J1-Ru probe solution obtained in 30 μ L steps (2) and is modified
Then 1h uses DNA buffer solution (25mM KCl, 1mM MgCl2, 50mM Tris-HCl (pH=8.0)) rinse remove absorption
Substance obtains TW/J1-Ru/4-ABA/GCE.
C. the 50mM Tris-HCl buffer (pH=8.0) that 30 μ L contain 0.05%BSA is dripped to what step b was modified
Electrode surface close 30min, to reduce any non-specific adsorption of electrode surface, after use DNA buffer solution (25mM
KCl、1mM MgCl2, 50mM Tris-HCl (pH=8.0)) rinse electrode surface to get to detectable lung cancer marker (NAP-
2) electrochemical luminescence biosensor.
In the preparation process of above-mentioned p-aminobenzoic acid (4-ABA) modified electrode:
The covering in the 4-ABA molecular layer of electrode face finish can be controlled by the circle number of control loop voltammetric scan
Density, the oxidation peak of first lap carries out charge integration according to fig. 2, and the 4-ABA molecular layer modified in embodiment one is calculated
Coverage density be about 3.26 × 10-8mol·cm-2。
Embodiment 2
A kind of preparation method for the electrochemical luminescence biosensor detecting early stage of lung cancer marker, sensor detection
Lung cancer marker is NAP-2 albumen, and specific preparation process is as follows:
1) preparation of p-aminobenzoic acid (4-ABA) modified electrode
A. first 0.0343g 4-ABA (concentration 10mM) is dissolved in 25mL 0.5M HCl solution, is added
0.0172gNaNO2(concentration 10mM).Under dark condition, which stirs in ice-water bath and reacts under conditions of leading to nitrogen
5min obtains the p-aminobenzoic acid solution containing diazonium cations.
B. by the glass-carbon electrode that diameter is 3mm successively use 1.0 μM, 0.3 μM, at 0.05 μM of aluminum oxide powder polishing
Reason, it is then successively clean spare with ethyl alcohol and deionized water ultrasonic cleaning again.With p-aminophenyl first diazotizing in above-mentioned steps a
Acid solution is electrolyte, and using glass-carbon electrode as working electrode, platinum filament is to electrode, and Ag/AgCl is reference electrode (saturation KCl) group
At three-electrode system carry out cyclic voltammetry scan, potential range be+0.5V~+2.0V, with 50mV.S-1Sweep fast scan round
2 circles.Diazotizing p-aminobenzoic acid covalent modification by way of electrochemical reduction is set to obtain 4- in glassy carbon electrode surface
ABA/GCE。
(2) preparation of NAP-2 protein-specific identification probe
With DNA buffer solution (25mM KCl, 1mM MgCl2, 50mM Tris-HCl (pH=8.0)) 100 μ L are respectively configured
Its isometric mixing is heated 2 minutes in 60 DEG C of water-baths and is hybridized to TW/J1-Ru, slowly by 1.0 μM of TW and 1.1 μM of Ru-J1
Continue hybridization 10h after being cooled to room temperature and obtain NAP-2 protein-specific identification probe, which is named as TW/J1-Ru.It is involved
And DNA sequence dna it is as follows:
TW:5 '-NH2-(CH2)6-O-TCT CCA GCG TCG AGG TTT TCT GGT ATT ATA CCA GCC GTT
ACG CAA TCC TTC TCG AGC TAA A-3'
J1:Ru-HN-(CH2)6-O-5'-TTT AGC TCG AGA CGA CGC TGG AGA-3'
(3) assembling of electrochemical luminescence sensor
A. the glass-carbon electrode modified of 4-ABA in step (1) is immersed in the 50mM that 30 μ L contain 8mM NHS and 5mM EDC
0.5h is activated in Tris-HCl buffer (pH=8.0), is then eluted, is obtained with 50mM Tris-HCl buffer (pH=8.0)
The 4-ABA/GCE of activation.
B. the 4-ABA/GCE after activation is immersed in TW/J1-Ru probe solution obtained in 30 μ L steps (2) and is modified
Then 1h uses DNA buffer solution (25mM KCl, 1mM MgCl2, 50mM Tris-HCl (pH=8.0)) rinse remove absorption
Substance obtains TW/J1-Ru/4-ABA/GCE.
