CN117554344A - DNase I/Apt/rGO-based combined multichannel microfluidic chip detection method and application - Google Patents
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Abstract
The invention relates to the field of molecular detection, and discloses a DNase I/Apt/rGO-based combined multichannel microfluidic chip detection method and application. The detection method comprises the following steps: s1, adding a sample to be detected into a buffer solution containing fluorogenic group-containing nucleic acid aptamer and biological reduction graphene oxide with a final concentration of 15-30 mug/mL for incubation, then adding deoxyribonuclease with a final concentration of 5-40U/mL for reaction, centrifuging, and collecting supernatant; s2, detecting the supernatant by adopting a microfluidic chip, and quantifying the concentration of the target detection object in the sample to be detected according to the fluorescent signal intensity. The detection method can realize simultaneous detection of protein and nucleic acid on the same chip, wherein the detection limit of the protein target substance can reach 4.5pg/mL, the detection limit of the nucleic acid target substance can reach 1.3fM, and the detection method can be used for detecting low-concentration markers in serum.
Description
Technical Field
The invention relates to the field of molecular detection, in particular to a DNase I/Apt/rGO-based multi-channel micro-fluidic chip detection method and application.
Background
Hepatocellular carcinoma (Hepatocellular Carcinoma, HCC) is one of the most common malignant tumors in the world, with its morbidity and mortality rising in the past 10 years. Early diagnosis of HCC includes non-invasive imaging methods, invasive needle biopsies and pathology detection, and detection methods for serological tumor markers such as AFP. Currently, tissue biopsies and pathology examinations are gold standard for diagnosing HCC, which, although highly accurate, are not easily accepted by patients due to their invasive nature. Imaging methods require large-scale equipment and are therefore difficult to popularize in areas where resources are scarce. Compared with the traditional tumor tissue detection technology, the liquid biopsy has the advantages of noninvasive detection, convenient operation, easily obtained samples and the like, becomes a new star for tumor detection, and is applied to early diagnosis of tumors.
In the related technology, serum alpha fetoprotein AFP is the most commonly used serum tumor marker for screening and diagnosing liver cancer at present, and has been widely applied in clinic. However, most liver cancer patients fail to be found at an early stage due to insufficient sensitivity and specificity of the monitoring tool, which highlights the need for more accurate biomarkers to improve early diagnosis of liver cancer. At present, many methods for detecting liver cancer biomarkers, such as enzyme-linked immunoassay, electrochemical method, fluorescence method, surface-enhanced raman spectroscopy, PCR, loop-mediated isothermal amplification and the like, have been developed, however, these methods have low sensitivity, and can only detect single protein biomarkers or nucleic acid biomarkers, and cannot realize simultaneous detection of protein and nucleic acid biomarkers.
Therefore, it is highly desirable to establish a high-specificity and high-sensitivity detection method, which provides accurate judgment basis for clinical diagnosis of HCC patients, and can perform treatment as soon as possible, thereby improving survival rate of patients.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, the invention provides a DNase I/Apt/rGO-based combined multichannel microfluidic chip detection method, which is based on a double-signal amplification strategy, can be used for simultaneously detecting the contents of a plurality of target substances in a sample to be detected, can reach 4.5pg/mL for the detection limit of protein target substances, can reach 1.3fM for the detection limit of nucleic acid target substances, and can be used for detecting serum low-concentration markers.
The invention also provides an application of the detection method based on the DNase I/Apt/rGO combined multichannel microfluidic chip in biomarker detection.
The invention provides a DNase I/Apt/rGO-based combined multichannel microfluidic chip detection method, which comprises the following steps of:
s1, adding a sample to be detected into a buffer solution containing fluorogenic group-containing nucleic acid aptamer and biological reduction graphene oxide with a final concentration of 15-30 mug/mL for incubation, then adding deoxyribonuclease with a final concentration of 5-40U/mL for reaction, centrifuging, and collecting supernatant;
s2, detecting the supernatant by adopting a microfluidic chip, and quantifying the concentration of a target detection object in the sample to be detected according to the fluorescent signal intensity;
the detection method is used for non-disease diagnostic purposes.
The detection method provided by the embodiment of the invention has at least the following beneficial effects:
(1) The method develops a high-efficiency detection method based on DNase I/Apt/rGO combined multi-pass microfluidic enrichment chip based on a double-signal amplification strategy, can detect multiple biomarkers simultaneously, and only needs 30 minutes for detecting protein and nucleic acid simultaneously on the same chip. In addition, taking the detection of the hepatocellular carcinoma biomarker as an example, the detection Limits (LOD) of AFP, CEA and miR-21 respectively reach 37.0pg/mL, 4.5pg/mL and 1.3fM by adopting the detection method disclosed by the invention, so that the detection of the serum low-concentration marker of an HCC patient can be satisfied.
