CN105823768B - Detection chip based on surface enhanced Raman scattering technology, preparation method and kit - Google Patents
Detection chip based on surface enhanced Raman scattering technology, preparation method and kit Download PDFInfo
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- CN105823768B CN105823768B CN201610262077.5A CN201610262077A CN105823768B CN 105823768 B CN105823768 B CN 105823768B CN 201610262077 A CN201610262077 A CN 201610262077A CN 105823768 B CN105823768 B CN 105823768B
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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
- G01N21/62—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
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
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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
- G01N21/62—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
Abstract
The invention provides a detection chip based on a surface enhanced Raman scattering technology, which comprises: a nano-metal substrate; two or more than two signal molecules are uniformly distributed on the nano metal substrate; and two or more probe sequences for detecting tumor markers, which are respectively fixed on different signal molecules. The invention also provides a preparation method of the detection chip and a detection kit containing the detection chip. The detection chip and the detection kit of the invention can realize one-step, simultaneous, sensitive and quantitative detection of various tumor markers by fixing different Raman signal molecules in different micro-regions and combining the nano-stress sensing technology, and have great application potential.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a detection chip based on a surface enhanced Raman scattering technology, a preparation method thereof and a kit containing the detection chip.
Background
The tumor markers microRNAs (miRNAs) show different expression levels along with the processes of tumor generation, growth, metastasis and treatment. The quantitative and high-sensitivity detection of miRNA can be used for diagnosing early stage tumor and improving the survival rate of patients, and has very important significance in the aspects of tumor diagnosis and treatment. The existing quantitative detection methods for miRNAs comprise Northern blotting, QT-PCR, a fluorescence method, a microarray method, an SERS method and the like.
At present, the two-temperature hybridization method in the SERS (surface enhanced Raman scattering) method can only detect one miRNA at a time, and has more steps. The process is as follows: and connecting a capture probe partially complementary with the target object to be detected on the SERS substrate for capturing the target object to be detected, and connecting a signal probe on the residual segment of the object to be detected to generate an SERS signal. However, in the detection by the SERS method, the SERS signal of the target analyte miRNA itself is very weak, and when the target analyte miRNA is directly detected, the number of interference peaks in a spectrogram is extremely large, and the structure and concentration information of the target analyte miRNA is not easily obtained. Therefore, most of the existing methods convert the concentration and structural information of miRNA into the intensity or displacement information of signal molecules by designing reasonable biochips, but most of the detection methods can only be used for detecting single miRNA, and the research on simultaneous detection of two or more markers is not reported.
Cancer is a complex disease, the same marker may appear in different tumors, and a tumor may also contain multiple markers, and the detection of only one marker hardly reflects the development and treatment status of the tumor. Therefore, the combined detection of multiple markers can greatly improve the accuracy of tumor diagnosis, and has important significance for early diagnosis of cancer.
Disclosure of Invention
In order to overcome the defect that the combined detection of multiple tumor markers cannot be realized in the prior art, the invention aims to provide a detection chip based on a Surface Enhanced Raman Scattering (SERS) technology, which can detect multiple markers simultaneously and has better accuracy and sensitivity.
The invention also aims to provide a preparation method of the detection chip and a detection kit containing the detection chip.
The invention provides a detection chip based on a surface enhanced Raman scattering technology, which comprises the following components:
a nano-metal substrate;
two or more than two signal molecules are uniformly distributed on the nano metal substrate; and
two or more probe sequences for detecting tumor markers are respectively fixed on different signal molecules.
In the detection chip, the nano metal substrate has better sensitivity, can be a common nano substrate in the field of detection chips, and can also be prepared in real time according to the existing method, including but not limited to substrates of nano silver, nano gold or nano copper; preferably a nanosilver substrate.
In the detection chip of the invention, the selection of the signal molecule needs to satisfy the following requirements: 1) the surface enhanced Raman scattering characteristic peaks can be obviously distinguished from each other; 2) can be printed on a nano metal substrate and has active groups connected with probe sequences. The signal molecule includes, but is not limited to, p-mercaptobenzoic acid, 5' -dithiobis (succinimidyl-2-nitrobenzoic acid), p-mercaptoaniline, p-mercaptophenylboronic acid, 2-nitro-5-mercaptobenzoic acid, or 2-amino-6-mercaptopurine.
