CN109900906B - Freeze-drying preservation method of wet paper-based sensor and application thereof - Google Patents
Freeze-drying preservation method of wet paper-based sensor and application thereof Download PDFInfo
- Publication number
- CN109900906B CN109900906B CN201910185625.2A CN201910185625A CN109900906B CN 109900906 B CN109900906 B CN 109900906B CN 201910185625 A CN201910185625 A CN 201910185625A CN 109900906 B CN109900906 B CN 109900906B
- Authority
- CN
- China
- Prior art keywords
- paper
- temperature
- drying
- based sensor
- sensor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 238000000034 method Methods 0.000 title claims abstract description 44
- 238000004108 freeze drying Methods 0.000 title claims abstract description 27
- 238000004321 preservation Methods 0.000 title claims abstract description 26
- 238000001514 detection method Methods 0.000 claims abstract description 33
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 31
- 201000011510 cancer Diseases 0.000 claims abstract description 27
- 238000001035 drying Methods 0.000 claims abstract description 25
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 9
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 9
- 238000007710 freezing Methods 0.000 claims abstract description 8
- 230000008014 freezing Effects 0.000 claims abstract description 8
- 238000010438 heat treatment Methods 0.000 claims abstract description 8
- 239000003223 protective agent Substances 0.000 claims abstract description 7
- 230000000630 rising effect Effects 0.000 claims abstract description 6
- 238000001816 cooling Methods 0.000 claims abstract description 5
- 230000009477 glass transition Effects 0.000 claims abstract description 5
- 238000004806 packaging method and process Methods 0.000 claims abstract description 3
- 238000005086 pumping Methods 0.000 claims abstract description 3
- 239000000427 antigen Substances 0.000 claims description 27
- 102000036639 antigens Human genes 0.000 claims description 27
- 108091007433 antigens Proteins 0.000 claims description 27
- 239000002048 multi walled nanotube Substances 0.000 claims description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- 239000002184 metal Substances 0.000 claims description 11
- 229910052751 metal Inorganic materials 0.000 claims description 11
- 238000003860 storage Methods 0.000 claims description 9
- 239000011521 glass Substances 0.000 claims description 7
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 5
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims description 5
- 239000000853 adhesive Substances 0.000 claims description 5
- 230000001070 adhesive effect Effects 0.000 claims description 5
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims description 5
- 201000002528 pancreatic cancer Diseases 0.000 claims description 5
- 208000008443 pancreatic carcinoma Diseases 0.000 claims description 5
- 238000007789 sealing Methods 0.000 claims description 5
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 4
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims description 4
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 4
- 229910052709 silver Inorganic materials 0.000 claims description 4
- 239000004332 silver Substances 0.000 claims description 4
- 229910052802 copper Inorganic materials 0.000 claims description 3
- 239000010949 copper Substances 0.000 claims description 3
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 2
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 2
- 229930006000 Sucrose Natural products 0.000 claims description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 2
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 claims description 2
- 239000008103 glucose Substances 0.000 claims description 2
- 239000003292 glue Substances 0.000 claims description 2
- 230000009467 reduction Effects 0.000 claims description 2
- 239000005720 sucrose Substances 0.000 claims description 2
- 125000000647 trehalose group Chemical group 0.000 claims description 2
- 238000009777 vacuum freeze-drying Methods 0.000 abstract description 12
- 230000000694 effects Effects 0.000 abstract description 10
- 230000007774 longterm Effects 0.000 abstract description 6
- 238000005516 engineering process Methods 0.000 abstract description 5
- 239000000523 sample Substances 0.000 description 26
- 239000000243 solution Substances 0.000 description 16
- 239000000126 substance Substances 0.000 description 10
- 238000002360 preparation method Methods 0.000 description 7
- 238000000859 sublimation Methods 0.000 description 7
- 230000008022 sublimation Effects 0.000 description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 6
- 239000002041 carbon nanotube Substances 0.000 description 6
- 229910021393 carbon nanotube Inorganic materials 0.000 description 6
- 239000013078 crystal Substances 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000003756 stirring Methods 0.000 description 5
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 4
- 229920001213 Polysorbate 20 Polymers 0.000 description 4
- 238000004140 cleaning Methods 0.000 description 4
- 238000002425 crystallisation Methods 0.000 description 4
- 230000008025 crystallization Effects 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 4
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 230000002035 prolonged effect Effects 0.000 description 3
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 2
- 229960001212 bacterial vaccine Drugs 0.000 description 2
- 238000010923 batch production Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 230000021523 carboxylation Effects 0.000 description 2
