CN110568044A - Microfluidic filter paper chip sensor based on aptamer and preparation method thereof - Google Patents
Microfluidic filter paper chip sensor based on aptamer and preparation method thereof Download PDFInfo
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Abstract
The present disclosure provides an aptamer-based microfluidic filter paper chip sensor and a method for manufacturing the same, the sensor comprising: the sample injection area comprises a sample injection hole, a microfluidic channel and a filter hole which are connected in sequence; the diffusion and reaction area is positioned below the sample injection area and comprises a diffusion filter hole corresponding to the filter hole and a plurality of reaction areas, and the reaction areas are respectively connected with the diffusion filter hole through a diffusion microfluidic channel; the diffusion and reaction areas are used for diffusing the samples to be detected after being filtered by the filtering holes to the plurality of reaction areas; the working electrode area is positioned below the diffusion and reaction area and comprises a plurality of working electrodes respectively corresponding to the reaction areas, and the working electrodes are provided with nano composite materials and aptamers; and the counter electrode and the reference electrode area are positioned on the diffusion and reaction area and comprise a plurality of sets of counter electrode and reference electrode groups respectively corresponding to the reaction areas.
Description
Technical Field
The disclosure relates to the fields of preparation and modification of novel nano composite materials, design and preparation of microfluidic filter paper chips, direct label-free electrochemical rapid detection technology and the like, in particular to an aptamer-based microfluidic filter paper chip sensor and a preparation method thereof.
Background
aptamers are small fragments that have been screened from a specific pool of oligonucleotides using the exponential enrichment of ligand evolution technology (SELEX) for oligonucleotides (DNA or RNA) that specifically bind to a target molecule. There are two types in total, an antisense nucleotide strand and a random nucleotide strand. An antisense nucleotide strand typically comprises 15-20 nucleotides, the base composition of which is complementary to the corresponding target sequence. However, in practical use, antisense nucleotides have various problems such as being easily degraded by nucleases, being poorly recognized to a specific target site, and having some toxicity. What has been developed on the basis of the antisense nucleotide chain is a random nucleotide chain, and the target sequence for the action of the random nucleotide chain includes not only nucleic acids but also such as proteins, cells, viruses, vitamins, allergens, and the like, as compared with the antisense nucleotide chain. And the length of the probe is generally 10-100, a stable three-dimensional structure is formed through pairing, electrostatic interaction or hydrogen bond interaction of some complementary bases in a chain, and the probe is combined with a target molecule more specifically and more stably.
aptamers have many properties similar to antibodies and have the following advantages over antibodies: 1) compared with an antibody, the aptamer is easier to obtain, can be quickly synthesized in vitro in a large amount by an exponential enrichment ligand system evolution technology, and is lower in cost; 2) the aptamer has higher stability and no immunogenicity, and can be stored at room temperature, which provides great convenience for storage; 3) the aptamer corresponding to the target can be screened out aiming at various targets of different types, and the application range of the aptamer is greatly widened; 4) aptamers have an affinity for the target molecule of interest that is comparable to or even higher than that of antibodies.
six sex hormones tested in the hospital include estradiol (E2), progesterone (P), folliculogenesis hormone (FSH), Luteinisation Hormone (LH), testosterone (T), Prolactin (PRL). Wherein the function of FSH is primarily to promote development and maturation of follicles in the ovaries of females; e2 functions primarily in two ways, namely promoting endometrial proliferation and promoting development of a second female characteristic; LH, in turn, functions to promote ovulation and luteal formation in synergy with FSH and secretion of progestin, respectively. However, the following problems exist in the current detection of sex hormones: 1) the content of sex hormone in the body is very low, so the prepared sensor is difficult to solve the scientific problems of quantitative detection of ultra-trace sex hormone, preparation and modification of nano materials and the like; 2) at present, a paper chip adopting a label-free electrochemical rapid detection method is directly dripped on an electrode when in use, and the sample consumption of the method is too large. Especially, in multi-parameter detection, the scientific problems of reducing the required sample amount, avoiding mutual interference in different parameter detection and the like need to be solved.
