CN110749585B - Biosensor based on surface enhanced Raman technology and preparation method thereof - Google Patents
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
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Abstract
The invention provides a biosensor based on a surface enhanced Raman technology and a preparation method thereof, wherein the biosensor is of a three-layer sandwich structure and comprises an upper layer structure: a gold core silver shell nanorod bonded with the Raman detection molecule and the detection antibody; the structure of the middle layer: an antigen; the following layer structure: the sandwich immunoassay biosensor based on the surface enhanced Raman technology greatly enhances detection signals through the combination of Raman detection molecules and the gold-core silver-shell nanorod, obviously improves the sensitivity of detection of ultra-trace substances and single molecules, and can reach fg/ml at the lowest limit.
Description
Technical Field
The invention relates to the field of Raman scattering, in particular to a sandwich immunoassay biosensor based on a surface enhanced Raman technology.
Background
At present, the commonly used immunoassay method is enzyme-linked immunosorbent assay (ELISA), the method detects trace substances in body fluid by the specific combination of an antibody and an enzyme complex and then color development detection, the detection limit of the method can reach pg/ml, but substances with higher sensitivity requirements cannot be detected.
Therefore, in order to reliably and accurately detect ultra-trace or even single-molecule level analytes, Surface Enhanced Raman (SERS) has been gradually developed and widely applied to various fields such as chemical sensing, biological analysis, biosensing, and early cancer diagnosis. Typical SERS immunoassay detection methods are based on sandwich immune complexes capable of capturing antibodies immobilized on a solid substrate. The corresponding antigen was captured from the sample solution and detected by characteristic raman spectroscopy of SERS-labeled antibodies after sandwich formation. Quantitative information was obtained from dose-dependent SERS experiments.
Schl ü cker and coworkers developed a sandwich-type SERS immunoassay method for detecting interleukin-6 (IL-6) on the basis of a gold/silver nanoshell-glass slice sandwich structure, and the limit of detection (LOD) of the SERS immunoassay method is 1 pg/mL. In recent years, based on a silver-gold bimetal surface-gold nanoparticle sandwich structure, simultaneous multiple recognition of IL-6, IL-8 and IL-18 is realized under picogram detection sensitivity. In addition, multiplex detection of CA19-9, MMP7 and MUC4 in serum samples from pancreatic cancer patients has been demonstrated on a gold substrate-gold nanoparticle sandwich platform with detection limits as low as 2 ng/mL. However, the conventional sandwich immunoassay design method mostly adopts a smooth macroscopic glass, gold, silver film or a double-metal film substrate thereof, resulting in limited sensitivity.
In order to improve the sensitivity of SERS detection, currently, a substrate material for SERS detection is bonded with Raman detection molecules, wherein the best effect is P-ATP molecules, and the sensitivity is ng/ml-pg/ml.
Disclosure of Invention
The invention provides a sandwich immunoassay biosensor based on a surface enhanced Raman technology, aiming at solving the problem of limited sensitivity of a sandwich type SERS immunoassay method in the prior art, comprising:
the top layer is a gold-core silver-shell nanorod bonded vitamin K molecule, a functional modifier and a detection antibody; and the substrate is a gold-core silver-shell nanorod bonding capture antibody.
Further, the functional modifier comprises glutaraldehyde and cysteamine.
Further, the biosensor can form a sandwich structure of detection antibody-antigen to be detected-capture antibody during working.
