CN115508331A - Raman sensor for rapidly detecting new coronavirus based on integrated optical flow control type microstructure optical fiber and SERS substrate - Google Patents

Abstract

The invention provides 1. A Raman sensor for rapidly detecting new coronavirus based on integration of an optical flow control type microstructure optical fiber and an SERS substrate, which comprises the following steps: step one, preparing nano silver sol by a sol-gel method; connecting nano graphene oxide by a chemical bonding method; step three, growing the nano gold particles; step four, modifying the microstructure optical fiber; step five, modifying the microstructure optical fiber by the receptor domain; and step six, preparing a Raman detection device. The invention prepares an SERS substrate specifically combined with SARS-CoV-2 spike protein, and combines with a hollow optical fiber to form a light flow control Raman detector, thereby realizing the rapid detection of SARS-CoV-2 spike protein. The microfluidic Raman SARS-CoV-2 spike protein sensor has the advantages of high integration degree, high sensitivity, high response speed, low cost and simple operation.

Description

A new rapid detection method based on optofluidic microstructured optical fiber and SERS substrate integration Coronavirus Raman Sensor

technical field

The present invention relates to a Raman sensor based on an optofluidic microstructured optical fiber and a SERS substrate integrated rapid detection of the new coronavirus, especially a nano-silver/nano-graphite oxide functionalized with ACE2 based on a natural microfluidic optical fiber As a special SERS substrate, alkene/nano-gold particles are integrated with a rapid detection Raman sensor that can specifically bind to SARS-CoV-2 and has a flow sampling function.

Background technique

Currently, methods for detecting viruses include reverse transcription polymerase chain reaction (RT-PCR), fluorescence detection, and immunological detection methods. However, the above methods require a long detection time and need to label the samples in advance, the operation is cumbersome and time-consuming, and the detection results are easily affected by many factors such as specimen quality, virus infection site and expression level. In contrast, the surface-enhanced Raman scattering SERS detection method has the advantages of label-free, fast, and high sensitivity, and has good application prospects in rapid and accurate diagnosis and prevention of disease transmission.

During the detection of the new coronavirus, in order to avoid the leakage of samples during the detection process and avoid the spread of the virus in a large area, a very small number of detection samples should be taken during the detection. Optical fiber sensors have attracted extensive attention due to their high sensitivity, resistance to electromagnetic interference, and small size. Especially some optical fibers with special structures, such as hollow optical fibers, have microstructured channels inside. Due to its special internal structure and flexible characteristics, it can be widely used in the detection of biological and chemical trace samples.

At present, gold and silver nanoparticles are widely used in Raman detection as common SERS substrates. At the same time, some new two-dimensional nanomaterials such as graphene oxide can be combined with nanoparticles to form a composite SERS substrate due to its special internal structure, which can greatly increase the detection effect of SERS.

Contents of the invention

The purpose of the present invention is: in order to solve the problems of expensive, cumbersome and time-consuming detection methods for the new coronavirus, a Raman sensor for rapid detection of the new coronavirus based on optofluidic microstructured optical fibers and SERS substrates is proposed, especially a A rapid detection system based on micro-structured optical fiber, with nano-silver/nano-graphene oxide/nano-gold particles functionalized with ACE2 as a special SERS substrate, which can specifically bind to SARS-CoV-2 and has the function of flow sampling Raman sensor. The proposed sensor combines the advantages of Raman sensors and fiber optic sensors, and has the advantages of high sensitivity, fast response, small sample consumption, strong anti-interference ability, and flow sampling.

The present invention is achieved like this:

An integrated Raman sensor based on an optofluidic microstructured optical fiber and a SERS substrate, including: an optical path, a SERS substrate, a Raman detection system, a flow sampling system, and the like. The optical path consists of the light source of the portable Raman spectrometer, the hollow fiber with suspended cores and microstructure channels inside; the SERS substrate is constructed by chemical bonding on the surface of the suspended core inside the hollow fiber; the Raman detection system includes the portable Raman spectrometer Spectral detection device, Raman spectral analysis software; the flow sampling function is realized through a micro-injection pump.