BRIEF SUMMARY OF THE PRESENT DISCLOSURE
Technical problem to be solved
Based on the problems, the disclosure provides an aptamer-based microfluidic filter paper chip sensor and a preparation method thereof, so as to alleviate the technical problems that the detection sensor in the prior art is difficult to solve scientific problems such as quantitative detection of ultra-trace sex hormones, preparation and modification of nano materials and the like, the amount of a detection sample is large, and mutual interference is caused during detection of different parameters.
(II) technical scheme
In one aspect of the present disclosure, there is provided an aptamer-based microfluidic filter paper chip sensor, comprising: the sample injection area comprises a sample injection hole, a microfluidic channel and a filter hole which are connected in sequence; the sample to be measured enters from the sample inlet hole and enters the filter hole through the microfluidic channel for filtering; the diffusion and reaction area is positioned below the sample injection area and comprises a diffusion filter hole corresponding to the filter hole and a plurality of reaction areas, and the reaction areas are respectively connected with the diffusion filter hole through a diffusion microfluidic channel; the diffusion and reaction areas are used for diffusing the samples to be detected after being filtered by the filtering holes to the plurality of reaction areas; the working electrode area is positioned below the diffusion and reaction area and comprises a plurality of working electrodes respectively corresponding to the reaction areas, and the working electrodes are provided with nano composite materials and aptamers; and the counter electrode and the reference electrode area are positioned on the diffusion and reaction area and comprise a plurality of sets of counter electrode and reference electrode groups respectively corresponding to the reaction areas.
In an embodiment of the present disclosure, each set of counter and reference electrodes includes a pair of electrodes and a reference electrode.
in an embodiment of the present disclosure, the working electrode is made of a material including: carbon slurry, nanocomposite, aptamer, and mercaptoethanol solution.
In the disclosed embodiment, the mercaptoethanol solution blocks excess active sites on the working electrode.
In an embodiment of the present disclosure, the nanocomposite comprises: conductive material, electroactive material and nanogold.
In an embodiment of the present disclosure, the conductive substance includes: at least one of aminated single-walled carbon nanotubes, aminated graphene, multi-walled carbon nanotubes or poly 3, 4 ethylene dioxythiophene.
In embodiments of the present disclosure, the electroactive species comprises: at least one of thionine, new methylene blue, hydroquinone, prussian blue, polyglutamic acid or potassium ferricyanide.
In an embodiment of the present disclosure, the sample to be tested includes: sex hormones, proteins, cells, viruses, vitamins or allergens.
in another aspect of the present disclosure, there is provided a method for preparing an aptamer-based microfluidic filter paper chip sensor, for preparing the aptamer-based microfluidic filter paper chip sensor according to any one of the above, the method comprising:
Step S1: selecting proper filter paper according to the characteristics of the object to be detected;
Step S2: preparing a hydrophobic area on the filter paper according to a design drawing, wherein the rest part is a hydrophilic area; the hydrophilic area comprises a sample introduction area, a diffusion and reaction area, a working electrode preparation area, a counter electrode preparation area and a reference electrode preparation area;
Step S3: preparing a working electrode in the working electrode preparation area prepared in the step S2 to finish the preparation of the working electrode area;
Step S4: preparing a counter electrode and a reference electrode in the counter electrode preparation area and the reference electrode preparation area prepared in the step S2, completing the preparation of the counter electrode and the reference electrode area, and further completing the preparation of the microfluidic filter paper chip based on the aptamer; and
Step S5: and arranging the microfluidic filter paper chip based on the aptamer according to the mode that the sample introduction region is on the surface, and the diffusion and reaction region is positioned between the working electrode region and the counter electrode and reference electrode region, so as to finish the preparation of the microfluidic filter paper chip sensor based on the aptamer.
In an embodiment of the present disclosure, the step S3 includes:
Step S31: printing conductive carbon paste on a preparation area of the counter working electrode corresponding to the working electrode;
step S32: modifying a nanocomposite material on the conductive carbon paste; and
Step S33: preparing an aptamer on the nano composite material, and sealing by using a mercaptoethanol solution to finish the preparation of the working electrode.
In yet another aspect of the present disclosure, an aptamer-based microfluidic filter paper chip for simultaneously testing different target substances of interest in a sample to be tested by folding or stacking comprises a sample entry region, a diffusion and reaction region, a working electrode region, and a counter electrode and a reference electrode region.
