CN117802268A - Ratio fluorescent paper-based sensor, application and kit for RNA virus detection - Google Patents
Ratio fluorescent paper-based sensor, application and kit for RNA virus detection Download PDFInfo
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Abstract
The invention discloses a ratio fluorescent paper-based sensor, application and a kit for detecting RNA viruses, which lead silicon dioxide B-SiO which emits blue fluorescence 2 And red fluorescent CdTe QDs modified silica/dopamine DPA-QDs@SiO 2 Labeling onto cellulose paper to make a ratiometric fluorescent paper-based sensor. The ratio fluorescencePaper-based sensors are used for visual or quantitative detection of RNA viruses. The nucleic acid detection kit comprises: aptamer modified magnetic beads, padlock probes, dNTPs, DNA polymerase, hemin solution and a ratio fluorescent paper-based sensor. The detection kit combines isothermal RCA amplification technology with proportional fluorescent paper-based sensor technology, and can detect RNA viruses such as SARS-CoV-2 with high sensitivity and accuracy; and the specificity is strong, the pollution rate is low, and the method is suitable for on-site rapid quantitative detection.
Description
Technical Field
The invention belongs to the technical field of biomedical detection, and particularly relates to preparation of a ratio fluorescent paper-based sensor, design and preparation of magnetic DNAzyme, which are used for detecting RNA viruses by high-sensitivity POC.
Background
The transmission speed of the virus is unprecedented, so timely diagnosis is required by contact tracking and routine detection to limit the transmission of the virus. However, current detection methods have limited ability to provide sensitive, rapid, accurate in-situ diagnostics and epidemiological monitoring. For example, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) technology has been used as the gold standard for diagnosis of viral infections due to its high specificity and sensitivity. However, this technique requires expensive equipment, skilled technicians, and complex procedures, and is not suitable for point-of-care diagnostics (POC) of SARS-CoV-2. Immunological methods for detecting antigens or antibodies are inherently less specific and, while enabling rapid point-of-care (POC), do not yield relatively reliable results. To solve these problems, recent studies on RNA virus detection have explored the use of biosensors based on isothermal nucleic acid amplification technology and advanced functional materials. These studies include Rolling Circle Amplification (RCA) detection of electrochemical methods, CRISPR-Cas12a assisted Strand Displacement (SDA) and colorimetric detection, exponential amplification reaction (EXPAR) and digital microfluidic-chemiluminescent analysis, electrochemical sensors based on triangular prismatic DNA nanostructures. However, in isothermal amplification techniques, RCA is of great interest in biosensing, drug delivery, and clinical diagnostic applications. One of the main advantages of the RCA-based approach is that it uses target RNA as a primer to completely match the RCA padlock probe initiation reaction. This makes the reaction element simpler, makes the experimental operation easier, and reduces background noise. However, RCA-based biosensors tend to have limited sensitivity and fail to meet the requirements for sensitive detection of RNA viruses.
Disclosure of Invention
The invention aims to solve the problems of complex operation, high cost and the like of an RNA virus in-situ detection process, and provides a method for accurately detecting the RNA virus in situ.
The ratio fluorescence sensor with the double-emission fluorescence characteristic has a self-calibration function, so that the stability and the accuracy of the fluorescence sensor can be improved, and the sensitivity of the fluorescence sensor can be greatly improved. In order to successfully construct a ratiometric fluorescence sensor, it is important to design an appropriate fluorescent probe to further improve detection accuracy. Silica has the characteristics of water-tolerant property, chemical stability, optical transparency, easiness in chemical modification and the like, is one of the most commonly used brackets for preparing compositions with fluorescent substances so as to obtain higher single-particle brightness, and is also an ideal candidate material for fluorescent probes. At present, the QDs modified silica has the advantages of wide raw material source, low cost and the like, and is applied to a response fluorescent probe. Since the emission spectrum of QDs modified silica coincides with the uv-vis curve of dopa chrome, its emission can be controlled by horseradish peroxidase (HRP) and H based on the Internal Filter Effect (IFE) 2 O 2 Catalyzing the quenching of dopachrome produced by dopamine oxidation. Since Hemin/G-quatelex has high HRP-like activity, G-quatelex DNA and H 2 O 2 Can be produced by target-induced transduction reactions, and thus, QDs-modified silica/dopamine systems can be used for biosensing G-quaterlex DNA and other biomarkers. In addition, the target induces the generation of DNase, and the combination of the target and the ratio fluorescence sensor can carry out high-sensitivity, rapid and visual quantitative detection on the RNA target.