In the detection chip, a bridging group is included between the probe sequence and the signal molecule, and the bridging group can increase the flexibility of the probe sequence fixed on the substrate, so that the combination of the probe sequence and the target miRNA is not influenced. The bridging group may be a linear saturated alkyl group having 20 or less carbon atoms; preferably, the alkyl group is a linear saturated alkyl group having 3 to 6 carbon atoms.
The detection chip of the present invention preferably comprises the following components:
a nano-silver substrate;
two signal molecules, namely p-mercaptobenzoic acid and 5,5' -dithiobis (succinimidyl-2-nitrobenzoic acid), are uniformly distributed on the nano-silver substrate; and
two probe sequences for detecting tumor markers are respectively fixed on the two signal molecules.
The preparation method of the detection chip provided by the invention comprises the following steps:
s1: preparing a nano metal substrate;
s2: respectively printing two or more than two signal molecules on the nano metal substrate by using a micro-contact printing technology so as to enable the signal molecules to be uniformly distributed on the nano metal substrate;
s3: two or more probe sequences for detecting tumor markers are bound to different signal molecules, respectively.
In step S2, the microcontact printing technique may use the existing process technology or simple modification, and the different signal molecules are printed without sequential limitation, and preferably, the signal molecules with slower growth speed are printed first.
Further, the step S3 includes:
s31: modifying said probe sequence to contain a reactive group that binds to said signal molecule;
s32: and (c) reacting the probe sequences obtained in the step (S31) with different signal molecules through the active groups respectively so as to enable the probe sequences to be combined with the signal molecules.
Further, the step S31 further includes modifying the probe sequence to include a bridging group between the probe sequence and the active group.
In step S32, a signal molecule with high reactivity can be generally bound to the probe, the reaction site is closed after the reaction is completed, and then other signal molecules are reacted, and if necessary, a protecting group can be used to ensure that the unreacted signal molecule is not affected, thereby ensuring the specific binding of different signal molecules to different probe sequences.
In step S32, when the reactivity of the active group on the signal molecule is low, the activator may be used to increase the reactivity. The type of activator may be one commonly used in the field of organic reactions, depending on the reactive group of the signal molecule.
The invention also provides a kit for detecting tumor markers, which comprises the detection chip based on the surface enhanced Raman scattering technology in any one of the technical schemes.
The detection chip of the invention constructs different functional micro-regions on a nano-metal substrate by utilizing a micro-contact printing technology, prints different signal molecules with obviously distinguished SERS characteristic peaks, then fixes detection probe sequences corresponding to different markers on the corresponding signal molecules respectively, and detects target miRNA through specific interaction between the target miRNA to be detected and the probe sequences, wherein the target miRNA can affect certain Raman peak displacement(s) of the signal molecules before and after the target miRNA is specifically combined with the probe, and the concentration information of the miRNA can be obtained by analyzing the size of the Raman peak displacement.
The detection chip of the invention has the following advantages:
(1) the nano metal substrate with higher sensitivity and different types of signal molecules are selected, and various tumor markers can be simultaneously detected by distinguishing the peak positions of the characteristic peaks of the signal molecules, so that the combined detection is realized, and the detection efficiency is improved.
(2) By adopting the micro-contact printing technology, different signal molecules can be uniformly printed on the nano metal substrate to form different functional micro-regions, and the chemical bonding of the detection probe and the signal molecules cannot influence each other, so that the detection sensitivity and accuracy can be improved, and moreover, the uniform distribution of the signal molecules can also ensure that the concentration information of miRNA is converted into stable SERS signals with better reproducibility, so that the detection process has good reproducibility.
(3) By utilizing nano-stress sensing, the concentration information of miRNA is converted into the characteristic peak displacement of signal molecules, so that the absolute concentration of miRNA can be detected through the characteristic peak displacement, and the detection accuracy is higher.
(4) The preparation process has fewer steps, and excessive interference factors are avoided, so that the accuracy of the detection result is further ensured.
The detection chip and the detection kit of the invention can realize one-step, simultaneous, sensitive and quantitative detection of various markers by fixing different Raman signal molecules in different micro-regions and combining the nano-stress sensing technology, and have great application potential.
