WO2015109682A1 - 纸芯片、其制备方法及生物分子的检测方法 - Google Patents
纸芯片、其制备方法及生物分子的检测方法 Download PDFInfo
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- WO2015109682A1 WO2015109682A1 PCT/CN2014/076556 CN2014076556W WO2015109682A1 WO 2015109682 A1 WO2015109682 A1 WO 2015109682A1 CN 2014076556 W CN2014076556 W CN 2014076556W WO 2015109682 A1 WO2015109682 A1 WO 2015109682A1
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Classifications
-
- 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
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
- G01N33/521—Single-layer analytical elements
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
-
- 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
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
Definitions
- the invention belongs to the technical field of paper chips, and in particular relates to a paper chip, a preparation method thereof and a method for detecting biological molecules. Background technique
- the paper chip is a device proposed by the whitesides research group in 2007 to meet the development requirements of the POCT diagnostic platform. It is inexpensive, portable, biocompatible, simple and fast, with low reagent consumption and flexible design. It has the potential to develop into a new type of cheap detection platform, providing medical diagnosis and health monitoring that is not available to developing countries and remote areas, and greatly promoting the popularization of personalized medicine.
- paper chips There are many methods for fabricating paper chips. Common methods include photolithography, paraffin printing, lithography, mapping, inkjet printing, plasma etching, and cutting. So far, the production method of paper chips has become more and more mature, and the focus of research has shifted to the use of paper chips for biological detection. At present, most of the analytical documents on paper chips still remain on the detection of small molecules such as glucose, uric acid, lactic acid and NADH, and relatively few biomolecules are detected. In 2012, the whiteside team used paper chips as a device for the detection of indicators of liver disease. They also tested alkaline phosphatase, aspartate aminotransferase and total serum protein. The device combines sample preparation and analysis and detection.
- the filter in the device can directly filter out red blood cells and realize direct detection of whole blood.
- the detection sensitivity of the device is very low, and further research is needed for practical application.
- the Lei Ge group used a three-dimensional paper chip to construct a device based on electrochemiluminescence immunoassay, and at the same time realized the detection of four tumor markers of AFP, CA125, CA199 and CEA, but there are still some limitations in practical application.
- the Krull team applied quantum dots to paper chips to achieve solid-phase DNA hybridization. At the same time, using the characteristics of multi-color emission of quantum dots, a proportional sensor for detecting DNA hybridization on paper chips was established, which improved the detection sensitivity. Algar's group also uses the combination of quantum dots and paper chip technology to detect hydrolase activity within 5 to 60 minutes, and the detection limit can reach 1-2 ⁇ .
- Quantum dots have certain toxicity and are not suitable for clinical medical analysis, which limits the application of quantum dots in the development of paper chip methods. Summary of the invention
- An object of the present invention is to provide a paper chip, a method for preparing a paper chip, and a method for detecting a biomolecule.
- the paper chip provided by the present invention is used for detection of biomolecules, has a high reaction speed, a low detection cost, and a high sensitivity.
- the present invention provides a paper chip comprising a paper-based material, a fluorescent donor and a fluorescent quencher; wherein the paper-based material is provided with a detection zone; the fluorescent donor material is fixed to the paper-based material for detection In the region, the fluorescent donor material comprises an upconverting fluorescent nanomaterial and a surface marker labeled on the upconverting fluorescent nanomaterial;
- the fluorescent quencher includes an organic dye or a fluorescent acceptor.
- the organic dye is labeled on the surface marker.
- the up-converting fluorescent nanomaterial has an atomic ratio represented by the formula (I):
- the particle size of the up-converting fluorescent nano material is 30nm ⁇ 100nm.
- the surface marker is a single-stranded nucleic acid, protein or polypeptide.
- the organic dye is rhodamine, BHQ, Cy3, Cy5 or fluorescein.
- the fluorescent acceptor is layered graphene oxide or oxidized carbon sphere.
- the paper-based material is office printing paper.
- the detection zone is formed by printing a pattern directly on the office paper.
- the invention also provides a method for preparing a paper chip, comprising:
- the fluorescent donor material comprising an upconverting fluorescent nanomaterial and a surface marker labeled on the upconverting fluorescent nanomaterial; removing the fluorescent donor material solution a solvent to obtain a paper-based material to which a fluorescent donor material is immobilized;
- the paper-based material to which the fluorescent donor material is immobilized forms a paper chip with a fluorescent quencher; the fluorescent quencher includes an organic dye or a fluorescent acceptor.
- the paper-based material is office printing paper.
- the specific method for forming a detection zone on a paper-based material comprises:
- a pattern is printed on office paper using an ink containing a hydrophobic substance, and a detection area is formed on an office paper.
- the present invention also provides a method for detecting a biomolecule, comprising the steps of: providing a paper chip, the paper chip comprising a paper-based material, a fluorescent donor and a fluorescent quencher; wherein the paper-based material is provided with a detection a fluorescent donor material fixed in the paper-based material detection zone, the fluorescent donor material comprising an up-converting fluorescent nanomaterial and a surface marker labeled on the upconverting fluorescent nanomaterial; the fluorescent Quenchers include organic dyes or fluorescent acceptors;
- the standard curve of the concentration and the fluorescence intensity is established according to the following method: adding a fluorescent quencher solution to the detection zone of the paper-based material, respectively adding a series of standard samples for incubation, respectively measuring the fluorescence of the obtained product Intensity, a standard curve of concentration and fluorescence intensity is established based on the series concentration of the standard sample and the corresponding fluorescence intensity.
- the sample to be tested is whole blood or serum containing biomolecules to be tested.
- the biomolecule is a single-stranded nucleic acid, a saccharide, a protein, uric acid or lactic acid.
- the present invention also provides a method for detecting a biomolecule, comprising the steps of: providing a paper chip, the paper chip comprising a paper-based material, a fluorescent donor and a fluorescent quencher; wherein the paper-based material is provided with a detection a fluorescent donor material fixed in the paper-based material detection region, the fluorescent donor material comprising an up-converting fluorescent nanomaterial and a surface marker labeled on the up-converting fluorescent nanomaterial;
- the quencher is an organic dye; the fluorescent quencher is labeled on the surface marker;
- the sample to be tested is added to the detection zone of the paper-based material for incubation, and the fluorescence intensity of the obtained product is measured, and the concentration of the sample to be tested is calculated based on the fluorescence intensity and a standard curve of the concentration and the fluorescence intensity.
- the present invention also provides a detecting device for biomolecule detection, comprising the paper chip and the fluorescence detecting device described in the above technical scheme.
- the paper chip provided by the present invention comprises a paper-based material, a fluorescent donor and a fluorescent quencher; wherein the paper-based material is provided with a detection zone; the fluorescent donor material is fixed in the Within the paper-based material detection zone, the fluorescent donor material includes an up-converting fluorescent nanomaterial and a surface marker labeled on the up-converted fluorescent nanomaterial; the fluorescent quencher comprising an organic dye or a fluorescent acceptor.
- the invention combines up-conversion fluorescence analysis with paper chip technology, reduces the amount of reactants, reduces the detection cost, accelerates the reaction speed of the reactants, and shortens the reaction time of the conventional 2h ⁇ 3h to less than 1h, and increases the reaction time.
- the invention introduces up-conversion fluorescence analysis into the paper chip technology, can overcome the influence of the background fluorescence and scattered light of the biological sample, avoid the interference of the paper additive, realize the direct detection of the serum and the whole blood sample, and improve the sensitivity of the analysis and detection.
- the experimental results show that the inspection provided by the present invention The coincidence rate between the test results and the actual concentration is above 99.5%, and the accuracy rate is high.
- the present invention can use the office printing paper as the paper-based material, and directly print the pattern on the printing paper to obtain the detection area, without further processing the pattern, simplifying the preparation method of the paper chip, and reducing the cost of raw materials. . DRAWINGS
- 1 is a method for forming a detection area of a paper-based material according to an embodiment of the present invention
- FIG. 3 is a graph showing fluorescence intensity curves of different concentrations of a test substance according to Embodiment 1 of the present invention.
- Embodiment 4 is a standard curve provided by Embodiment 1 of the present invention.
- FIG. 5 is a graph showing fluorescence intensity curves of different concentrations of a test substance according to Embodiment 2 of the present invention.
- Figure 6 is a standard curve provided by Embodiment 2 of the present invention.
- FIG. 7 is a graph showing fluorescence intensity curves of different concentrations of a test substance according to Embodiment 3 of the present invention.
- Figure 8 is a standard curve provided in Example 3 of the present invention. detailed description
- the present invention provides a paper chip comprising a paper-based material, a fluorescent donor and a fluorescent quencher; wherein the paper-based material is provided with a detection zone; the fluorescent donor material is fixed to the paper-based material for detection Within the region, the fluorescent donor material includes an upconverting fluorescent nanomaterial and a surface marker labeled on the upconverting fluorescent nanomaterial; the fluorescent quencher comprising an organic dye or a fluorescent acceptor.
- the paper chip provided by the present invention comprises a paper-based material, which is also referred to as a paper-based carrier, and is the basic structure of the paper chip.
- the paper-based material of the present invention is not particularly limited, and may be a Whatman No. filter paper, an office printing paper, or the like, and is preferably an office printing paper.
- the paper chip For paper substrates In order to obtain a higher background signal, in order to obtain a more accurate measurement result, the paper chip generally uses paper with less additives such as whatmanl filter paper, but the present invention uses up-conversion fluorescence analysis and The combination of paper chip technology avoids the effects of additives in the paper, so it is possible to directly use paper that is cheaper and easier to obtain, such as office paper.
- the paper-based material is provided with a detection area.
- the method for setting the detection area is not particularly limited, and may be a photolithography method, a paraffin printing method, a lithography method, a drawing method, an inkjet method which are well known to those skilled in the art.
- a printing method, a plasma etching method, a dicing method, or the like after forming a pattern on the paper-based material by the above method, the pattern is subjected to a hydrophobic treatment to obtain a detection region on the paper-based material.
- the present invention prints a pattern directly on a paper-based material without directly performing a hydrophobic treatment to form a detection zone. In this case, it is preferred to perform printing using an ink containing a hydrophobic substance.