(III) advantageous effects
According to the technical scheme, the microfluidic filter paper chip sensor based on the aptamer and the preparation method thereof have at least one or part of the following beneficial effects:
(1) The functions of sample introduction, filtration, detection and the like are integrated, so that the operation flow of the sensor is greatly simplified, and the sensor device is more portable;
(2) the sensitivity and the detection limit of the device are improved, and the quantitative detection of the ultra-trace sex hormone is realized;
(3) The corresponding concentration can be obtained only by analyzing the peak current change of the differential pulse voltammetry, and the detection speed is greatly improved.
Drawings
FIG. 1 is a schematic diagram of the composition of an aptamer-based microfluidic filter paper chip sensor according to an embodiment of the present disclosure; wherein FIG. 1(a) is a schematic plan-view development; FIG. 1(b) is a schematic diagram of a hierarchical relationship.
Fig. 2 is a schematic flow chart of a preparation method of the microfluidic filter paper chip sensor based on the aptamer according to the embodiment of the disclosure.
fig. 3 is a schematic flow chart illustrating a process for preparing a working electrode of an aptamer-based microfluidic filter paper chip sensor according to an embodiment of the present disclosure.
fig. 4 is a schematic flow chart of a process for preparing an aptamer-based microfluidic filter paper chip sensor in a folding manner according to an embodiment of the present disclosure.
Detailed Description
the invention provides an aptamer-based microfluidic filter paper chip sensor and a preparation method thereof, and particularly relates to a microfluidic channel and stack layout structure designed based on the principle of aptamer-antigen specific binding, a working electrode of a microfluidic filter paper chip is directionally modified by a synthesized nano composite material, and the detection of a sample (sex hormone) to be detected is taken as an entry point to perform rapid and high-sensitivity instant detection of a target substance. The detection process takes less time, the consumption of the required sample is low, and the simultaneous detection of the three sex hormones can be realized.
The microfluidic filter paper chip sensor based on the aptamer takes the filter paper as the substrate, so that the microfluidic filter paper chip sensor is good in biocompatibility, easy to degrade, light in texture and capable of being used in a portable mode. In addition, the application of the microfluidic technology enables the sample injection, filtration, detection and other functions to be completed on one chip. And the micro-fluidic filter paper chip consumes very little sample amount, thereby being convenient for realizing the simultaneous detection of multiple parameters. The microfluidic filter paper chip sensor based on the aptamer has the advantages that the operating steps are simplified through the folding structure while the characteristics of the microfluidic filter paper chip are not affected by the ingenious folding structure, the microfluidic filter paper chip is combined with a label-free electrochemical rapid detection method, so that a label-free sample can be added on the chip to directly start electrochemical detection, and the instant diagnosis with controllability, excellent selectivity and repeatability, low detection limit and wide linear range is realized. The use of nanomaterials is to achieve more sensitive detection. The electrode modified by the nano material has larger specific surface area, higher catalytic activity and faster electron transfer capacity, so that more sensitive detection is realized, and the simultaneous detection of estradiol (E2), follicle-stimulating hormone (FSH) and Luteinizing Hormone (LH) can be realized. Meanwhile, the microfluidic filter paper chip can be combined with a plurality of different detection methods to form various detection devices based on different principles.
For the purpose of promoting a better understanding of the objects, aspects and advantages of the present disclosure, reference is made to the following detailed description taken in conjunction with the accompanying drawings.
in an embodiment of the present disclosure, there is provided an aptamer-based microfluidic filter paper chip sensor, as shown in fig. 1, the aptamer-based microfluidic filter paper chip sensor including:
The sample injection area comprises a sample injection hole, a microfluidic channel and a filter hole which are connected in sequence; the sample to be measured enters from the sample inlet hole and enters the filter hole through the microfluidic channel for filtering;
the diffusion and reaction area is positioned below the sample injection area and comprises a diffusion filter hole corresponding to the filter hole and a plurality of reaction areas, and the reaction areas are respectively connected with the diffusion filter hole through a diffusion microfluidic channel; the diffusion and reaction areas are used for diffusing the samples to be detected after being filtered by the filtering holes to the plurality of reaction areas;
the working electrode area is positioned below the diffusion and reaction area and comprises a plurality of working electrodes respectively corresponding to the reaction areas, and the working electrodes are provided with nano composite materials and aptamers; and
The counter electrode and the reference electrode area are positioned on the diffusion and reaction area and comprise a plurality of groups of counter electrode and reference electrode groups respectively corresponding to the reaction areas;
The sample introduction region may be integrated (in one layer) with the working or counter and reference electrode regions; or can be independently positioned on one layer;
the sample inlet, the microfluidic channel, the filter hole, the diffusion filter hole and the reaction area in the microfluidic filter paper chip sensor based on the aptamer are hydrophilic areas; the regions prepared by the working electrode, the counter electrode and the reference electrode are hydrophilic regions; the regions outside the hydrophilic regions are hydrophobic regions.