C. the 50mM Tris-HCl buffer (pH=8.0) that 30 μ L contain 0.05%BSA is dripped to what step b was modified
Electrode surface close 30min, to reduce any non-specific adsorption of electrode surface, after use DNA buffer solution (25mM
KCl、1mM MgCl2, 50mM Tris-HCl (pH 8.0)) rinse electrode surface to get to detectable lung cancer marker (NAP-
2) electrochemical luminescence biosensor.
Embodiment 3
A kind of preparation method for the electrochemical luminescence biosensor detecting early stage of lung cancer marker, sensor detection
Lung cancer marker is NAP-2 albumen, and specific preparation process is as follows:
(1) preparation of p-aminobenzoic acid (4-ABA) modified electrode
A. first 0.0343g 4-ABA (concentration 10mM) is dissolved in 25mL 0.5M HCl solution, adds 0.207g
NaNO2(concentration 12mM).Under dark condition, which stirs in ice-water bath and reacts 10min under conditions of leading to nitrogen, obtains
P-aminobenzoic acid solution containing diazonium cations.
B. by the glass-carbon electrode that diameter is 3mm successively use 1.0 μM, 0.3 μM, at 0.05 μM of aluminum oxide powder polishing
Reason, it is then successively clean spare with ethyl alcohol and deionized water ultrasonic cleaning again.With p-aminophenyl first diazotizing in above-mentioned steps a
Acid solution is electrolyte, and using glass-carbon electrode as working electrode, platinum filament is to electrode, and Ag/AgCl is reference electrode (saturation KCl) group
At three-electrode system carry out cyclic voltammetry scan, potential range be+0.5V~+2.0V, with 100mV.S-1Sweep speed circulation sweep
Retouch 4 circles.Diazotizing p-aminobenzoic acid covalent modification by way of electrochemical reduction is set to obtain 4- in glassy carbon electrode surface
ABA/GCE。
(2) preparation of NAP-2 protein-specific identification probe
With DNA buffer solution (25mM KCl, 1mM MgCl2, 50mM Tris-HCl (pH=8.0)) 100 μ L are respectively configured
Its isometric mixing is heated 10 minutes in 90 DEG C of water-baths and is hybridized to TW/J1-Ru, slowly by 1.0 μM of TW and 1.2 μM of Ru-J1
Continue hybridization 15h after being cooled to room temperature and obtain NAP-2 protein-specific identification probe, which is named as TW/J1-Ru.It is involved
And DNA sequence dna it is as follows:
TW:5 '-NH2-(CH2)6-O-TCT CCA GCG TCG AGG TTT TCT GGT ATT ATA CCA GCC GTT
ACG CAA TCC TTC TCG AGC TAA A-3'
J1:Ru-HN-(CH2)6-O-5'-TTT AGC TCG AGA CGA CGC TGG AGA-3'
(3) assembling of electrochemical luminescence sensor
A. the glass-carbon electrode modified of 4-ABA in step (1) is immersed in the 50mM that 30 μ L contain 8mM NHS and 5mM EDC
0.5h is activated in Tris-HCl buffer (pH=8.0), is then eluted, is obtained with 50mM Tris-HCl buffer (pH=8.0)
The 4-ABA/GCE of activation.
B. the 4-ABA/GCE after activation is immersed in TW/J1-Ru probe solution obtained in 30 μ L steps (2) and is modified
Then 1h uses DNA buffer solution (25mM KCl, 1mM MgCl2, 50mM Tris-HCl (pH=8.0)) rinse remove absorption
Substance obtains TW/J1-Ru/4-ABA/GCE.
C. the 50mM Tris-HCl buffer (pH 8.0) that 30 μ L contain 0.05%BSA is dripped into the electricity that step b is modified
Pole surface close 30min, to reduce any non-specific adsorption of electrode surface, after use DNA buffer solution (25mM
KCl、1mM MgCl2, 50mM Tris-HCl (pH 8.0)) rinse electrode surface to get to detectable lung cancer marker (NAP-
2) electrochemical luminescence biosensor.
Embodiment 4
A kind of preparation method for the electrochemical luminescence biosensor detecting early stage of lung cancer marker, sensor detection
Lung cancer marker is NAP-2 albumen, and specific preparation process is as follows:
(1) preparation of p-aminobenzoic acid (4-ABA) modified electrode
A. first 0.0343g 4-ABA (concentration 10mM) 4-ABA is dissolved in 25mL 0.5M HCl solution, is added
0.0138g (concentration 8mM) NaNO2.Under dark condition, which stirs in ice-water bath and reacts under conditions of leading to nitrogen
20min obtains the p-aminobenzoic acid solution containing diazonium cations.