- the present invention has no particular limitation on the number of the detection zones, and can be set by a person skilled in the art according to actual needs.
- the paper chip further includes a fluorescent donor, the fluorescent donor functioning to provide a fluorescent substance, and detecting the biomolecule by fluorescence quenching of the fluorescent substance.
- the fluorescent donor comprises an upconverting fluorescent nanomaterial and a surface marker labeled on the upconverting fluorescent nanomaterial.
- the up-converting fluorescent nanomaterial is preferably a water-soluble up-converting fluorescent nanomaterial, preferably having an atomic ratio represented by the formula (I):
- Ln is Er, Tm or Ho, preferably Er.
- the particle diameter of the up-converting fluorescent nanomaterial is preferably from 30 nm to 100 nm, and more preferably
- the source of the up-converting fluorescent nanomaterial of the present invention is not particularly limited, and may be commercially available or may be obtained by a method well known to those skilled in the art.
- the present invention prepares an up-converting fluorescent nanomaterial according to a method well known to those skilled in the art, and at the same time, an amino group or a carboxyl group is modified on the surface thereof, for example, a polyethyleneimine or a polyacrylic acid is modified to facilitate subsequent labeling of the surface. Mark.
- the surface marker is preferably a single-stranded nucleic acid, protein or polypeptide, such as a carcinoembryonic antigen aptamer or the like, which is labeled on the surface of the upconverting fluorescent nanomaterial.
- the surface marker of the present invention The manner in which the surface of the up-converting fluorescent nanomaterial is recorded is not particularly limited, and the labeling may be performed by a chemical reaction, for example, the upper converted fluorescent nanomaterial is modified with polyethyleneimine or polyacrylic acid, and then The surface markers are incubated together to obtain upconverting fluorescent nanomaterials with surface markers labeled with surface markers.
- the fluorescent donor is fixed in the detection zone of the paper-based material.
- the method for fixing the fluorescent donor in the detection region of the paper-based material is not particularly limited, and the fixing can be achieved by the following method: adding a fluorescent donor solution to the detection region of the paper-based material, and fluorescently The solvent in the donor solution is removed to immobilize the fluorescent donor solution within the detection zone.
- the paper chip provided by the present invention further comprises a fluorescent quencher, and the fluorescent quencher functions to cause fluorescence quenching of the fluorescent material, thereby realizing detection and analysis of the biomolecule.
- the fluorescent quencher comprises an organic dye or a fluorescent acceptor; the organic dye includes but is not limited to rhodamine, BHQ, Cy3, Cy5 or fluorescein, etc.; the fluorescent acceptor is preferably layered oxidation Graphene or oxidized carbon spheres, etc.
- the fluorescent quencher when the fluorescent quencher is an organic dye, it may be present alone, and then mixed with the fluorescent donor in the detection zone during use; or may be labeled on the surface marker, at this time, the organic dye mark On the surface marker, the surface marker is labeled on the upconverting fluorescent nanomaterial and the upconverting fluorescent nanomaterial is immobilized in the detection zone of the paper based material.
- the method for labeling the organic dye on the surface marker of the present invention is not particularly limited, and a surface marker labeled with an organic dye can be directly purchased.
- the fluorescent quencher is a fluorescent acceptor, it is preferably present alone and, when used, is mixed with a fluorescent donor in the detection zone.
- the source of the layered graphene oxide or oxidized carbon sphere of the present invention is not particularly limited and may be purchased directly or may be prepared according to a method well known to those skilled in the art.
- the paper chip provided by the present invention may include two types:
- the first type of paper chip is: a paper-based material, a fluorescent donor, and an organic dye.
- the up-converting fluorescent material is fixed in the detection area of the paper-based material, the surface-converted material is marked on the up-converting fluorescent material, and the surface marker is marked with organic Dye; when used, the test sample can be directly added to the detection zone of the paper-based material for incubation;
- the second paper chip is: a paper-based material, a fluorescent donor, and a fluorescent quencher; an up-converting fluorescent material is fixed in a detection area of the paper-based material, and a surface marker is marked on the up-converting fluorescent material;
- the fluorescent quencher is added to the detection zone of the paper-based material, and then the sample to be tested is added for incubation to achieve detection.
- the fluorescent quencher may be an organic dye or a fluorescent acceptor.
- the paper chip provided by the present invention needs to be combined with a solid phase fluorescence detector to detect the product obtained after the addition of the sample to be tested, thereby obtaining the concentration of the sample to be tested. That is, the paper chip provided by the present invention needs to be used in conjunction with a solid phase fluorescence detector.
- the solid phase fluorescence detector of the present invention is not particularly limited, and a commercially available detector can be used.
- the invention also provides a method for preparing a paper chip, comprising:
- the fluorescent donor material comprising an upconverting fluorescent nanomaterial and a surface marker labeled on the upconverted fluorescent nanomaterial; removing the fluorescent donor material solution a solvent to obtain a paper-based material to which a fluorescent donor material is immobilized;
- the paper-based material to which the fluorescent donor material is fixed forms a paper chip with a fluorescent quencher.
- the paper base material is a basic structure of a paper chip, and the paper base material is also called a paper base carrier, and may be a Whatmanl filter paper, an office printing paper or the like, and is preferably an office printing paper.
- the paper chip since the additive generates a high background signal, in order to obtain a more accurate measurement result, the paper chip generally uses paper with less additives such as whatmanl filter paper, but the present invention uses up-conversion. Fluorescence analysis combined with paper chip technology avoids the effects of additives in the paper, so it is possible to directly use paper that is cheaper and easier to obtain, such as office paper.
- the method of the present invention for forming a detection region on a paper-based material is not particularly limited, and may be a photolithography method, a paraffin printing method, a lithography method, a drawing method, an inkjet printing method, a plasma etching method, and a cutting method which are well known to those skilled in the art.
- the pattern is formed on the paper-based material by the above method, the pattern is subjected to a hydrophobic treatment to form a detection zone on the paper-based material.
- the invention is preferably straight
- the pattern is printed on the paper-based material, and the detection zone is directly formed without hydrophobic treatment.
- the ink containing the hydrophobic substance for printing, that is, the ink containing the hydrophobic substance can be printed on the office paper.
- the pattern, one-step method forms a detection area on the office paper.
- the present invention has no particular limitation on the number of the detection zones, and can be set by a person skilled in the art according to actual needs.
- Fig. 1 shows a method for forming a detection area of a paper-based material according to an embodiment of the present invention.
- the pattern is printed directly on paper, and the paper-based material provided with the detection area can be obtained in one step without further processing.
- the up-converting fluorescent nanomaterial is preferably a water-soluble up-converting fluorescent nanomaterial, preferably having an atomic ratio represented by the formula (I):
- Ln is Er, Tm or Ho, preferably Er.
- the particle diameter of the up-converting fluorescent nanomaterial is preferably from 30 nm to 100 nm, and more preferably
- the source of the up-converted fluorescent nanomaterial of the present invention is not particularly limited, and may be commercially available or may be obtained by a method well known to those skilled in the art.
- the present invention prepares an up-converting fluorescent nanomaterial according to a method well known to those skilled in the art, and at the same time, an amino group or a carboxyl group is modified on the surface thereof, for example, a polyethyleneimine or a polyacrylic acid is modified to facilitate subsequent labeling of the surface. Mark.
- the surface marker is preferably a single-stranded nucleic acid, protein or polypeptide, such as a carcinoembryonic antigen aptamer or the like, which is labeled on the surface of the upconverting fluorescent nanomaterial.
- the method for labeling the surface marker on the surface of the up-converting fluorescent nanomaterial is not particularly limited, and may be labeling by chemical reaction, for example, modifying the up-conversion with polyethyleneimine or polyacrylic acid.
- the fluorescent nanomaterial is then incubated with the surface marker to obtain an upconverting fluorescent nanomaterial having a surface marker labeled with a surface.
- the fluorescent donor solution is preferably formed by dispersing a fluorescent donor in a Tris-HCl buffer solution.
- the solution is added to the detection zone of the paper-based material to remove the solvent from the fluorescent donor solution, thereby immobilizing the fluorescent donor in the detection zone of the paper-based material.
- the method for removing the solvent in the present invention is not particularly limited, and may be a method well known to those skilled in the art such as evaporation.
- the fluorescent quencher comprises an organic dye or a fluorescent acceptor; the organic dye includes, but is not limited to, rhodamine, BHQ, Cy3, Cy5 or fluorescein, etc.; the fluorescent acceptor is preferably layered oxidation Graphene or oxidized carbon spheres, etc.
- the fluorescent quencher when the fluorescent quencher is an organic dye, it may be present alone, and then mixed with the fluorescent donor in the detection zone during use; or may be labeled on the surface marker, at this time, the organic dye mark On the surface marker, the surface marker is labeled on the upconverting fluorescent nanomaterial and the upconverting fluorescent nanomaterial is immobilized in the detection zone of the paper based material.
- the method for labeling the organic dye on the surface marker of the present invention is not particularly limited, and a surface marker labeled with an organic dye can be directly purchased.
- the fluorescent quencher is a fluorescent acceptor, it is preferably present alone and, in use, is mixed with a fluorescent donor in the detection zone.
- the source of the layered graphene oxide or oxidized carbon sphere of the present invention is not particularly limited and may be purchased directly or may be prepared according to a method well known to those skilled in the art.
- the method of preparing the paper chip is as follows:
- the fluorescent donor material comprising an upconverting fluorescent nanomaterial, a surface marker labeled on the upconverted fluorescent nanomaterial, and a label on the surface marker Organic dye
- the paper-based material to which the fluorescent donor material is fixed can be used as a paper chip.
- the fluorescent dye can be directly labeled on the surface marker, it can be directly fixed to the detection area of the paper-based material together with the surface marker, the up-converted fluorescent nanomaterial, etc., and other steps are The above preparation methods are the same, and the present invention will not be repeated herein.
- amino- or carboxyl-modified up-converted fluorescent nanomaterial can be prepared as follows:
- rare earth ions are 4 ethyl ions with a molar ratio of 80:18:2: ⁇ ion: ⁇ ion, 18 mL of absolute ethanol is added thereto, and then 0.9000 g of polyacrylic acid is added.