Each set of counter electrode and reference electrode set comprises a pair of electrodes and a reference electrode;
The preparation material of the working electrode comprises: carbon slurry, nanocomposite, aptamer, and mercaptoethanol solution;
and the mercaptoethanol solution seals redundant active sites on the working electrode.
The nano composite material is a mixture of a conductive substance, an electroactive substance and nano gold;
The conductive substance includes: at least one of aminated single-walled carbon nanotubes, aminated graphene, multi-walled carbon nanotubes or poly 3, 4 ethylene dioxythiophene;
The electroactive species comprises: at least one of thionine, new methylene blue, hydroquinone, prussian blue, polyglutamic acid or potassium ferricyanide;
The sample to be tested comprises: sex hormones (at least one of estradiol (E2), progesterone (P), Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH), testosterone (T), Prolactin (PRL)), proteins, cells, viruses, vitamins or allergens.
The preparation material of the counter electrode comprises: at least one of carbon paste, graphene conductive ink or conductive gold paste;
The preparation material of the reference electrode comprises: conductive silver paste;
The working electrode, the counter electrode and the reference electrode jointly form a three-electrode structure.
the aptamer type corresponds to the species to be detected, and different aptamers can be arranged on different working electrodes, so that various different species to be detected can be detected simultaneously.
In the present disclosure, there is also provided a method for preparing an aptamer-based microfluidic filter paper chip sensor, for preparing the above aptamer-based microfluidic filter paper chip sensor, as shown in fig. 2 to 4, the method comprising:
step S1: selecting proper filter paper according to the characteristics of the object to be detected;
step S2: preparing a hydrophobic area on the filter paper according to a design drawing, wherein the rest part is a hydrophilic area; the hydrophilic area comprises a sample introduction area, a diffusion and reaction area, a working electrode preparation area, a counter electrode preparation area and a reference electrode preparation area;
Step S3: preparing a working electrode in the working electrode preparation area prepared in the step S2 to finish the preparation of the working electrode area;
step S4: preparing a counter electrode and a reference electrode in the counter electrode preparation area and the reference electrode preparation area prepared in the step S2, completing the preparation of the counter electrode and the reference electrode area, and further completing the preparation of the microfluidic filter paper chip based on the aptamer;
Step S5: and arranging the microfluidic filter paper chip based on the aptamer according to the mode that the sample introduction region is on the surface, and the diffusion and reaction region is positioned between the working electrode region and the counter electrode and reference electrode region, so as to finish the preparation of the microfluidic filter paper chip sensor based on the aptamer.
In step S1, a suitable filter paper type is selected according to the physical or chemical characteristics of the object to be detected, and the layout and structure of the filter paper chip, including the layout and structure of the microfluidic channel and the layout and structure of the three electrodes (working electrode, counter electrode, reference electrode) are designed by drawing software in a computer.
in step S2, the designed pattern is printed on the filter paper using a wax-jet printer and baked in an oven at 60 ℃ for 50 min. After baking, the wax printed on the surface of the filter paper is melted and permeates into the filter paper, the area covered by the wax is changed into a hydrophobic area, and the area of the filter paper which does not pass through wax spraying is still kept as a hydrophilic area; through multiple experiments, the invention realizes the accurate control of the sizes of the hydrophilic area and the hydrophobic area of the filter paper chip by accurately controlling the heating temperature and the heating time.
the step S3 includes:
step S31: printing conductive carbon paste on a counter electrode preparation area corresponding to the working electrode;
step S32: modifying a nanocomposite material on the conductive carbon paste;
Step S33: preparing an aptamer on the nano composite material, and sealing by using a mercaptoethanol solution to finish the preparation of the working electrode.