(2) According to the detection method, reduced Graphene Oxide (GO) is adopted, so that the Graphene Oxide (GO) is generally considered to be an ideal biocompatible nanomaterial, and has extremely strong distance-dependent fluorescence quenching capacity. GO adsorbs single-stranded DNA (ssDNA) through pi-pi electron stacking interaction, so that fluorescence of FAM labeled DNA can be quenched, stability of single-stranded DNA adsorbed on the surface of the FAM can be protected, and enzyme digestion is prevented. However, reduced graphene oxide (rGO) has the property of better quenching single-stranded fluorescent DNA than GO.
In some embodiments of the invention, the sample to be tested comprises whole blood or serum.
In some embodiments of the invention, the nucleic acid aptamer comprises a nucleic acid aptamer for detecting a protein and/or a nucleic acid aptamer for detecting a nucleic acid.
The nucleic acid aptamer (Apt) is a novel recognition molecule, is a type of oligomeric DNA or RNA molecule obtained by in vitro screening by using a novel combination chemistry technology-systematic evolution of ligands by exponential enrichment (SELEX), has a special and stable three-dimensional structure, can be combined with different target molecules in a high-affinity and high-specificity manner through spatial configuration, is similar to the combination of antibodies and antigens, and is a special chemical antibody. Compared with the traditional protein antibody, the Apt has the advantages of high affinity, strong specificity, flexible screening conditions, wide target range, low cost, small molecular weight, easy synthesis and modification, low immunogenicity, good stability and the like.
In the related time, although the detection sensitivity is greatly improved based on the circulatory identification of the Apt, DNase I and the nano material to the target spot, the detection of the ultra-low concentration protein and miRNA in the whole blood is difficult to meet. The invention realizes the detection of ultra-low concentration protein and miRNA in whole blood through reasonable collocation and optimization.
In some embodiments of the invention, the nucleic acid aptamer comprises at least one of Apt-CEA, apt-AFP, and Apt-miR 21.
Serum Alpha Fetoprotein (AFP) is a currently accepted biomarker for HCC, with 30-40% of HCC patients having low abundance of AFP, resulting in insensitivity and non-specificity for early diagnosis of HCC; carcinoembryonic antigen (CEA) is a broad spectrum tumor marker that is elevated in the serum of a variety of malignant tumors. CEA, although not being a specific index for diagnosing a certain malignant tumor, has important clinical value in the aspects of differential diagnosis, disease monitoring, curative effect evaluation and the like of malignant tumors; micro-RNA (miRNA) is a non-coding RNA containing 18-23 nucleotides, plays an important role in tumorigenesis and metastasis, and is a new marker for predicting, diagnosing and monitoring cancer treatment. The detection method based on DNase I/Apt/rGO combined multi-channel microfluidic chip can be used for simultaneously detecting AFP, CEA and miR 21 in serum, can provide accurate judgment basis for clinical diagnosis of HCC patients, realizes early treatment and improves survival rate of the patients.
In some embodiments of the present invention, the nucleotide sequence information of the Apt-CEA is shown in SEQ ID NO. 1.
In some embodiments of the invention, the nucleotide sequence information of the Apt-AFP is shown in SEQ ID NO. 2.
In some embodiments of the invention, the nucleotide sequence information of the Apt-miR 21 is shown in SEQ ID NO. 3.
In some embodiments of the invention, the fluorescent group is selected from one of FAM, HEX, CY 5. Preferably, the fluorescent group is FAM.
In some embodiments of the invention, the fluorescent moiety is located at the 5' end of the nucleic acid aptamer.
In some embodiments of the invention, the concentration of the fluorophore-containing aptamer is 5 to 50nM.
In some preferred embodiments of the invention, the concentration of the fluorophore-containing aptamer is 5 to 30nM.
In some more preferred embodiments of the invention, the concentration of the fluorophore-containing nucleic acid aptamer is 5 to 15nM.
In some embodiments of the invention, the buffer further comprises 15-25 mM Tris, 80-120 mM NaCl, 3-8 mM MgCl 2 And 0.5 to 1.5mM CaCl 2 。
In some embodiments of the invention, the dnase comprises dnase I.
Deoxyribonuclease I (DNase I) cleaves either single-or double-stranded DNA at any site in the presence of magnesium ion, but does not cleave rGO-adsorbed ssDNA.
In some embodiments of the invention, the final concentration of the DNase is 10-30U/mL.
In some embodiments of the invention, the incubation time is 10 to 30 minutes.
In some embodiments of the invention, the reaction time is 10 to 120 minutes.
In some preferred embodiments of the invention, the reaction time is 30 to 120 minutes.
In some more preferred embodiments of the invention, the reaction time is 40 to 90 minutes.
In some embodiments of the invention, the reaction further comprises terminating the enzymatic reaction.
In some embodiments of the invention, the conditions for terminating the enzymatic reaction are heating at 70-80 ℃ for 10-20 min.