- 238000006473 carboxylation reaction Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000000151 deposition Methods 0.000 description 2
- 239000000645 desinfectant Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000005265 energy consumption Methods 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
- 238000003760 magnetic stirring Methods 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000000439 tumor marker Substances 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241000222336 Ganoderma Species 0.000 description 1
- 229920002730 Poly(butyl cyanoacrylate) Polymers 0.000 description 1
- 230000036760 body temperature Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 230000000475 sunscreen effect Effects 0.000 description 1
- 239000000516 sunscreening agent Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a freeze-drying preservation method of a wet paper-based sensor and application thereof, wherein the method comprises the following steps: adding a protein protective agent into the wet paper-based sensor to be stored, reducing the temperature of a cold trap to be below-30 ℃, and placing the wet paper-based sensor into the cold trap; setting the cooling speed of the cold trap to a preset value, and reducing the temperature in the cold trap until the temperature is-60 ℃; freezing at-60 deg.C; moving the frozen wet paper-based sensor into a temperature rising part of a cold trap, carrying out vacuum pumping operation, and starting temperature rising after the preset vacuum degree requirement is met; stopping heating to the glass transition temperature; heating to room temperature in an environment meeting the requirement of a preset vacuum degree, and continuously desorbing and drying for more than 8 hours; packaging and finishing preservation. The preservation method of the invention carries out vacuum freeze drying on the filter paper-based cancer detection sensor by the vacuum freeze drying technology, can keep the activity of biological tissues to the maximum extent, and can realize long-term preservation.
Description
Technical Field
The invention belongs to the technical field of paper-based sensor preservation, and particularly relates to a freeze-drying preservation method of a wet paper-based sensor and application thereof.
Background
Paper-based detection is favored by in vitro detection and related application detection due to its high detection rate, high selectivity, high sensitivity, relatively low cost and no pollution, and has great potential particularly in the detection of cancers, infectious diseases and other physiological diseases.
In the process of tumor detection, the biological substance is treated by a freeze-drying method, so that the advantages are outstanding, the main performance of the treated substance is kept unchanged, the volatile components are few, the rehydration performance is good, the activity of the biological tissue is kept to the maximum extent, and the long-term storage of the substance is facilitated. At present, a freeze-drying method is widely applied to a plurality of fields of medicine, food, novel materials and the like, for example, in the preparation process of active vaccines, the freeze-drying method is utilized to produce live bacterial vaccine, and the processing and the fresh keeping of medicinal materials are realized.
Nowadays, paper-based biosensors make a great contribution to tumor detection, but the timeliness is not satisfactory, and a good storage mode is not provided, so that the biosensor can only be prepared immediately for use, and certain biological substances are inactivated even after natural drying, so that the biological substances cannot be detected. The freeze-drying method is applied to the paper-based biosensor, so that the effective period of the detection activity of the paper-based biosensor is effectively prolonged, the commercialization of the paper-based biosensor is facilitated, and the sensor product can be stored for a long time. In view of the above, there is a need for a long-term preservation method capable of maintaining the activity of biological tissues.
Disclosure of Invention
The invention aims to provide a freeze-drying preservation method of a wet paper-based sensor and application thereof, so as to solve one or more of the technical problems. The paper-based sensor still has good detection performance in a low CA19-9 concentration range, and has high detection efficiency; the preservation method of the invention carries out vacuum freeze drying on the filter paper-based cancer detection sensor by the vacuum freeze drying technology, can keep the activity of biological tissues to the maximum extent, and can realize long-term preservation.
In order to achieve the purpose, the invention adopts the following technical scheme:
a freeze-drying preservation method of a wet paper-based sensor comprises the following steps:
and 6, sealing and packaging the paper-based sensor processed in the step 5 to finish storage.
Further, still include: and 7, taking out the preserved paper-based sensor during use, carrying out sealed cultivation at normal temperature of human body to combine the antigen and the antibody, drying at room temperature to remove redundant water, and carrying out antigen concentration detection.
Further, in the step 3, the temperature reduction speed is averagely kept at 1.5 ℃/min, and the freezing treatment is carried out for 6-10 h in the environment of-60 ℃; in step 4, the average temperature rise rate is maintained at 0.5 ℃/min.
Further, in step 1, the added protein protective agent is trehalose, sucrose, glucose or maltose.
Further, the vacuum degree at the time of temperature rise and drying is required to be 10Pa or less.