The invention provides a sandwich immunoassay biosensor based on a surface enhanced Raman technology, wherein a top layer and a substrate both adopt gold-core silver-shell nanorods as base materials, the gold-core silver-shell nanorods can remarkably improve electromagnetic signals in the surface Raman detection process, on the basis, the gold-core silver-shell nanorod at the top layer is firmly bonded with a diazotized sulfydryl vitamin K4 molecule through sulfydryl, so that the signal is further obviously enhanced, simultaneously, functional modified molecules of glutaraldehyde and cysteine are bonded to firmly fix the detection antibody by the top layer gold core silver shell nanorod and bond the capture antibody by the gold core silver shell nanorod serving as the substrate, the sandwich immunoassay biosensor based on the surface enhanced Raman technology can obviously improve the sensitivity of surface enhanced Raman detection by combining and fixing the antigen to be detected through specificity and integrating the structure.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
Drawings
Further objects, features and advantages of the present invention will become apparent from the following description of embodiments of the invention, with reference to the accompanying drawings, in which:
FIG. 1 shows a sandwich structure of "detection antibody-antigen to be detected-capture antibody" formed by a biosensor and an antigen to be detected in the present invention;
FIG. 2 shows a Raman spectrum of a SERS immunoassay with an IL-6 concentration of 1fg/mL and a blank sample in example 1;
FIG. 3 shows Raman intensity as a function of KIM1 concentration;
FIG. 4 shows 1145cm-1The raman intensity of the peak is plotted as a function of KIM1 concentration, and the linear dynamic response range is shown in the inset.
Detailed Description
The objects and functions of the present invention and methods for accomplishing the same will be apparent by reference to the exemplary embodiments. However, the present invention is not limited to the exemplary embodiments disclosed below; it can be implemented in different forms. The nature of the description is merely to assist those skilled in the relevant art in a comprehensive understanding of the specific details of the invention.
Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In the drawings, like reference characters designate like or similar parts, or like or similar steps.
Example 1
The embodiment provides a sandwich immunoassay biosensor based on a surface enhanced Raman technology, which is used for SERS immunoassay of an IL-6 solution with a concentration of 1fg/mL and a blank sample, and the specific process is as follows:
1. preparation of gold nanorods
1) 0.1mL of 25mM HAuCl in a 20mL glass vial4Diluting the solution to 5mL by using deionized water, and adding 5mL of 0.2M CTAB solution into the diluted solution to obtain a first solution;
2) adding 0.6mL0.01M NaBH4Solution is injected into solution one quickly, NaBH4The solution is prepared on site, the mixed solution is stirred by magnetic force at the speed of 1200rpm for 2min, and finally the obtained seed solution is kept stand for 30min at the temperature of 30 ℃ for standby;
3) dissolving 7.0g of CTAB and 1.234g of sodium oleate in 250mL of 50 ℃ water, naturally cooling to 30 ℃, adding 18mL of 4.0mM silver nitrate solution, and keeping the temperature for 1min to obtain a solution II;
4) 250ml of 1.0mM HAuCl was injected into the second solution while magnetically stirring4Stirring the solution at 700rpm for 90min, changing the second magnetic stirring speed to 400rpm, adding 2.1ml of 37 wt% HCl solution while stirring, stirring for 15min, finally adding 1.25 ml of 0.064M ascorbic acid solution, stirring for the third time at 1200rpm, and stirring for 30s to obtain a growth solution;
5) injecting 0.4mL of seed solution into the growth solution, stirring at 1500rpm for 30s, standing the mixed solution at 30 ℃ for 10h, centrifuging the standing mixed solution at 8000r/min for 10min, collecting precipitate, and dispersing the precipitate in 80mM CTAC solution;
6) repeating the step 5) for three times, storing the obtained precipitate in a CTAC solution to obtain a gold nanorod solution
2. Preparation of gold-core silver-shell nanorod
Diluting 0.5mL of gold nanorod solution to 4mL with water, adding 2.5mL of 10 mM silver nitrate solution into the diluent, carrying out ultrasonic treatment for 2min at the frequency of 1000Hz, then adding 2.5mL of 0.1M ascorbic acid solution, preserving in a water bath at 65 ℃ for 4h, centrifuging at 8000r/min for 10min, collecting precipitate, and dispersing in 1mL of deionized water to obtain the gold-core-silver-shell nanorod suspension.
3. Raman detection of molecular bonding
2 mu L of 5mM vitamin K4 ethanol solution is diazotized and sulfydryl is introduced into the solution, then the solution is added into 1.0ml gold core silver shell nano rod suspension to obtain a mixture, and the mixture is gently shaken for 2h to obtain the vitamin K4 marked gold core silver shell nano rod solution.