And printing the conductive carbon paste in a working electrode preparation area corresponding to the working electrode and a counter electrode preparation area corresponding to the counter electrode by a screen printing technology. And printing conductive silver paste on a reference electrode preparation area corresponding to the reference electrode. And preparing a novel nano composite material modified working electrode, and respectively fixing the aptamers corresponding to the detection object on the corresponding working electrodes. And the excess active sites on the electrode were blocked using a mercaptoethanol solution.
In the embodiment of the disclosure, as shown in fig. 1, the microfluidic filter paper chip based on the aptamer has three sets of electrochemical three-electrode systems, so that three detection objects in the same sample to be detected can be detected simultaneously, wherein three working electrodes are provided. Three sets of counter electrodes are printed in pairs with reference electrodes. When the filter paper chip is used, an object to be detected enters the filter paper chip through the sample inlet hole and flows into the filter hole through the microfluidic channel, and the sample to be detected is filtered. The sample to be measured after being filtered by the filtering holes downwards reaches the diffusion and reaction areas and flows to the three reaction areas through the diffusion filtering holes, so that the sample to be measured respectively reacts with the aptamers fixed on the working electrode. In the reaction process, the counter electrode and the reference electrode form a three-electrode structure with the working electrode through the reaction area. After the reaction is finished, the microfluidic filter paper chip sensor based on the aptamer is connected with an external electrochemical workstation through the working electrode, the counter electrode and the reference electrode, the working electrode is scanned by adopting a differential pulse voltammetry method, and the concentration of the substances to be detected (E2, FSH and LH) in the sample to be detected is obtained according to the variation of the current peak value.
In step S5, the microfluidic filter paper chip sensor based on the aptamer may be prepared by folding or stacking; in the embodiment of the present disclosure, as shown in fig. 1 and fig. 4, the prepared aptamer-based microfluidic filter paper chip is processed into a final aptamer-based microfluidic filter paper chip sensor by folding. In fig. 4, the part marked 1 comprises a sample introduction region, a counter electrode and a reference electrode region, the part marked 2 is a working electrode region, and the part marked 3 is a diffusion and reaction region, the diffusion and reaction region is firstly folded onto the working electrode region, and then the part marked 1 is folded onto the diffusion and reaction region, so that the preparation of the microfluidic filter paper chip sensor based on the aptamer is completed in a folding mode. The parts marked 1, 2 and 3 can also be processed respectively and then stacked to be processed into the microfluidic filter paper chip sensor based on the aptamer.
simultaneously detecting multiple sex hormones E2, FSH and LH, and measuring the concentration of each substance to be detected; and thus can determine whether the patient has an endocrine disorder. For example, lower concentrations of E2 are found in diseases such as ovarian hypofunction, premature ovarian failure, Sheehan's syndrome, etc. If the concentrations of FSH and LH are high, ovarian failure can be diagnosed.
in the embodiment of the disclosure, the microfluidic filter paper chip sensor based on the aptamer can detect E2, FSH, and LH simultaneously, specifically as follows:
(1) respectively screening aptamers corresponding to E2, FSH and LH by an exponential enrichment ligand system evolution technology, and modifying sulfydryl at the 5' end of the aptamers; and preparing the microfluidic chip provided by the invention.