Drawings
FIG. 1 is a schematic diagram of a process for preparing a detection chip according to an embodiment of the present invention (two signal molecules);
FIG. 2A is a SERS spectrum of a signal molecule during detection according to an embodiment of the present invention; FIG. 2B is an enlarged view of the characteristic peak of the signal molecule p-mercaptobenzoic acid (MBA); FIG. 2C is an enlarged view of the characteristic peak of the signal molecule 5,5' -dithiobis (succinimidyl-2-nitrobenzoic acid) (DSNB); FIG. 2D shows the Raman signal peak of the C-S bond; wherein: curve a represents a SERS graph after printing the signal molecule MBA, curve b represents a SERS graph after connecting the second signal molecule DSNB to the blank of the SERS substrate, curve c represents a SERS graph after connecting the probe, and curve d represents a SERS graph after detecting miRNA;
FIGS. 3A and 3B are the relationship between the concentrations of two miRNAs and the peak shifts of different signal molecules (error bar is the standard deviation of the results obtained in 5 experiments) in the example of the present invention;
FIG. 4 is a SERS spectrum of 2-amino-6-mercaptopurine printed on a nano silver substrate in an example of the invention.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Examples
As shown in fig. 1, for example, two markers are detected, a Polydimethylsiloxane (PDMS) stamp with a microarray structure on the surface is used to print a signal molecule MBA on a silver island substrate. Then growing signal molecules DSNB in other areas to form a surface functional micro-area structure with a plurality of signal molecules distributed uniformly and at intervals; controlling reaction conditions to enable the probe 1 and the probe 2 to be specifically combined with the signal molecule 1 and the signal molecule 2 in sequence; and finally, the probe 1 and the probe 2 are respectively hybridized with the target object to be detected in a specificity mode, the structure of the signal molecules at the bottom is affected, and therefore peak displacement is generated. The specific process is as follows:
1. silver island substrate preparation
1×1cm2Respectively performing ultrasonic treatment on the silicon wafer/glass sheet in ethanol and acetone for 10min, blow-drying, and placing in piranha washing solution (H)2SO4/H2O23:1, v/v), heated to 95 ℃ and washed for 40 min. After the silicon wafer/glass plate was thoroughly cleaned with ultrapure water, it was blow-dried and modified for 8 hours in a toluene solution of (3-mercaptopropyl) trimethoxysilane (2%). And respectively using toluene, acetone and ethanol to clean the silicon wafer/glass sheet for later use.
440mg of silver nitrate is weighed into an 80mL beaker, 50mL of water is added, and the mixture is stirred uniformly. 135. mu.L NaOH (10%) was slowly added to the silver nitrate solution with rapid stirring to form a grayish green precipitate. 2mL of 10% aqueous ammonia was added dropwise to the beaker until the precipitate disappeared. The solution was cooled in an ice-water bath. Adding 300 mu L of glutaraldehyde solution (25%) into the cooled solution, reacting for 10s, placing in a water bath at 90 ℃, and placing the modified silicon wafer/glass plate in the solution in an adherent manner. Reacting for 4min, and growing silver nano particles on the silicon wafer/glass sheet to form the silver island substrate. The islands of silver were immediately removed and placed in pre-cooled ethylene glycol to terminate growth. And (4) removing the silver nano particles which are not specifically attached by ultrasonic treatment for 20s for standby.
2. Stamp preparation
And ultrasonically cleaning the Si microarray templates arranged in a 7-micron multiplied by 7-micron sequence for 10min by using ethanol and acetone respectively, drying, putting the cleaned Si microarray templates into piranha cleaning solution, heating to 95 ℃, cleaning for 40min, cleaning with ultrapure water, and drying. The template was placed in a 20. mu.L stream of perfluorosilane, modified for 15min, and fixed at the opening of the disposable cuvette (microarray facing inwards). The disposable color comparison vessel is opened at the bottom for standby. Uniformly stirring polydimethylsiloxane and curing agent according to the proportion of 10:1, standing until bubbles disappear, and slowly injecting from the bottom of a disposable color comparison dish. And (5) placing the cuvette in an oven at 60 ℃, and curing for 2 hours to obtain the seal.
3. Construction of surface micro-nano structure
And (3) dropwise adding 20 mu L of MBA ethanol solution on the surface of the stamp, standing for 1min and drying. The stamp is contacted with the silver island for 1min, so that the MBA on the stamp grows on the silver island. And then placing the silver island in an acetonitrile solution of 5mM DSNB, reacting for 4h to grow the DSNB on the blank area of the silver island where the MBA does not grow, and obtaining two modified silver islands with uniformly distributed signal molecule micro-areas.