- the surface mark of the water-soluble up-converting fluorescent nano material can be carried out as follows:
- polyacrylic acid or polyethyleneimine modified upconverting fluorescent nanomaterial is dissolved in 2mL ⁇ 5mL MES buffer (10mM, pH 5.5) or HEPES buffer ( ⁇ , pH 7.4), adding 0.8mg ⁇ 3.2mg EDC ⁇ HC1 2.2mg ⁇ 6.6mg Sulfo-NHS or lmg ⁇ 5mg sulfo-SMCC, incubate for 0.5h ⁇ 2h at 30°C under slight shaking to activate the carboxyl or amino group on the surface of the up-converting fluorescent nanomaterial. Upon completion of the activation, the activated polyacrylic acid or polyethyleneimine modified upconverting fluorescent nanomaterial was centrifuged and washed three times with high purity water.
- the washed precipitate was dispersed in 2 mL ⁇ 5 mL HEPES buffer (1 OmM, pH 7.2), and 1.5 uM-4 uM single-stranded DNA or 1 mg ⁇ 3 mg of dye-labeled polypeptide was added thereto at 30 ° C with slight shaking conditions. Incubate 21! After 24 hours, 50 mg of Tris was added to block excess NHS. After centrifugation, the resulting precipitate was washed three times with high purity water to remove excess single-stranded DNA or polypeptide, and after washing, it was dispersed in 2.5 ml of Tris-HCl (10 mM, 150 mM NaCl, pH 7.4).
- the layered graphene oxide can be prepared according to the following method: 50 mL of concentrated sulphuric acid is heated to 90 ° C, 10 g of K 2 S 2 0 8 and 10 g of P 2 0 5 are added thereto, and the temperature is lowered to 80.
- the above methods are merely illustrative of upconverting the fluorescent nanomaterial, labeling the surface marker on the surface of the upconverting nanomaterial, and preparing the layered graphene oxide, and those skilled in the art can prepare the above materials by other known methods.
- the present invention also provides a method for detecting a biomolecule, comprising the steps of: providing a paper chip, the paper chip comprising a paper-based material, a fluorescent donor and a fluorescent quencher; wherein the paper-based material is provided with a detection a fluorescent donor material fixed in the paper-based material detection zone, the fluorescent donor material comprising an up-converting fluorescent nanomaterial and a surface marker labeled on the upconverting fluorescent nanomaterial; the fluorescent Quenchers include organic dyes or fluorescent acceptors
- the method for detecting the biomolecule comprises the steps of:
- the paper chip comprising a paper-based material, a fluorescent donor and a fluorescent quencher; wherein the paper-based material is provided with a detection zone; the fluorescent donor material is fixed in the paper-based material detection zone Internally, the fluorescent donor material includes an upconverting fluorescent nanomaterial and a surface marker labeled on the upconverting fluorescent nanomaterial; the fluorescent quencher is labeled on the surface marker; the fluorescence quenching The agent is an organic dye; The sample to be tested is added to the detection zone of the paper-based material for incubation, and the fluorescence intensity of the obtained product is measured, and the concentration of the sample to be tested is calculated according to the fluorescence intensity and a standard curve of the concentration and the fluorescence intensity.
- the first detection method will be described as an example.
- the paper chip prepared by the above-mentioned technical solution or the above-mentioned preparation method is used as a detecting device for detecting biomolecules.
- the specific structure of the paper chip and the preparation process thereof are as described above, and the present invention is no longer Narration.
- the invention first selects the optimal fluorescence quencher concentration, and the specific method is as follows:
- Different concentrations of the fluorescent quencher solution were added to the detection zone of the paper-based material, and the fluorescence intensity of the obtained reaction product was respectively detected after the reaction, and the concentration of the fluorescent quencher solution was selected as the optimal concentration when the fluorescence quenching efficiency was maximum.
- the fluorescent quencher is an organic dye and is labeled on a surface marker, this step may not be performed.
- the specific method is as follows:
- the fluorescence intensity of the sample solution concentration is recorded as F
- the fluorescence intensity of the blank solution is recorded as F0
- the standard of (F-FO) / F0 is plotted on the ordinate
- the concentration is plotted on the abscissa. Curve, the appropriate detection concentration of the detection method is obtained.
- the sample to be tested formed by the biomolecule in whole blood or serum can be used as a test object, and the establishment of a standard curve and the detection of the sample to be tested at an unknown concentration are performed.
- the curve is built as follows:
- the concentration of the series is in the above-mentioned suitable detection concentration range
- F fluorescence intensity of the sample solution concentration
- F0 fluorescence intensity of the blank solution
- F0 fluorescence intensity of the blank solution
- F0 fluorescence intensity of the blank solution
- the process parameters of the reaction, the process parameters of the incubation, and the detected parameters are similar, and the specifics may be: After the fluorescent quencher and the fluorescent donor are mixed in the detection zone, preferably placed at 20 ° C ⁇ 30 ° C for 3 min ⁇ 10 min, The fluorescent quencher and the fluorescent donor are reacted; the temperature for incubation is preferably 20 ° C to 30 ° C, and the time is preferably 10 min to 30 min; preferably, the fluorescence intensity of the product obtained after the incubation is detected under a 980 nm laser.
- the paper can be directly used to establish the standard curve of the sample to be tested in the buffer solution and the establishment of a standard curve in the whole blood or serum of the sample to be tested. Then, the detection of the unknown concentration sample can be directly performed, and it is not necessary to select the concentration of the fluorescent quencher at the optimum concentration.
- the detection method described in the above method there is basically no difference from the detection method described in the above method, and those skilled in the art can refer to the method described above, and the present invention will not be repeated herein.
- FIG. 2 is a detection mechanism of a detection method provided by the present invention.
- a fluorescent quencher, a surface marker and an up-converted fluorescent nano material are sequentially connected; after the test object is added, the test object is Separating the fluorescent quencher from the surface marker and/or the upconverting fluorescent nanomaterial to cause fluorescence quenching of the upconverting fluorescent nanomaterial, thereby establishing a relationship between the fluorescence intensity and the concentration of the analyte, and realizing the object to be tested Detection.
- the paper chip provided by the present invention comprises a paper-based material, a fluorescent donor and a fluorescent quencher; wherein the paper-based material is provided with a detection zone; the fluorescent donor material is fixed in the paper-based material detection zone, The fluorescent donor material includes an upconverting fluorescent nanomaterial and a surface marker labeled on the upconverting fluorescent nanomaterial; the fluorescent quencher comprising an organic dye or a fluorescent acceptor.
- the invention combines up-conversion fluorescence analysis with paper chip technology, reduces the amount of reactants, reduces the detection cost, accelerates the reaction speed of the reactants, and shortens the reaction time of the conventional 2h ⁇ 3h to less than 1h, and increases the reaction time. The possibility of paper chips being used for clinical testing.
- the invention adopts up-conversion fluorescence analysis into the paper chip technology, can overcome the influence of the background fluorescence and scattered light of the biological sample, avoid the interference of the paper additive, realize the direct detection of the serum and the whole blood sample, and improve the analysis and detection. Sensitivity.
- polyacrylic acid modified upconverting fluorescent nanomaterial 5 mg was dissolved in 5 mL of 10 mM MES buffer with a pH of 5.5, and 3.2 mg of EDC ⁇ HC1 and 6.6 mg of Sulfo-NHS were added, and incubated at 30 ° C: under slight shaking conditions. .5h, activated polyacrylic acid modified upconverting fluorescent nanomaterial was centrifuged and washed three times with high purity water.
- the washed precipitate was dispersed in 3 mL of 10 mM HEPES buffer at pH 7.2, to which was added 2 mg of TAMRA-labeled polypeptide purchased from Shanghai Junyi Biotechnology Co., Ltd., sequence TAMRA-GPLGVRC, After incubating for 20 h at 30 ° C with gentle shaking, 50 mg of Tris was added, and the resulting precipitate was washed three times with high-purity water. After washing, it was dispersed in 2.5 mL of 10 mM, 150 mM NaCl, and pH 7.4 Tris-HCl. UCP-peptide-TAMRA solution;
- FIG 3 is a graph showing fluorescence intensity curves of different concentrations of the test substance provided in Example 1 of the present invention.
- the degree of recovery of the fluorescence intensity F of the sample and the fluorescence intensity F Q of the sample without the target is obtained by plotting the target concentration as the abscissa and (FF 0 ) /F 0 as the ordinate.
- Figure 4 A standard curve provided for Embodiment 1 of the present invention.
- rare earth ions are 4 ethyl ion, cerium ion and cerium ion with a molar ratio of 80:18:2, and 18 mL of absolute ethanol is added thereto, and then 0.3400 g of polyethylene is added.
- polyethyleneimine modified upconverting fluorescent nanomaterial 5 mg was dissolved in 5 mL of 10 mM MES buffer with H value of 5.5, 3.2 mg of EDC ⁇ HC1 and 6.6 mg of Sulfo-NHS were added, and incubated at 30 ° C under slight shaking conditions.
- the activated polyethyleneimine-modified upconverting fluorescent nanomaterial was isolated by centrifugation and washed three times with high purity water. The washed precipitate was dispersed in 3 mL of 10 mM HEPES buffer at pH 7.2, and 3 uM of the carcinoembly antigen aptamer was added thereto.
- the dried product is further ultrasonicated for 2 h, and the graphene oxide is gradually peeled off into layered graphene oxide;
- Using oily pen ink as printing ink print the pattern on A4 printing paper to form the detection area; take 4 L 0.05mg/mL UCP-CEA aptamer solution into the above detection area, leave lOmin at room temperature, add 4 L after drying
- Concentrated layered graphene oxide solution at concentrations of 0 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, and 0.5 mg/mL, at room temperature for 10 min, at 980 nm laser
- the fluorescence intensity of the upconversion is measured to obtain a fluorescence quenching curve, and the concentration of the layered graphene oxide is 0.1 mg/mL when the fluorescence quenching efficiency is maximized;
- FIG. 5 is a graph showing fluorescence intensity curves of different concentrations of the test substance according to Embodiment 2 of the present invention; calculating the fluorescence intensity F of the sample to which the target is added and the fluorescence intensity F Q of the sample containing no target substance
- the standard curve is obtained by plotting the target concentration as the abscissa and (FF 0 ) /F 0 as the ordinate.