Further, for protecting paper-based cancer antigen detection sensors;
the paper-based cancer antigen detection sensor comprises: the biosensor comprises a filter paper base, a biosensor element and two metal electrodes;
the biosensor element is deposited on the filter paper base;
the biosensor element includes: carboxylated multi-walled carbon nanotubes; the surface of the carboxylated multi-wall carbon nanotube is characteristically combined with a specific CA19-9 antibody of pancreatic cancer cells;
the two metal electrodes are respectively adhered to the filter paper base through conductive adhesive, and are respectively electrically connected with the filter paper base loaded with the carboxylated multi-walled carbon nanotubes; the two metal electrodes are connected with lead-out copper wires.
Further, in the paper-based cancer antigen detection sensor for protection, the filter paper base adopts microporous filter paper.
Further, the paper-based cancer antigen detection sensor for protection also comprises: a second substrate; the filter paper base is packaged between the two second substrates, and the filter paper base is adhered to the glass slide through the silver adhesive.
Further, the paper-based cancer antigen detection sensor for protection can detect the cancer antigen in the range of 0-40U/ml of CA19-9 concentration.
Compared with the prior art, the invention has the following beneficial effects:
in the sensor, a specific antibody CA19-9 of pancreatic cancer cells is added on the surface of a multiwall carbon nanotube (MWCNT), the multiwall carbon nanotube is deposited on the surface of test paper with a fixed size, and finally the MWCNT is connected with a metal electrode for electrical test representation, so that antigen concentration information can be converted into a directly measurable electric signal (namely, the U-I characteristic of resistance can be reflected), the sensor has the advantages of low manufacturing cost, high precision and high stability, and can realize economical and rapid cancer antigen screening.
The preservation method of the invention can realize the long-term preservation of the paper-based cancer detection sensor element by carrying out vacuum freeze drying on the paper-based cancer detection sensor element by using a vacuum freeze drying technology, and has extremely high market value. The concrete expression is as follows: the invention uses vacuum freeze-drying to process the detector, which is to freeze the wet detector into solid state, then sublimate the ice crystal in the detector into water vapor under proper temperature and vacuum degree, to remove the water and dry it. Because the free water in the sensor is frozen into the ice crystals during drying, pores can be left in the dried sensor, so that the structure in the sensor can not be damaged, the physical, chemical and biological properties of the sensor are basically unchanged, and the phenomena of breakage, folding and the like in the drying process can not occur. In addition, the medical vacuum freeze drying equipment can meet the requirements of on-site cleaning and on-line sterilization, ensures that the paper-based cancer detector is not polluted in the manufacturing process, is simple to operate, has low energy consumption and can realize large-scale batch production. The detector after vacuum freeze drying can be stored for a long time under sealed package, and the transportation and storage of the paper-based cancer detector are convenient; the rehydration performance is excellent, and the antigen is only required to be dripped and moistened again when the disinfectant is used; the operation is simple and quick, and the application value is very high.
Drawings
FIG. 1 is a schematic diagram of the structure of a paper-based cancer antigen detection sensor of the present invention;
FIG. 2 is a schematic block flow diagram of a method for freeze-drying preservation of a wet paper-based sensor of the present invention;
FIG. 3 is a graph showing the comparison of the results of paper-based sensors after preservation using the preservation method of the present invention with those of paper-based sensors after the same number of days of natural drying;
in fig. 1, copper wire; 2. a glass slide; 3. microporous filter paper; 4. a metal electrode; 5. CA19-9 antibody-multi-walled carbon nanotubes.
Detailed Description
The invention is described in further detail below with reference to the figures and specific examples.
Referring to fig. 1, a wet paper-based sensor for cancer detection according to an embodiment of the present invention includes: the filter paper base adopts microporous filter paper 3; depositing a plurality of carboxylated multi-walled carbon nanotubes on a filter paper base, wherein the carboxylated multi-walled carbon nanotubes are added with a preset tumor marker with preservation activity; for example, CA19-9 antibody-multi-walled carbon nanotube 5, i.e., CA19-9, a specific antibody that characteristically binds to a predetermined tumor marker that is pancreatic cancer cells; the two metal electrodes 4 are respectively and electrically connected with the carboxylated carbon nano tube through conductive adhesive. The filter paper base is fixed by silver glue and is packaged between the two glass slides 2, and the two metal electrodes 4 are led out through the copper lead 1. In the sensor, a specific antibody CA19-9 of pancreatic cancer cells is added on the surface of a multiwall carbon nanotube (MWCNT), the multiwall carbon nanotube is deposited on the surface of test paper with a fixed size, and finally the multiwall carbon nanotube is connected with a metal electrode 4 to carry out electrical test characterization, so that antigen concentration information can be converted into a directly measurable electrical signal (namely the U-I characteristic of resistance can be reflected), the sensor has the advantages of low manufacturing cost, high precision and strong stability, and can realize economical and rapid cancer antigen screening.