The preparation process of the optofluidic Raman sensor for detecting the new coronavirus of the present invention comprises the following steps:

1. Preparation of nano-silver sol by sol-gel method: First, 30 mg of silver nitrate was dissolved in 100 mL of deionized water and heated. Next, dissolve 20 mg of sodium citrate in 2 mL of deionized water to make a sodium citrate solution. When the silver nitrate solution is heated to boiling, slowly add the sodium citrate solution dropwise. Finally, after heating with residual temperature for 20 minutes, it was cooled to room temperature to obtain silver nanoparticles with a size of about 80 nm.

Optionally, the residual temperature heating time may be 10-30 minutes.

Alternatively, 20 mg of sodium citrate was dissolved in 3 mL of deionized water.

2. Connecting nano-graphene oxide by chemical bonding: Mix the silver nano-solution and graphene oxide solution in the above step 1 at a ratio of 1:10. Single-layer nano-graphene oxide (GO) was selected. First, a GO solution with a concentration of 0.5 mg/mL was prepared using deionized water. Before the two solutions are mixed, the GO solution needs to be pretreated. That is, 96 mg of polyvinylpyrrolidone (PVP) was added to 24 mL of GO solution, and dissolved in 12 mL of deionized water after centrifugation. Then, 3mL PVP modified GO solution was added to 12mL PDDA (30wt%) solution and KCl mixed solution to obtain PDDA functionalized graphene oxide (GO/PDDA) solution, which was dissolved in 6mL deionized water after centrifugation. Finally, the GO/Ag NPs SERS substrate was obtained by mixing the GO/PDDA solution and Ag NPs and stirring for 3 min.

Optionally, the silver nanometer solution is mixed with the graphene oxide solution at a ratio of 1:8.

Optionally, the GO/PDDA solution and Ag NPs were mixed and stirred for 2–5 min.

3. Growth of nano-gold particles: Mix the above step 2 nano-silver/nano-graphene oxide solution with gold nano-solution in a ratio of 1:2 to prepare a nano-silver/nano-graphene oxide/nano-gold SERS-enhanced substrate . Mix 1 mL of 1% PVP with 100 μL of 100 mM ascorbic acid and 100 μL of 200 mM NaOH solution. After stirring for 5 min, add 50 μL of nanosilver/nanographene oxide and 100 μL of ₂ mM concentration of HAuCl solution to the above solution. And keep shaking in the dark for 3h. Finally, the mixture was centrifuged and the precipitate was dissolved in 50 μL of ultrapure water to prepare the nanosilver/nanographene oxide/nanogold SERS substrate.

Optionally, the nano silver/nano graphene oxide solution is mixed with the gold nano solution at a ratio of 1:4 under the action of PVP.

Optionally, keep shaking in the dark for 2-4 hours.

4. Modification of micro-structured optical fiber: the SERS substrate composed of nano-silver/nano-graphene oxide/nano-gold synthesized in the above step 3 is grown on the surface of the suspended core in the micro-structured optical fiber. To construct the in-fiber Raman signal-enhancing substrate around the suspended core, nanosilver/nanographene oxide/nanogold are fixed around the suspended core by chemical bonds. The pretreatment process of the hollow fiber is that the piranha solution (4 volumes of H ₂ SO ₄ and 1 volume of H ₂ O ₂ ) was first injected into the MHF; then the hydroxyl groups (-OH) on the surface of the suspended core were exposed. Subsequently, 3-aminopropyltriethoxysilane (APTES, the volume ratio of APTES to ethanol is 1:15) was injected to form amino groups (—NH ₂ ). Amino groups and exposed hydroxyl groups combine to serve as bridges connecting nanosilver/nanographene oxide/nanogold. Finally, through the reaction between amino groups and nanosilver/nanographene oxide/nanogold, nanosilver/nanographene oxide/nanogold were deposited on the inner surface of MHF. This last process was repeated several times in order to allow a large number of particles to grow to the suspended core surface. Then, the optical fiber optofluidic online SERS sensing fiber was prepared after washing with deionized water.

Optionally, the volume ratio of H ₂ SO ₄ and H ₂ O ₂ in the piranha solution may be 2:1.

Alternatively, the volume ratio of APTES to ethanol may be 1:10.