(2) Synthesis of nanocomposite (aminated Single-walled carbon nanotubes/SinaMethyl blue/nano gold solution), a working electrode in the microfluidic filter paper chip based on the aptamer is modified, so as to improve the conductive capability of the working electrode and facilitate fixing the aptamer corresponding to E2 on the surface of the electrode, and the method specifically comprises the following steps: dissolving 2mg of aminated single-walled carbon nanotube into 1mL of aqueous solution, and collecting 1mL of solution with a concentration of 2mg mL-1The aminated single-walled carbon nanotube solution and 1mL of the solution with the concentration of 2mg mL-1The new methylene blue solution of (2) was mixed and stirred vigorously in a magnetic stirrer for 24 h. And (3) mixing 20uL of the mixed solution with 100uL of nano-gold solution with the particle size of 15nm, and stirring vigorously for 12 hours to obtain the aminated single-walled carbon nanotube/new methylene blue/nano-gold compound. 10 mu L of the prepared aminated single-walled carbon nanotube/new methylene blue/nanogold composite mixed solution is dripped on the surface of a working electrode, and then 10 mu L of the aminated single-walled carbon nanotube/new methylene blue/nanogold composite mixed solution with the concentration of 28 mu gmL is dripped on the surface of the working electrode-1the aptamer of E2 was applied to the electrode surface, and left to stand in a refrigerator set at 4 ℃ for 6 hours for binding the aptamer, and finally, 10. mu.L of a 1% mercaptoethanol solution was dropped onto the electrode surface at room temperature to block the remaining active sites, thereby completing the modification of the working electrode (detection electrode) of E2;
(3) Synthesizing another nano composite material (polyetherimide/multi-wall carbon nano tube/thionine/nano gold solution), and modifying another working electrode in the filter paper chip, in order to improve the conductive capability of the working electrode and facilitate fixing an aptamer corresponding to FSH on the surface of the working electrode, the method specifically comprises the following steps: 6mg of polyetherimide was dissolved in 1mL of the aqueous solution to obtain 6mg mL of a solution-1The polyetherimide solution of (1). Dissolving 2mg of multi-walled carbon nanotube into 1mL of aqueous solution to obtain 2mg mL of solution-1the multi-walled carbon nanotube solution of (1). Taking 1mL of the solution with the concentration of 6mg mL-1and 1mL of a polyetherimide solution having a concentration of 2mg mL-1the multi-wall carbon nano tube solution is mixed and is subjected to ultrasonic treatment in an ultrasonic instrument for 1 hour to obtain the polyetherimide/multi-wall carbon nano tube mixed solution. Taking 1mL of polyetherimide/multi-wall carbon nano tube mixed solution and 1mL of 2mg mL of solution with concentration-1mixing the thionine solution, stirring vigorously in a magnetic stirrer for 24 hr, mixing 20uL of the above mixed solution with 100uL of nano gold solution with particle size of 15nm, and stirring vigorouslyStirring for 12h to obtain the polyetherimide/multi-wall carbon nano tube/thionine/nano gold solution. Taking 10 mu L of prepared polyetherimide/multi-wall carbon nano tube/thionine/nano gold solution to be dripped on the surface of a working electrode, then taking 10 mu L of FSH aptamer on the surface of the working electrode, placing the working electrode in a refrigerator at 4 ℃ for 6h for binding the aptamer, finally dripping 10 mu L of 1% mercaptoethanol solution on the surface of the electrode at room temperature to seal the rest active sites, and finishing the modification of the FSH working electrode;
(4) synthesizing another nanocomposite material (graphene/polyphenolic acid/nanogold solution), and modifying another working electrode in the filter paper chip, so as to improve the conductive capability of the working electrode and facilitate fixing an aptamer corresponding to LH to the surface of the working electrode, specifically comprising: dissolving 2mg of graphene into 1mL of aqueous solution, and taking 1mL of graphene with the concentration of 2mg mL-1And 1mL of the graphene solution with a concentration of 2mg mL-1The solution of polyphenolic acid was mixed and stirred vigorously in a magnetic stirrer for 24 h. And (3) mixing 20uL of the mixed solution with 100uL of nano-gold solution with the particle size of 15nm, and stirring vigorously for 12 hours to obtain the graphene/polyphenolic acid/nano-gold compound. Dripping 10 mu L of prepared graphene/polyphenolic acid/nano-gold solution on the surface of the working electrode, then dripping 10 mu L of LH aptamer on the surface of the electrode, placing the electrode in a refrigerator at 4 ℃ for 6 hours for binding the aptamer, and finally dripping 10 mu L of 1% mercaptoethanol solution on the surface of the electrode at room temperature to seal the rest active sites, thereby finishing the modification of the LH working electrode (detection electrode);
(5) processing the prepared filter paper chip into an aptamer-based microfluidic filter paper chip sensor, and simultaneously detecting sex hormones E2, FSH and LH, wherein the specific operations are as follows: and taking 15 mu L of clinical serum solution, flowing into the sensor from the sample inlet hole, and filtering to reach the diffusion and reaction area. Standing at room temperature for 25min to make the sex hormone to be detected and the corresponding aptamer on the surface of the working electrode fully combined. And then, connecting the sensor with an electrochemical workstation through a rear-end interface, scanning the working electrode by adopting a differential pulse voltammetry method, and obtaining the concentrations of E2, FSH and LH in the sample to be detected according to the variation of the current peak value.