- FIG. 6 is a standard curve provided by Embodiment 2 of the present invention.
- a 80 ng/mL CEA sample was prepared, and the fluorescence intensity was measured according to the above procedure, and the concentration was calculated according to the above standard curve. The results showed that the calculated concentration was 79.6 ng/mL, and the coincidence rate with the known concentration was 99.5%.
- the washed precipitate was dispersed in 3 mL of 10 mM HEPES buffer with a H value of 7.2, and 3 uM of the carotenoid antigen aptamer was added thereto, and after incubation at 30 ° C for 2 hours under gentle shaking, 50 mg of Tris was added and centrifuged.
- the obtained precipitate was washed three times with high-purity water, and after washing, it was dispersed in 2.5 ml of 0 mM, 150 mM NaCl, and pH 7.4 in Tris-HCl to obtain a UCP-CEA aptamer solution;
- FIG. 7 is a graph showing the fluorescence intensity of the different concentrations of the test substance according to the third embodiment of the present invention; calculating the fluorescence intensity F of the sample to which the target is added and containing no target Sample fluorescence intensity F ⁇ recovery Degree, with the target concentration as the abscissa, with (F-FQ ) /F.
- a standard curve is obtained by plotting the ordinate, see Fig. 8, which is a standard curve provided in Example 3 of the present invention.
- a 60 ng/mL CEA sample was prepared, and the fluorescence intensity was measured according to the above procedure, and the concentration was calculated according to the above standard curve. The results showed that the calculated concentration was 59.9 ng/mL, and the coincidence rate with the known concentration was 99.9%.
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Abstract
一种纸芯片、其制备方法及生物分子的检测方法，其中，纸芯片包括纸基材料、荧光供体和荧光淬灭剂，纸基材料上设置有检测区，荧光供体材料固定在纸基材料检测区内，荧光供体材料包括上转换荧光纳米材料和标记在该上转换荧光纳米材料上的表面标记物，荧光淬灭剂包括有机染料或荧光受体。将上转换荧光分析与纸芯片技术相结合，减少了反应物的用量，降低了检测成本，加快了反应物的反应速度，增加了纸芯片应用于临床检测的可能。
Description
纸芯片、 其制备方法及生物分子的检测方法
本申请要求于 2014 年 01 月 27 日提交中国专利局、 申请号为 201410040579.4、 发明名称为 "纸芯片、 其制备方法及生物分子的检测方 法" 的中国专利申请的优先权， 其全部内容通过引用结合在本申请中。 技术领域
本发明属于纸芯片技术领域，尤其涉及一种纸芯片、其制备方法及生 物分子的检测方法。 背景技术
纸芯片是 whitesides课题组于 2007年提出的一种满足 POCT诊断平 台发展要求的器件，具有价格低廉、可便携式、生物相容性好、简单快速、 试剂消耗量小和设计灵活的等点，极有潜力发展成为一种新型廉价的检测 平台，给发展中国家以及偏远地区提供原本无法获得的医疗诊断及健康监 测， 对个性化医疗的普及化起到巨大的推动作用。
纸芯片的制作方法有很多，常见的方法主要包括光刻法、石蜡打印法、 平版印刷法、 绘图法、 喷墨打印法、 等离子体蚀刻法和切割法等。 到目前 为止纸芯片的制作方法已经日趋成熟，研究的重点转移到利用纸芯片进行 生物检测上来。目前纸芯片上的分析检测文献还大多数还停留在对小分子 的检测上， 例如葡萄糖、 尿酸、 乳酸和 NADH等， 关于生物大分子的检 测相对较少。 2012年， whiteside课题组将纸芯片做成器件用于肝类疾病 指示物的检测，他们同时检测了碱性磷酸酶、天冬氨酸转氨酶和总血清蛋 白。该器件将样品前处理和分析检测结合于一体，器件中的滤膜可以直接 过滤掉血红细胞， 实现全血的直接检测， 但是该器件的检测灵敏度很低， 需进一步研究才能用于实际应用。 Lei Ge课题组利用三维纸芯片构建了 基于电化学发光免疫检测的器件， 同时实现了 AFP、 CA125、 CA199、 CEA 四种肿瘤标志物的检测， 但在实际应用方面还是存在一定局限性。
Krull课题组将量子点应用于纸芯片中， 实现了固相 DNA杂交的检测，
同时利用量子点多色发射的特点， 建立了纸芯片上检测 DNA杂交的比例 型传感器， 提高了检测灵敏度。 Algar课题组同样利用量子点与纸芯片技 术的结合在 5-60min内实现水解酶活性的检测， 检测限可以达到 1-2ηΜ。
但是， 这些方法都存在一定的缺陷：
( 1 )纸芯片上生物大分子的检测通常是利用酶联免疫反应进行检测， 需要进行洗涤分离等， 操作繁瑣、 耗时， 同时， 酶联免疫过程还需要使用 抗原抗体等蛋白， 价格昂贵， 检测成本高；
( 2 )纸张基底中的添加剂会产生相当高的背景信号， 目前纸芯片上 的荧光检测方法无法避免这一缺陷， 限制其灵敏度的提高；
( 3 )量子点具有一定的毒性， 不适宜用于临床医学分析， 限制了量子点 在开发纸芯片方法中的应用。 发明内容
本发明的目的在于提供一种纸芯片、纸芯片的制备方法及生物分子的 检测方法， 本发明提供的纸芯片用于生物分子的检测时， 反应速度快、检 测成本低、 灵敏度较高。
本发明提供了一种纸芯片， 包括纸基材料、 荧光供体和荧光猝灭剂； 其中，所述纸基材料上设置有检测区； 所述荧光供体材料固定在所述纸基 材料检测区内，所述荧光供体材料包括上转换荧光纳米材料和标记在所述 上转换荧光纳米材料上的表面标记物；
所述荧光猝灭剂包括有机染料或荧光受体。
优选的， 所述有机染料标记在所述表面标记物上。
优选的， 所述荧光供体材料中， 所述上转换荧光纳米材料具有式（I ) 所示的原子比：
NaYF4: Yb, Ln ( I );
其中， Ln为 Er、 Tm或 Ho。
优选的， 所述荧光供体材料中， 所述上转换荧光纳米材料的粒径为
30nm~100nm„
优选的， 所述表面标记物为单链核酸、 蛋白质或多肽。
优选的， 所述有机染料为罗丹明、 BHQ、 Cy3、 Cy5或荧光素。 优选的， 所述荧光受体为层状氧化石墨烯或氧化碳球。
优选的， 所述纸基材料为办公室用打印纸。
优选的， 所述检测区通过直接在办公室用打印纸上打印图案形成。 本发明还提供了一种纸芯片的制备方法， 包括：
在纸基材料上形成检测区；
向所述检测区内加入荧光供体材料溶液，所述荧光供体材料包括上转 换荧光纳米材料和标记在所述上转换荧光纳米材料上的表面标记物； 去除所述荧光供体材料溶液中的溶剂，得到固定有荧光供体材料的纸 基材料；
所述固定有荧光供体材料的纸基材料与荧光猝灭剂形成纸芯片； 所述荧光猝灭剂包括有机染料或荧光受体。
优选的， 所述纸基材料为办公室用打印纸。
优选的， 所述在纸基材料上形成检测区的具体方法包括：
用含疏水性物质的油墨在办公室用打印纸上打印图案 ,在办公室用打 印纸上形成检测区。
本发明还提供了一种生物分子的检测方法， 包括以下步骤： 提供纸芯片， 所述纸芯片包括纸基材料、 荧光供体和荧光猝灭剂； 其 中，所述纸基材料上设置有检测区； 所述荧光供体材料固定在所述纸基材 料检测区内，所述荧光供体材料包括上转换荧光纳米材料和标记在所述上 转换荧光纳米材料上的表面标记物；所述荧光猝灭剂包括有机染料或荧光 受体；