In some embodiments of the invention, the rotational speed of the centrifugation is 12000-13500 r/min.
In some embodiments of the invention, the centrifugation time is from 5 to 15 minutes.
In some embodiments of the invention, the microfluidic chip comprises 1 to 4 parallel microchannels.
In some preferred embodiments of the invention, the microfluidic chip comprises 3 parallel microchannels.
In some embodiments of the invention, the parallel microchannels are each fed with a target analyte degradation supernatant of a different sample to be tested.
In some embodiments of the invention, the width of the microchannel is from 100 to 700 μm.
In some preferred embodiments of the invention, the width of the microchannel is 200 to 600 μm.
In some more preferred embodiments of the invention, the width of the microchannel is 300 to 600 μm.
In some embodiments of the invention, the height of the micro-channels is 20 to 200 μm.
In some preferred embodiments of the invention, the height of the microchannels is 40 to 200 μm.
In some more preferred embodiments of the invention, the height of the microchannels is 40 to 100 μm.
In some embodiments of the invention, the microchannel is flanked by buffer channels.
In some embodiments of the present invention, the method for preparing a microfluidic chip includes:
s11, placing trimethyl chlorosilane and a silicon wafer template in a vacuum pump, and performing silanization treatment;
s12, repeatedly cleaning with isopropanol and water, and then drying with nitrogen to obtain a silicon wafer template after silanization treatment;
s13, mixing PDMS with a curing agent, pouring the mixture on the silicon wafer template subjected to silanization treatment, curing, and assembling the mixture with a slide glass with a Nafion film patterned and fixed after punching.
In some embodiments of the invention, the volume ratio of PDMS to curing agent is 8 to 12:1.
in some embodiments of the invention, the temperature of the curing is 90 to 100 ℃ and the time of the curing is 2 to 4 hours.
In some embodiments of the invention, the Nafion membrane has a width of 300 to 600 μm and a depth of 40 to 200 μm;
preferably, the Nafion film has a width of 300-500 μm and a depth of 40-100 μm;
more preferably, the Nafion membrane has a width of 400 μm and a depth of 45 μm.
In some embodiments of the present invention, the detecting the supernatant using a microfluidic chip specifically includes: firstly, modifying the micro-fluidic chip micro-channel by bovine serum albumin, and then, after containing CH 3 Detection in PBS buffer of CN.
In some embodiments of the invention, the PBS buffer is CH 3 The concentration of CN is 1-10% (v/v).
In some preferred embodiments of the invention, the PBS buffer is CH 3 The concentration of CN is 2-6% (v/v).
In some embodiments of the invention, the PBS buffer has a pH of 7.2 to 7.6.
In some embodiments of the invention, the PBS buffer is a1 x PBS buffer or a 1.5 x PBS buffer or a 2 x PBS buffer.
In some preferred embodiments of the invention, the PBS buffer is a1 XPBS buffer.
In some embodiments of the invention, the detected voltage is 25-35V.
In some embodiments of the invention, the detected current is a direct current.
In some embodiments of the invention, the detection method is used for non-disease diagnostic purposes.
The second aspect of the invention provides an application of the detection method based on the DNase I/Apt/rGO combined multi-channel microfluidic chip in the first aspect in biomarker detection.
In some embodiments of the invention, the biomarker comprises a liver cancer biomarker.
In some embodiments of the invention, the biomarker comprises at least one of CEA, AFP, and miR 21.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The invention is further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a schematic diagram of detection of a FAM-Apt/rGO/DNase I combined microfluidic chip, wherein A is the principle of amplification of FAM-Apt/rGO/DNase I signals; b is a workflow for simultaneous detection of multiple biomarkers (CEA, AFP, miRA-21), F 0 : blank value, FI: fluorescence intensity induced by the target.
Fig. 2 is a schematic diagram of a multichannel microfluidic chip according to the present invention.
FIG. 3 shows the effect of the concentration of Dnase I and the incubation time on the fluorescence intensity according to the present invention, wherein A is the effect of the concentration of Dnase I on the fluorescence intensity, B is the effect of the incubation time of Dnase I on the fluorescence intensity, F 0 : blank value, FI: fluorescence intensity induced by the target.
FIG. 4 shows the results of the detection of the effect of rGO concentration on the reaction efficiency, F 0 : blank value, FI: target-induced fluorescence intensity。
FIG. 5 shows the effect of the run buffer PBS and Nafion membrane on the enrichment effect, wherein A is the effect of the concentration of the run buffer PBS on the enrichment effect; b is the influence of adding CH3CN with different concentrations into PBS on enrichment effect; c is the influence of different Nafion film widths on the enrichment effect, and D is the influence of different Nafion film thicknesses on the enrichment effect. F: fluorescence intensity induced by target, F 0 : background signal.