The paper-based sensor element for detecting cancer of the embodiment of the invention is prepared by the following steps:
according to the mass parts, the carboxylated multi-wall carbon nanotube accounts for 2 parts, EDC is more than or equal to 77 parts, and NHSS is more than or equal to 22 parts;
specifically, reagents and materials required by the preparation process are prepared, wherein the reagents and materials comprise 2mg of carboxylated multi-wall carbon nanotubes, at least 4M L (0.1M) MES solution, 77mg of EDC powder, 22mg of NHSS powder, at least 100M L of PBS solution, 10u L of Tween20 solution (with the concentration of 0.05%), 100u L of BSA solution (with the concentration of 1%), two pieces of filter paper, two pieces of glass slides, gel and 0.5M L of antibody solution (with the concentration of 0.01mg/M L).
The preparation process comprises the following steps:
1) weighing 2mg of carboxylated carbon nanotubes, and pouring the powder into a large centrifuge tube; the carboxylation step can also be designed by itself, and the binding capacity of the antibody and the carbon nano tube can be enhanced through carboxylation.
2) 2m of the MES solution L was dropped into the large centrifugal tube by a dropper to improve the dispersibility of the carbon nanotubes.
3) Placing the large centrifuge tube into an ultrasonic oscillator, oscillating for 1min, and mixing the solution with a mixing instrument.
4) 77mg of EDC is weighed and poured into a centrifuge tube, 1m of MES L is added, the tube wall is cleaned, and then ultrasonic oscillation is carried out for 10-15 min.
5) Weigh and add 22mg NHSS to the large centrifuge tube to protect EDC from hydrolysis before binding to antigen, add MES to 4m L and sonicate for 40 min.
6) After shaking, the tube wall is washed by MES, the volume is constant, the tube wall is placed into a centrifuge for centrifugation (5000r/min) for 10min, and after centrifugation, the supernatant is sucked out by a dropper.
7) PBS was added to 7.5m L in a large centrifuge tube to neutralize residual EDC and NHSS, followed by sonication for 1min, followed by centrifugation (5000r/min) for 10min after completion, and the supernatant was aspirated with a pipette after completion.
8) Adding PBS to 10m L, or adding more PBS to make the carbon nanotubes disperse more uniformly, ultrasonically shaking for 1min, pouring into a magnetic rotor bottle, cleaning the tube wall of the centrifugal tube with PBS, and pouring the cleaning solution into a magnetic stirring bottle.
9) Taking out the antibody and thawing at room temperature; the antibody was poured into a magnetic rotor flask and the remaining antibody was washed out with PBS.
10) Stirring with magnetic stirrer for a day and night at 20 deg.C or below to add CA19-9 into the functionalized multi-wall carbon nanotubes.
11) After stirring, 10u L of Tween20 and 100Ul of BSA are added into the large centrifuge tube by using a pipette gun, wherein Tween20 is a nonionic surfactant and is used for clearing the binding sites of CA19-9 antibody, and the BSA is used for preventing all non-characteristic binding, and then the large centrifuge tube is kept standing for one hour at room temperature.
12) Centrifuging for 10min (5000r/min), removing supernatant, adding PBS to constant volume of 7.5m L, and ultrasonic oscillating for 1min to remove excessive CA19-9 antibody and BSA.
13) A piece of filter paper was taken out and a 2mm × 5mm rectangle was drawn at 8 circularly symmetric positions.
14) The liquid in the large centrifuge tube was suction filtered onto another piece of filter paper.
15) Two sheets of filter paper were overlaid together and 8 rectangular paper-based sensor elements were cut out.
16) And (3) respectively sticking a copper wire on two ends of each of the 8 rectangular paper-based sensors by using conductive silver adhesive, and sealing and storing to finish the preparation.
The paper-based sensor testing method comprises the steps of dropping 100 mu L CA19-9 containing PBS solution on a filter paper unit, keeping a piece of sensor unit without dropping CA19-9 as a control, keeping the temperature at 37 ℃ for 1.5h in a plastic dish with a sealing cover, and drying in the air for 30 min.
Under the environmental condition, the U-I characteristic is measured, the resistance value of the sensor unit is measured, and the ratio of the resistance value of the sensor unit without adding CA19-9 satisfies the following linear relation, for example:
antigen concentration (U/m L) ═ 90.513 · (R/R)0)-87.636
According to the measurement result, the concentration of the antigen to be added can be calculated. Under the condition that reagents are slightly different, values of linear relation functions are probably slightly different, so in order to be more accurate, in practical use, the first batch of samples are manufactured according to the preparation process, the function relation is calculated, and then detection is carried out.
