SERS biomimetic taste sensing chip for cell secretion, preparation and detection method

Abstract

The application discloses a SERS biomimetic taste sensing chip for cell secretion and a preparation and detection method thereof. The biological detection chip is composed of a SERS sensing array and a fixing device thereof. The chip can be directly hung on a cell culture dish. A clamping groove beside the chip is used for fixing a sensor substrate. A SERS sensing array on the substrate is composed of a dense monolayer of metal nanoparticles and an array of receptor molecules. The cell secretion can be quantitatively detected by collecting and analyzing the SERS spectrum of the array unit. The application can be adapted to the cell culture dish. In the case of not affecting the cell culture, the real-time, high-sensitivity and automatic quantitative analysis of various secretions outside the measured cell can be realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of spectroscopy and biological analysis, in particular to a SERS biomimetic taste sensing chip design and application for cell secretion. BACKGROUND

[0002] Cell secretion is used as an important parameter for cell state evaluation, and is involved in regulating many biological processes such as cell growth, signal transduction, material production, metabolism, etc., and is closely related to many pathophysiological processes. Compared with normal cells, cancer cells usually produce more active oxygen, etc. How to realize the rapid, high-sensitivity analysis and detection of the content in cell secretion is a difficult problem to be solved.

[0003] So far, people have proposed various secretion detection methods, including fluorescence method, electrochemical method, high performance liquid chromatography and colorimetric method, etc., but there are disadvantages in terms of anti-photobleaching, self-quenching, single molecule detection limit and multiplex detection. Therefore, it is an urgent problem to develop a rapid, accurate and convenient cell secretion content detection technology.

[0004] Surface enhanced Raman scattering (SERS) is an ultra-sensitive analysis technology. Under the irradiation of incident laser, when the molecules to be measured approach or adsorb on the surface of noble metal (such as gold, silver, etc.) nanoparticles, the Raman scattering signal will be improved by 6-14 orders of magnitude. SERS spectrum retains many advantages of Raman spectrum, such as strong molecular fingerprint characteristics, non-destructive in-situ analysis, short analysis time, high sensitivity, wide detection range, etc., and the instrument is portable, easy to operate, and does not require complex sample pretreatment steps. It has been successfully applied to the fields of material structure analysis, biochemical detection, environmental pollution monitoring, food safety, etc. SUMMARY

[0005] Technical problem: The purpose of the present application is to overcome the shortcomings of the prior art, provide a SERS biomimetic taste sensing chip for cell secretion and a preparation and detection method, and realize rapid, high-sensitivity, accurate quantitative analysis and detection of various extracellular secretions.

[0006] Technical solution: In order to achieve the above purpose, the technical solution of the present application is as follows:

[0007] The SERS biomimetic taste sensing chip for cell secretion of the present application comprises a SERS sensing array and a substrate.

[0008] The SERS sensing array is located on a substrate and comprises a plurality of SERS sensing units, each of which comprises a dense monolayer of metal nanoparticles and corresponding receptor molecules, the receptor molecules being combined with the upper surface of the dense monolayer of metal nanoparticles through chemical bonds or electrostatic adsorption, and the dense monolayer of metal nanoparticles can amplify the SERS signal of the receptor molecules. After the cell secretion interacts with the receptor molecules, the vibration state of the chemical bonds of the corresponding receptor molecules changes, thereby causing a change in the SERS spectrum of the receptor molecules, and the cell secretion can be quantitatively detected by collecting and analyzing the SERS spectrum of the array unit. Different types of cell secretion can cause changes in different receptor molecules, so that the SERS sensing array can be used to simultaneously detect a plurality of cell secretions.

[0009] Further, the metal nanoparticles are gold core-silver shell nanorods.

[0010] Further, the receptor molecules are Raman markers that are easily inserted into the surface of the metal nanoparticles through chemical bonds or adsorbed by electrostatic action.