5. Modification of the acceptor domain to the microstructured optical fiber: the Raman sensor device of the SERS substrate with nano-silver/nano-graphene oxide/nano-gold grown on the surface of the suspended core in the micro-structured optical fiber in the above step 4 was thoroughly rinsed with deionized water and rinsed with deionized water. Dry using _N2 . Subsequently, 1 μL of ACE2 solution (30 pg) was passed into the microstructure channel, and placed in an incubator under constant temperature and humidity conditions (25° C.; 75%, w/w) for 4 hours. A Raman sensor for detecting the new coronavirus based on microstructured optical fiber and SERS integration was prepared.

Alternatively, 0.5 μL of ACE2 solution (30 pg) was passed through the microstructured channels.

Optionally, the incubation time in the incubator can be 2-5 hours.

6. Preparation of Raman detection device: use the nano-silver/nano-graphene oxide/nano-gold particles functionalized with ACE2 in the above step 5 as the microstructure of the special SERS substrate that can specifically bind to SARS-CoV-2 Microholes are etched on the surface of the optical fiber, and the CO ₂ laser is used to etch the micropores without damaging the suspension core so that the detection sample solution can flow out, and a microfluidic Raman sensor device is prepared. At the same time, a gold film was coated on the end of the optical fiber with an optical fiber coating machine to prepare a reflective layer to construct a reflective microfluidic Raman sensor device.

Optionally, an optical fiber profile grinder can be used to grind micropores on the surface of the optical fiber.

Optionally, silver-plated and aluminum films can be used as reflective layers at the end of the optical fiber.

The present invention proposes a microfluidic on-line Raman sensor based on microstructure optical fiber with high sensitivity, fast response speed, low cost and simple operation. The most significant advantage lies in the integration and miniaturization of the device. The miniature novel coronavirus Raman sensor of the present invention is expected to be applied in rapid and efficient detection of novel coronavirus.

Compared with prior art, the beneficial effect of the present invention is:

The invention prepares a SERS substrate specifically bound to the SARS-CoV-2 spike protein, and combines it with a hollow optical fiber to form an optofluidic Raman detector to realize rapid detection of the SARS-CoV-2 spike protein. The microfluidic Raman SARS-CoV-2 spike protein sensor of the present invention has the advantages of high integration, high sensitivity, fast response, low cost and simple operation.

Description of drawings

Fig. 1 is the synthesizing process schematic diagram of nano-silver/nano-graphene oxide/nano-gold;

Figure 2 is a schematic diagram of the process of ACE2 modification and growth of nano-silver/nano-graphene oxide/nano-gold microstructure hollow fiber;

Fig. 3 is the connection diagram of each part of the microstructure optical fiber microfluidic Raman sensor;

Figure 4 is a schematic diagram of the construction process of the microstructure fiber optic microfluidic Raman sensor;

Fig. 5 is a Raman spectrum of the detection of rhodamine solution based on the microstructure optical fiber microfluidic Raman sensor.

detailed description

Please refer to Figure 1 and Figure 2 for the process of constructing a microstructured hollow fiber with ACE2 modification and growth of nano-silver/nano-graphene oxide/nano-gold. Please refer to Figure 3 for the connection method of each part of the microstructure optical fiber microfluidic Raman sensor proposed by the present invention, and please refer to Figure 4 for the construction process. The labels of each part represent: microstructured hollow fiber[1], inner wall of hollow fiber[1-1], microstructure channel of hollow fiber[1-2], suspended core of hollow fiber[1-3], surface growth of suspended core ACE2 modified SERS substrate[1-4], ACE2 layer in SERS substrate[1-5], graphene oxide layer in SERS substrate[1-6], silver nanoparticle layer in SERS substrate[1-7 ], the gold nanoparticle layer in the SERS substrate [1-8], and the sampling hole [2].

The present invention relates to a preparation method of a Raman sensor for rapid detection of novel coronavirus based on an optofluidic microstructured optical fiber and a SERS substrate. Specific examples are as follows:

1. Preparation of nano-silver sol by sol-gel method: First, dissolve 30 mg of silver nitrate in 100 mL of deionized water, and heat under the action of an electric furnace. Next, dissolve 20 mg of sodium citrate in 2 mL of deionized water to make a sodium citrate solution. When the silver nitrate solution is heated to boiling, slowly add 0.1 mL of sodium citrate solution dropwise, and after 2 minutes, slowly and gradually drop the remaining sodium citrate solution into the silver nitrate solution, and react for 10 minutes. Finally, turn off the power, heat with residual temperature for 20 minutes, put the solution into a magnetic stirrer, stir and cool the solution to room temperature to prepare silver nanoparticles with a size of about 80nm.