B. by the glass-carbon electrode that diameter is 3mm successively use 1.0 μM, 0.3 μM, at 0.05 μM of aluminum oxide powder polishing
Reason, it is then successively clean spare with ethyl alcohol and deionized water ultrasonic cleaning again.With p-aminophenyl first diazotizing in above-mentioned steps a
Acid solution is electrolyte, and using glass-carbon electrode as working electrode, platinum filament is to electrode, and Ag/AgCl is reference electrode (saturation KCl) group
At three-electrode system carry out cyclic voltammetry scan, potential range be+0.5V~+2.0V, with 20mV.S-1Sweep fast scan round
8 circles.Diazotizing p-aminobenzoic acid covalent modification by way of electrochemical reduction is set to obtain 4- in glassy carbon electrode surface
ABA/GCE。
(2) preparation of NAP-2 protein-specific identification probe
With DNA buffer solution (25mM KCl, 1mM MgCl2, 50mM Tris-HCl (pH 8.0)) 100 μ L are respectively configured
Its isometric mixing is heated 5 minutes in 80 DEG C of water-baths and is hybridized to TW/J1-Ru, slowly by 1.0 μM of TW and 1.5 μM of Ru-J1
Continue hybridization 12h after being cooled to room temperature and obtain NAP-2 protein-specific identification probe, which is named as TW/J1-Ru.It is involved
And DNA sequence dna it is as follows:
TW:5 '-NH2-(CH2)6-O-TCT CCA GCG TCG AGG TTT TCT GGT ATT ATA CCA GCC GTT
ACG CAA TCC TTC TCG AGC TAA A-3'
J1:Ru-HN-(CH2)6-O-5'-TTT AGC TCG AGA CGA CGC TGG AGA-3'
(3) assembling of electrochemical luminescence sensor
A. the glass-carbon electrode modified of 4-ABA in step (1) is immersed in the 50mM that 30 μ L contain 8mM NHS and 5mM EDC
1h is activated in Tris-HCl buffer (pH=8.0), is then eluted, is lived with 50mM Tris-HCl buffer (pH=8.0)
The 4-ABA/GCE of change.
B. the 4-ABA/GCE after activation is immersed in TW/J1-Ru probe solution obtained in 30 μ L steps (2) and is modified
Then 0.5h uses DNA buffer solution (25mM KCl, 1mM MgCl2, 50mM Tris-HCl (pH=8.0)) rinse remove absorption
Substance, obtain TW/J1-Ru/4-ABA/GCE.
C. the 50mM Tris-HCl buffer (pH=8.0) that 30 μ L contain 0.1%BSA is dripped into the electricity that step b is modified
Pole surface close 45min, to reduce any non-specific adsorption of electrode surface, after use DNA buffer solution (25mM
KCl、1mM MgCl2, 50mM Tris-HCl (pH=8.0)) rinse electrode surface to get to detectable lung cancer marker (NAP-
2) electrochemical luminescence biosensor.
Embodiment 5
A kind of preparation method for the electrochemical luminescence biosensor detecting early stage of lung cancer marker, sensor detection
Lung cancer marker is NAP-2 albumen, and specific preparation process is as follows:
(1) preparation of p-aminobenzoic acid (4-ABA) modified electrode
A. first 0.0343g 4-ABA (concentration 10mM) is dissolved in 25mL 0.5M HCl solution, adds 0.0138g
(concentration 8mM) NaNO2.Under dark condition, which stirs in ice-water bath and reacts 20min under conditions of leading to nitrogen, obtains
To the p-aminobenzoic acid solution containing diazonium cations.