将荧光猝灭剂溶液加入到纸基材料的检测区内，再加入待测样品进行 孵育，测定得到的产物的荧光强度，根据所述荧光强度以及浓度与荧光强 度的标准曲线计算所述待测样品的浓度。
优选的， 所述浓度与荧光强度的标准曲线按照以下方法建立： 将荧光猝灭剂溶液加入到纸基材料的检测区内，分别加入系列浓度的 标准样品进行孵育，分别测定得到的产物的荧光强度，根据标准样品的系 列浓度以及相对应的荧光强度建立浓度与荧光强度的标准曲线。
优选的， 所述待测样品为含有待测生物分子的全血或血清。
优选的， 所述生物分子为单链核酸、 糖类、 蛋白质、 尿酸或乳酸。 本发明还提供了一种生物分子的检测方法， 包括以下步骤： 提供纸芯片， 所述纸芯片包括纸基材料、 荧光供体和荧光猝灭剂； 其 中，所述纸基材料上设置有检测区； 所述荧光供体材料固定在所述纸基材 料检测区内，所述荧光供体材料包括上转换荧光纳米材料和标记在所述上 转换荧光纳米材料上的表面标记物； 所述荧光猝灭剂为有机染料； 所述荧 光猝灭剂标记在所述表面标记物上；
将待测样品加入到纸基材料的检测区内进行孵育，测定得到的产物的 荧光强度，根据所述荧光强度以及浓度与荧光强度的标准曲线计算所述待 测样品的浓度。
本发明还提供了一种用于生物分子检测的检测装置，包括上述技术方 案所述的纸芯片和荧光检测装置。
与现有技术相比，本发明提供的纸芯片包括纸基材料、荧光供体和荧 光猝灭剂； 其中， 所述纸基材料上设置有检测区； 所述荧光供体材料固定 在所述纸基材料检测区内，所述荧光供体材料包括上转换荧光纳米材料和 标记在所述上转化荧光纳米材料上的表面标记物；所述荧光猝灭剂包括有 机染料或荧光受体。本发明将上转换荧光分析与纸芯片技术相结合，减少 了反应物的用量， 降低了检测成本， 加快了反应物的反应速度， 将传统的 2h~3h的反应时间缩短到 lh以内，增加了纸芯片应用于临床检测的可能。 同时，本发明将上转换荧光分析引入纸芯片技术中，可以克服生物样品本 底荧光和散射光的影响， 同时避免纸张添加剂的干扰， 实现血清和全血样 品的直接检测，提高分析检测的灵敏度。 实验结果表明， 本发明提供的检
测方法的检测结果与实际浓度的吻合率在 99.5%以上， 准确率较高。
进一步的，本发明可以使用办公室用打印纸作为纸基材料，直接在该 打印纸上打印出图案即可得到检测区，无需对图案进行进一步处理， 简化 了纸芯片的制备方法， 降低了原材料成本。 附图说明
图 1为本发明实施例提供的纸基材料检测区形成的方法；
图 2为本发明提供的检测方法的检测机理；
图 3为本发明实施例 1提供的不同浓度的待测物质的荧光强度曲线 图；
图 4为本发明实施例 1提供的标准曲线；
图 5为本发明实施例 2提供的不同浓度的待测物质的荧光强度曲线 图；
图 6为本发明实施例 2提供的标准曲线；
图 7为本发明实施例 3提供的不同浓度的待测物质的荧光强度曲线 图；
图 8为本发明实施例 3提供的标准曲线。 具体实施方式
本发明提供了一种纸芯片， 包括纸基材料、 荧光供体和荧光猝灭剂； 其中，所述纸基材料上设置有检测区； 所述荧光供体材料固定在所述纸基 材料检测区内，所述荧光供体材料包括上转换荧光纳米材料和标记在所述 上转换荧光纳米材料上的表面标记物；所述荧光猝灭剂包括有机染料或荧 光受体。
本发明提供的纸芯片包括纸基材料， 所述纸基材料又称为纸基载体， 是纸芯片的基础结构。 本发明对所述纸基材料没有特殊限制， 可以为 whatmanl号滤纸、 办公用打印纸等， 优选为办公用打印纸。 对于纸基材
料而言， 由于其中的添加剂会产生较高的背景信号，为了获得较为准确的 测定结果，纸芯片一般釆用 whatmanl号滤纸等添加剂较少的纸张，但是， 本发明釆用上转换荧光分析与纸芯片技术相结合,避免了纸张中的添加剂 的影响，因此，可以直接使用办公室打印纸等较为便宜、容易获得的纸张。
所述纸基材料上设置有检测区，本发明对所述检测区的设置方法没有 特殊限制， 可以为本领域技术人员熟知的光刻法、石蜡打印法、 平板印刷 法、 绘图法、 喷墨打印法、 等离子蚀刻法和切割法等， 釆用上述方法在纸 基材料上形成图案后，对所述图案进行疏水处理，从而在纸基材料上获得 检测区。本发明优选直接在纸基材料上打印出图案，不对其进行疏水处理 直接形成检测区， 此时， 优选使用含疏水性物质的油墨进行打印。 本发明 对所述检测区的个数没有特殊限制，本领域技术人员可以根据实际需要进 行设定。
所述纸芯片还包括荧光供体， 所述荧光供体的作用在于提供荧光物 质，并通过荧光物质的荧光猝灭现象实现对生物分子的检测。在本发明中， 所述荧光供体包括上转换荧光纳米材料和标记在所述上转换荧光纳米材 料上的表面标记物。其中，所述上转换荧光纳米材料优选为水溶性上转换 荧光纳米材料， 优选具有式（I ) 所示的原子比：
NaYF4: Yb, Ln ( I );
其中， Ln为 Er、 Tm或 Ho, 优选为 Er。
所述上转换荧光纳米材料的粒径优选为 30nm~100nm , 更优选为
50nm~80nm。 本发明对所述上转换荧光纳米材料的来源没有特殊限制， 可以为市场上购买得到， 也可以按照本领域技术人员熟知的方法制备得 到。本发明优选在按照本领域技术人员熟知的方法制备上转换荧光纳米材 料， 同时， 在其表面修饰有氨基或者羧基， 例如釆用聚乙烯亚胺或聚丙烯 酸等对其进行修饰， 以便后续标记表面标记物。
所述表面标记物优选为单链核酸、蛋白质或多肽，如癌坯抗原适配体 等，其标记在所述上转换荧光纳米材料表面。本发明对所述表面标记物标
记在所述上转换荧光纳米材料表面的方式没有特殊限制，可以为通过化学 反应进行连接实现标记，例如，釆用聚乙烯亚胺或聚丙烯酸修饰所述上转 化荧光纳米材料，然后将其与表面标记物共同孵育，得到表面标记有表面 标记物的上转换荧光纳米材料。
在本发明中，所述荧光供体固定在所述纸基材料的检测区内。本发明 对所述荧光供体固定在所述纸基材料的检测区内的方式没有特殊限制，可 以釆用以下方法实现固定： 将荧光供体溶液加入到纸基材料的检测区内， 将荧光供体溶液中的溶剂去除， 即可将荧光供体溶液固定于所述检测区 内。
本发明提供的纸芯片还包括荧光猝灭剂，所述荧光猝灭剂的作用是使 荧光材料发生荧光猝灭， 从而实现对生物分子的检测分析。 在本发明中， 所述荧光猝灭剂包括有机染料或荧光受体；所述有机染料包括但不限于罗 丹明、 BHQ、 Cy3、 Cy5或荧光素等； 所述荧光受体优选为层状氧化石墨 烯或氧化碳球等。 在本发明中， 所述荧光猝灭剂为有机染料时， 可以单独 存在，使用时再与检测区内的荧光供体混合；也可以标记在所述表面标记 物上， 此时，有机染料标记在表面标记物上，表面标记物标记在上转换荧 光纳米材料上，上转化荧光纳米材料固定在纸基材料的检测区内。本发明 对所述有机染料在表面标记物上的标记方法没有特殊限制 ,可以直接购买 标记有有机染料的表面标记物。所述荧光猝灭剂为荧光受体时，优选单独 存在，使用时再与检测区内的荧光供体混合。本发明对所述层状氧化石墨 烯或氧化碳球的来源没有特殊限制，可以直接购买得到，也可以按照本领 域技术人员熟知的方法制备得到。
具体而言， 本发明提供的纸芯片可以包括两种：
第一种纸芯片为： 纸基材料、 荧光供体和有机染料， 纸基材料的检测 区内固定有上转换荧光材料，上转换荧光材料上标记有表面标记物，表面 标记物上标记有有机染料；使用时，直接将待测样品加入到纸基材料的检 测区内进行孵育即可实现检测；
第二种纸芯片为：， 纸基材料、 荧光供体和荧光猝灭剂， 纸基材料的 检测区内固定有上转换荧光材料， 上转换荧光材料上标记有表面标记物； 使用时，将荧光猝灭剂加入到纸基材料的检测区内，再加入待测样品进行 孵育即可实现检测。 此时， 荧光猝灭剂可以为有机染料， 也可以为荧光受 体。
同时，本发明提供的纸芯片需要配合使用固相荧光检测器对加入待测 样品孵育后得到的产物进行检测， 从而获得待测样品的浓度。 即， 本发明 提供的纸芯片需要配合固相荧光检测器使用。本发明对所述固相荧光检测 器没有特殊限制， 市场上购买的检测器即可。
本发明还提供了一种纸芯片的制备方法， 包括：
在纸基材料上形成检测区；
向所述检测区内加入荧光供体材料溶液，所述荧光供体材料包括上转 换荧光纳米材料和标记在所述上转化荧光纳米材料上的表面标记物； 去除所述荧光供体材料溶液中的溶剂，得到固定有荧光供体材料的纸 基材料；
所述固定有荧光供体材料的纸基材料与荧光猝灭剂形成纸芯片。 本发明以纸基材料为纸芯片的基础结构 ,所述纸基材料又称为纸基载 体， 可以为 whatmanl号滤纸、 办公用打印纸等， 优选为办公用打印纸。 对于纸基材料而言， 由于其中的添加剂会产生较高的背景信号，为了获得 较为准确的测定结果， 纸芯片一般釆用 whatmanl号滤纸等添加剂较少的 纸张，但是， 本发明釆用上转换荧光分析与纸芯片技术相结合， 避免了纸 张中的添加剂的影响， 因此， 可以直接使用办公室打印纸等较为便宜、 容 易获得的纸张。
本发明对在纸基材料上形成检测区的方法没有特殊限制，可以为本领 域技术人员熟知的光刻法、 石蜡打印法、 平板印刷法、 绘图法、 喷墨打印 法、 等离子蚀刻法和切割法等， 釆用上述方法在纸基材料上形成图案后， 对所述图案进行疏水处理， 即可在纸基材料上形成检测区。本发明优选直
接在纸基材料上打印出图案，不对其进行疏水处理直接形成检测区，此时， 优选使用含疏水性物质的油墨进行打印，即可以用含疏水性物质的油墨在 办公室用打印纸上打印图案，一步法在办公室用打印纸上形成检测区。本 发明对所述检测区的个数没有特殊限制，本领域技术人员可以根据实际需 要进行设定。