Referring to fig. 2, a method for storing a filter paper-based sensor for cancer detection according to an embodiment of the present invention includes the following steps:
(1) freezing at low temperature; adding a protein protective agent (such as trehalose) with a common dosage into a sensor sample which is deposited on filter paper, waiting for the temperature of a cold trap to be reduced to be below-30 ℃, and directly transferring the sample into the cold trap from a normal temperature environment to realize rapid crystallization. Thereafter, an appropriate average cooling rate (e.g., 1.5 ℃/min) is maintained until the temperature is reduced to-60 ℃. The method ensures that the sample is frozen overnight at the temperature of-60 ℃, only low-temperature treatment is carried out on the sample at the stage, a treatment mode of temperature shock is adopted, and vacuumizing operation is not carried out, so that the antibody is prevented from being damaged due to too low crystallization speed, the internal pressure of the cold trap can be reduced to about a few Pa at most in a low-temperature state, the internal pressure of the cold trap is close to one percent of the internal pressure of the cold trap in a normal-temperature state, the sublimation speed of water is accelerated, and the residual quantity of the water is reduced.
(2) Sublimation drying; and (3) moving the sample out of the cold trap, placing the sample on an iron stand above the cold trap, turning on a vacuum pump, and starting to heat when the vacuum pump is vacuumized to about a few Pa. At this time, the temperature of the sample begins to rise as the sample leaves the cold trap, and since the experiment is carried out at a lower ambient temperature, a suitable average temperature rise rate (e.g., at 0.5 ℃/min) can be maintained, and the sublimation drying is finished when the temperature rises to be close to the glass transition temperature, and the process lasts for about 2 hours.
(3) Desorbing and drying; after sublimation drying was complete, the sample temperature was now near 0 ℃ and continued to rise slowly to room temperature. Keeping the environmental pressure of the sample at about 10Pa, and continuously desorbing and drying for more than 8 hours. After the step is finished, the medicine can be packaged, and is stored in a dry environment at a proper temperature (such as room temperature) in a dark place for use.
(4) Moistening and reviving; when a sensor sample needs to be used, the sample is taken out, a proper amount of sample solution to be detected is dripped to moisten the sensor sample, the sample is dried for 0.5h at room temperature after being sealed and cultured at the human body temperature for 1.5h, and then the subsequent measurement can be carried out.
In conclusion, the existing wet paper-based cancer detector has extremely limited storage time, and loses activity once dried, thereby greatly restricting the practical value. According to the preservation method provided by the embodiment of the invention, the detector is processed by using vacuum freeze drying, the wet detector is frozen into a solid state, and then the ice crystals in the detector are sublimated into water vapor at a proper temperature and a proper vacuum degree, so that the water in the ice crystals is removed and the ice crystals are dried. Because the free water in the sensor is frozen into the ice crystals during drying, pores can be left in the dried sensor, so that the structure in the sensor can not be damaged, the physical, chemical and biological properties of the sensor are basically unchanged, and the phenomena of breakage, folding and the like in the drying process can not occur. In addition, the medical vacuum freeze drying equipment can meet the requirements of on-site cleaning and on-line sterilization, ensures that the paper-based cancer detector is not polluted in the manufacturing process, is simple to operate, has low energy consumption and can realize large-scale batch production. The detector after vacuum freeze drying can be stored for a long time under the condition of sealed package, and the transportation and the storage of the paper-based cancer detector are convenient. The rehydration performance is excellent, and the antigen is only needed to be dripped and moistened again when the disinfectant is used. The operation is simple and quick, and the application value is very high.
The freeze drying method is applied to the paper-based biosensor, so that the product can be realized, and the effect of long-time storage can be achieved. In the process of tumor detection, the biological substance is treated by a freeze-drying method, so that the advantages are outstanding, the main performance of the treated substance is kept unchanged, the volatile components are few, the rehydration performance is good, the activity of the biological tissue is kept to the maximum extent, and the long-term storage of the substance is facilitated. At present, a freeze-drying method is widely applied to a plurality of fields of medicine, food, novel materials and the like, such as the production of live bacterial vaccine and the processing and the fresh keeping of medicinal materials by utilizing the freeze-drying method in the preparation process of active vaccine. Combining this technology with paper-based biosensors would be a very important attempt.