B. by the glass-carbon electrode that diameter is 3mm successively use 1.0 μM, 0.3 μM, at 0.05 μM of aluminum oxide powder polishing
Reason, it is then successively clean spare with ethyl alcohol and deionized water ultrasonic cleaning again.With p-aminophenyl first diazotizing in above-mentioned steps a
Acid solution is electrolyte, and using glass-carbon electrode as working electrode, platinum filament is to electrode, and Ag/AgCl is reference electrode (saturation KCl) group
At three-electrode system carry out cyclic voltammetry scan, potential range be+0.5V~+2.0V, with 20mV.S-1Sweep fast scan round
8 circles.Diazotizing p-aminobenzoic acid covalent modification by way of electrochemical reduction is set to obtain 4- in glassy carbon electrode surface
ABA/GCE。
(2) preparation of NAP-2 protein-specific identification probe
With DNA buffer solution (25mM KCl, 1mM MgCl2, 50mM Tris-HCl (pH=8.0)) 100 μ L are respectively configured
Its isometric mixing is heated 5 minutes in 80 DEG C of water-baths and is hybridized to TW/J1-Ru, slowly by 1.0 μM of TW and 1.5 μM of Ru-J1
Continue hybridization 12h after being cooled to room temperature and obtain NAP-2 protein-specific identification probe, which is named as TW/J1-Ru.It is involved
And DNA sequence dna it is as follows:
TW:5 '-NH2-(CH2)6-O-TCT CCA GCG TCG AGG TTT TCT GGT ATT ATA CCA GCC GTT
ACG CAA TCC TTC TCG AGC TAA A-3'
J1:Ru-HN-(CH2)6-O-5'-TTT AGC TCG AGA CGA CGC TGG AGA-3'
(3) assembling of electrochemical luminescence sensor
A. the glass-carbon electrode modified of 4-ABA in step (1) is immersed in the 50mM that 30 μ L contain 8mM NHS and 5mM EDC
2h is activated in Tris-HCl buffer (pH=8.0), is then eluted, is lived with 50mM Tris-HCl buffer (pH=8.0)
The 4-ABA/GCE of change.
B. the 4-ABA/GCE after activation is immersed in TW/J1-Ru probe solution obtained in 30 μ L steps (2) and is modified
Then 2h uses DNA buffer solution (25mM KCl, 1mM MgCl2, 50mM Tris-HCl (pH 8.0)) rinse remove absorption
Substance obtains TW/J1-Ru/4-ABA/GCE.
C. the 50mM Tris-HCl buffer (pH 8.0) that 30 μ L contain 1%BSA is dripped into the electrode that step b is modified
Surface-closed 60min, to reduce any non-specific adsorption of electrode surface, after with DNA buffer solution (25mM KCl,
1mM MgCl2, 50mM Tris-HCl (pH 8.0)) rinse electrode surface to get to detectable lung cancer marker (NAP-2)
Electrochemical luminescence biosensor.
Application example
Example 1NAP-2 Protein Detection
A kind of application method for the biosensor detection NAP-2 albumen detecting early stage of lung cancer marker, including walk as follows
It is rapid:
(1) the electrochemical luminescence bio-sensing for the detection early stage of lung cancer marker being prepared with above-mentioned preparation embodiment 1
Device is working electrode, and Ag/AgCl electrode is reference electrode, and platinum electrode is to construct three-electrode system to electrode；
(2) in electrochemical luminescence test fluid, electrochemical luminescence intensity I is tested0, the electrochemical method of use: circulation volt
An Fa；Scanning range: 0.2V-1.35V；Sweep speed: 0.1VS-1；The electrochemical luminescence test fluid is tripropyl amine (TPA) containing 50mM
(TPA) DNA buffer solution (25mM KCl, 1mM MgCl2, 50mM Tris-HCl (pH=8.0)).
(3) again by working electrode prepared by embodiment 1 be successively immersed in 50 μ L concentration be respectively 0.01pM, 0.02pM,
In the solution to be measured of the NAP-2 albumen of 0.03pM, 0.04pM, 0.05pM, 0.07pM, 0.1pM, 0.5pM, 1.0pM, after 20min,
It is cleaned with DNA buffer solution (pH=8.0), in electrochemical luminescence test solution, tests electrochemical luminescence when various concentration
Intensity I；
(4) according to test result, it is found that electrochemical luminescence intensity I is within the scope of 0.01-1.0pM in NAP-2 protein concentration
Y=-19884x+1732.8 in a linear relationship, R=0.9921 (the corresponding electrochemical luminescence letter of the NAP-2 albumen of various concentration
Number, as shown in Figure 3).
(5) working electrode prepared by embodiment 1 is immersed in the solution to be measured of 50 μ L albumen containing NAP-2,20min
Afterwards, it is cleaned with DNA buffer solution (pH=8.0), in electrochemical luminescence test solution, tests electrochemical luminescence intensity；According to
Linear relationship between electrochemical luminescence intensity and NAP-2 protein concentration, the concentration value C of obtained NAP-2 albumenNAP-2, unit
For pM.