参见图 1 , 图 1为本发明实施例提供的纸基材料检测区形成的方法， 直接在纸上打印图案，无需进行其他处理即可一步得到设置有检测区的纸 基材料。
形成检测区后，向所述检测区加入荧光供体材料溶液，所述荧光供体 材料包括上转换荧光纳米材料和标记在所述上转换荧光纳米材料上的表 面标记物。其中，所述上转换荧光纳米材料优选为水溶性上转换荧光纳米 材料， 优选具有式（I ) 所示的原子比：
NaYF4: Yb, Ln ( I );
其中， Ln为 Er、 Tm或 Ho, 优选为 Er。
所述上转换荧光纳米材料的粒径优选为 30nm~100nm , 更优选为
50nm~80nm。 本发明对所述上转化荧光纳米材料的来源没有特殊限制， 可以为市场上购买得到， 也可以按照本领域技术人员熟知的方法制备得 到。本发明优选在按照本领域技术人员熟知的方法制备上转化荧光纳米材 料， 同时， 在其表面修饰有氨基或者羧基， 例如釆用聚乙烯亚胺或聚丙烯 酸等对其进行修饰， 以便后续标记表面标记物。
所述表面标记物优选为单链核酸、蛋白质或多肽，如癌坯抗原适配体 等，其标记在所述上转换荧光纳米材料表面。本发明对所述表面标记物标 记在所述上转换荧光纳米材料表面的方式没有特殊限制，可以为通过化学 反应进行连接实现标记，例如，釆用聚乙烯亚胺或聚丙烯酸修饰所述上转 换荧光纳米材料，然后将其与表面标记物共同孵育，得到表面标记有表面 标记物的上转换荧光纳米材料。
所述荧光供体溶液优选为将荧光供体分散于 Tris-HCl緩冲液中形成
的溶液， 将其加入到纸基材料的检测区内， 去除荧光供体溶液中的溶剂， 即可将荧光供体固定在纸基材料的检测区内。本发明对去除溶剂的方法没 有特殊限制， 可以为蒸发等本领域技术人员熟知的方法。
得到检测区固定有荧光供体材料的纸基材料后，将其与荧光猝灭剂配 套组合， 即可得到纸芯片。将该纸芯片与固相荧光检测器配套组合， 即可 实现对生物分子的检测。在本发明中，所述荧光猝灭剂包括有机染料或荧 光受体； 所述有机染料包括但不限于罗丹明、 BHQ、 Cy3、 Cy5或荧光素 等； 所述荧光受体优选为层状氧化石墨烯或氧化碳球等。 在本发明中， 所 述荧光猝灭剂为有机染料时，可以单独存在，使用时再与检测区内的荧光 供体混合； 也可以标记在所述表面标记物上， 此时，有机染料标记在表面 标记物上，表面标记物标记在上转换荧光纳米材料上，上转化荧光纳米材 料固定在纸基材料的检测区内。本发明对所述有机染料在表面标记物上的 标记方法没有特殊限制，可以直接购买标记有有机染料的表面标记物。所 述荧光猝灭剂为荧光受体时，优选单独存在，使用时再与检测区内的荧光 供体混合。 本发明对所述层状氧化石墨烯或氧化碳球的来源没有特殊限 制，可以直接购买得到，也可以按照本领域技术人员熟知的方法制备得到。
在本发明中， 当有机染料标记在表面标记物上时，纸芯片的制备方法 下：
在纸基材料上形成检测区；
向所述检测区内加入荧光供体材料溶液，所述荧光供体材料包括上转 换荧光纳米材料、标记在所述上转化荧光纳米材料上的表面标记物和标记 在所述表面标记物上的有机染料；
去除所述荧光供体材料溶液中的溶剂，得到固定有荧光供体材料的纸 基材料；
所述固定有荧光供体材料的纸基材料即可作为纸芯片使用。
由于荧光染料可直接标记在表面标记物上， 因此，其可以与表面标记 物、上转换荧光纳米材料等直接固定在纸基材料的检测区内，其他步骤与
上述制备方法相同， 本发明在此不再赘述。
在本发明中，所述氨基或羧基修饰的上转化荧光纳米材料可以按照以 下方法制备：
取 2mL0.25mol/L稀土硝酸盐溶液， 其中， 稀土离子为摩尔比为 80: 18: 2的 4乙离子： 镱离子： 铒离子， 向其中加入 18mL无水乙醇， 再加入 含 0.9000g聚丙烯酸（聚丙烯酸与稀土离子摩尔比为 1:1 )或 0.3400g聚 乙烯亚胺的水溶液 8mL,搅拌 lOmin;然后加入含 0.2100g氟化钠（F7Ln3+ 摩尔比为 10： 1 )或0.1260 氟化钠（F7Ln3+摩尔比为 6： 1 )的水溶液 8mL, 搅拌 20min后， 置于高压反应釜中， 在搅拌条件下于 200°C反应 4h~10h; 停止加热并保持搅拌冷却至室温， 离心分离出固体产物，用无水乙醇和超 纯水各洗 3次，室温下真空干燥 12h得到聚丙烯酸或聚乙烯亚胺修饰的上 转换荧光纳米材料。
所述水溶性上转换荧光纳米材料的表面标记可以按如下方法进行：取
5mg 聚丙烯酸或聚乙烯亚胺修饰的上转换荧光纳米材料溶于 2mL~5mLMES緩冲（ 10mM, pH 5.5 )或者是 HEPES緩冲（ ΙΟηΜ, pH 7.4 ) 中， 加入 0.8mg~3.2mg EDC · HC1、 2.2mg~6.6mg Sulfo-NHS 或者是 lmg~5mg sulfo-SMCC, 在 30°C、 轻微振荡条件下孵育 0.5h~2h以活化上 转换荧光纳米材料表面的羧基或氨基。活化完成后离心分离得活化的聚丙 烯酸或聚乙烯亚胺修饰的上转换荧光纳米材料，将其用高纯水洗三次。洗 完的沉淀分散于 2mL~5mL HEPES緩冲（ 1 OmM , pH 7.2 )中， 向其中加 入 1.5uM-4uM的单链 DNA或 lmg~3mg的染料标记的多肽， 在 30°C、轻 微振荡条件下孵育 21！〜 24h后加入 50mgTris以封闭过量的 NHS。 离心分 离， 将得到的沉淀用高纯水洗三次以除去过量的单链 DNA或多肽， 洗完 后分散于 2.5mlTris-HCl ( 10mM, 150mM NaCl, pH 7.4 ) 中。
在本发明中， 所述层状氧化石墨烯可以按照以下方法制备： 取 50mL 浓石克酸加热至 90 °C , 向其中加入 10g K2S208和 10g P205 , 降温至 80 °C , 待 K2S208和 P205完全溶解后緩慢加入 12g石墨粉， 30min内加完， 80 °C
条件下反应 4h~5h, 然后加入 2L水， 静置一夜后过滤， 离心洗涤以除去 酸， 将得到的预氧化产物进行干燥； 取 460mL浓硫酸至于水水浴中， 加 入所述预氧化产物并搅拌， 然后保持反应液温度不超过 10°C下緩慢加入 60g高锰酸钾， 于 35 °C反应 2h后緩慢加入 1L蒸馏水， 使其温度不超过 50 °C , 继续搅拌 2h后加入 3L去离子水和 50mL 30%H2O2,静置一天后弃 去上清液，残留液先用 10%HC1洗再用蒸馏水洗，将得到的氧化产物进行 干燥； 将干燥后产物继续超声 2h, 使氧化石墨烯逐渐剥落成层状氧化石 墨烯。
以上方法只是对上转换荧光纳米材料、在上转换纳米材料表面标记表 面标记物、制备层状氧化石墨烯的举例说明，本领域技术人员可以用其他 已知方法制备上述物质。
本发明还提供了一种生物分子的检测方法， 包括以下步骤： 提供纸芯片， 所述纸芯片包括纸基材料、 荧光供体和荧光猝灭剂； 其 中，所述纸基材料上设置有检测区； 所述荧光供体材料固定在所述纸基材 料检测区内，所述荧光供体材料包括上转换荧光纳米材料和标记在所述上 转换荧光纳米材料上的表面标记物；所述荧光猝灭剂包括有机染料或荧光 受体
将荧光猝灭剂溶液加入到纸基材料的检测区内，再加入待测样品进行 孵育，测定得到的产物的荧光强度，根据所述荧光强度以及浓度与荧光强 度的标准曲线计算所述待测样品的浓度。
当所述荧光猝灭剂标记于所述表面标记物时，所述生物分子的检测方 法包括以下步骤：
提供纸芯片， 所述纸芯片包括纸基材料、 荧光供体和荧光猝灭剂； 其 中，所述纸基材料上设置有检测区； 所述荧光供体材料固定在所述纸基材 料检测区内，所述荧光供体材料包括上转换荧光纳米材料和标记在所述上 转换荧光纳米材料上的表面标记物；所述荧光猝灭剂标记在所述表面标记 物上； 所述荧光猝灭剂为有机染料；
将待测样品加入到纸基材料的检测区内进行孵育，测定得到的产物的 荧光强度,根据所述荧光强度以及浓度与荧光强度的标准曲线计算所述待 测样品的浓度。
除了荧光猝灭剂的加入方式不同，上述两种检测方法无其他区别， 因 此， 以第一种检测方法为例进行说明。
本发明以上述技术方案所述的纸芯片或者上述制备方法制备得到的 纸芯片为检测装置对生物分子进行检测，纸芯片的具体结构及其制备工艺 参见上文所述， 本发明在此不再赘述。
将荧光猝灭剂溶液加入到纸基材料的检测区内，使荧光猝灭剂与荧光 供体发生反应，然后加入待测样品进行孵育，测定得到的产物的荧光强度， 根据所述荧光强度以及浓度与荧光强度的标准曲线即可计算所述待测样 品的浓度。
本发明首先选择最佳荧光猝灭剂浓度， 具体方法如下：
向纸基材料的检测区内分别加入不同浓度的荧光猝灭剂溶液，反应后 分别检测得到的反应产物的荧光强度，选取荧光猝灭效率最大时荧光猝灭 剂溶液的浓度作为最优浓度。
当荧光猝灭剂为有机染料且标记在表面标记物上时，可以不进行该步 操作。
本发明优选以生物分子在緩冲溶液中形成的待测样品作为检测对象 进行检测浓度的确定， 具体方法如下：
向纸基材料的检测区内分别加入最优浓度的荧光猝灭剂溶液，反应后 加入系列浓度的生物分子在緩冲溶液中形成的待测样品溶液，孵育后分别 检测得到的反应产物的荧光强度， 待测样品溶液浓度的荧光强度记为 F , 空白溶液的荧光强度记为 F0 , 以 （F-FO ) /F0为纵坐标、 浓度为横坐标制 作待测物质在緩冲溶液中的标准曲线， 获得该检测方法的适宜检测浓度。
获得适宜检测浓度后，可以生物分子在全血或血清中形成的待测样品 作为检测对象，进行标准曲线的建立以及未知浓度待测样品的检测，标准