4. Preparation of top layer structure with labeled detection antibody
mu.L of 5mM ethanol solution of biotin K4 was diazotized to thiolate to introduce a thiol group, and added to 1.0mL of gold core-silver shell nanorod solution, and then the mixture was gently shaken for 2h to give vitamin K4-labeled gold core-silver shell nanorod solution, which was then centrifuged at 7000rpm for 10 minutes and dispersed into 1.0mL of deionized water. Next, 2. mu.L of a 25% wt glutaraldehyde solution was added to 1.0mL of a vitamin K4-labeled gold-core silver-shell nanorod solution, gently shaken for 1.5h, the resultant was centrifuged at 6000rpm for 10min, and then dispersed in 1mL of a 9. mu.g/mL detection antibody solution for IL-6, and the mixed solution was stored at 4 ℃ for 12h, then centrifuged and purified with 1.0mL of 1 XPBS buffer, and stored at 4 ℃ for use.
5. Preparation of substrate Structure with Capture antibody
Firstly, carrying out amino functionalization treatment on a gold-core silver-shell nanorod solution by using a 25mM cysteamine solution, then adding 2 mu L of 25% wt glutaraldehyde solution to functionalize the gold-core silver-shell nanorod solution, then adding 2mL of capture antibody solution of 20 mu g/mL IL-6 into the functionalized gold-core silver-shell nanorod solution, preserving at the temperature of 4 ℃ for 12 hours, next, adding 2mL of 1% BSA solution into the preserved solution, and standing for 1 hour to block non-specific binding active sites.
6. Immobilization of IL-6 antigen
mu.L, 1fg/mL of IL-6 antigen and blank samples, respectively, were dropped onto the substrate with capture antibody and incubated at room temperature for 1 h.
7. Sandwich structure assembly of detection antibody-antigen to be detected-capture antibody
200 mu L of vitamin K4-labeled gold core silver shell nanorod solution bonded with the IL-6 detection antibody is dripped into the substrate solution fixed with the antigen to be detected, and a detection antibody-antigen to be detected-capture antibody sandwich structure is formed after specific binding.
Example 2
The embodiment provides a sandwich immunoassay biosensor based on a surface enhanced raman technique, which is used for measuring KIM1 in artificial urine, and comprises the following specific steps:
using the structure of the sandwich immunoassay biosensor based on the surface enhanced raman technique in example 1, the top layer was bonded with KIM1 detection antibody and the substrate was bonded with KIM1 capture antibody as follows:
first, 6.0mL of a gold core silver shell nanorod solution labeled with vitamin K4 was taken, centrifuged at 7000rpm for 10 minutes and dispersed into 6.0mL of deionized water. Next, 12. mu.L of a 25% wt glutaraldehyde solution was added to 6.0mL of a vitamin K4-labeled gold-core silver-shell nanorod solution, gently shaken for 1.5h, the resultant was centrifuged at 6000rpm for 10min, the precipitate was dispersed in 6mL of a 9. mu.g/mL KIM1 detection antibody solution to obtain a top layer structure solution, and the mixture was stored at 4 ℃ for 12h, then centrifuged and purified with 1.0mL of 1 XPBS buffer, and stored at 4 ℃ for use.
Then, the gold-core silver-shell nanorod solution was subjected to amino functionalization treatment with 150mM cysteamine solution, then, 12. mu.L of 25% wt glutaraldehyde solution was added to functionalize the gold-core silver-shell nanorod solution, then, 12mL of 20. mu.g/mL KIM1 capture antibody solution was added to the functionalized gold-core silver-shell nanorod solution, stored at 4 ℃ for 12 hours, next, 12mL of 1% BSA solution was added to the stored solution, and left to stand for 1 hour to block non-specific binding active sites, to obtain a substrate structure solution.
And (3) taking 200 mu L of each 6 parts of artificial urine solution, respectively adding KIM1 with the concentrations of 1ng/mL, 10pg/mL, 100fg/mL, 1fg/mL, 0.1fg/mL and 0fg/mL into the 6 parts of artificial urine solution, and uniformly mixing to obtain the urine to be detected for later use.
Equally dividing the substrate solution into 6 parts, respectively adding 6 kinds of urine to be tested into the 6 parts of substrate solution, and incubating for 1h at room temperature to obtain KIM1 urine to be tested.
And finally, adding 200 mu L of top layer structure solution into each KIM1 urine to be detected, and forming a detection antibody-antigen to be detected-capture antibody sandwich structure solution to be detected after specific binding.
Test example 1
The raman spectrum of the sample of example 1 was collected on a come microscope using a Renishaw inVia confocal raman spectrometer with a 20-fold objective lens and a 785nm laser as excitation source. The spectrum is 800-1800cm-1In the range, the exposure time is 10s, and the detection result is shown in FIG. 2, which shows that the detection limit of the biosensor provided by the invention on IL-6 can reach fg/ml.
Test example 2
The sample in example 2 was collected on a Leica microscope by using a Renishaw inVia confocal Raman spectrometer with a 20-fold objective lens and a 785nm laser as excitation sourcesA product raman spectrum. The spectrum is 800-1800cm-1In the range of 10s exposure time, the results are shown in FIGS. 3-4, and it can be seen from FIG. 3 that the Raman intensity shows a monotone increasing trend with the increase of the concentration of KIM1, and from FIG. 4 that 1145cm is seen-1The Raman intensity of the peak varies with the concentration of KIM1, and the inset in FIG. 4 shows its broad linear dynamic response range, from 1ng/mL to 0.1 fg/mL. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
Claims (1)
1. A sandwich immunoassay biosensor based on surface enhanced raman technology, the biosensor comprising: the top layer is a gold-core silver-shell nanorod bonded Raman detection molecule, a functional modifier and a detection antibody; and a substrate, which is a gold core silver shell nanorod bonding capture antibody;
the Raman detection molecule is vitamin K4, and the vitamin K4 molecule is introduced into sulfydryl through diazotization sulfydryl;
the functional modifier comprises glutaraldehyde and cysteamine;
the biosensor can form a sandwich structure of 'detection antibody-antigen to be detected-capture antibody' in the working process.
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| CN1444045A (en) * | 2003-04-15 | 2003-09-24 | 吉林大学 | Surface enhancement Raman scattering labelling immunodetection method |
| CN103116019A (en) * | 2013-01-16 | 2013-05-22 | 宁波大学 | Preparation method of immune base and antigen or antibody immunoassay method |
| CN105307976A (en) * | 2013-03-06 | 2016-02-03 | 南洋理工大学 | Monolayer of nanorods on a substrate and method of forming the same |
Family Cites Families (1)
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|---|---|---|---|---|
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1444045A (en) * | 2003-04-15 | 2003-09-24 | 吉林大学 | Surface enhancement Raman scattering labelling immunodetection method |
| CN103116019A (en) * | 2013-01-16 | 2013-05-22 | 宁波大学 | Preparation method of immune base and antigen or antibody immunoassay method |
| CN105307976A (en) * | 2013-03-06 | 2016-02-03 | 南洋理工大学 | Monolayer of nanorods on a substrate and method of forming the same |
Non-Patent Citations (3)
| Title |
|---|
| A SERS-based immunoassay with highly increased sensitivity using gold/silver core-shell nanorods;Lei Wu等;《Biosensors and Bioelectronics》;20120516;第38卷;摘要，第95页 * |
| 二元纳米离子-金属耦合体系的SERS研究及其在免疫分析中的应用;韦超;《中国博士学位论文全文数据库（工程科技Ⅰ辑）》;20190115(第1期);第20-21页 * |
| 维生素K3的表面增强拉曼光谱研究;杜银霄 等;《光谱学与光谱分析》;20031231;第23卷(第4期);摘要，引言，第1-2节，图1-4，表1 * |
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