FIG. 6 shows the feasibility verification of the detection of biomarkers according to the invention, wherein A and B are respectively corresponding fluorescent bands and FI values under different conditions, a: rGO+FAM-Apt; rGO+FAM-Apt+DNase I; rGO+FAM-Apt+CEA100 pg/mL; rGO+FAM-Apt+CEA100pg/mL+DNase I, FI: fluorescence intensity induced by the target.
FIG. 7 shows the results of the present invention for simultaneous detection of fluorescence bands and FI values induced by the high, medium, and low concentration targets (CEA, AFP, miR-21), where A is the result of the fluorescence band and B is the result of the FI value, F: fluorescence intensity induced by target, F 0 : background signal.
FIG. 8 shows the result of repeatability verification of the detection method based on DNase I/Apt/rGO combined multi-channel microfluidic chip.
FIG. 9 shows the result of specificity verification based on DNase I/Apt/rGO combined multi-channel microfluidic chip detection method of the present invention, wherein A is the specificity of CEA detection, and B is the specificity of AFP detection; c is the specificity of detecting miR-21, FI: fluorescence intensity induced by the target.
FIG. 10 shows fluorescence bands and corresponding calibration curves for different target standard solutions of the present invention, wherein A is CEA detection and B is AFP detection; c is miR-21 detection, F: fluorescence intensity induced by target, F 0 : background signal.
FIG. 11 shows the accuracy detection result of the DNase I/Apt/rGO-based multi-channel micro-fluidic chip detection method.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
When a range of values is disclosed herein, the range is considered to be continuous and includes both the minimum and maximum values for the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range description features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to include any and all subranges subsumed therein.
In the description of the present invention, the descriptions of the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
In the description of the present invention, the culture buffer comprises 20mM Tris, 100mM NaCl, 5mM MgCl 2 And 1mM CaCl 2 ，pH 7.6。
In the description of the present invention, all the fluorescence bar charts were analyzed using Image J, fluorescence values were calculated, the resulting fluorescence values were analyzed using GraphPad Prism 8 pair, and the data were expressed as mean ± SD.
The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Detection principle based on DNase I/Apt/rGO combined multichannel microfluidic chip
The detection principle schematic diagram based on DNase I/Apt/rGO combined multi-channel microfluidic chip provided by the invention is shown in figure 1, is based on a dual signal amplification strategy and mainly comprises the following steps:
(1) Taking the detection of lung cell cancer markers as an example, FAM-Apt, DNase I and rGO are realized in the primary signal amplification stage, and FAM-Apt of different targets including CEA, AFP and miR-21 is rapidly adsorbed by rGO through hydrophobic and pi-pi stacking effects, so that significant fluorescence quenching is caused, and in this case, DNase I degradation is limited due to the existence of steric hindrance.
(2) When the target occurs, FAM-Apt on the rGO surface is recognized, and a target-FAM-Apt complex is formed. Subsequently, the complex is released from rGO into solution, resulting in its FAM-Apt fluorescence recovery. In the absence of rGO protection, FAM-Apt is degraded in solution by DNase I into oligonucleotide fragments, releasing the target, the released target binds to another FAM-Apt on the rGO surface, and the FAM molecule remains in solution.
(3) The accumulation of free FAM molecules eventually results from the cyclic dissociation of FAM-Apts on the rGO surface in homogeneous solution. In contrast, unreleased FAM-Apt was retained on the rGO surface and removed by centrifugation. The supernatant containing FAM molecules is then added to the reservoir of the multichannel chip for further enrichment to form secondary signal amplification. Under the application of an electric field, a quantifiable fluorescent band is formed.
In this example, the detection of a marker for lung cell cancer is taken as an example, and the sequence information related thereto is shown in table 1.
Table 1: the invention relates to a sequence information table
(II) preparation of multichannel microfluidic chip
The structure of the multichannel microfluidic chip is shown in figure 2, the chip is provided with three parallel microchannels, each of which is 400 mu m wide and 45 mu m high, each of which is provided with a liquid storage tank, different targets can be detected respectively, and each of which is provided with a single inlet, 3 channels share an outlet, and when lung cell cancer markers are detected, the three inlets on the chip are filled with DNase I degradation supernatant of CEA, AFP and miR-21 respectively. Buffer solution channels are arranged on two sides of the three channels, and after a template with micro channels is prepared on a silicon wafer by using SU-8 photoresist and a standard wet etching process, a PDMS micro-channel chip is prepared by adopting a molding method.
The preparation method of the multichannel microfluidic chip comprises the following steps:
(1) The PDMS gel was easily removed by placing 25mL of trimethylchlorosilane solution with the silicon template in a vacuum pump, silanizing for 1 hour to prevent adhesion.
(2) Repeatedly cleaning the silanized silicon wafer template with isopropanol and ultrapure water, and drying by blowing nitrogen.
(3) And fully mixing PDMS and a curing agent in a volume ratio of 10:1, degassing in a vacuum pump until gas disappears, slowly pouring the mixture on a silicon wafer, curing for 3 hours at 95 ℃, and then stripping the PDMS from the silicon wafer to transfer the microstructure on the silicon wafer to the PDMS with elasticity.