[0011] Further, the SERS biomimetic taste sensing chip for cell secretion of the application further comprises a fixing device; the fixing device is obtained by 3D printing template reverse molding. The fixing device comprises a stage with a suspension device, the suspension device is a downwardly open groove structure, and is used for directly suspending the fixing device on a cell culture dish; and the substrate is located on the stage.

[0012] The preparation method of the SERS biomimetic taste sensing chip for cell secretion of the application comprises the following steps:

[0013] Step 1, taking the metal nanoparticles as a substrate, assembling the metal nanoparticles on an oxygen plasma treated glass substrate to form a dense monolayer;

[0014] Step 2, modifying the receptor molecules

[0015] The PDMS mask with an array structure is attached to the substrate, different receptor molecules are labeled in different array units, and a SERS sensing array for recognizing a plurality of secretions is prepared;

[0016] By labeling different receptor molecules, a SERS sensing array for distinguishing different tumor cell subtypes can also be prepared.

[0017] The PDMS mask is obtained by 3D printing template reverse molding.

[0018] Further, in step 1, the metal nanoparticles are taken as a substrate, and the metal nanoparticles are assembled on an oxygen plasma treated glass substrate to form a dense monolayer; the specific steps are as follows:

[0019] Step 1.1. Preparation of gold core silver shell nanorods.

[0020] Step 1.1.1. Preparation of gold seeds

[0021] Mix 2.5 mL of 0.2 M CTAB solution with 1.5 mL of 1.0 mM tetrachloroauric acid solution at 25 °C, add 0.6 mL of 0.01 M ice-cold sodium borohydride solution with vigorous stirring and allow to stir for 2 minutes to obtain a brownish yellow seed solution.

[0022] Step 1.1.2. Preparation of growth solution,

[0023] Mix 12.5 mL of 0.2 M CTAB solution with 0.187 mL of 15 mM silver nitrate solution at 27-30 °C, add reagents in the following order with vigorous stirring: 1.25 mL of 15 mM tetrachloroauric acid solution, 11.25 mL of deionized water, and stir slowly until uniform.

[0024] Subsequently, add 0.1 M ascorbic acid dropwise until the solution becomes colorless, then continue to add 1 / 4 to 1 / 2 of the total amount.

[0025] Finally, add 20 uL of seed solution and allow to stand for 10-20 min to obtain a gold nanorod solution.

[0026] Centrifuge 20 mL of the above solution to remove the surface CTAB, disperse it in 20 mL of water, stir, and add the following reagents in the following order: 0.728 g of CTAB, 40 mL of deionized water, 1.3 mL of 0.1 M ascorbic acid, 1.2 mL of 15 mM silver nitrate solution, and 2.4 mL of 0.1 M sodium hydroxide, to obtain a deep red solution. Centrifuge once, remove the supernatant, and re-disperse in 20 mL of water.

[0027] Step 1.2. Conversion of CTAB ligand to PVP ligand.

[0028] Centrifuge the above gold core silver shell nanorod solution, disperse it in a 1% PVP solution, and ultrasonicate the mixed solution for 2-3 h, then centrifuge and re-disperse in ethanol. Repeat the centrifugation and re-dispersion steps at least 3 times to remove excess PVP from the solution and retain only the PVP on the surface of the nanoparticles. After the final re-dispersion step, PVP ligated gold core silver shell nanorods are obtained.

[0029] Step 1.3. Oxygen plasma treatment of glass substrate

[0030] Treat the glass substrate with oxygen plasma for 7-10 min at a power of 100 W and an inlet gas flow rate of 80 seem.

[0031] First, take three 5mL centrifuge tubes, respectively, 1mL dichloromethane, 2-3mL n-hexane, 1.8mL deionized water, only the centrifuge tube filled with deionized water is washed with deionized water in advance, the remaining two centrifuge tubes only need to be simply blown with nitrogen to remove the dust in the tube.

[0032] Step 1.4. Forming a dense monolayer on the glass substrate

[0033] Then 100uL gold core silver shell nanorods are added to dichloromethane, gently shake and quickly pour into 1.8mL deionized water, then tightly cover the cap and shake vigorously for about 1min, and then slowly float the nanoparticles to the water-air interface and form a dense gold yellow film.