Example 1
The preparation method of the biosensor element for detecting cancer according to the embodiment of the present invention comprises the steps of preparing 2mg carboxylated multi-walled carbon nanotubes (purchased from Shanghai Michelin Biochemical technology Co., Ltd., inner diameter of 5-12nm, outer diameter of 30-50nm, length of <10nm), adding 0.4mmol EDC and 0.1mmol NHSS, dispersing in 4m L of 0.1mol MES solution, subjecting the dispersion solution to ultrasonic bath in an ultrasonic instrument (New glossy ganoderma SB-3200OTD) for about 40min, mixing uniformly with a homogenizer (SCI L OGEX ICC8), centrifuging for 10min (3000rp/m) with a centrifuge (plain instrument TD 25), removing supernatant, adding PBS buffer solution into the precipitate after finishing, centrifuging for 10min (3000rp/m), removing supernatant, repeating the above PBS washing process for at least 5 times to remove excess amounts of the precipitate, adding 0.5m 2 of CA19-9 antibody (0.01m, 0.01mg of modified PBS buffer solution into 0.01m, adding the PBS buffer into a magnetic stirring bottle, adding the modified PBCA 389-9 into a vial, stirring at room temperature, adding the biological technology EDC mixed solution under stirring at room temperature, adding 3-300 mg MCT.
Tween20 solution with a concentration of 0.05% was mixed in 1% BSA of 100. mu. L, which was then added to the solution obtained after stirring overnight, after the mixture solution was allowed to stand at room temperature for one hour, it was centrifuged for 10min (5000rp/m) with a centrifuge (plain instrument TD6), the supernatant was removed, the precipitate was washed and centrifuged 5 times with PBS buffer to remove excess CA19-9 antibody and BSA, the product of this process was called CA19-9 antibody-multiwalled carbon nanotubes.
Dispersing the obtained product in water solution, and mixing with water bath ultrasound in ultrasonic instrument (Xinzhi SB-3200 OTD). Our biosensor element was prepared by depositing CA19-9 antibody-multi-walled carbon nanotubes on microporous filter paper using a syringe filtration method and cutting the carbon nanotube cake filter paper into 5mmX2mm format (the filter should be cut from center to circumference).
And then carrying out a freeze drying step. Adding a protein protective agent (such as trehalose) with a common dosage into a sensor sample which is deposited on filter paper, waiting for the temperature of a cold trap to be reduced to be below-30 ℃, and directly transferring the sample into the cold trap from a normal temperature environment to realize rapid crystallization. Thereafter, an appropriate average cooling rate (e.g., 1.5 ℃/min) is maintained until the temperature is reduced to-60 ℃. The method ensures that the sample is frozen overnight at the temperature of-60 ℃, only low-temperature treatment is carried out on the sample at the stage, a treatment mode of temperature shock is adopted, and vacuumizing operation is not carried out, so that the antibody is prevented from being damaged due to too low crystallization speed, the internal pressure of the cold trap can be reduced to about a few Pa at most in a low-temperature state, the internal pressure of the cold trap is close to one percent of the internal pressure of the cold trap in a normal-temperature state, the sublimation speed of water is accelerated, and the residual quantity of the water is reduced.
And (3) moving the sample out of the cold trap, placing the sample on an iron stand above the cold trap, turning on a vacuum pump, and starting to heat when the vacuum pump is vacuumized to about a few Pa. At this time, the temperature of the sample begins to rise as the sample leaves the cold trap, and since the experiment is carried out at a lower ambient temperature, a suitable average temperature rise rate (e.g., at 0.5 ℃/min) can be maintained, and the sublimation drying is finished when the temperature rises to be close to the glass transition temperature, and the process lasts for about 2 hours.
After sublimation drying was complete, the sample temperature was now near 0 ℃ and continued to rise slowly to room temperature. Keeping the environmental pressure of the sample at about 10Pa, and continuously desorbing and drying for more than 8 hours. After the step is finished, the medicine can be packaged, and is stored in a dry environment at a proper temperature (such as room temperature) in a dark place for use.
The test method of the freeze-dried paper-based sensor comprises the steps of taking out a sensor sample when the sensor sample is used, dropping 100 mu L CA19-9 containing PBS solution on a filter paper unit, keeping a sensor unit without dropping CA19-9 as a control, keeping the sensor unit in a plastic dish with a sealing cover at 37 ℃ for 1.5h, and drying the sensor unit in air for 30 min.
Under ambient conditions, the measurement of the U-I characteristic was performed and the resistance of the sensor cell was measured, the ratio of which to the resistance of the sensor cell without CA19-9 should still satisfy a linear relationship and be close to the wet drying results of the same batch. If the antibody is directly and naturally dried without adopting freeze drying, the antibody is inactivated, and the antibody can not be used any more as shown in an experimental result. Therefore, the freeze-drying method is very effective for extending the shelf life.