(4) Nafion film was fixed on a glass slide by patterning, the Nafion film having a width of 400 μm and a depth of 45. Mu.m.
(5) Punching holes (with the diameter of 1.5 mm) at the sample inlet and the sample outlet, then placing the PDMS chip and the Nafion film patterning fixed glass slide into a plasma cleaning machine for surface modification treatment, and irreversibly bonding the PDMS chip onto the glass slide, wherein the chip channel and the Nafion film are mutually perpendicular to each other.
(III) optimization based on microfluidic chip detection method
1. DNase I concentration and incubation time optimization
In order to obtain the best detection sensitivity, the concentration of DNase I and the incubation time were optimized in this section, and the selection of (F-F in this section 0 ) The specific optimization method using the/F ratio as the sensitivity index is as follows:
(1) CEA (100 pg/mL) was added to culture buffer (containing 20mM Tris, 100) containing the corresponding FAM-Apt (10 nM) and rGO (20. Mu.g/mL)mM NaCl、5mM MgCl 2 、1mM CaCl 2 pH 7.6), incubated at 37℃for 20min.
(2) DNase I (1U/mL, 2U/mL, 5U/mL, 10U/mL, 20U/mL, and 30U/mL) was added to the above mixture at different final concentrations, respectively, and the final volume of the mixture was 100. Mu.L. After the mixed solution is incubated at 37 ℃ for 5 min-1.5 h, the enzymatic reaction is stopped by heating at 75 ℃ for 15min.
(3) rGO was removed by centrifugation at 13000r/min for 10min and the supernatant was used for chip detection. Before detection, the passivated microchannels were modified with 1% Bovine Serum Albumin (BSA) for 10min to prevent non-specific adhesion, after which the channels were rinsed three times with ultrapure water and filled with 1 x PBS buffer for use. A10. Mu.L pipette tip with the tip cut was inserted into the chip well as a reservoir, and then the electrode was inserted into the reservoir and connected to a DC power supply. In all experiments a dc voltage of 30V was applied.
The results of the assay are shown as A and B in FIG. 3, which show that increasing DNase I concentration and extending degradation time favors the cyclic dissociation of FAM-Apts. As DNase I concentration increased to 20U/mL, (F-F0)/F0 was gradually increased, then decreased at higher concentrations. This is because high concentrations of DNase I replace FAM-Apts on rGO surfaces, resulting in higher background signals. The (F-F0)/F0 value increased with increasing DNase I degradation time, reaching plateau after 60min incubation. Thus, 20U/mL DNase I incubation for 1h was optimally selected as the optimal condition.
2. rGO concentration optimization
In order to obtain the best detection sensitivity, the concentration of rGO is optimized in this section, and the concentration of rGO is selected from the optimization of this section (F-F 0 ) The specific optimization method using the/F ratio as the sensitivity index is as follows:
(1) CEA (100 pg/mL) was added separately to culture buffers (containing 20mM Tris, 100mM NaCl, 5mM MgCl) containing the corresponding FAM-Apt (10 nM) and varying concentrations of rGO (5. Mu.g/mL, 10. Mu.g/mL, 15. Mu.g/mL, 20. Mu.g/mL, 30. Mu.g/mL) 2 、1mM CaCl 2 pH 7.6), incubated at 37℃for 20min.
(2) DNase I (20U/mL) was added to each of the above mixtures, and the final volume of the mixture was 100. Mu.L. After incubation of the mixed solution at 37℃for 1h, the enzymatic reaction was stopped by heating at 75℃for 15min.
(3) rGO was removed by centrifugation at 13000r/min for 10min and the supernatant was used for chip detection. Before detection, the passivated microchannels were modified with 1% Bovine Serum Albumin (BSA) for 10min to prevent non-specific adhesion, after which the channels were rinsed three times with ultrapure water and filled with 1 x PBS buffer for use. A10. Mu.L pipette tip with the tip cut was inserted into the chip well as a reservoir, and then the electrode was inserted into the reservoir and connected to a DC power supply. In all experiments a dc voltage of 30V was applied.
As shown in FIG. 4, rGO concentration affects background and binding efficiency of target to Apt, (F-F0)/F0 reaches maximum at rGO concentration of 20. Mu.g/mL. Thus, 20. Mu.g/mLrGO was optimally chosen as the optimal condition.
3. Buffer PBS concentration optimization
In order to obtain the best detection sensitivity, the run buffer PBS concentration was optimized in this section, and the concentration of the sample buffer PBS was selected in this section (F-F 0 ) The specific optimization method using the/F ratio as the sensitivity index is as follows:
(1) CEA (100 pg/mL) was added to culture buffers (containing 20mM Tris, 100mM NaCl, 5mM MgCl) containing the corresponding FAM-Apt (10 nM) and rGO (20. Mu.g/mL), respectively 2 、1mM CaCl 2 pH 7.6), incubated at 37℃for 20min.