By the example 1 it is found that electrochemical luminescence intensity value and NAP-2 protein concentration are in 0.01pM~1.0pM concentration range
It is interior in a linear relationship, therefore, 3 σ of calculation formula/S is limited according to detection, is computed the detection lung cancer early stage that embodiment 1 is prepared
The detection of the electrochemical luminescence sensor detection NAP-2 albumen of marker is limited up to 0.008pM, this illustrates electrochemistry of the invention
The sensitivity of luminescence sensor is very high.
2 selectivity test of example:
The electrochemical luminescence biosensor for the detection early stage of lung cancer marker being prepared with above-mentioned preparation embodiment 1 is
Working electrode, experiment condition is identical as examples detailed above 1, respectively detect 0.2pM common interference albumen: MMP14, CD44, PEX-14,
Chymotrypsin (CT), Albumin, Mb and 0.01pM NAP-2 albumen and the mixing of the above-mentioned several albumen of 0.2pM are to be measured
Liquid, as a result as shown in Figure 4.
The result shows that: in addition to target protein NAP-2, after remaining interference albumen and the interaction of ECL biosensor, Δ I/
I0Without generation significant change (Δ I=I0-I).In addition to this, ECL sensor is in the mixing containing the above-mentioned common proteins of 0.2pM
The Δ I/I of 0.01pM NAP-2 is detected in liquid0Value is with detection error when independent 0.01pM NAP-2 less than 11%.This explanation
200 times of common interference albumen does not influence to detect, and ECL biosensor has preferable selectivity.This is primarily due to give birth to
Object sensor is to induce TW/J1-Ru probe conformation to change by the specific binding of NAP-2 albumen and its aptamers.
3 stability test of example
The electrochemical luminescence biosensor for the detection early stage of lung cancer marker being prepared with above-mentioned preparation embodiment 1 is
Working electrode, experiment condition is identical as examples detailed above 1, after ECL biosensor and 0.03pM NAP-2 protein-interacting,
Continuous scanning 20 encloses ECL response signal under constant potential+1.35V, as a result as shown in Figure 5.The ECL signal of ECL biosensor
The relative standard deviation that intensity continuous scanning 20 is enclosed is 2.5%.The result shows that the present invention is using the covalent coupled method preparation of C-N
ECL biosensor is with good stability.
Only several embodiments of the present invention are expressed for above-described embodiment, and the description thereof is more specific and detailed, but cannot manage
Solution is limitations on the scope of the patent of the present invention.It should be pointed out that for those of ordinary skill in the art, not departing from
Under the premise of present inventive concept, various modifications and improvements can be made, and these are all within the scope of protection of the present invention, the present invention
Protection scope be subject to claims.
Claims (10)
1. a kind of preparation method for the electrochemical luminescence biosensor for detecting early stage of lung cancer marker NAP-2 albumen, feature
It is, includes the following steps:
Step 1, working electrode is placed in the p-aminobenzoic acid solution with diazonium cations, passes through cyclic voltammetry scan legal system
It is standby to obtain p-aminobenzoic acid modified electrode；
Step 2, preparation NAP-2 protein-specific identifies probe:
TW and J1-Ru is added in DNA buffer and is mixed, heating is hybridized to TW/J1-Ru, then cools to room temperature and continues to hybridize
NAP-2 protein-specific identification probe is obtained, TW/J1-Ru probe is named as；
Step 3, the assembling of electrochemical luminescence sensor；
Step 3.1, p-aminobenzoic acid modified electrode in step 1 is immersed in the Tris-HCl buffer containing NHS and EDC
Activation, the 4-ABA modified electrode activated；
Step 3.2, the 4-ABA modified electrode after activation is immersed in TW/J1-Ru probe solution obtained in step 2 and is modified,
Obtain TW/J1-Ru/4-ABA modified electrode；
Step 3.3, the DNA buffer containing bovine serum albumin is dripped into step 3.2 gained TW/J1-Ru/4-ABA modified electrode
Surface is closed, and electrochemical luminescence biosensor is obtained.