Referring to fig. 3, the freeze-drying method for extending the shelf life of paper-based sensors according to the present invention compares with the natural drying method. After the shelf life is prolonged by the freeze-drying method, the antibody is moistened and reactivated after three days, and the ratio of the resistance to the antigen concentration still presents a good linear relation, which shows that the freeze-drying method maintains the activity of the antibody protein. After the paper-based sensor is manufactured, if no measures are taken, the paper-based sensor is naturally dried for three days only in a dustproof and sunscreen place; in the subsequent tests, under the condition of antigen concentration change, the resistance has no obvious linear change, which indicates that most of the antibody protein is denatured and can not be used for detection any more. Through a plurality of groups of experiments, the paper-based sensor after the shelf life is prolonged by using the freeze drying method can ensure the detection effectiveness after being used again within two weeks, and compared with the paper-based sensor which is dried by adopting a wet method and has the shelf life of only a few hours, the paper-based sensor greatly improves the shelf life and is beneficial to actual production and use.
Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can make modifications and equivalents to the embodiments of the present invention without departing from the spirit and scope of the present invention, which is set forth in the claims of the present application.
Claims (5)
1. A freeze-drying preservation method of a wet paper-based sensor is characterized by comprising the following steps:
step 1, adding a protein protective agent into a wet paper-based sensor to be stored;
step 2, reducing the temperature of the cold trap to be below-30 ℃, and placing the wet paper-based sensor processed in the step 1 into the cold trap;
step 3, setting the cooling speed of the cold trap to a preset value, and reducing the temperature in the cold trap until the temperature is-60 ℃; freezing at-60 deg.C;
step 4, moving the paper-based sensor subjected to the freezing treatment in the step 3 into a temperature rising part of a cold trap, performing vacuum pumping operation, and starting temperature rising after the preset vacuum degree requirement is met; stopping heating to the glass transition temperature;
step 5, heating the paper-based sensor processed in the step 4 to room temperature in an environment meeting the requirement of preset vacuum degree, and continuously desorbing and drying for more than 8 hours;
step 6, sealing and packaging the paper-based sensor processed in the step 5 to finish storage;
step 7, when in use, the paper-based sensor after preservation treatment is taken out, sealed cultivation is carried out at normal temperature of human body to combine antigen and antibody, and excess water is removed by drying at room temperature to carry out antigen concentration detection;
in the step 3, the temperature reduction speed is averagely kept at 1.5 ℃/min, and the freezing treatment is carried out for 6-10 h in the environment of-60 ℃; in step 4, the average heating speed is kept at 0.5 ℃/min;
in the step 1, the added protein protective agent is trehalose, sucrose, glucose or maltose;
the vacuum degree during heating and drying is required to be less than or equal to 10 Pa.
2. Use of the method of freeze-drying preservation of a wet-process paper-based sensor of claim 1 for protecting a paper-based cancer antigen detection sensor;
the paper-based cancer antigen detection sensor comprises: the biosensor comprises a filter paper base, a biosensor element and two metal electrodes;
the biosensor element is deposited on the filter paper base;
the biosensor element includes: carboxylated multi-walled carbon nanotubes; the surface of the carboxylated multi-wall carbon nanotube is characteristically combined with a specific CA19-9 antibody of pancreatic cancer cells;
the two metal electrodes are respectively adhered to the filter paper base through conductive adhesive, and are respectively electrically connected with the filter paper base loaded with the carboxylated multi-walled carbon nanotubes; the two metal electrodes are connected with lead-out copper wires.
3. The application of the freeze-drying preservation method of the wet paper-based sensor in claim 2, wherein the filter paper base is microporous filter paper for the protected paper-based cancer antigen detection sensor.
4. The use of the method for freeze-drying preservation of a wet-process paper-based sensor according to claim 2, for use in a protected paper-based cancer antigen detection sensor, further comprising: a glass slide;
the filter paper base is packaged between the two glass slides and is adhered to the glass slides through silver glue.