A high-sensitivity field detection technology based on magnetic DNAzyme and a paper-based ratio fluorescence sensor is used for rapidly and highly specifically detecting SARS-CoV-2. Blue fluorescent silica (B-SiO) 2 ) And CdTe QDs modified silica/dopamine (DPA-QDs@SiO) 2 Or R-SiO 2 ) The system was marked onto cellulose paper to make a ratiometric fluorescent paper-based sensor for visual detection of SARS-CoV-2.
The detection method involves rolling circle amplification induced by target RNA to produce a magnetic DNase that is detectable by a ratiometric fluorescent paper-based sensor. Magnetic DNase quenching paperQDs modified silica/dopamine (R-SiO) in base ratio fluorescence sensor 2 ) The paper-based ratio fluorescence sensor changes color from pink to blue under uv conditions as the target RNA induces more magnetic dnase. Meanwhile, a smart phone auxiliary device is integrated and used for recording and analyzing fluorescent images.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a method for preparing a ratio fluorescent paper-based sensor comprises the steps of preparing blue fluorescent silicon dioxide B-SiO 2 And red fluorescent CdTe QDs modified silica/dopamine DPA-QDs@SiO 2 Integrating the sensor with cellulose paper to prepare a ratio fluorescent paper-based sensor; the DPA-QDs@SiO 2 And B-SiO 2 (1-5): 2 by volume. Preferably, the blue fluorescent silica B-SiO 2 Is prepared from the following steps:
s1, adoptingMethod for preparing silicon dioxide nano-particles SiO 2 ；
S2, adding APTES into the mixture containing 7-hydroxycoumarin and S1 to prepare SiO 2 Is stirred in PBS solution, then NH is added 3 .H 2 O, obtaining blue fluorescent silica particles B-SiO 2 ；
The red fluorescent CdTe QDs modified silica/dopamine DPA-QDs@SiO 2 Is prepared from the following steps:
s3, mixing a borate buffer solution of CdTe QDs with GSH and TMAH solution, and purifying to obtain a CdTe QDs solution of the GSH coating;
s4, preparing SiO from the S1 2 With ethanol, IPS and NH 3 .H 2 O is mixed and stirred for reaction, and finally IPS functionalized SiO is obtained 2 ；
S5, performing IPS functionalization on CdTe QDs of the GSH coating prepared by the S3 and the IPS prepared by the S4 2 Mixing to obtain QD@SiO 2 ；
S6, QD@SiO prepared in S5 is prepared 2 Mixing with PBS buffer containing EDC/NHS, adding dopamine into the mixed solution, and stirring to obtainDPA-QD@SiO of red fluorescence 2 (R-SiO 2 )；
Preparation of a ratiometric fluorescent paper-based sensor:
DPA-QDs@SiO prepared by S6 2 And B-SiO prepared by S2 2 Proportionally mixing, and reacting the mixed solution with cellulose paper to obtain the ratiometric fluorescent paper-based sensor.