[0034] Subsequently, 0.4mL n-hexane is quickly added along the tube wall, the centrifuge tube is tilted at about 45°, and the tube is repeatedly rotated to make the nanoparticles near the dichloromethane-water interface float up and form a layer on the tube wall. Part of the n-hexane is carefully sucked away with a syringe, and the remaining n-hexane is allowed to volatilize naturally for two minutes.

[0035] Finally, the treated glass sheet is carefully inserted into the film with tweezers and slowly taken out and placed on an inclined dust-free cloth to dry naturally. Generally, assemble 1-2 times, and the liquid surface will be loose. At this time, only n-hexane extraction needs to be added again until the water layer becomes clear and transparent.

[0036] The SERS biomimetic taste detection method for cell secretions of the application comprises the following steps: first, a substrate with a SERS sensing array is prepared based on the preparation method of the SERS biomimetic taste sensing chip; the substrate with the SERS sensing array is placed on the object table of a fixing device, and then the substrate and the object table are placed in a culture dish of cells to be detected; and the cell secretions can be quantitatively detected through the collection and analysis of the SERS spectrum of the array unit.

[0037] Advantages: the advantages of the application are as follows:

[0038] 1. The detection chip of the application can be directly hung on a commercially available cell culture dish, and the SERS spectrum can be collected in real time without affecting the cell culture, so that the cell secretions can be monitored in real time and quantitatively.

[0039] 2. The chip uses the biomimetic taste sensing principle, can realize the accurate detection of target molecules in a complex environment by monitoring the specific molecular structure changes caused by the interaction between the receptor molecules and the substances to be detected through the SERS spectrum.

[0040] 3. The SERS sensing array of the chip can simultaneously detect multiple secretions with high throughput through spectral and spatial joint coding. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 is the structural diagram of the SERS biomimetic taste sensor chip for cell secretion of the present application.

[0042] Figure 2 is the SERS sensor array diagram.

[0043] 1, cell culture dish; 2, fixing device; 21, carrier table; 22, suspension device; 3, substrate; 4, SERS sensor array; 41, dense monolayer of metal nanoparticles. DETAILED DESCRIPTION

[0044] The present application will be further described below in conjunction with the drawings.

[0045] As shown in the drawings, Figure 1 A SERS biomimetic taste sensor chip for cell secretion is composed of a SERS sensor array 4, a substrate 3 and its fixing device 2.

[0046] The fixing device includes a carrier table 21 with a suspension device 22, which is a downwardly open groove structure for directly suspending the fixing device 2 on the cell culture dish 1; the substrate 3 is located on the carrier table 21.

[0047] As shown in the drawings, Figure 2 The SERS sensor array 4 on the substrate 3 is assembled by a dense monolayer 41 of metal nanoparticles and an array of receptor molecules, and the cell secretion can be quantitatively detected by collecting and analyzing the SERS spectrum of the array unit.

[0048] A preparation and detection method of a SERS biomimetic taste sensor chip for cell secretion, the specific steps are as follows:

[0049] (1) Take metal nanoparticles as the substrate, assemble them on the glass substrate 3 treated by oxygen plasma to form a dense monolayer 41;

[0050] (2) Take a PDMS mask with an array structure and paste it on the substrate 3 in step (1), mark different receptor molecules in different arrays, and prepare a SERS sensor array 4 for recognizing different kinds of secretion;

[0051] (3) Put the sensor substrate 3 in step (2) into the fixing device 2, then hang the detection chip on the culture dish of the cell to be detected, and quantitatively detect the cell secretion by collecting and analyzing the SERS spectrum of the array unit.

[0052] Further, the metal nanoparticles are gold core silver shell nanorods, the surface labeled Raman molecules are Raman labels which are easy to be inserted by chemical bonds or adsorbed by electrostatic action on the surface of the metal nanoparticles, and the fixing device 2 and the PDMS mask are obtained by 3D printing template reverse molding.