曲线的建立方法如下：
向纸基材料的检测区内分别加入最优浓度的荧光猝灭剂溶液，反应后 加入系列浓度的生物分子在血清或全血中形成的待测样品溶液，该系列浓 度在上述适宜检测浓度范围内，孵育后分别检测得到的反应产物的荧光强 度， 待测样品溶液浓度的荧光强度记为 F, 空白溶液的荧光强度记为 F0 , 以 （ F-F0 ) /F0为纵坐标、 浓度为横坐标制作待测物质在全血或血清中的 标准曲线。
对于未知浓度待测样品的检测而言， 具体方法如下：
向纸基材料的检测区内加入最优浓度的荧光猝灭剂溶液,反应后加入 生物分子在血清或全血中形成的待测样品溶液，孵育后检测得到的反应产 物的荧光强度，根据该荧光强度以及待测物质在全血或血清中的标准曲线 计算该待测样品的浓度即可；
或者可以为：
向纸基材料的检测区内加入最优浓度的荧光猝灭剂溶液,反应后加入 生物分子在緩冲溶液中形成的待测样品溶液，孵育后检测得到的反应产物 的荧光强度，根据该荧光强度以及待测物质在緩冲溶液中的标准曲线计算 该待测样品的浓度即可。
在上述最优浓度荧光猝灭剂溶液的确定、 緩冲溶液中标准曲线的建 立、 全血或血清中标准曲线的建立以及未知浓度待测样品的检测过程中 , 荧光猝灭剂和荧光供体进行反应的工艺参数、孵育的工艺参数以及检测的 参数等相似， 具体可以为： 荧光猝灭剂和荧光供体在检测区混合后，优选 在 20°C~30°C下放置 3min~10min, 使荧光猝灭剂和荧光供体进行反应； 进行孵育的温度优选为 20 °C~30 °C , 时间优选为 10min~30min; 优选为 980nm激光器下检测孵育后得到的产物的荧光强度。 为生物大分子， 也可以为生物小分子， 包括但不限于单链核酸、 糖类、 蛋 白质、 尿酸或乳酸等。
当荧光猝灭剂为有机染料且标记在表面标记物表面时，得到纸芯片后 即可直接进行待测样品在緩冲溶液中标准曲线的建立以及待测样品全血 或血清中标准曲线的建立，然后直接进行未知浓度样品的检测即可，无需 选择最适宜浓度的荧光猝灭剂浓度。除此之外，与上述方法所述的检测方 法基本无差别，本领域技术人员可以参考上文所述的方法，本发明在此不 再赘述。
参见图 2, 图 2为本发明提供的检测方法的检测机理， 在加入待测物 之前， 荧光猝灭剂、表面标记物和上转化荧光纳米材料依次连接； 加入待 测物之后， 待测物使荧光猝灭剂与表面标记物和 /或上转换荧光纳米材料 分离，使上转换荧光纳米材料发生荧光猝灭，从而建立荧光强度与待测物 的浓度之间的关系， 实现对待测物的检测。
本发明提供的纸芯片包括纸基材料、 荧光供体和荧光猝灭剂； 其中， 所述纸基材料上设置有检测区；所述荧光供体材料固定在所述纸基材料检 测区内，所述荧光供体材料包括上转换荧光纳米材料和标记在所述上转换 荧光纳米材料上的表面标记物； 所述荧光猝灭剂包括有机染料或荧光受 体。本发明将上转换荧光分析与纸芯片技术相结合，减少了反应物的用量， 降低了检测成本， 加快了反应物的反应速度， 将传统的 2h~3h 的反应时 间缩短到 lh以内， 增加了纸芯片应用于临床检测的可能。 同时， 本发明 将上转换荧光分析 )入纸芯片技术中，可以克服生物样品本底荧光和散射 光的影响，同时避免纸张添加剂的干扰，实现血清和全血样品的直接检测， 提高分析检测的灵敏度。
为了进一步说明本发明， 以下结合实施例对本发明提供的纸芯片、其 实施例 1
取 2mL0.25mol/L稀土硝酸盐溶液， 其中， 稀土离子为摩尔比为 80:
18: 2的 4乙离子、 镱离子和铒离子， 向其中加入 18mL无水乙醇， 再加入 含 0.9000g聚丙烯酸（聚丙烯酸与稀土离子摩尔比为 1 : 1 )的水溶液 8mL,
搅拌 lOmin; 然后加入含 0.2100g氟化钠 （F7稀土离子摩尔比为 10： 1 ) 的水溶液 8mL, 搅拌 20min后， 置于高压反应釜中， 在搅拌条件下于 200 °C反应 10h; 停止加热并保持搅拌冷却至室温， 离心分离出固体产物， 用 无水乙醇和超纯水各洗 3次，室温下真空干燥 12h得到聚丙烯酸修饰的上 转换荧光纳米材料；
取 5mg聚丙烯酸修饰的上转换荧光纳米材料溶于 5mL10mM, pH值 为 5.5的 MES緩冲液中， 加入 3.2mg EDC · HC1和 6.6mgSulfo-NHS, 在 30°C：、轻微振荡条件下孵育 l.5h, 离心分离得活化的聚丙烯酸修饰的上转 换荧光纳米材料， 将其用高纯水洗三次。 洗完的沉淀分散于 3mL 10mM、 pH值为 7.2的 HEPES緩冲液中， 向其中加入 2mgTAMRA标记的多肽， 所述 TAMRA 标记的多肽购买自上海俊怡生物科技有限公司， 序列为 TAMRA-GPLGVRC,在 30°C、轻微振荡条件下孵育 20h后加入 50mgTris, 离心分离， 将得到的沉淀用高纯水洗三次， 洗完后分散于 2.5mL10mM、 150mM NaCl、 pH值为 7.4的 Tris-HCl中， 得到 UCP-peptide-TAMRA溶 液；
以油性笔油墨作为打印油墨，在 A4打印纸上打印图案，形成检测区； 取 4 L 0.05mg/mL的 UCP-peptide-TAMRA溶液加入到上述检测区中， 室 温放置 lOmin, 干燥后分别加入 4 L含 1000倍稀释的血清的不同浓度的 MMP-2溶液， 浓度分别为 O pg/mL、 50 pg/mL、 500 pg/mL, 800 pg/mL、 1000 pg/mL、 2000 pg/mL和 5000pg/mL, 室温孵育 30min, 在 980nm激 光器下测其上转换荧光强度， 参见图 3 , 图 3为本发明实施例 1提供的不 同浓度的待测物质的荧光强度曲线图；计算加入了目标物的样品荧光强度 F与不含目标物的样品荧光强度 FQ的恢复程度， 以目标物浓度为横坐标， 以（F-F0 ) /F0为纵坐标作图得到标准曲线， 参见图 4, 图 4为本发明实施 例 1提供的标准曲线。
配制 2500pg/mL的 MMP-2血清样品，按照上述步骤检测其荧光强度， 并根据上述标准曲线计算其浓度， 结果表明， 计算得到的浓度为
2470pg/mL, 与已知浓度的吻合率为 99.8%。
实施例 2
取 2mL0.25mol/L稀土硝酸盐溶液， 其中， 稀土离子为摩尔比为 80: 18: 2的 4乙离子、 镱离子和铒离子， 向其中加入 18mL无水乙醇， 再加入 含 0.3400g聚乙烯亚胺的水溶液 8mL, 搅拌 lOmin; 然后加入含 0.1260g 氟化钠 （F7稀土离子摩尔比为 6 : 1 ) 的水溶液 8mL, 搅拌 20min后， 置 于高压反应釜中，在搅拌条件下于 200 °C反应 4h;停止加热并保持搅拌冷 却至室温， 离心分离出固体产物， 用无水乙醇和超纯水各洗 3次， 室温下 真空干燥 12h得到聚乙烯亚胺修饰的上转换荧光纳米材料；
取 5mg聚乙烯亚胺修饰的上转换荧光纳米材料溶于 5mL10mM, H 值为 5.5的 MES緩冲液中， 加入 3.2mg EDC · HC1和 6.6mgSulfo-NHS, 在 30°C、 轻微振荡条件下孵育 1.5h, 离心分离得活化的聚乙烯亚胺修饰 的上转换荧光纳米材料， 将其用高纯水洗三次。 洗完的沉淀分散于 3mL 10mM、 pH值为 7.2的 HEPES緩冲液中， 向其中加入 3uM的癌坯抗原适 配体， 在 30°C、 轻微振荡条件下孵育 2h后加入 50mgTris, 离心分离， 将 得到的沉淀用高纯水洗三次， 洗完后分散于 2.5mL10mM、 150mM NaCl、 pH值为 7.4的 Tris-HCl中， 得到 UCP-CEA aptamer溶液；
取 50mL浓硫酸加热至 90 °C , 向其中加入 1 Og K2S208和 10g P205 , 降温至 80 °C , 待 K2S208和 P205完全溶解后緩慢加入 12g石墨粉， 30min 内加完， 80°C条件下反应 5h, 然后加入 2L水， 静置一夜后过滤， 离心洗 涤以除去酸， 将得到的预氧化产物进行干燥； 取 460mL浓硫酸至于水水 浴中， 加入所述预氧化产物并搅拌， 然后保持反应液温度不超过 10°C下 緩慢加入 60g高锰酸钾， 于 35°C反应 2h后緩慢加入 1L蒸馏水， 使其温 度不超过 50 °C , 继续搅拌 2h后加入 3L去离子水和 50mL 30%H2O2, 静 置一天后弃去上清液，残留液先用 10%HC1洗再用蒸馏水洗，将得到的氧 化产物进行干燥； 将干燥后产物继续超声 2h, 使氧化石墨烯逐渐剥落成 层状氧化石墨烯；
以油性笔油墨作为打印油墨，在 A4打印纸上打印图案，形成检测区； 取 4 L 0.05mg/mL的 UCP-CEA aptamer溶液加入到上述检测区中， 室温 放置 lOmin,干燥后加入 4 L不同浓度的层状氧化石墨烯溶液，浓度分别 为 0 mg/mL、 0.1 mg/mL、 0.2 mg/mL、 0.3 mg/mL、 0.4 mg/mL和 0.5mg/mL, 室温放置 lOmin, 在 980nm激光器下测其上转换荧光强度， 得到荧光猝 灭曲线，其荧光猝灭效率达到最大时层状氧化石墨烯的浓度为 O.lmg/mL;