Preferably, the specific steps of step S1 are: TEOS is added into the mixture of ethanol and water, and then NH is added into the mixed solution 3 .H 2 O, stirring the mixed solution for 48+/-12 hours, and centrifugally separating SiO 2 Finally, siO is treated with 2 Eluting to remove impurities; the TEOS is mixed with ethanol, water and NH 3 .H 2 The volume ratio of O is (0.01-0.1): 1: (0.01-0.1): (0.01-0.1);
the specific steps of the step S2 are as follows: APTES is added into the mixture containing 7-hydroxycoumarin and SiO 2 After stirring for 1.+ -. 0.5 hours, NH was added 3 .H 2 O and TEOS, stirring for 12+/-6 hours; the APTES and NH 3 .H 2 The volume ratio of O to TEOS is 1: (1-4): (1-4); 7-hydroxycoumarin, siO 2 The mass volume ratio of the APTES to the APTES is (0.1-1) g: (0.05-0.5) g:1mL;
the mass volume ratio of the CdTe QDs to the GSH to the TMAH in the step S3 is (0.1-0.5) mg (100-500) mg is 1mL;
step S4 of SiO 2 With ethanol, IPS and NH 3 .H 2 The mass volume ratio of O is 10mg: (0.1-5) mL: (0.01-0.1) mL: (0.01-0.1) mL, stirring and reacting for 5+/-3 h, and centrifugally washing after the reaction;
CdTe QDs and IPS functionalized SiO of GSH coating in step S5 2 The mass ratio of (2) is (10-3) 1;
step S6 the QD@SiO 2 The mass ratio of EDC, NHS and dopamine is 1: (1-0.25): (1.25-0.25): (0.1-0.05), and stirring for 30-60min.
Preferably, the DPA-QDs@SiO 2 And B-SiO 2 3:2 by volume.
The ratiometric fluorescent paper-based sensor is used for RNA virus nucleic acid visualization or quantitative detection.
A nucleic acid detection kit for detecting RNA viruses comprising: aptamer-modified magnetic beads, padlock probes, dNTPs, DNA polymerase, hemin (Hemin) solution, and ratiometric fluorescent paper-based sensors.
Preferably, the primers are as follows
The polymerase comprises phi29 polymerase with the working concentration of: 1+ -0.5U/. Mu.L;
the padlock probe working concentration is as follows: 400.+ -.100 nM.
The nucleic acid detection kit further comprises an amplification buffer.
The application method of the nucleic acid detection specifically comprises the following steps:
(1) Simulating RNA of a sample to be tested;
(2) The magnetic bead modified aptamer, padlock probes, dNTPs, phi29 polymerase and Hemin in the nucleic acid kit are adopted, RCA amplification is carried out on the padlock probes, and then magnetic DNAzyme is formed.
(3) As a preferred embodiment of the invention, the amplification in step (1) is carried out at a temperature of 37℃to 41℃and may be, for example, 37℃38℃39℃40℃41℃and preferably 41 ℃.
Preferably, the amplification time in the step (2) is 60-150min, for example, 60min,90min,120min,150min, etc. Preferably 150min.
A system for visually detecting RNA viruses is provided with a smart phone provided with a fluorescence detection application program, a darkroom, a test paper bearing platform positioned in the darkroom, an ultraviolet lamp and a light filtering system;
the target RNA is specifically combined with padlock probes and magnetic beads modified by an aptamer by adopting the kit, then RCA is amplified, and then magnetism is formedDNAzyme of (A), magnetic separation of DNAzyme into H-containing form 2 O 2 The obtained solution is dripped on a ratio fluorescent paper-based sensor; and then the ratio fluorescent paper-based sensor is placed on a test paper bearing platform, ultraviolet light emitted by an ultraviolet lamp irradiates a detection area of the test paper bearing platform, and generated fluorescent signals are transmitted to the intelligent mobile phone for fluorescent detection after background noise is reduced by a light filtering system, and fluorescent images are recorded and analyzed by a fluorescent detection application program of the intelligent mobile phone.
Target RNA virus sequence induced rolling circle amplification to generate magnetic DNAzyme, and magnetic DNAzyme excitation ratio fluorescent paper-based sensor red fluorescent light source DPA/QD@SiO 2 Quenching; the ultraviolet lamp is a light-emitting diode, and lambda=365 nm; the filtering system filters visible light with the wavelength of < 460 nm.