[0053] The present application will be further described in conjunction with specific embodiments, but the examples are only exemplary and do not constitute any limitation on the scope of the present application. It should be understood that the details and forms of the technical solutions can be improved and polished without deviating from the scope of the embodiments, but these improvements and polishing do not deviate from the protection scope of the present application.

[0054] Embodiment: Example 1, gold core silver shell nanorods are used to prepare SERS detection substrate, and the detection of extracellular secretion content is realized by using the specific reaction between receptor molecules and the detected substances.

[0055] Example 2, gold core silver shell nanorods are used to prepare SERS detection substrate, and the tumor cell subtypes are distinguished by using the specific reaction between receptor molecules and the detected substances.

[0056] Example 1: Gold core silver shell nanorods are used to prepare SERS detection substrate, and the detection of extracellular secretion content is realized by using the specific reaction between receptor molecules and the detected substances.

[0057] Step 1, preparation of gold core silver shell nanorods.

[0058] Step 1.1, preparation of gold seeds

[0059] 2.5 mL of 0.2M cetyltrimethylammonium bromide (CTAB) solution and 1.5 mL of 1.0 mM tetrachloroauric acid solution are mixed at 25℃, and 0.6 mL of 0.01M ice-cold sodium borohydride solution is added, and after stirring for 2 minutes, a brown-yellow seed solution is obtained.

[0060] Step 1.2, preparation of growth solution,

[0061] 12.5 mL of 0.2M CTAB solution and 0.187 mL of 15mM silver nitrate solution are mixed at 27-30℃, and after stirring, the following reagents are added in order: 1.25 mL of 15mM tetrachloroauric acid solution, 11.25 mL of deionized water, and slowly stirring until uniform.

[0062] Then 0.1M ascorbic acid is added dropwise until the solution becomes colorless, and then 1 / 4-1 / 2 of the total amount is added.

[0063] Finally, 20uL of seed solution is added, and after standing for 10-20min, a gold nanorod solution is obtained.

[0064] The obtained gold nanorods have a size of about 15 nm x 45 nm.

[0065] The above solution of 20 mL was centrifuged to remove the surface CTAB, and was dispersed in 20 mL of water, stirred and sequentially added with 0.728 g of CTAB, 40 mL of deionized water, 1.3 mL of 0.1 M ascorbic acid, 1.2 mL of 15 mM silver nitrate solution, 2.4 mL of 0.1 M sodium hydroxide, to obtain a deep red solution, which was centrifuged once, the supernatant was removed and was re-dispersed in 20 mL of water.

[0066] Step 2, CTAB ligand to PVP ligand.

[0067] The metal nanoparticles with CTAB ligand cannot be directly self-assembled, and need to be converted into PVP ligand. The above gold core silver shell nanorod solution was centrifuged and dispersed in a 1% PVP solution, the mixed solution was ultrasonically treated for 2-3 h, then was centrifuged and re-dispersed in ethanol, and the process of centrifugation and re-dispersion was repeatedly performed for at least 3 times to remove the excess PVP in the solution and retain only the PVP on the surface of the nanoparticles. After the last re-dispersion, the gold core silver shell nanorods with PVP ligand were obtained. During the repeated centrifugation, the solution was gradually concentrated, and finally was concentrated by at least 50 times.

[0068] Step 3, self-assembly of metal nanoparticles on the surface of glass slices.