(2) DNase I (20U/mL) was added to each of the above mixtures, and the final volume of the mixture was 100. Mu.L. After incubation of the mixed solution at 37℃for 1h, the enzymatic reaction was stopped by heating at 75℃for 15min.
(3) rGO was removed by centrifugation at 13000r/min for 10min and the supernatant was used for chip detection. Before detection, the passivated microchannels were modified with 1% Bovine Serum Albumin (BSA) for 10min to prevent non-specific adhesion, after which the channels were rinsed three times with ultra pure water and each with 4% (v/v) CH 3 Different concentrations of CN in PBS buffer (0.1×,0.5×,1×,1.5×,2×) were filled for use. A10. Mu.L pipette tip with the tip cut was inserted into the chip well as a reservoir, and then the electrode was inserted into the reservoir and connected to a DC power supply. In all experiments a dc voltage of 30V was applied.
The results are shown as a in fig. 5, which shows that the enrichment efficiency increases with increasing concentration of PBS, and that the enrichment efficiency decreases when 1 x PBS reaches a maximum value and exceeds 1 x PBS. This is because too high or too low an ionic strength of the buffer is detrimental to sample enrichment, so 1 XPBS was chosen for further investigation.
4. Optimization of CH in buffer PBS 3 Optimization of CN concentration
CH3CN is often used as an additive for on-line sample pre-concentration, allowing rapid accumulation of nucleic acids by transient pseudo isotachophoresis, and in order to obtain optimal detection sensitivity, the concentration of CH3CN in the run buffer PBS is optimized, and the concentration of CH3CN in this part is selected (F-F 0 ) The specific optimization method using the/F ratio as the sensitivity index is as follows:
(1) CEA (100 pg/mL) was added to culture buffer (containing 20mM Tris, 100mM NaCl, 5mM MgCl) containing the corresponding FAM-Apt (10 nM) and rGO (20. Mu.g/mL) 2 、1mM CaCl 2 pH 7.6), incubated at 37℃for 20min.
(2) DNase I (20U/mL) was added to each of the above mixtures, and the final volume of the mixture was 100. Mu.L. After incubation of the mixed solution at 37℃for 1h, the enzymatic reaction was stopped by heating at 75℃for 15min.
(3) rGO was removed by centrifugation at 13000r/min for 10min and the supernatant was used for chip detection. Before detection, the passivated microchannels were modified with 1% Bovine Serum Albumin (BSA) for 10min to prevent non-specific adhesion, after which the channels were washed three times with ultra pure water and with different concentrations (0%, 1%, 2%, 4%, 6%, 8%, V/V) of CH, respectively 3 The CN 1 XPBS buffer was filled for use. A10. Mu.L pipette tip with the tip cut was inserted into the chip well as a reservoir, and then the electrode was inserted into the reservoir and connected to a DC power supply. In all experiments a dc voltage of 30V was applied. The optimal CH3CN concentration was determined by comparing the fluorescence intensities of the fluorescent bands on the chip.
As shown in B in FIG. 5, it was revealed that the enrichment efficiency of the microfluidic chip was significantly improved when the CH3CN concentration was 4% (v/v), thus selecting 4% (v/v) CH 3 CN as the optimal concentration.
5. Optimization of Nafion film width and thickness
Triggered by 100pg/mL CEAThe FAM molecules produced by DNase I cycles of (c) were used as the target for optimization validation. The enrichment effect of chips of different width Nafion membranes (100 μm,200 μm, 400 μm, 600 μm and 700 μm) and different thickness Nafion membranes (20 μm,45 μm,100 μm,200 μm) was examined. The buffer solution contains 1 XPBS, pH7.4,4% (V/V) CH 3 CN, dc voltage 30V.
The results, shown as C and D in fig. 5, show that the width and depth of the Nafion membrane have a significant effect on the sensitivity of detection. As the size of the Nafion film increases, the chip enrichment efficiency increases and then decreases, and when the width of the Nafion film is 400 mu m and the depth of the Nafion film is 45 mu m, the chip enrichment efficiency is highest, so that the width of the Nafion film is 400 mu m and the depth of the Nafion film is 45 mu m as the optimal detection conditions.
In summary, the optimization conditions are as follows: incubation for 1h with 20U/mL DNase I, 20. Mu.g/mLrGO, 4% (v/v) CH 3 CN 1 XPBS (pH 7.4), nafion membrane width 400 μm,45 μm deep.