2. the electrochemical luminescence biosensor of detection early stage of lung cancer marker NAP-2 albumen according to claim 1
Preparation method, which is characterized in that step 1 includes the following steps:
Step 1.1, first p-aminobenzoic acid is dissolved in HCl solution, adds NaNO2, stir under ice-water bath and logical condition of nitrogen gas
5-20min is reacted, the p-aminobenzoic acid solution with diazonium cations is obtained；P-aminobenzoic acid and NaNO2Molar ratio be
1:(0.8-1.2)；
Step 1.2, using step 1.1 prepare the p-aminobenzoic acid solution with diazonium cations as electrolyte, with working electrode,
The three-electrode system formed to electrode and reference electrode carries out cyclic voltammetry scan, obtains p-aminobenzoic acid modified electrode.
3. the electrochemical luminescence biosensor of detection early stage of lung cancer marker NAP-2 albumen according to claim 1
Preparation method, which is characterized in that in step 1, working electrode is in glass-carbon electrode, graphite electrode, ITO electrode and noble metal electrode
One kind.
4. the electrochemical luminescence biosensor of detection early stage of lung cancer marker NAP-2 albumen according to claim 1
Preparation method, which is characterized in that in step 2, the molar ratio of TW and J1-Ru are 1:(1.1-1.5).
5. the electrochemical luminescence biosensor of detection early stage of lung cancer marker NAP-2 albumen according to claim 1
Preparation method, which is characterized in that in step 2,
TW:5 '-NH2-(CH2)6-O-TCT CCA GCG TCG AGG TTT TCT GGT ATT ATA CCA GCC GTT ACG
CAA TCC TTC TCG AGC TAAA-3'；
J1-Ru:Ru-HN-(CH2)6-O-5'-TTT AGC TCG AGA CGA CGC TGG AGA-3'。
6. the electrochemical luminescence biosensor of detection early stage of lung cancer marker NAP-2 albumen according to claim 1
Preparation method, which is characterized in that in step 2, first under the conditions of 60-90 DEG C heating hybridization 2-10min, then cool to room temperature after
Continuous hybridization 10-15h.
7. the electrochemical luminescence biosensor of detection early stage of lung cancer marker NAP-2 albumen according to claim 1
Preparation method, which is characterized in that in step 3.1, activation time 0.5-2h.
8. the electrochemical luminescence biosensor of detection early stage of lung cancer marker NAP-2 albumen according to claim 1
Preparation method, which is characterized in that in step 3.2, the concentration of TW/J1-Ru probe solution is 0.01-2 μM, and the modification time is 0.5-
2h。
9. the electrochemical luminescence biosensor of detection early stage of lung cancer marker NAP-2 albumen according to claim 1
Preparation method, which is characterized in that in step 3.3, bovine serum albumen solution mass concentration is 0.05%-1%, and off-period is
30-60min。
10. the detection early stage of lung cancer marker NAP-2 albumen that the described in any item preparation methods of claim 1-9 are prepared
Electrochemical luminescence biosensor.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102507689A (en) * | 2011-10-19 | 2012-06-20 | 青岛科技大学 | Manufacturing method and application of electrochemiluminescence sensor for detecting thrombin |
| CN104655616A (en) * | 2015-01-23 | 2015-05-27 | 宁波大学 | Preparation method and application of electrochemiluminescence aptamer sensor for detecting tumor marker MUC1 |
-
2019
- 2019-01-30 CN CN201910094175.6A patent/CN109856210B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102507689A (en) * | 2011-10-19 | 2012-06-20 | 青岛科技大学 | Manufacturing method and application of electrochemiluminescence sensor for detecting thrombin |
| CN104655616A (en) * | 2015-01-23 | 2015-05-27 | 宁波大学 | Preparation method and application of electrochemiluminescence aptamer sensor for detecting tumor marker MUC1 |
Non-Patent Citations (2)
| Title |
|---|
| FEN MA ET AL.: "Electrogenerated chemiluminescence biosensor for detection of mercury (II) ion via target-triggered manipulation of DNA three-way junctions", 《TALANTA》 * |
| JASON M. THOMAS ET AL.: "Analyte-Driven Switching of DNA Charge Transport: De Novo Creation of Electronic Sensors for an Early Lung Cancer Biomarker", 《J. AM. CHEM. SOC.》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116908265A (en) * | 2023-09-11 | 2023-10-20 | 常州先趋医疗科技有限公司 | Preparation method of electrochemical biosensor for detecting LAMP amplification products of nucleic acids |
| CN116908265B (en) * | 2023-09-11 | 2023-12-12 | 常州先趋医疗科技有限公司 | Preparation method of electrochemical biosensor for detecting LAMP amplification products of nucleic acids |
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