[0069] The glass slices were treated with oxygen plasma for 7-10 min, and the parameters were selected as 100 w power and 80 sccm gas inlet amount. First, 3 centrifuge tubes of 5 mL were taken, and 1 mL of dichloromethane, 2-3 mL of n-hexane and 1.8 mL of deionized water were respectively added into the centrifuge tubes. Only the centrifuge tube with deionized water was washed with deionized water in advance, and the remaining two centrifuge tubes only needed to be simply blown with nitrogen to remove the dust in the tubes. Then, 100 uL of gold core silver shell nanorods were added into the dichloromethane, which was gently shaken and quickly poured into 1.8 mL of deionized water. Subsequently, the cap was tightly closed and shaken vigorously for about 1 min, and then the centrifuge tube was left to stand to allow the nanoparticles to slowly float to the water-air interface and form a dense golden yellow film. Then, 0.4 mL of n-hexane was quickly added along the wall of the tube, and the centrifuge tube was tilted at about 45° and repeatedly rotated to make the nanoparticles near the dichloromethane-water interface float up and form a layer of film on the wall of the tube. Some n-hexane was carefully sucked away with a pipette, and the remaining n-hexane was allowed to volatilize naturally for two minutes. Finally, the treated glass slices were carefully inserted under the film with tweezers, and then slowly taken out and placed on the inclined dust-free cloth to dry naturally. Generally, the assembly was performed for 1-2 times, and loose pages appeared at this time, which needed to be extracted again with n-hexane until the water layer became clear and transparent, and then the assembly was not suitable.

[0070] Step 4, modification of receptor molecules.

[0071] A piece of PDMS mask with array structure was attached to the assembled glass substrate 3, and 100 uL of 10 mM different receptor molecules were dropped into each unit. After natural drying, the PDMS mask was removed.

[0072] Step 5, SERS spectrum collection.

[0073] The sensor substrate 3 prepared in step 4) was placed in the clamping groove of the fixing device 2, and then the detection chip was hung on the cell culture dish 1. During detection, the chip was taken down, washed with deionized water, and then the SERS spectra of each unit of the substrate were collected in sequence by a Raman spectrometer. The content of the secretions in the sample to be detected was measured by interpreting the SERS signals.

[0074] Example 2: Gold core-silver shell nanorods were used to prepare a SERS detection substrate, and the specificity between the receptor molecules and the analyte was used to realize the differentiation of tumor cell subtypes.

[0075] Step 1, preparation of gold core-silver shell nanorods.

[0076] Step 1.1, preparation of gold seeds

[0077] At 25°C, 2.5 mL of 0.2M cetyltrimethylammonium bromide (CTAB) solution was mixed with 1.5 mL of 1.0 mM tetrachloroauric acid solution, and 0.6 mL of 0.01 M ice-cold sodium borohydride solution was added. After stirring for 2 minutes, a brown-yellow seed solution was obtained.

[0078] Step 1.2, preparation of growth solution,

[0079] At 27-30°C, 12.5 mL of 0.2M CTAB solution was mixed with 0.187 mL of 15 mM silver nitrate solution, and after vigorous stirring, the following reagents were added in sequence: 1.25 mL of 15 mM tetrachloroauric acid solution, 11.25 mL of deionized water, and slowly stirred until uniform.

[0080] Then 0.1 M ascorbic acid was added dropwise until the solution turned colorless, and then 1 / 4-1 / 2 of the total amount was added.

[0081] Finally, 20 uL of seed solution was added, and the gold nanorod solution was obtained after standing for 10-20 min.

[0082] The obtained gold nanorods were about 15 nm x 45 nm in size.

[0083] Take 20 mL of the above solution to centrifuge to remove the surface CTAB, disperse back into 20 mL of water, stir and add 0.728 g of CTAB, 40 mL of deionized water, 1.3 mL of 0.1 M ascorbic acid, 1.2 mL of 15 mM silver nitrate solution, 2.4 mL of 0.1 M sodium hydroxide, to obtain a deep red solution, centrifuge once, remove the supernatant and re-disperse into 20 mL of water.

[0084] Step 2, CTAB ligand to PVP ligand.

[0085] Metal nanoparticles with CTAB ligand cannot be directly self-assembled, and need to be converted to PVP ligand. Centrifuge the above gold core silver shell nanorod solution and disperse it into a 1% PVP solution. After ultrasonic mixing of the solution for 2-3 h, centrifuge and re-disperse in ethanol. Repeat the centrifugation and re-dispersion steps at least 3 times to remove excess PVP from the solution and retain only the PVP on the surface of the nanoparticles. After the last re-dispersion, the PVP ligand gold core silver shell nanorods are obtained. Concentrate gradually during the repeated centrifugation process, and finally concentrate at least 50 times.