取 4 L 0.05mg/mL的 UCP-CEA aptamer溶液加入到上述检测区， 室 温放置 lOmin, 干燥后加入 4 L0.1mg/mL的层状氧化石墨烯溶液， 干燥 后加入 4 L不同浓度的 CEA溶液， CEA的浓度分别为 0 ng/mL 、 0.5 ng/mL 、 10 ng/mL 、 50 ng/mL 、 100 ng/mL和 500 ng/mL,室温孵育 30min, 在 980nm激光器下测其上转换荧光强度， 参见图 5 , 图 5为本发明实施 例 2提供的不同浓度的待测物质的荧光强度曲线图；计算加入了目标物的 样品荧光强度 F与不含目标物的样品荧光强度 FQ的恢复程度， 以目标物 浓度为横坐标， 以 （F-F0 ) /F0为纵坐标作图得到标准曲线， 参见图 6 , 图 6为本发明实施例 2提供的标准曲线。
配制 80ng/mL的 CEA样品， 按照上述步骤检测其荧光强度， 并根据 上述标准曲线计算其浓度， 结果表明， 计算得到的浓度为 79.6ng/mL, 与 已知浓度的吻合率为 99.5%。
实施例 3
取 2mL0.25mol/L稀土硝酸盐溶液， 其中， 稀土离子为摩尔比为 80:
18: 2的 4乙离子、 镱离子和铒离子， 向其中加入 18mL无水乙醇， 再加入 含 0.3400g聚乙烯亚胺的水溶液 8mL, 搅拌 lOmin; 然后加入含 0.1260g 氟化钠 （F7稀土离子摩尔比为 6 : 1 ) 的水溶液 8mL, 搅拌 20min后， 置 于高压反应釜中，在搅拌条件下于 200 °C反应 4h;停止加热并保持搅拌冷 却至室温， 离心分离出固体产物， 用无水乙醇和超纯水各洗 3次， 室温下 真空干燥 12h得到聚乙烯亚胺修饰的上转换荧光纳米材料；
取 5mg聚乙烯亚胺修饰的上转换荧光纳米材料溶于 5mL10mM, H
值为 5.5的 MES緩冲液中， 加入 3.2mg EDC · HC1和 6.6mgSulfo-NHS, 在 30°C、 轻微振荡条件下孵育 1.5h, 离心分离得活化的聚乙烯亚胺修饰 的上转换荧光纳米材料， 将其用高纯水洗三次。 洗完的沉淀分散于 3mL 10mM、 H值为 7.2的 HEPES緩冲液中， 向其中加入 3uM的癌坯抗原适 配体， 在 30°C、 轻微振荡条件下孵育 2h后加入 50mgTris, 离心分离， 将 得到的沉淀用高纯水洗三次， 洗完后分散于 2.5mll0mM、 150mM NaCl、 pH值为 7.4的 Tris-HCl中， 得到 UCP-CEA aptamer溶液；
取一支新买蜡烛点燃后用洗净玻璃片收集蜡烛灰，将其刮下后冷却称 取 8 mg溶于 15 ml 6 mol/L的硝酸溶液中，架上球形冷凝管，于 115 °C下 加热 10 h。 加热结束后， 待溶液冷至室温， 用新制碳酸钠溶液调 pH至中 性， 再在 12000 rpm/min下离心 20 min, 得到黑色沉淀。 将黑色沉淀用高 纯水洗 3次， 最后用高纯水将氧化好的碳球定溶至 4 mL的 PE管里， 超 声 1 h待用， 所得氧化碳球浓度为 2 mg/mL
以油性笔油墨作为打印油墨，在 A4打印纸上打印图案，形成检测区； 取 4 L 0.05mg/mL的 UCP-CEA aptamer溶液加入到上述检测区中， 室温 放置 lOmin, 干燥后加入 4 L 不同浓度的氧化碳球溶液， 浓度分别为 0 mg/mL、 0.001 mg/mL , 0.002 mg/mL、 0.005 mg/mL、 O.Olmg/mL , 0.02 mg/mL, 0.05 mg/mL, 0.08 mg/mL和 O.lOmg/mL, 室温放置 lOmin, 在 980nm 激光器下测其上转换荧光强度， 得到荧光猝灭曲线， 其荧光猝灭 效率达到最大时氧化碳球的浓度为 0.15mg/mL;
取 4 L 0.05mg/mL的 UCP-CEA aptamer溶液加入到上述检测区， 室 温放置 lOmin, 干燥后加入 4 L0.15mg/mL的氧化碳球溶液， 干燥后加入 4 L不同浓度的 CEA溶液， CEA的浓度分别为 O ng/mL 、 0.5 ng/mL 、 1 ng/mL、 10 ng/mL 、 20 ng/mL、 50 ng/mL 、 80 ng/mL和 100 ng/mL, 室 温孵育 30min, 在 980nm激光器下测其上转换荧光强度， 参见图 7, 图 7 为本发明实施例 3提供的不同浓度的待测物质的荧光强度曲线图；计算加 入了目标物的样品荧光强度 F与不含目标物的样品荧光强度 F 々恢复程
度， 以目标物浓度为横坐标， 以 （F-FQ ) /F。为纵坐标作图得到标准曲线， 参见图 8, 图 8为本发明实施例 3提供的标准曲线。
配制 60ng/mL的 CEA样品， 按照上述步骤检测其荧光强度， 并根据 上述标准曲线计算其浓度， 结果表明， 计算得到的浓度为 59.9ng/mL, 与 已知浓度的吻合率为 99.9%。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的 普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进 和润饰， 这些改进和润饰也应视为本发明的保护范围。
Claims
1、 一种纸芯片， 包括纸基材料、 荧光供体和荧光猝灭剂； 其中， 所 述纸基材料上设置有检测区；所述荧光供体材料固定在所述纸基材料检测 区内，所述荧光供体材料包括上转换荧光纳米材料和标记在所述上转换荧 光纳米材料上的表面标记物；
所述荧光猝灭剂包括有机染料或荧光受体。
2、 根据权利要求 1所述的纸芯片， 其特征在于， 所述有机染料标记 在所述表面标记物上。
3、 根据权利要求 1~2任意一项所述的纸芯片， 其特征在于， 所述荧 光供体材料中， 所述上转换荧光纳米材料具有式（I ) 所示的原子比：
NaYF4: Yb, Ln ( I );
其中， Ln为 Er、 Tm或 Ho。
4、 根据权利要求 3所述的纸芯片， 其特征在于， 所述荧光供体材料 中， 所述上转换荧光纳米材料的粒径为 30nm~100nm。
5、 根据权利要求 1~2任意一项所述的纸芯片， 其特征在于， 所述表 面标记物为单链核酸、 蛋白质或多肽。
6、 根据权利要求 1~2任意一项所述的纸芯片， 其特征在于， 所述有 机染料为罗丹明、 BHQ、 Cy3、 Cy5或荧光素。
7、 根据权利要求 1~2任意一项所述的纸芯片， 其特征在于， 所述荧 光受体为层状氧化石墨烯或氧化碳球。
8、 根据权利要求 1~2任意一项所述的纸芯片， 其特征在于， 所述纸 基材料为办公室用打印纸。
9、 根据权利要求 8所述的纸芯片， 其特征在于， 所述检测区通过直 接在办公室用打印纸上打印图案形成。
10、 一种纸芯片的制备方法， 包括：
在纸基材料上形成检测区；
向所述检测区内加入荧光供体材料溶液，所述荧光供体材料包括上转 换荧光纳米材料和标记在所述上转换荧光纳米材料上的表面标记物； 去除所述荧光供体材料溶液中的溶剂，得到固定有荧光供体材料的纸 基材料；
所述固定有荧光供体材料的纸基材料与荧光猝灭剂形成纸芯片； 所述荧光猝灭剂包括有机染料或荧光受体。
11、 根据权利要求 10所述的制备方法， 其特征在于， 所述纸基材料 为办公室用打印纸。
12、 根据权利要求 10所述的制备方法， 其特征在于， 所述在纸基材 料上形成检测区的具体方法包括：
用含疏水性物质的油墨在办公室用打印纸上打印图案 ,在办公室用打 印纸上形成检测区。
13、 一种生物分子的检测方法， 包括以下步骤：
提供纸芯片， 所述纸芯片包括纸基材料、 荧光供体和荧光猝灭剂； 其 中，所述纸基材料上设置有检测区； 所述荧光供体材料固定在所述纸基材 料检测区内，所述荧光供体材料包括上转换荧光纳米材料和标记在所述上 转换荧光纳米材料上的表面标记物；所述荧光猝灭剂包括有机染料或荧光 受体；
将荧光猝灭剂溶液加入到纸基材料的检测区内，再加入待测样品进行 孵育，测定得到的产物的荧光强度，根据所述荧光强度以及浓度与荧光强 度的标准曲线计算所述待测样品的浓度。
14、 根据权利要求 13所述的检测方法， 其特征在于， 所述浓度与荧 光强度的标准曲线按照以下方法建立：
将荧光猝灭剂溶液加入到纸基材料的检测区内，分别加入系列浓度的 标准样品进行孵育，分别测定得到的产物的荧光强度，根据标准样品的系 列浓度以及相对应的荧光强度建立浓度与荧光强度的标准曲线。
15、 根据权利要求 14所述的检测方法， 其特征在于， 所述待测样品
为含有待测生物分子的全血或血清。
16、 根据权利要求 15所述的检测方法， 其特征在于， 所述生物分子 为单链核酸、 糖类、 蛋白质、 尿酸或乳酸。
17、 一种生物分子的检测方法， 包括以下步骤：
提供纸芯片， 所述纸芯片包括纸基材料、 荧光供体和荧光猝灭剂； 其 中，所述纸基材料上设置有检测区； 所述荧光供体材料固定在所述纸基材 料检测区内，所述荧光供体材料包括上转换荧光纳米材料和标记在所述上 转换荧光纳米材料上的表面标记物； 所述荧光猝灭剂为有机染料； 所述荧 光猝灭剂标记在所述表面标记物上；
将待测样品加入到纸基材料的检测区内进行孵育，测定得到的产物的 荧光强度,根据所述荧光强度以及浓度与荧光强度的标准曲线计算所述待 测样品的浓度。
18、 一种用于生物分子检测的检测装置， 包括权利要求 1~9任意一 项所述的纸芯片和荧光检测装置。
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| CN111351925A (zh) * | 2019-07-29 | 2020-06-30 | 中南大学 | 一种可同时检测多种氨基糖苷类抗生素纸芯片、制备及其应用 |
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