(IV) feasibility verification based on DNase I/Apt/rGO combined multichannel microfluidic chip detection method
1. Feasibility verification of DNase I cycle dissociation and degradation of FAM-Apt
The part adopts the optimized conditions to verify the feasibility of DNase I cycle dissociation and FAM-Apt degradation, and the specific method is as follows:
and (3) respectively designing four groups of experiments by adopting the microfluidic chip detection method after the optimization of the third part, wherein the group a is rGO+FAM-Apt (without adding targets CEA and DNase I), the group b is rGO+FAM-Apt+DNase I, the group c is rGO+FAM-Apt+CEA100pg/mL, and the group d is rGO+FAM-Apt+CEA100pg/mL+DNase I.
The detection results are shown as A and B in FIG. 6, and show that in the absence of targets, fluorescence bands a and B are negligible, indicating that FAM-Apts is adsorbed on the rGO surface and prevents DNase I degradation. As expected, in the presence of target (CEA as model analyte), a fluorescent band (as indicated by band c) appeared, and when target and DNase I were present at the same time, a prominent fluorescent band (as indicated by band d) was detected, indicating the feasibility of DNase I cycling to dissociate and degrade FAM-Apt.
2. Feasibility verification for detecting CEA, AFP and miR-21
The feasibility of simultaneously detecting CEA, AFP and miR-21 is verified by adopting the optimized conditions, and the specific method is as follows:
and (3) adopting the microfluidic chip detection method after the optimization of the third part, respectively adding a high-concentration target (1000 pg/mL CEA, 2000pg/mL AFP, 5000fg/mL miR-21), a medium-concentration target (100 pg/mL CEA, 500pg/mL AFP, 500fg/mL miR-21) and a low-concentration target (50 pg/mL CEA, 150pg/mL AFP, 50fg/mL miR-21) in the step (1), and judging the feasibility of simultaneously detecting CEA, AFP and miR-21 through fluorescence intensity.
The results of the assay are shown in FIG. 7, which shows that these 3 different concentrations of targets induced F-F 0 The regularity of the values was enhanced, indicating that it is feasible to detect CEA, AFP and miR-21 simultaneously on the chip.
3. Repeatability detection
The repeatability of the simultaneous detection of CEA, AFP and miR-21 is detected by adopting the optimized conditions, and the specific method is as follows:
and (3) adopting the microfluidic chip detection method after the optimization of the third part, respectively adding the targets (100 pg/mL CEA, 300pg/mL AFP and 100fg/mL miR-21) with the same concentration in the step (1) for repeated detection, and analyzing the repeatability of CEA, AFP and miR-21 in the day (n=6) and in the daytime (n=15) of F-F0 values in 4 days.
The results of the assay are shown in fig. 8, showing that the Relative Standard Deviation (RSD) within CEA day (n=6) and daytime (n=15) is 3.6% and 5.1%, AFP is 5.2% and 5.4%, and miR-21 is 3.5% and 4.4%, respectively, indicating that the method has high reproducibility (precision).
4. Specific detection
The specificity of CEA, AFP and miR-21 is detected by adopting the optimized conditions, and the specific method is as follows:
adopting the microfluidic chip detection method after the optimization of the third part, respectively adding different targets (100 pg/mL CEA, 300pg/mL AFP and 100fg/mL miR-21) in the step (1), and taking HSA, AFP, CRP, IL-6 and Her-2 as interfering molecules for detecting CEA under the same detection condition; HSA, CEA, CRP, IL-6 and Her-2 are used as interfering molecules for detecting AFP; miR-141, miR-375, single base mismatch (mis-1) and a random RNA sequence are used as interfering molecules of miR-21. FI values measured for 3 targets are compared with FI values of common interfering molecules, and the specificity of the method is verified.
As shown in FIG. 9, the specific detection results show that the FI values of CEA (100 pg/mL), AFP (300 pg/mL) and miR-21 (100 fM) are significantly higher than those of the interferents. The FI values of the interfering substances are lower than blank +3SD (n=11), which shows that the method has higher selectivity, and targets (CEA, AFP and miR-21) and F-F with different concentrations (x) 0 (y) have a good linear relationship between them.
5. Accuracy detection
The accuracy of CEA, AFP and miR-21 is detected by adopting the optimized conditions, and the specific method is as follows:
and (3) preparing CEA (100 mug/mL), AFP (100 mug/mL) and miR-21 (100 mug) standard stock solutions in deionized water by adopting a microfluidic chip detection method after the optimization of the third part. The stock solution was diluted appropriately with deionized water to prepare standard solutions of different concentrations. CEA, AFP and miR-21 standard solutions of different concentrations were added to incubation buffers (20 mM Tris, 100mM NaCl, 5mM MgCl) containing the corresponding FAM-apt (10 nM) and rGO (20. Mu.g/mL), respectively 2 、1mM CaCl 2 pH 7.6), incubation was carried out at 37℃for 20min, followed by addition of DNase I (20U/mL) to the above mixture. After incubation of the mixed solution for 1h at 37℃the mixture was heated for 15min at 75℃and finally centrifuged at 13000r/min for 10min to remove rGO. The chip is used for detecting the fluorescence of the supernatant induced by targets with different concentrations.