In the invention, under the existence of target RNA, aptamer modified magnetic beads are specifically combined with padlock probes and target RNA, rolling circle amplification is induced under the action of phi29 polymerase to form magnetic DNAzyme containing a large number of G4-Hemin structures, and finally, the magnetic DNAzyme is magnetically separated to induce a ratio fluorescent paper-based sensor DPA-QD@SiO 2 Quenching red fluorescence; at this time, the paper-based ratio fluorescent sensor changes its color from pink to blue under ultraviolet conditions.
Meanwhile, the detection method of the invention not only can detect SARS-CoV-2, but also can detect other RNA virus nucleic acid by only designing and replacing corresponding primers.
The rolling circle amplification signal amplification technology can further accurately and specifically reduce red fluorescent signals, and the method is simple in one-step formation of the magnetic DNAzyme, can magnetically separate, prevents false positive results, and is high in accuracy.
Compared with the prior art, the invention has the following beneficial effects:
(1) In the present invention, a ratiometric fluorescent paper-based sensor is used, in which a blue fluorescent source (B-SiO 2 ) As a reference, DPA-QD@SiO on a paper substrate is realized by utilizing the action of magnetic DNAzyme generated by RNA induction and a ratiometric fluorescent paper substrate sensor in the presence of an RNA target object 2 Is quenched by fluorescence, solidHigh-sensitivity POC detection of ultrasensitive RNA viruses is now being performed. Magnetic DNase and DPA-QD@SiO on cellulose paper base 2 The effect induced a red fluorescence quench and finally, quantitative analysis was performed by using a portable smartphone auxiliary device.
(2) The detection kit combines isothermal RCA amplification technology with proportional fluorescent paper-based sensor technology, has portability, high sensitivity, good stability, rapidness and accuracy, strong specificity and low pollution rate, and can be used for rapidly detecting RNA viruses in the environment on site.
Drawings
FIG. 1 is a schematic diagram showing the flow of RNA virus detection according to the present invention.
FIG. 2 shows DPA-QD@SiO in the present invention 2 Preparing a flow chart (a); siO (SiO) 2 (b) Scanning Electron Microscopy (SEM) and (c) Transmission Electron Microscopy (TEM); QD@SiO 2 (d) Scanning Electron Microscopy (SEM) and (e) Transmission Electron Microscopy (TEM); (f) QD@SiO 2 A high angle annular dark field scanning TEM image (HAADF-STEM) and an elemental map.
FIG. 3 shows the different volume ratios of B-SiO on the paper base in the present invention 2 And R-SiO 2 (DPA-QDs@SiO 2 ) And the effect of different concentrations of target RNA on the magnetic DNase on the contrast ratio fluorescent paper-based sensor. (a) Reaction mechanism of magnetic dnase and ratiometric fluorescent paper-based sensor; different volume ratios of B-SiO 2 And R-SiO 2 A fluorescence image (b) for a ratiometric fluorescent paper-based sensor and a resulting red value (c).
FIG. 4 is a schematic diagram of a portable fluorescence reading device according to the present invention, (a) a smart phone; (b) A CIE chromaticity diagram of the ratiometric fluorescence sensing system when different concentrations of the target are added; (c) The color shift distance of the ratiometric fluorescent paper-based sensor varies with the concentration of the target.
Detailed Description
The invention is described in further detail below with reference to the drawings in the specific examples.
Unless otherwise indicated, all reagents, methods and apparatus used in the present invention are conventional. Materials and reagents used in the present invention are commercially available unless otherwise specified.
Example 1
A method for preparing a ratio fluorescent paper-based sensor, comprising the steps of:
step S1: TEOS (4.3 mL) was added to a mixture of ethanol (66.7 mL) and water (3.2 mL). Then, 4.2mL of NH was added to the above mixed solution 3 .H 2 O (aq, 28%). The mixed solution was stirred at room temperature for 48 hours, and the SiO was separated by centrifugation (8000 rmp;20 minutes, 25 ℃ C.) 2 . Finally, siO is carried out 2 Eluting for 3 times, removing impurities, and collecting SiO 2 Dispersing into PBS solution, and storing at room temperature for standby.