4. Probe modification
The signal molecule-modified silver islands were placed in 10. mu.M solution of aminated modified probe 1(SEQ ID NO: 1, molecular structure shown in Table 1, BIONEER Co.) (3 XSSC (sodium citrate buffer solution), pH 7.4, containing 0.04% sodium dodecyl sulfate) and reacted at room temperature for 4 hours to bind probe 1 to DSNB and immobilize the same on the substrate. The substrate was transferred to a 100. mu.M solution of butylamine (3 XSSC, pH 7.4) and the reaction continued for 40min at room temperature, blocking unreacted DSNB sites. The islands of silver were transferred to phosphate buffer (pH 6.8, 10mM) containing NHS (10mM) and EDC (50mM) and reacted for 1h to activate the carboxyl group at the terminus of MBA. After washing with 2 XSSC (pH 7.4, containing 0.1% sodium dodecyl sulfate), probe 2 was immobilized on the substrate in combination with MBA by transferring it to a solution (3 XSSC, pH 7.4, pH 7.4, containing 0.04% sodium dodecyl sulfate) containing 10. mu.M of an amino-modified probe 2(SEQ ID NO: 2, molecular structure shown in Table 1, available from BIONEER). And measuring the SERS spectrogram of the substrate to obtain the signal molecule displacement before detection.
MiRNA detection
The substrates modified by the probes are respectively arranged at 10-6-10-18M, andputting the miRNA-containing 5 XSSC solution (pH 7.4, containing 0.01% sodium dodecyl sulfate) at 42 ℃ for hybridization for 16h, and measuring an SERS spectrogram of the substrate to obtain the detected signal molecule displacement.
6. Analysis of results
The spectral changes of the signal molecules on the substrate are shown in FIGS. 2A-2D. Firstly, a signal molecule MBA is printed by utilizing a micro-contact printing technology, an SERS spectrogram is detected and found to be 1068cm-1And 1581cm-1A characteristic peak of MBA is formed; after growth of the second signal molecule DSNB, at 1334cm-1A characteristic peak of DSNB is detected; after the signal molecule is connected with the probe molecule, the characteristic peaks of MBA and DSNB are both red-shifted; after specific binding of the two miRNAs, characteristic peaks of MBA and DSNB are red-shifted again, and the displacement is related to the concentration of the miRNAs. Therefore, the two miRNAs can be quantitatively detected at the same time.
FIGS. 3A and 3B show the relationship between miRNA concentration and signal molecule peak shift. The concentration of miRNA 1(SEQ ID NO: 3, sequence shown in Table 1, BIONEER company) is reacted by the displacement of the characteristic peak of signal molecule MBA, and the detection limit can reach 10-16mol/L; the concentration of miRNA 2(SEQ ID NO: 4, sequence shown in Table 1, BIONEER company) is reacted by the displacement of signal molecule DSNB characteristic peak, and the detection limit can be 10-16mol/L。
MiRNAs and their corresponding probe sequences used in the examples of Table 1
7. Three signal molecules
On the basis of the above, three signal molecules such as MBA, DSNB and 2-amino-6-mercaptopurine (structural formula shown below) can be added.
As shown in FIG. 4, 2-amino-6-mercaptopurine printed on the nano-silver substrate has a Raman spectrum with a characteristic peak of 1300cm-1In conjunction with FIGS. 2A-2D, it can be seen that: 2-amino-6-mercaptopurineThe characteristic peak of (A) can be obviously distinguished from the characteristic peaks of MBA and DSNB, so that a detection chip for simultaneously detecting three tumor markers can be prepared.
Unless otherwise defined, all terms used herein have the meanings commonly understood by those skilled in the art.
The described embodiments of the present invention are for illustrative purposes only and are not intended to limit the scope of the present invention, and those skilled in the art may make various other substitutions, alterations, and modifications within the scope of the present invention, and thus, the present invention is not limited to the above-described embodiments but only by the claims.