so far, the embodiments of the present disclosure have been described in detail with reference to the accompanying drawings. It is to be noted that, in the attached drawings or in the description, the implementation modes not shown or described are all the modes known by the ordinary skilled person in the field of technology, and are not described in detail. Further, the above definitions of the various elements and methods are not limited to the various specific structures, shapes or arrangements of parts mentioned in the examples, which may be easily modified or substituted by those of ordinary skill in the art.
from the above description, those skilled in the art should clearly recognize that the aptamer-based microfluidic filter paper chip sensor and the preparation method thereof according to the present disclosure.
in summary, the present disclosure provides an aptamer-based microfluidic filter paper chip sensor and a preparation method thereof, based on the principle of aptamer-antigen specific binding, a microfluidic channel and a stacking layout structure are designed, a working electrode of the microfluidic filter paper chip is directionally modified by a synthesized nanocomposite, and a target object to be detected in a sample to be detected is used as an entry point to perform rapid and high-sensitivity real-time detection of the target substance. The detection process takes less time, the consumption of the required sample is low, and the simultaneous detection of a plurality of target substances to be detected can be realized.
It should also be noted that directional terms, such as "upper", "lower", "front", "rear", "left", "right", and the like, used in the embodiments are only directions referring to the drawings, and are not intended to limit the scope of the present disclosure. Throughout the drawings, like elements are represented by like or similar reference numerals. Conventional structures or constructions will be omitted when they may obscure the understanding of the present disclosure.
And the shapes and sizes of the respective components in the drawings do not reflect actual sizes and proportions, but merely illustrate the contents of the embodiments of the present disclosure. Furthermore, in the claims, any reference signs placed between parentheses shall not be construed as limiting the claim.
Unless otherwise indicated, the numerical parameters set forth in the specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the present disclosure. In particular, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about". Generally, the expression is meant to encompass variations of ± 10% in some embodiments, 5% in some embodiments, 1% in some embodiments, 0.5% in some embodiments by the specified amount.
Furthermore, the word "comprising" does not exclude the presence of elements or steps not listed in a claim. The word "a" or "an" preceding an element does not exclude the presence of a plurality of such elements.
The use of ordinal numbers such as "first," "second," "third," etc., in the specification and claims to modify a corresponding element does not by itself connote any ordinal number of the element or any ordering of one element from another or the order of manufacture, and the use of the ordinal numbers is only used to distinguish one element having a certain name from another element having a same name.
in addition, unless steps are specifically described or must occur in sequence, the order of the steps is not limited to that listed above and may be changed or rearranged as desired by the desired design. The embodiments described above may be mixed and matched with each other or with other embodiments based on design and reliability considerations, i.e., technical features in different embodiments may be freely combined to form further embodiments.
Those skilled in the art will appreciate that the modules in the device in an embodiment may be adaptively changed and disposed in one or more devices different from the embodiment. The modules or units or components of the embodiments may be combined into one module or unit or component, and furthermore they may be divided into a plurality of sub-modules or sub-units or sub-components. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and all of the processes or elements of any method or apparatus so disclosed, may be combined in any combination, except combinations where at least some of such features and/or processes or elements are mutually exclusive. Each feature disclosed in this specification (including any accompanying claims, abstract and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Also in the unit claims enumerating several means, several of these means may be embodied by one and the same item of hardware.
Similarly, it should be appreciated that in the foregoing description of exemplary embodiments of the disclosure, various features of the disclosure are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various disclosed aspects. However, the disclosed method should not be interpreted as reflecting an intention that: that is, the claimed disclosure requires more features than are expressly recited in each claim. Rather, as the following claims reflect, disclosed aspects lie in less than all features of a single foregoing disclosed embodiment. Thus, the claims following the detailed description are hereby expressly incorporated into this detailed description, with each claim standing on its own as a separate embodiment of this disclosure.
The above-mentioned embodiments are intended to illustrate the objects, aspects and advantages of the present disclosure in further detail, and it should be understood that the above-mentioned embodiments are only illustrative of the present disclosure and are not intended to limit the present disclosure, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present disclosure should be included in the scope of the present disclosure.