Step S2:35 mu L APTES was added to the mixture containing 7-hydroxycoumarin (20 mg) and SiO 2 (10 mg) in 20mL of PBS. After stirring for 1 hour, NH was added 3 .H 2 O (56. Mu.L) and TEOS (56. Mu.L) were stirred for 12 hours. Finally, the obtained matrix is eluted with PBS solution for three times to remove 7-hydroxycoumarin, and blue fluorescent silica particles (B-SiO) 2 ) The method comprises the steps of carrying out a first treatment on the surface of the And dispersed into PBS solution for further experiments.
Step S3: cdTe QDs (100. Mu.L, 3 mg/mL) were diluted with borate buffer (900. Mu.L, 50mM, pH 8.5) and mixed with GSH (80 mg in 300. Mu.L TMAH) for 12 hours at room temperature. The GSH coated QDs solution is then purified using dialysis bags to remove excess reagents.
Step S4: siO prepared by S1 2 (10 mg), 1mL of ethanol with IPS (100. Mu.L, 50% v/v IPS in ethanol) and 40. Mu.L of NH 3 .H 2 O (aq, 28%) were mixed. The mixed solution was shaken at room temperature for 5 hours, and IPS-functionalized SiO was prepared 2 Centrifugation (10,000 rmp,20 min), washing 3 times with borate buffer. Finally, siO is carried out 2 Redispersed in borate buffer (1 mL).
Step S5: cdTe QDs of GSH coating prepared by S3 and IPS functionalized SiO prepared by S4 2 Mixing the materials in a mass ratio of 3:1 to obtain QD@SiO 2 。
Step S6: QD@SiOprepared by S5 2 (0.5 mL) and EDC/NHS (0.5 mg mL) -1 ) PBS buffer (pH 5.8,2.5mL,10mmol L) -1 ) Mix for 10 minutes at room temperature. Then, 50. Mu.L moreBalamine (0.1 mol L) -1 ) Added to the mixed solution and shaken for 30 minutes. Finally, the obtained DPA-QD@SiO 2 (R-SiO 2 ) Washed 3 times and redispersed in 2mL of PBS buffer for further use.
Step S7: R-SiO prepared by S6 2 (DPA-QDs@SiO 2 ) And B-SiO prepared by S2 2 The mixed solution was prepared in 3:2, and the mixed solution reacts with cellulose paper to be used as a signal response ratio fluorescent paper-based sensor.
Referring to FIG. 1, an RNA virus detection process comprises the following steps: the target RNA was added to the kit and incubated for 2.5 hours, then the Hemin solution was added and incubated for 30 minutes at room temperature. Magnetically separating the prepared DNase to H-containing 2 O 2 The obtained solution was added dropwise to a ratiometric fluorescent paper-based sensor, and the obtained fluorescent image was recorded and analyzed by a smart phone accessory for quantitative detection of SARS-CoV-2.
Example 2
Referring to FIG. 2, DPA-QD@SiO 2 Preparation flow diagram (FIG. 2 a) and p-SiO 2 And SiO 2 The @ QDs are morphologically characterized. Characterization by scanning electron microscopy and transmission electron microscopy, siO obtained in example 2 2 SiO 2 Morphology characterization of @ QDs As shown in FIG. 2, siO 2 Scanning Electron Microscope (SEM) images before (FIG. 2 b) and after (FIG. 2 d) marking QDs, and SiO 2 Transmission Electron Microscope (TEM) images before (FIG. 2 c) and after (FIG. 2 e) marking QDs, siO can be seen 2 After marking the QDs, the QDs quantum dots are uniformly distributed on the SiO 2 A surface. Furthermore, siO 2 The morphology and composition of @ QDs was also characterized by high angle annular dark field scanning transmission electron microscopy (HAADF-STEM). And to SiO 2 The corresponding element map of @ QDs was also analyzed. As shown in HAADF-STEM image (FIG. 2 f), siO 2 The @ QDs diameter is 260.+ -.20 nm. It can also be seen from FIG. 2f that Si and O elements are uniformly dispersed in SiO 2 In @ QDs, it was shown that SiO was formed 2 And (3) particles. Meanwhile, the Cd element is uniformly distributed in FIG. 2f, which illustrates that the QDs are embedded in the SiO 2 And (3) upper part.