[0086] Step 3, assemble metal nanoparticles on the surface of glass slices.

[0087] Treat the glass slices with oxygen plasma for 7-10 min, with parameters of 100 w power and 80 sccm gas flow. First, take 3 5 mL centrifuge tubes, each containing 1 mL of dichloromethane, 2-3 mL of n-hexane, and 1.8 mL of deionized water. Only the centrifuge tube containing deionized water is pre-washed with deionized water, and the remaining two centrifuge tubes only need to be blown with nitrogen to remove dust. Then add 100 uL of gold core silver shell nanorods to the dichloromethane, shake gently, and quickly pour into 1.8 mL of deionized water. Then tightly cap the tube and shake vigorously for about 1 min. Let the nanoparticles slowly float to the water-air interface and form a dense golden yellow film. Then quickly add 0.4 mL of n-hexane along the tube wall, tilt the centrifuge tube at about 45°, and rotate the tube repeatedly to make the nanoparticles near the dichloromethane-water interface float up and form a layer on the tube wall. Use a pipette to carefully remove some of the n-hexane, and let the remaining n-hexane evaporate naturally for two minutes. Finally, carefully insert the treated glass slices under the film with tweezers, and slowly remove them and place them on a tilted dust-free cloth to dry naturally. Generally assemble 1-2 times, and the page will appear loose. At this time, you only need to add n-hexane again to extract it, until the water layer becomes clear and transparent. At this time, it is not suitable for assembly

[0088] Step 4, modify the receptor molecules.

[0089] A piece of PDMS mask with array structure is attached to the assembled glass substrate 3, 100 uL of 10 mM different receptor molecules are dropped into each unit in sequence, and the PDMS mask is removed after natural drying.

[0090] Step 5, SERS spectrum acquisition.

[0091] The sensor substrate 3 in step 4) is placed in the clamping groove of the fixing device 2, and then each detection chip is hung on the culture dish of different tumor cells. During detection, each chip is taken down, cleaned with deionized water, and then the SERS spectrum of each unit of each chip is acquired in sequence by using a Raman spectrometer. Through interpretation of the SERS signal, the subtypes of tumor cells can be distinguished.

[0092] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A SERS biomimetic taste sensing chip for cellular secretions, characterized in that, Including SERS sensor array and substrate; The SERS sensing array is located on the substrate. Each sensing unit in the SERS sensing array includes a dense monolayer of metal nanoparticles and different acceptor molecules. The acceptor molecules are bonded to the upper surface of the dense monolayer of metal nanoparticles through chemical bonds or electrostatic adsorption. The metal nanoparticles are gold-core, silver-shell nanorods. It also includes a fixation device, which includes a stage with a suspension device. The suspension device is a downward-opening groove structure for directly suspending the fixation device on the cell culture dish; the substrate is located on the stage.

2. The SERS biomimetic taste sensor chip for cell secretions according to claim 1, characterized in that, The acceptor molecule is a Raman label that is easily adsorbed onto the surface of metal nanoparticles through chemical bonding or electrostatic interaction.

3. A method for fabricating a SERS biomimetic taste sensor chip for cell secretions as described in claim 1 or 2, characterized in that, Includes the following steps: Step 1: Using metal nanoparticles as a substrate, metal nanoparticles are assembled onto a glass substrate treated with oxygen plasma to form a dense monolayer. Step 2, modify receptor molecules; A PDMS mask with an array structure is attached to a substrate, and different receptor molecules are labeled in different array units to prepare a SERS sensing array for recognizing different types of secretions.

4. A SERS-based biomimetic taste detection method for cellular secretions, characterized in that, Based on the method for fabricating the SERS biomimetic taste sensor chip for cell secretions as described in claim 3, a substrate with a SERS sensor array is prepared. A substrate with a SERS sensing array is placed on a stage of a fixed device. The substrate and stage are then placed together in a culture dish of cells to be tested. Cell secretions are quantitatively detected by acquiring and analyzing the SERS spectra of the array units.

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