Claims (11)
1. An aptamer-based microfluidic filter paper chip sensor comprising:
the sample injection area comprises a sample injection hole, a microfluidic channel and a filter hole which are connected in sequence; the sample to be measured enters from the sample inlet hole and enters the filter hole through the microfluidic channel for filtering;
The diffusion and reaction area is positioned below the sample injection area and comprises a diffusion filter hole corresponding to the filter hole and a plurality of reaction areas, and the reaction areas are respectively connected with the diffusion filter hole through a diffusion microfluidic channel; the diffusion and reaction areas are used for diffusing the samples to be detected after being filtered by the filtering holes to the plurality of reaction areas;
The working electrode area is positioned below the diffusion and reaction area and comprises a plurality of working electrodes respectively corresponding to the reaction areas, and the working electrodes are provided with nano composite materials and aptamers; and
and the counter electrode and the reference electrode area are positioned on the diffusion and reaction area and comprise a plurality of sets of counter electrode and reference electrode groups respectively corresponding to the reaction areas.
2. The aptamer-based microfluidic filter paper chip sensor according to claim 1, wherein each set of counter and reference electrodes comprises a pair of electrodes and a reference electrode.
3. the aptamer-based microfluidic filter paper chip sensor according to claim 1, wherein the working electrode is prepared from a material comprising: carbon slurry, nanocomposite, aptamer, and mercaptoethanol solution.
4. The aptamer-based microfluidic filter paper chip sensor according to claim 3, wherein the mercaptoethanol solution blocks excess active sites on the working electrode.
5. The aptamer-based microfluidic filter paper chip sensor according to claim 1, wherein the nanocomposite material comprises: conductive material, electroactive material and nanogold.
6. The aptamer-based microfluidic filter paper chip sensor according to claim 5, wherein the conductive substance comprises: at least one of aminated single-walled carbon nanotubes, aminated graphene, multi-walled carbon nanotubes or poly 3, 4 ethylene dioxythiophene.
7. The aptamer-based microfluidic filter paper chip sensor according to claim 5, the electroactive species comprising: at least one of thionine, new methylene blue, hydroquinone, prussian blue, polyglutamic acid or potassium ferricyanide.
8. The aptamer-based microfluidic filter paper chip sensor according to claim 1, wherein the sample to be tested comprises: sex hormones, proteins, cells, viruses, vitamins or allergens.
9. A method for preparing an aptamer-based microfluidic filter paper chip sensor, which is used for preparing the aptamer-based microfluidic filter paper chip sensor according to any one of claims 1 to 8, and comprises the following steps:
Step S1: selecting proper filter paper according to the characteristics of the object to be detected;
Step S2: preparing a hydrophobic area on the filter paper according to a design drawing, wherein the rest part is a hydrophilic area; the hydrophilic area comprises a sample introduction area, a diffusion and reaction area, a working electrode preparation area, a counter electrode preparation area and a reference electrode preparation area;
Step S3: preparing a working electrode in the working electrode preparation area prepared in the step S2 to finish the preparation of the working electrode area;
Step S4: preparing a counter electrode and a reference electrode in the counter electrode preparation area and the reference electrode preparation area prepared in the step S2, completing the preparation of the counter electrode and the reference electrode area, and further completing the preparation of the microfluidic filter paper chip based on the aptamer; and
step S5: and arranging the microfluidic filter paper chip based on the aptamer according to the mode that the sample introduction region is on the surface, and the diffusion and reaction region is positioned between the working electrode region and the counter electrode and reference electrode region, so as to finish the preparation of the microfluidic filter paper chip sensor based on the aptamer.
10. The method for preparing an aptamer-based microfluidic filter paper chip sensor according to claim 9, wherein the step S3 comprises:
step S31: printing conductive carbon paste on a preparation area of the counter working electrode corresponding to the working electrode;
step S32: modifying a nanocomposite material on the conductive carbon paste; and
Step S33: preparing an aptamer on the nano composite material, and sealing by using a mercaptoethanol solution to finish the preparation of the working electrode.
11. the microfluidic filter paper chip based on the aptamer simultaneously tests different target substances in a sample to be tested after being folded or stacked, and comprises a sample inlet area, a diffusion and reaction area, a working electrode area, a counter electrode and a reference electrode area.
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