Example 3
Referring to FIG. 3, by marking B-SiO on a paper substrate 2 And R-SiO 2 (DPA-QDs@SiO 2 ) A ratiometric fluorescent paper-based sensor was prepared. 7-hydroxycoumarin is embedded in silica nanoparticles to fluoresce blue, and for reference, the red-fluorescing QDs are covalently linked to SiO 2 The surface acts as a signal reporting unit. Dopamine (DPA) is then attached to QDs@SiO by a simple covalent bond 2 A surface. In magnetic DNase and H 2 O 2 In the presence of QDs@SiO 2 The interface is oxidized to dopamine quinine, and then the electron transfer channel (ET) is opened due to the efficient electron acceptance of dopamine quinine. Thus, DPA-QDs@SiO on ratiometric fluorescent paper-based sensors 2 Is significantly quenched, while B-SiO 2 The blue fluorescence intensity of (a) remains unchanged (fig. 3 a). For the design of a ratiometric fluorescent paper-based sensor, B-SiO on paper 2 And R-SiO 2 (DPA-QDs@SiO 2 ) The volume ratio of (2) and the magnetic dnase induced by different concentrations of target RNA have a great influence on the sensitivity of the test, since these conditions affect the intensity and dynamic range of signal readout. To test the effect of a proportional fluorescent paper-based sensor, we first studied modified B-SiO on cellulose paper 2 And R-SiO 2 Is different in volume ratio and concentration of RdRp gene (0, 10 1 、10 2 、10 3 pM) induces the effect of a magnetic dnase reaction. As shown in FIGS. 3B, c, rdRp gene induced magnetic DNase to different volume ratios of B-SiO 2 And R-SiO 2 The marked paper-based reaction, R value of the proportional fluorescent paper-based sensor follows the B-SiO used for the paper-based 2 And R-SiO 2 The volume ratio decreases and increases. B-SiO in ratiometric fluorescent paper-based sensors when incubated with RdRp gene-induced magnetic DNase at different concentrations 2 And R-SiO 2 The two red values with higher volume ratios (2:1 and 2:2) are relatively low, making it difficult to determine the low concentration level of the RdRp gene by visual inspection and smartphone image analysis. In addition, two lower volume ratios of B-SiO 2 And R-SiO 2 Ratio fluorescent paper-based sensors (2:4 and 2:5) in the absence of RdRp gene-induced magnetic dnaseThe R value of these ratio fluorescent paper-based sensor samples up to and exceeding 10, produces a strong fluorescent output 3 The pM RdRp gene concentration was not decreased until after the stimulated magnetic DNase reaction. In all tested B-SiO 2 And R-SiO 2 In the volume ratio, B-SiO 2 And R-SiO 2 The R value of the fluorescent paper-based sensor with the volume ratio of 2:3 is reduced even at low concentration of RdRp genes, and the visualization effect is more obvious.
Example 4
Referring to fig. 4a, a system for analyzing a ratiometric fluorescent paper-based sensor, the system consisting of five parts: the intelligent mobile phone comprises a darkroom, a paper-based bearing platform, an optical module (ultraviolet lamp), a light filtering system and an intelligent mobile phone auxiliary support.
The optical module is equipped with a light emitting diode (λ=365 nm);
the filtering system filters visible light with the wavelength less than 460 nm;
the intelligent mobile phone auxiliary platform further comprises a power supply, a switch, an intelligent mobile phone, a fluorescence detection application program (APP) and the like. The input parameters of the power supply are 5-12V, and the switch controls the power supply on-off of the auxiliary equipment of the whole intelligent mobile phone. The detection test paper is placed on a test paper bearing platform, ultraviolet light emitted by an optical module irradiates a detection area of the test paper bearing platform, generated fluorescent signals are transmitted to the smart phone for fluorescence detection through a light filtering system to reduce background noise, fluorescent images are generated, the fluorescent images are recorded and analyzed by a smart phone application program developed by Java, and the images can be converted into digital values of red (R), green (G) and blue (B) colors.
The darkroom is obtained by 3D printing of a photosensitive resin composite material with a black light-shielding function, and a required dark closed environment can be provided for a detection process.
Further, the detection sensitivity
The prepared detection system is used for sensitivity test of SARS-CoV-2RdRp gene: the sensitivity calculation was achieved by measuring the effect of RdRp induced magnetic dnase at different concentrations on the fluorescence intensity of the ratiometric fluorescent paper-based sensor. RdRp of 0.1pM to 1000pM induced magnetic DNase reacted with a ratiometric fluorescent paper-based sensor. After processing, the resulting fluorescence image was recorded and analyzed by the smartphone auxiliary device, as shown in fig. 4b, c, the ratiometric fluorescent paper-based sensor showed a continuous fluorescent color conversion from pink to blue as the concentration of the SARS-CoV-2RdRp gene increased (fig. 4 b). And the intelligent mobile phone application software is used for acquiring the distribution emission intensity of the red, green and blue channels in the image. The top of fig. 4c is an image showing the ratiometric fluorescent paper-based sensor detection zone captured by the portable smartphone accessory. To quantify the color change associated with SARS-CoV-2RdRp gene concentration, we calculated the (x, y) chromaticity coordinates of the ratiometric fluorescent paper-based sensor. The chromaticity coordinates of the ratiometric fluorescent paper-based sensor are linearly shifted with increasing RdRp gene content, thus constructing a calibration curve for target quantification. The net color shift distance (square root of Δx square and Δy square, Δx and Δy refer to the net color shift distance of chromaticity coordinates on the x-axis and y-axis, respectively) of the ratiometric fluorescent paper-based sensor is a good linear relationship with the logarithm of the RdRp gene concentration (0.1 pM to 1000 pM). The regression equation for the calibration curve is sqrt (Δx2+Δy2) =0.074lgctarget+0.089 (r2=996). Thus, the calculated limit of detection (LODs, 3 δ) of the RdRp gene target RNA was 30fM.
Claims (10)
1. A method for preparing a ratio fluorescent paper-based sensor is characterized in that blue fluorescent silicon dioxide B-SiO 2 And red fluorescent CdTe QDs modified silica/dopamine DPA-QDs@SiO 2 Labeling on cellulose paper to prepare a ratio fluorescent paper-based sensor;
the DPA-QDs@SiO 2 And B-SiO 2 (1-5): 2 by volume.
2. The method of claim 1, wherein the blue fluorescent silica B-SiO 2 Is prepared from the following steps:
s1, adoptingMethod for preparing silicon dioxide nano-particles SiO 2 ；
S2, adding APTES into the mixture containing 7-hydroxycoumarin and S1 to prepare SiO 2 Is stirred in PBS solution, then NH is added 3 .H 2 O, obtaining blue fluorescent silica particles B-SiO 2 ；
The red fluorescent CdTe QDs modified silica/dopamine DPA-QDs@SiO 2 Is prepared from the following steps:
s3, mixing a borate buffer solution of CdTe QDs with GSH and TMAH solution, and purifying to obtain a CdTe QDs solution of the GSH coating;
s4, preparing SiO from the S1 2 With ethanol, IPS and NH 3 .H 2 O is mixed and stirred for reaction, and finally IPS functionalized SiO is obtained 2 ；
S5, preparing CdTe QDs of GSH coating by S3 and preparing IPS functionalized SiO by S4 2 Mixing to obtain QD@SiO 2 ；
S6, preparing QD@SiO by using S5 2 Mixing with PBS buffer solution containing EDC/NHS, adding dopamine into the mixed solution, and stirring to obtain red fluorescent DPA-QD@SiO 2 ；
Preparation of a ratiometric fluorescent paper-based sensor:
preparation of DPA-QDs@SiO by S6 2 And S2 preparation of B-SiO 2 Proportionally mixing, and reacting the mixed solution with cellulose paper to obtain the ratiometric fluorescent paper-based sensor.
3. The preparation method according to claim 1, wherein the specific steps of step S1 are: TEOS is added into the mixture of ethanol and water, and then NH is added into the mixed solution 3 .H 2 O, stirring the mixed solution for 48+/-12 hours, and centrifugally separating SiO 2 Finally, siO is treated with 2 Eluting to remove impurities; the TEOS is mixed with ethanol, water and NH 3 .H 2 The volume ratio of O is (0.01-0.1): 1: (0.01-0.1): (0.01-0.1);
the specific steps of the step S2 are as follows: APTES is added into the mixture containing 7-hydroxycoumarin and SiO 2 After stirring for 1.+ -. 0.5 hours, NH was added 3 .H 2 O and TEOS, stirring for 12+/-6 hours; the APTES and NH 3 .H 2 The volume ratio of O to TEOS is 1: (1-4): (1-4); 7-hydroxycoumarin, siO 2 The mass volume ratio of the APTES to the APTES is (0.1-1) g: (0.05-0.5) g:1mL;
the mass volume ratio of the CdTe QDs to the GSH to the TMAH in the step S3 is (0.1-0.5) mg (100-500) mg is 1mL;
step S4 of SiO 2 With ethanol, IPS and NH 3 .H 2 The mass volume ratio of O is 10mg: (0.1-5) mL: (0.01-0.1) mL: (0.01-0.1) mL, stirring and reacting for 5+/-3 h, and centrifugally washing after the reaction;
CdTe QDs and IPS functionalized SiO of GSH coating in step S5 2 The mass ratio of (2) is (10-3) 1;
step S6 the QD@SiO 2 The mass ratio of EDC, NHS and dopamine is 1: (1-0.25): (1.25-0.25): (0.1-0.05), and stirring for 30-60min.
4. A method according to claim 1, 2 or 3, wherein the DPA-qds@sio 2 And B-SiO 2 3:2 by volume.
5. A ratiometric fluorescent paper-based sensor made by the method of any one of claims 1-4.
6. The use of a ratiometric fluorescent paper-based sensor of claim 5, wherein the ratiometric fluorescent paper-based sensor is used for RNA virus nucleic acid detection.
7. A nucleic acid detection kit for detecting RNA viruses, the nucleic acid detection kit comprising: an aptamer-modified magnetic bead, padlock probe, dNTPs, DNA polymerase, hemin solution, the ratiometric fluorescent paper-based sensor of claim 5.
8. The kit of claim 7, wherein the primers are as follows
The polymerase comprises phi29 polymerase with the working concentration of: 1+ -0.5U/. Mu.L;
the padlock probe working concentration is as follows: 400.+ -.100 nM.
9. A system for visually detecting RNA viruses, which is characterized in that the system is provided with a smart phone, a darkroom, a test paper bearing platform, an ultraviolet lamp and a light filtering system, wherein the smart phone is provided with a fluorescence detection application program;
specific binding of target RNA to padlock probe and aptamer modified magnetic bead to induce RCA amplification, and magnetic DNAzyme formation to separate magnetically DNAzyme into H-containing magnetic substance 2 O 2 The obtained solution is dripped on a ratio fluorescent paper-based sensor; and then the ratio fluorescent paper-based sensor is placed on a test paper bearing platform, ultraviolet light emitted by an ultraviolet lamp irradiates a detection area of the test paper bearing platform, and generated fluorescent signals are transmitted to the intelligent mobile phone for fluorescent detection after background noise is reduced by a light filtering system, and fluorescent images are recorded and analyzed by a fluorescent detection application program of the intelligent mobile phone.
10. The system of claim 9, wherein the target RNA virus sequence induces rolling circle amplification to produce magnetic DNAzyme, which excites a ratiometric fluorescent paper-based sensor red fluorescent light source DPA-qd@sio 2 Quenching; the ultraviolet lamp is a light-emitting diode, and lambda=365 nm; the filtering system filters visible light with the wavelength of < 460 nm.
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