Preferably, in this embodiment, the residual temperature heating time is 20 minutes.

Preferably, in this example, 20 mg of sodium citrate is dissolved in 2 mL of deionized water.

2. Connecting nano-graphene oxide by chemical bonding: Mix the silver nano-solution and graphene oxide solution in the above step 1 at a ratio of 1:10. Selection of single-layer nanographene oxide (GO) First, a GO solution with a concentration of 0.5 mg/mL was prepared using deionized water. Before the two solutions are mixed, the GO solution needs to be pretreated. That is, 96 mg of polyvinylpyrrolidone (PVP) was added to 24 mL of GO solution, and after stirring for 1.5 h with a magnetic stirrer, impurities were removed by centrifugation three times and dissolved in 12 mL of deionized water. Then, 3mL PVP modified GO solution was added to 12mL PDDA (30wt%) solution and KCl mixed solution, and stirred for 30 minutes to obtain PDDA functionalized graphene oxide (GO/PDDA) solution, after 2 times of centrifugation to remove excess impurities Dissolve in 6mL deionized water. Finally, GO/Ag NPs SERS substrate was obtained by mixing GO/PDDA solution and Ag NPs at a ratio of 10:1 and stirring for 3 min.

Preferably, in this embodiment, the silver nano solution is mixed with the graphene oxide solution at a ratio of 1:10.

Preferably, in this example, the mixing and stirring time of GO/PDDA solution and Ag NPs is 3 minutes.

3. Growth of nano-gold particles: Mix the above step 2 nano-silver/nano-graphene oxide solution with gold nano-solution in a ratio of 1:2 to prepare a nano-silver/nano-graphene oxide/nano-gold SERS-enhanced substrate . Mix 1 mL of 1% PVP with 100 μL of 100 mM ascorbic acid and 100 μL of 200 mM NaOH solution. After stirring with a magnetic stirrer for 5 min, add 50 μL of nanosilver/nanographene oxide and 100 μL of ₂ mM concentration of HAuCl solution to the above solution. And keep shaking in the dark for 3h. Finally, the mixture was centrifuged 5 times to remove excess impurities, and the concentrated precipitate was dissolved in 50 μL deionized water to prepare a SERS substrate of nano-silver/nano-graphene oxide/nano-gold.

Preferably, in this embodiment, the nano-silver/nano-graphene oxide solution is mixed with the gold nano-solution at a ratio of 1:2 under the action of PVP.

Preferably, in this embodiment, the time for shaking in the dark is 3 hours.

4. Modification of microstructured optical fiber: the SERS substrate composed of nano-silver/nano-graphene oxide/nano-gold synthesized in the above step 3 is grown on the surface of the suspended core in the microstructured optical fiber. To construct the in-fiber Raman signal-enhancing substrate around the suspended core, nanosilver/nanographene oxide/nanogold are fixed around the suspended core by chemical bonds. The pretreatment process of the hollow fiber is that the piranha solution (4 volumes of H ₂ SO ₄ and 1 volume of H ₂ O ₂ ) is firstly injected into the MHF; in order to fully expose the surface of the suspended core to the hydroxyl group (-OH) on the surface of the suspended core , the piranha solution was repeatedly passed into the hollow fiber for 3 times, and the solution stayed inside for 30 minutes after each passage. Subsequently, 3-aminopropyltriethoxysilane (APTES, the volume ratio of APTES to ethanol is 1:15) was injected 4 times and stayed inside for 30 minutes each time to form amino group (—NH ₂ ). Amino groups and exposed hydroxyl groups combine to serve as bridges connecting nanosilver/nanographene oxide/nanogold. The treated microstructured hollow fiber was repeatedly rinsed with deionized water and dried. Finally, through the reaction between amino groups and nanosilver/nanographene oxide/nanogold, nanosilver/nanographene oxide/nanogold were deposited on the inner surface of MHF. This last process is repeated several times in order to grow a large number of particles into the fiber. Then, the photofluidic online SERS sensing fiber in the fiber was prepared after being rinsed with deionized water.

Preferably, in this embodiment, the volume ratio of the piranha solution H ₂ SO ₄ and H ₂ O ₂ may be 4:1.

Preferably, in this embodiment, the volume ratio of APTES to ethanol may be 1:15.

5. Modification of the acceptor domain to the microstructured optical fiber: the Raman sensor device of the SERS substrate with nano-silver/nano-graphene oxide/nano-gold grown on the surface of the suspended core in the micro-structured optical fiber in the above step 4 was thoroughly rinsed with deionized water and rinsed with deionized water. Dry using _N2 . Subsequently, 1 μL of ACE2 solution (30pg) was injected into the microstructure channel. In order to make ACE2 fully cover and grow on the surface of the SERS substrate, ACE2 was repeatedly injected after the first ACE2 injection for 1 hour, and finally placed at a constant temperature. Keep in an incubator under constant humidity conditions (25°C; 75%, w/w) for 4 hours. A Raman sensor for detecting the new coronavirus based on microstructured optical fiber and SERS integration was prepared.

Preferably, in this example, 1 μL of ACE2 solution (30 pg) is injected into the microstructure channel.

Preferably, in this embodiment, the incubation time in the incubator may be 4 hours.

6. Preparation of Raman detection device: use the nano-silver/nano-graphene oxide/nano-gold particles functionalized with ACE2 in the above step 5 as the microstructure of the special SERS substrate that can specifically bind to SARS-CoV-2 Microhole etching is carried out on the surface of the optical fiber, and the _CO2 laser is used to etch the microhole without damaging the suspension core. Microfluidic Raman sensor devices. At the same time, the tail end of the optical fiber was coated with a layer of gold film with a fiber coating machine, and the coating time was 5 minutes, which was used to prepare the reflective layer and construct a reflective microfluidic Raman sensor device.

Preferably, in this embodiment, a _CO2 laser is used to etch the microholes without damaging the suspended cores.

Preferably, in this embodiment, a gold film is plated on the end of the optical fiber as a reflective layer.

Please refer to Figure 5 for the Raman spectrum of the detection of rhodamine solution based on the microstructure fiber optic microfluidic Raman sensor. Injecting a rhodamine solution with a concentration of 10-14M, the Raman characteristic peak of rhodamine can be clearly detected. It is proved that the integrated Raman sensor based on optofluidic microstructure fiber and SERS substrate of the present invention has good sensitivity and detection ability.

The working principle of the new coronavirus Raman sensor based on optofluidic microstructured optical fiber and SERS substrate integration proposed by the present invention is as follows: the cell receptor angiotensin-converting enzyme 2 (ACE2) is modified on the surface of the suspended core of the microstructured hollow optical fiber. When the SARS-CoV-2 spike protein sample passes through the microstructure channel inside the hollow optical fiber, it can bind to the ACE2-modified composite SERS substrate on the surface of the suspended core to form the recognition and receptor binding of the SARS-CoV-2 spike protein domain (RBD). At the same time, the evanescent field can collect the Raman signal into the suspended core. This binding domain successfully inhibited ACE2, which had a distinct Raman signature. The SARS-CoV-2 spike protein RBD targets short β5 and β6 strands, α4 and α5 helices and loops on ACE2, which are rich in phenylalanine and amide III for C–N stretching and N–H bending. Quenching of SERS signal intensity occurs throughout the entire spectrum.

Gold and silver nanoparticles are widely used as common SERS enhancement substrates. The Raman enhancement principle is based on the LSPR effect. By changing the size of the particles and the arrangement spacing between the particles, the electromagnetic field can be changed, that is, the Raman enhancement effect. Synthesizing a composite SERS substrate with nano-gold and nano-silver can have a stronger Raman enhancement effect than the SERS substrate composed of a single particle. At the same time, using the special structure inside the two-dimensional material graphene oxide, nano-silver and nano-gold are embedded on the surface of nano-graphene oxide, which means that the distance between nanoparticles is greatly shortened, so that stronger Raman enhancement can be obtained The effect is conducive to the detection of more trace and lower concentration test samples.

Since the hollow fiber is a purely natural optofluidic device, it has a suspended core and a microstructure channel inside, and the detection sample can flow in from the microstructure channel and directly contact the suspension core. At the same time, the suspension core can use chemical methods to grow special materials on its surface to achieve a series of sensing effects. That is to say, when SARS-CoV-2 flows into the hollow fiber, it can contact the ACE2-modified nano-silver/nano-graphene oxide/nano-gold SERS substrate grown on the surface of the suspended core, and the excited Raman signal passes through the suspension core. The evanescent field is collected and transmitted to the detector. Thus, a Raman spectrum is obtained.

Claims (5)

1. An integrated rapid detection novel coronavirus Raman sensor based on optofluidic microstructured optical fiber and SERS substrate, characterized in that: comprising the following steps: Step 1. Prepare nano-silver sol by sol-gel method: add sodium citrate solution to boiling silver nitrate solution by oxidation-reduction method until the two solutions completely react and cool to room temperature to obtain silver nano-sol with a size of about 80nm. particle; Step 2, chemical bonding method to connect nano-graphene oxide: mix the above-mentioned step one silver nano-solution with the graphene oxide solution modified with polyvinylpyrrolidone (PVP) and PDDA (30wt%) solution in a ratio of 1:10, fully Stir for 3 minutes to obtain the nano-silver/nano-graphene oxide SERS substrate; Step 3. Nano-gold particle growth: Mix the above-mentioned step 2 nano-silver/nano-graphene oxide solution with the gold nano-solution in a ratio of 1:2 to prepare the SERS enhancement of nano-silver/nano-graphene oxide/nano-gold base; Step 4. Modification of microstructured optical fiber: the SERS substrate composed of nano-silver/nano-graphene oxide/nano-gold synthesized in the above step 3 is grown on the surface of the suspended core in the micro-structured optical fiber under the action of chemical bonding, and deionized water is used to Prepare the optofluidic online SERS sensing fiber in the fiber after washing; Step 5. Modification of the acceptor domain to the microstructured optical fiber: the Raman sensor device of the SERS substrate with nano-silver/nano-graphene oxide/nano-gold grown on the surface of the suspended core in the above-mentioned step 4 micro-structured optical fiber is thoroughly rinsed with deionized water And after drying with N2, pass through ACE2 solution and let it stand for 3 hours to prepare a Raman sensor for detecting new coronavirus based on microstructured optical fiber and SERS integration; Step 6. Preparation of Raman detection device: use the nano-silver/nano-graphene oxide/nano-gold particles functionalized with ACE2 in the above step 5 as a special SERS substrate capable of specifically binding to SARS-CoV-2. Microholes are etched on the surface of the structured optical fiber, and the CO ₂ laser is used to etch the microholes without damaging the suspension core, so that the detection sample solution can flow out, and a microfluidic Raman sensor device is prepared. 2. A kind of Raman sensor based on optofluidic microstructured optical fiber and SERS substrate integrated rapid detection of new coronavirus according to claim 1, characterized in that: a natural microfluidic optical fiber device with a suspended core inside And the microstructure channel can realize the online monitoring of trace samples; at the same time, this kind of optical fiber is beneficial to the integration and miniaturization of the device. 3. A Raman sensor based on optofluidic microstructured optical fiber and SERS substrate integrated rapid detection of new coronavirus according to claim 1, characterized in that: in this microstructured optical fiber, the prepared nanometer Silver/nano-graphene oxide/nano-gold are grown on the surface of the suspended core to form an optofluidic Raman sensor device with Raman enhancement effect. 4. A kind of Raman sensor based on optofluidic microstructure optical fiber and SERS substrate integrated rapid detection of new coronavirus according to claim 1, characterized in that: the microstructure channel inside the sensor can allow the sample solution to flow from one end to the other. Surface micropore outflow, with flow sampling function, realize optofluidic Raman sensor device. 5. A kind of Raman sensor based on optofluidic microstructured optical fiber and SERS substrate integrated rapid detection of new coronavirus according to claim 1, characterized in that: construct this kind of nano-silver/nano-graphite oxide functionalized with ACE2 As a special SERS substrate, alkene/nano-gold particles can specifically bind to SARS-CoV-2, and have high sensitivity and rapid detection characteristics without external interference.

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