Claims (7)
1. A detection chip based on a surface enhanced Raman scattering technology comprises the following components:
a nano-metal substrate;
two or more than two signal molecules are uniformly distributed on the nano metal substrate; two or more probe sequences for detecting miRNA tumor markers are respectively fixed on different signal molecules, and a linear saturated alkyl group with 3-6 carbon atoms is contained between the probe sequences and the signal molecules as a bridging group; the probe sequence is modified to contain a reactive group that binds to the signal molecule and a bridging group between the probe sequence and the reactive group.
2. The detection chip according to claim 1, wherein the nano metal substrate is selected from a substrate of nano silver, nano gold or nano copper.
3. The detection chip according to claim 1, wherein the signal molecule is selected from the group consisting of p-mercaptobenzoic acid, 5' -dithiobis (succinimidyl-2-nitrobenzoic acid), p-mercaptoaniline, p-mercaptophenylboronic acid, 2-nitro-5-mercaptobenzoic acid, and 2-amino-6-mercaptopurine.
4. The detection chip according to any one of claims 1 to 3, wherein the detection chip comprises the following components:
a nano-silver substrate;
two signal molecules, namely p-mercaptobenzoic acid and 5,5' -dithiobis (succinimidyl-2-nitrobenzoic acid), are uniformly distributed on the nano-silver substrate; and
two probe sequences for detecting miRNA tumor markers are respectively fixed on the two signal molecules, and a linear saturated alkyl group with 3-6 carbon atoms is contained between the probe sequences and the signal molecules as a bridging group.
5. The method for preparing the detection chip of any one of claims 1 to 4, comprising the steps of:
s1: preparing a nano metal substrate;
s2: respectively printing two or more than two signal molecules on the nano metal substrate by using a micro-contact printing technology so as to enable the signal molecules to be uniformly distributed on the nano metal substrate;
s3: respectively combining two or more probe sequences for detecting miRNA tumor markers with different signal molecules;
the step S3 includes:
s31: modifying the probe sequence to contain a reactive group that binds to the signal molecule and a bridging group between the probe sequence and the reactive group;
s32: and (c) reacting the probe sequences obtained in the step (S31) with different signal molecules through the active groups respectively so as to enable the probe sequences to be combined with the signal molecules.
6. The method according to claim 5, wherein the step S32 is carried out in the presence of an activator.
7. A kit for detecting a tumor marker, comprising the detection chip based on the surface-enhanced Raman scattering technique according to any one of claims 1 to 4.
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| PCT/CN2017/081420 WO2017186056A1 (en) | 2016-04-25 | 2017-04-21 | Surface enhanced raman scattering technique based detection chip, preparation method therefor, kit and tumour detection method |
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| CN105823768B (en) * | 2016-04-25 | 2020-03-27 | 中国科学院高能物理研究所 | Detection chip based on surface enhanced Raman scattering technology, preparation method and kit |
| CN109997042B (en) * | 2016-09-22 | 2022-08-19 | 高丽大学校产学协力团 | Nanoplasmon biosensor and method of detecting disease marker using the same |
| CN106634532A (en) * | 2016-12-13 | 2017-05-10 | 沈荣存 | High-brightness high-heat-dissipation-property intelligent street lamp |
| CN107460462B (en) * | 2017-08-17 | 2019-03-19 | 西安工业大学 | The preparation method of silver nano-grain compacted zone on silicon wafer |
| CN108845130B (en) * | 2018-06-21 | 2021-06-01 | 天津大学 | Tumor marker miRNA detection probe and detection chip |
| CN109337982A (en) * | 2018-11-29 | 2019-02-15 | 天津大学 | A kind of tumor markers Circulating tumor DNA detection chip and kit |
| CN109781697B (en) * | 2018-12-27 | 2021-03-02 | 西安交通大学 | Flexible SERS substrate, preparation method thereof and application of hydrogen peroxide SERS spectrum detection |
| CN110646399B (en) * | 2019-09-29 | 2021-08-13 | 吉林大学 | SERS-based high-throughput protein detection chip |
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| WO2011030286A1 (en) * | 2009-09-14 | 2011-03-17 | Koninklijke Philips Electronics N.V. | Highly sensitive immunoassay with large particle labels |
| KR101059896B1 (en) * | 2009-10-12 | 2011-08-29 | 한국과학기술원 | Detection of biochemicals using surface enhanced Raman scattering |
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| WO2014189960A1 (en) * | 2013-05-22 | 2014-11-27 | University Of Notre Dame Du Lac | Method and apparatus for a nanopipette biosensor |
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