The calibration curve results are shown in fig. 10 and table 2.
Table 2: CEA, AFP, miR-21 regression equation and related parameters
The results show that the relative coefficients (r) of CEA, AFP and miR-21 are 0.9945, 0.9981 and 0.9937, respectively, indicating good linear correlation. The detection limits (S/n=3) for CEA, AFP and miR-21 were 4.5pg/mL, 37.0pg/mL and 1.3fM, respectively.
Further, the accuracy of the evaluation method is recovered by labeling. 3 different target concentrations (CEA: 20pg/mL, 250pg/mL, 850pg/mL, AFP:60pg/mL, 600pg/mL, 1800pg/mL, miR-21:10fM, 250fM, 2500 fM) were added to 1% serum samples, respectively, and their recovery rates were analyzed. The serum sample was prepared by taking 1ml of whole blood without anticoagulant from the elbow vein of volunteer, standing at room temperature for 2 hours, and centrifuging at 3000rpm for 15 minutes, and collecting the supernatant serum for storage at-20deg.C. The results are shown in FIG. 11 and Table 3.
Table 3: labeling results of CEA, AFP and miRNA-21 in human serum
The results show that the average recovery rates of CEA, AFP and miR-21 are 106.5%, 108.2% and 109.4%, respectively, which indicate that the detection method has excellent accuracy.
In summary, the invention provides a DNase I/Apt/rGO-based multi-channel micro-fluidic chip detection method and application. According to the method, a novel strategy for simultaneously detecting multiple biomarkers is developed through the combination of FAM-Apt/rGO/DNase I and a multi-pass microfluidic enrichment chip. The method has high efficiency, and only 30 minutes are needed for detecting the protein and the nucleic acid simultaneously on the same chip. Under the dual signal amplification strategy, the detection Limits (LOD) of AFP, CEA and miR-21 are respectively 37.0pg/mL, 4.5pg/mL and 1.3fM, so that the detection of serum low-concentration markers of HCC patients can be satisfied.
While the embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (10)
1. The DNase I/Apt/rGO-based combined multichannel microfluidic chip detection method is characterized by comprising the following steps of:
s1, adding a sample to be detected into a buffer solution containing fluorogenic group-containing nucleic acid aptamer and biological reduction graphene oxide with a final concentration of 15-30 mug/mL for incubation, then adding deoxyribonuclease with a final concentration of 5-40U/mL for reaction, centrifuging, and collecting supernatant;
s2, detecting the supernatant by adopting a microfluidic chip, and quantifying the concentration of a target detection object in the sample to be detected according to the fluorescent signal intensity;
the detection method is used for non-disease diagnostic purposes.
2. The detection method according to claim 1, wherein the nucleic acid aptamer comprises a nucleic acid aptamer for detecting a protein and/or a nucleic acid aptamer for detecting a nucleic acid;
preferably, the nucleic acid aptamer comprises at least one of Apt-CEA, apt-AFP and Apt-miR 21;
preferably, the nucleotide sequence information of the Apt-CEA is shown in SEQ ID NO. 1;
preferably, the nucleotide sequence information of the Apt-AFP is shown as SEQ ID NO. 2;
preferably, the nucleotide sequence information of the Apt-miR 21 is shown in SEQ ID NO. 3.
3. The method according to claim 1, wherein the concentration of the fluorescent group-containing aptamer is 5 to 50nM.
4. The method according to claim 1, wherein the buffer solution further comprises 15-25 mM Tris, 80-120 mM NaCl, 3-8 mM MgCl 2 And 0.5 to 1.5mM CaCl 2 。
5. The method of claim 1, wherein the dnase comprises dnase I.
6. The method according to claim 1, wherein the incubation time is 10 to 30min;
preferably, the reaction time is 10-120 min;
preferably, the rotational speed of the centrifugation is 12000-13500 r/min;
preferably, the centrifugation time is 5 to 15min.
7. The method according to claim 1, wherein the microfluidic chip comprises 1 to 4 parallel microchannels;
preferably, the width of the micro-channel is 100-700 μm, and the height is 20-200 μm;
preferably, buffer channels are arranged on two sides of the micro-channel.
8. The method according to claim 1, wherein the detecting the supernatant using a microfluidic chip specifically comprises: firstly, modifying the micro-fluidic chip micro-channel by bovine serum albumin, and then, after containing CH 3 Detecting in PBS buffer solution of CN;
preferably, the CH in the PBS buffer 3 The concentration of CN is 1-10% (v/v);
preferably, the pH value of the PBS buffer solution is 7.2-7.6;
preferably, the detected voltage is 25-35V.
9. The use of a detection method based on DNase I/Apt/rGO combined multichannel microfluidic chip according to any one of claims 1 to 8 for the detection of biomarkers.
10. The use of claim 9, wherein the biomarker comprises a liver cancer biomarker.
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