5. The application of the freeze-drying preservation method of the wet paper-based sensor in claim 2, wherein the paper-based cancer antigen detection sensor for protection can detect the CA19-9 concentration in the range of 0-40U/ml.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910185625.2A CN109900906B (en) | 2019-03-12 | 2019-03-12 | Freeze-drying preservation method of wet paper-based sensor and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910185625.2A CN109900906B (en) | 2019-03-12 | 2019-03-12 | Freeze-drying preservation method of wet paper-based sensor and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109900906A CN109900906A (en) | 2019-06-18 |
| CN109900906B true CN109900906B (en) | 2020-07-28 |
Family
ID=66947117
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910185625.2A Expired - Fee Related CN109900906B (en) | 2019-03-12 | 2019-03-12 | Freeze-drying preservation method of wet paper-based sensor and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109900906B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108709914A (en) * | 2018-05-28 | 2018-10-26 | 华东理工大学 | A kind of method of quick detection alpha-fetoprotein |
-
2019
- 2019-03-12 CN CN201910185625.2A patent/CN109900906B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108709914A (en) * | 2018-05-28 | 2018-10-26 | 华东理工大学 | A kind of method of quick detection alpha-fetoprotein |
Non-Patent Citations (3)
| Title |
|---|
| A preliminary study on the stabilization of blood typing antibodies sorbed into paper;Liyun Guan et al;《Cellulose》;20131208;第21卷;摘要，第719页右栏， Fig 1 * |
| Detection of early stage prostate cancer by using a simple carbon nanotube@paper biosensor;Sungkyung Ji et al;《Biosensors and Bioelectronics》;20171110;第102卷;摘要，Scheme 1-2，第2节 * |
| 冷冻干燥的工艺流程及其应用;霍贞;《干燥技术与设备》;20071231;第5卷(第5期);第1和3节 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109900906A (en) | 2019-06-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20090075354A1 (en) | Nanofiber structures for supporting biological materials | |
| JP5501053B2 (en) | Method for producing effective microorganism-coated seeds requiring a decompression step | |
| Cai et al. | High-water-absorbing calcium alginate fibrous scaffold fabricated by microfluidic spinning for use in chronic wound dressings | |
| CN1156967A (en) | Method of preventing aggregation of various substances upon rehydration or thawing and compositions obtained thereby | |
| JPH02265984A (en) | Storage of substance | |
| JPH05293354A (en) | Process for storage of material | |
| JP2007295936A (en) | Stable biotinylated biomolecule composition and method | |
| CN104024841A (en) | Glycated protein measurement sensor and portable glycated protein measurement apparatus including same | |
| CN204369906U (en) | A kind of test kit extracting and preserve nucleic acid | |
| CN111205351A (en) | PD-1 targeted blocking peptide and application thereof | |
| CN109900906B (en) | Freeze-drying preservation method of wet paper-based sensor and application thereof | |
| CN106176620A (en) | A kind of Graphene medicament slow-release microsphere and preparation method thereof | |
| CN107722321A (en) | The method of two kinds of phosphoryl choline polymer bionic coating Chitosan films containing epoxy and amino | |
| CN110585122B (en) | Injectable natural triterpenoid antibacterial hydrogel and preparation method thereof | |
| CN105504328A (en) | Method for improving chitosan membrane blood compatibility through one-step coating at room temperature | |
| CN114262373B (en) | Albumin HSA-hydrohopbic-IIB with self-assembly performance and application thereof | |
| CN114249814B (en) | Albumin HSA-hydrohopbic-IIIB with self-assembly performance and application thereof | |
| JPS62185027A (en) | Recombined interleukin-2 composition and manufacture | |
| CN112135641A (en) | Protection of bioactive molecules during radiation sterilization | |
| CN116284207A (en) | Fluorinated modified antibacterial peptide and preparation method and application thereof | |
| CN101991857B (en) | Stable pharmaceutical preparation and preparation method thereof | |
| CN106011123B (en) | Application of the epiphyte hydrophobic protein HGFI in trypsase immobilization | |
| US7604807B2 (en) | Use of pullulan to isolate and preserve biological material | |
| CN112858676A (en) | Fluorescent microsphere identification and detection card for African swine fever virus antibody | |
| Phillips et al. | A study of water binding in lyophilized viral vaccine systems |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| CB03 | Change of inventor or designer information |
Inventor after: Jiang Tianshu Inventor after: Yang Dengtian Inventor after: Yi Wenhui Inventor after: Jin Weiqiu Inventor after: Zhang Ruotong Inventor after: Jia Minglong Inventor after: Li Chenguang Inventor after: Li Yanru Inventor after: Huang Zhiqi Inventor after: Yang Qicheng Inventor before: Yi Wenhui Inventor before: Yang Dengtian Inventor before: Jin Weiqiu Inventor before: Jiang Tianshu Inventor before: Zhang Ruotong Inventor before: Jia Minglong Inventor before: Li Chenguang Inventor before: Li Yanru Inventor before: Huang Zhiqi Inventor before: Yang Qicheng |
|
| CB03 | Change of inventor or designer information | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20200728 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |