CN117554344A - DNase I/Apt/rGO-based combined multichannel microfluidic chip detection method and application - Google Patents

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

A detection method based on DNase I/Apt/rGO combined with multi-channel microfluidic chip and its application

Technical Field

The present invention relates to the field of molecular detection, and in particular to a detection method and application based on DNase I/Apt/rGO combined multi-channel microfluidic chip.

Background Art

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world, and its morbidity and mortality have been on the rise in the past 10 years. The early diagnosis of HCC includes non-invasive imaging methods, invasive puncture biopsy and pathological testing, and detection methods of serological tumor markers such as AFP. At present, tissue biopsy and pathological examination are the gold standard for diagnosing HCC. Although this method is highly accurate, it is not easy for patients to accept it because of its trauma. Imaging methods require large instruments, so they are difficult to popularize in resource-deficient areas. Compared with traditional tumor tissue detection technology, liquid biopsy has the advantages of non-invasive examination, easy operation, and easy specimen acquisition. It has become a new star in tumor detection and is used for early diagnosis of tumors.

In the related technologies, serum alpha-fetoprotein AFP is currently the most commonly used serum tumor marker for liver cancer screening and diagnosis, and has been widely used in clinical practice. However, due to the insufficient sensitivity and specificity of monitoring tools, most liver cancer patients are not detected in the early stages, which highlights the need for more accurate biomarkers to improve the early diagnosis of liver cancer. At present, many methods for detecting liver cancer biomarkers have been developed, such as enzyme-linked immunosorbent assay, electrochemical method, fluorescence method, surface-enhanced Raman spectroscopy, PCR, loop-mediated isothermal amplification, etc. However, these methods have low sensitivity and can only detect a single protein biomarker or nucleic acid biomarker, and cannot achieve simultaneous detection of protein and nucleic acid biomarkers.

Therefore, there is an urgent need to establish a highly specific and sensitive detection method to provide an accurate basis for the clinical diagnosis of HCC patients, to initiate treatment as early as possible, and to improve the survival rate of patients.

Summary of the invention

The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a detection method based on DNase I/Apt/rGO combined with a multi-channel microfluidic chip, which is based on a dual signal amplification strategy and can simultaneously detect the contents of multiple target substances in the sample to be tested. The detection limit for protein target substances can reach 4.5pg/mL, and the detection limit for nucleic acid target substances can reach 1.3fM, which can be used for the detection of low-concentration serum markers.

The present invention also proposes an application of the above-mentioned detection method based on DNase I/Apt/rGO combined with a multi-channel microfluidic chip in detecting biomarkers.

In a first aspect of the present invention, a detection method based on DNase I/Apt/rGO combined with a multi-channel microfluidic chip is provided, comprising the following steps:

S1. Add the sample to be tested to a nucleic acid aptamer containing a fluorescent group and a buffer solution with a final concentration of 15 to 30 μg/mL of bioreduced graphene oxide, and then add a deoxyribonuclease reaction with a final concentration of 5 to 40 U/mL, centrifuge, and collect the supernatant;

S2, using a microfluidic chip to detect the supernatant, and then quantifying the concentration of the target detection substance in the sample to be tested according to the intensity of the fluorescence signal;

The detection method is used for non-disease diagnosis purposes.

The detection method according to the embodiment of the present invention has at least the following beneficial effects:

(1) The method of the present invention develops an efficient detection method based on a dual signal amplification strategy based on DNase I/Apt/rGO combined with a multi-channel microfluidic enrichment chip, which can simultaneously detect multiple biomarkers, and it only takes 30 minutes to detect proteins and nucleic acids on the same chip. In addition, taking the detection of hepatocellular carcinoma biomarkers as an example, using the detection method of the present invention, the detection limits (LOD) of AFP, CEA and miR-21 reached 37.0pg/mL, 4.5pg/mL and 1.3fM, respectively, which can meet the detection of low-concentration serum markers in HCC patients.

(2) Reduced graphene oxide is used in the detection method of the present invention. Graphene oxide (GO) is generally considered to be an ideal biocompatible nanomaterial with extremely strong distance-dependent fluorescence quenching ability. GO adsorbs single-stranded DNA (ssDNA) through π-π electron stacking interaction, which can not only quench the fluorescence of FAM-labeled DNA, but also protect the stability of single-stranded DNA adsorbed on its surface and prevent enzyme cleavage. However, compared with GO, reduced graphene oxide (rGO) has better properties of quenching single-stranded fluorescent DNA.

In some embodiments of the present invention, the sample to be tested includes whole blood or serum.

In some embodiments of the present invention, the nucleic acid aptamer includes a nucleic acid aptamer for detecting a protein and/or a nucleic acid aptamer for detecting a nucleic acid.

Nucleic acid aptamers (Apt) are a new type of recognition molecules. They are oligomeric DNA or RNA molecules obtained by in vitro screening using a new combinatorial chemistry technology - Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Apt has a special and stable three-dimensional structure, and can bind to different target molecules with high affinity and high specificity through spatial configuration. This binding mode is similar to the binding of antibodies and antigens, and is a special "chemical antibody". Compared with traditional protein antibodies, Apt has many advantages such as high affinity, strong specificity, flexible screening conditions, wide target range, low cost, small molecular weight, easy synthesis and modification, low immunogenicity and good stability.

In the related field, although the detection sensitivity is greatly improved based on the circular recognition of targets by Apt, DNase I and nanomaterials, it is still difficult to meet the detection of ultra-low concentration proteins and miRNAs in whole blood. The present invention realizes the detection of ultra-low concentration proteins and miRNAs in whole blood through reasonable matching and optimization.

In some embodiments of the present invention, the nucleic acid aptamer includes at least one of Apt-CEA, Apt-AFP and Apt-miR21.

Serum alpha-fetoprotein (AFP) is a currently recognized biomarker for HCC. 30-40% of HCC patients have low abundance of AFP, which leads to insensitivity and nonspecificity in the 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. Although CEA cannot be used as a specific indicator for diagnosing a certain malignant tumor, it still has important clinical value in the differential diagnosis, disease monitoring, and efficacy evaluation of malignant tumors. Micro-RNA (miRNA) is a class of non-coding RNA containing 18-23 nucleotides, which plays an important role in tumor occurrence and metastasis. It is a new biomarker for predicting, diagnosing and monitoring cancer treatment. The DNase I/Apt/rGO combined multi-channel microfluidic chip detection method of the present invention can simultaneously detect AFP, CEA and miR 21 in serum, which can provide an accurate judgment basis for the clinical diagnosis of HCC patients, achieve early treatment, and improve patient survival rate.

In some embodiments of the present invention, the nucleotide sequence information of Apt-CEA is shown as SEQ ID NO.1.

In some embodiments of the present invention, the nucleotide sequence information of the Apt-AFP is shown as SEQ ID NO.2.

In some embodiments of the present invention, the nucleotide sequence information of Apt-miR 21 is shown as SEQ ID NO.3.

In some embodiments of the present invention, the fluorescent group is selected from one of FAM, HEX, and CY5. Preferably, the fluorescent group is FAM.

In some embodiments of the present invention, the fluorescent group is located at the 5' end of the nucleic acid aptamer.

In some embodiments of the present invention, the concentration of the fluorescent group-containing nucleic acid aptamer is 5 to 50 nM.

In some preferred embodiments of the present invention, the concentration of the nucleic acid aptamer containing a fluorescent group is 5 to 30 nM.

In some more preferred embodiments of the present invention, the concentration of the nucleic acid aptamer containing a fluorescent group is 5 to 15 nM.

In some embodiments of the present invention, the buffer further comprises 15-25 mM Tris, 80-120 mM NaCl, 3-8 mM MgCl ₂ and 0.5-1.5 mM CaCl ₂ .

In some embodiments of the invention, the deoxyribonuclease comprises deoxyribonuclease I.

Deoxyribonuclease I (DNase I) can cleave any site of single-stranded or double-stranded DNA in the presence of magnesium ions, but cannot cleave ssDNA adsorbed by rGO.

In some embodiments of the present invention, the final concentration of the deoxyribonuclease is 10 to 30 U/mL.

In some embodiments of the present invention, the incubation time is 10 to 30 minutes.

In some embodiments of the present invention, the reaction time is 10 to 120 minutes.

In some preferred embodiments of the present invention, the reaction time is 30 to 120 minutes.

In some more preferred embodiments of the present invention, the reaction time is 40 to 90 minutes.

In some embodiments of the present invention, the reaction further comprises terminating the enzymatic reaction.

In some embodiments of the present invention, the condition for terminating the enzymatic reaction is heating at 70-80° C. for 10-20 min.

In some embodiments of the present invention, the centrifugal rotation speed is 12000-13500 r/min.

In some embodiments of the present invention, the centrifugation time is 5 to 15 minutes.

In some embodiments of the present invention, the microfluidic chip comprises 1 to 4 parallel microchannels.

In some preferred embodiments of the present invention, the microfluidic chip comprises three parallel microchannels.

In some embodiments of the present invention, the parallel microchannels are respectively introduced with target detection substance degradation supernatants of different samples to be detected.

In some embodiments of the present invention, the width of the microchannel is 100-700 μm.

In some preferred embodiments of the present invention, the width of the microchannel is 200-600 μm.

In some more preferred embodiments of the present invention, the width of the microchannel is 300-600 μm.

In some embodiments of the present invention, the height of the microchannel is 20-200 μm.

In some preferred embodiments of the present invention, the height of the microchannel is 40-200 μm.

In some more preferred embodiments of the present invention, the height of the microchannel is 40-100 μm.

In some embodiments of the present invention, both sides of the microchannel are buffer channels.

In some embodiments of the present invention, the method for preparing the microfluidic chip comprises:

S11, placing trimethylsilyl chloride and the silicon wafer template in a vacuum pump for silanization treatment;

S12, repeatedly washing with isopropanol and water, and then blowing and drying with nitrogen to obtain a silicon wafer template after silanization treatment;

S13, mixing PDMS with a curing agent, pouring the mixture onto the silanized silicon wafer template, curing the mixture, punching holes, and assembling the mixture with the glass slide fixed with a Nafion membrane pattern.

In some embodiments of the present invention, the volume ratio of the PDMS to the curing agent is 8 to 12:1.

In some embodiments of the present invention, the curing temperature is 90-100° C., and the curing time is 2-4 hours.

In some embodiments of the present invention, the width of the Nafion membrane is 300 to 600 μm and the depth is 40 to 200 μm;

Preferably, the width of the Nafion membrane is 300-500 μm and the depth is 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 detection of the supernatant using a microfluidic chip specifically comprises: firstly modifying the microchannel of the microfluidic chip with bovine serum albumin, and then detecting in a PBS buffer containing CH ₃ CN.

In some embodiments of the present invention, the concentration of CH ₃ CN in the PBS buffer is 1-10% (v/v).

In some preferred embodiments of the present invention, the concentration of CH ₃ CN in the PBS buffer is 2-6% (v/v).

In some embodiments of the present invention, the pH value of the PBS buffer is 7.2-7.6.

In some embodiments of the present invention, the PBS buffer is 1×PBS buffer or 1.5×PBS buffer or 2×PBS buffer.

In some preferred embodiments of the present invention, the PBS buffer is 1×PBS buffer.

In some embodiments of the present invention, the detected voltage is 25-35V.

In some embodiments of the present invention, the detected current is a direct current.

In some embodiments of the present invention, the detection method is used for non-disease diagnosis purposes.

The second aspect of the present invention provides the application of the detection method based on DNase I/Apt/rGO combined with a multi-channel microfluidic chip described in the first aspect in detecting biomarkers.

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.

Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

Figure 1 is a schematic diagram of FAM-Apt/rGO/DNase I combined microfluidic chip detection of the present invention, wherein A is the principle of FAM-Apt/rGO/DNase I signal amplification; B is the workflow of simultaneous detection of multiple biomarkers (CEA, AFP, miRA-21), _F0 : blank value, FI: target-induced fluorescence intensity.

FIG. 2 is a schematic diagram of a multi-channel microfluidic chip of the present invention.

FIG3 shows the effect of DNase I concentration and incubation time on fluorescence intensity of the present invention, wherein A is the effect of DNase I concentration on fluorescence intensity, B is the effect of DNase I incubation time on fluorescence intensity, F ₀ : blank value, FI: target-induced fluorescence intensity.

FIG. 4 is a test result of the effect of rGO concentration on reaction efficiency of the present invention, F ₀ : blank value, FI: target-induced fluorescence intensity.

Figure 5 shows the effects of the running buffer PBS and Nafion membrane on the enrichment effect of the present invention, wherein A is the effect of the running buffer PBS concentration on the enrichment effect; B is the effect of adding different concentrations of CH3CN to PBS on the enrichment effect; C is the effect of different Nafion membrane widths on the enrichment effect, and D is the effect of different Nafion membrane thicknesses on the enrichment effect. F: target-induced fluorescence intensity, F ₀ : background signal.

Figure 6 is a feasibility verification of the present invention for detecting biomarkers, where A and B are the corresponding fluorescence bands and FI values under different conditions, respectively, a: rGO+FAM-Apt; b: rGO+FAM-Apt+DNase I; c: rGO+FAM-Apt+CEA 100pg/mL; d: rGO+FAM-Apt+CEA 100pg/mL+DNase I, FI: target-induced fluorescence intensity.

7 shows the fluorescence bands and FI value results of the present invention for simultaneous detection of high, medium and low concentration targets (CEA, AFP, miR-21), wherein A is the fluorescence band result, B is the FI value result, F: target induced fluorescence intensity, F ₀ : background signal.

FIG8 is a result of repeatability verification of the detection method based on DNase I/Apt/rGO combined with multi-channel microfluidic chip of the present invention.

Figure 9 shows the specificity verification results of the detection method based on DNase I/Apt/rGO combined with multi-channel microfluidic chip of the present invention, wherein A is the specificity of detecting CEA, B is the specificity of detecting AFP; C is the specificity of detecting miR-21, FI: target-induced fluorescence intensity.

FIG. 10 shows the fluorescence bands of different target standard solutions of the present invention and the corresponding calibration curves, wherein A is for detecting CEA, B is for detecting AFP, C is for detecting miR-21, F: target-induced fluorescence intensity, F ₀ : background signal.

FIG. 11 is a result of the accuracy test of the detection method based on DNase I/Apt/rGO combined with multi-channel microfluidic chip of the present invention.

DETAILED DESCRIPTION

The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

In the description of the present invention, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

In the description of the present invention, the culture buffer contains 20 mM Tris, 100 mM NaCl, 5 mM MgCl ₂ and 1 mM CaCl ₂ , pH 7.6.

In the description of the present invention, all fluorescence band graphs were analyzed and processed using Image J to calculate the fluorescence values, and the obtained fluorescence values were analyzed and processed using GraphPad Prism 8, and the data were expressed as mean ± SD.

If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased from the market.

(I) Detection principle based on DNase I/Apt/rGO combined with multi-channel microfluidic chip

The schematic diagram of the detection principle of the DNase I/Apt/rGO combined multi-channel microfluidic chip provided by the present invention is shown in FIG1 , which is based on a dual signal amplification strategy and mainly includes the following processes:

(1) Taking the detection of lung cell carcinoma markers as an example, the primary signal amplification stage is achieved by FAM-Apt, DNase I and rGO. FAM-Apt of different targets including CEA, AFP and miR-21 is rapidly adsorbed by rGO through hydrophobic and π-π stacking effects, resulting in significant fluorescence quenching. At this time, the degradation of DNase I is limited due to the presence of steric hindrance.

(2) When the target appears, it recognizes the FAM-Apt on the rGO surface and forms a target-FAM-Apt complex. Subsequently, the complex is released from rGO into the solution, resulting in the recovery of its FAM-Apt fluorescence. In the absence of rGO protection, its FAM-Apt is degraded into oligonucleotide fragments by DNase I in the solution, releasing the target, and the released target binds to another FAM-Apt on the rGO surface, and the FAM molecule remains in the solution.

(3) The cyclic dissociation of FAM-Apts on the rGO surface in a homogenous solution eventually leads to the accumulation of free FAM molecules. In contrast, the unreleased FAM-Apt is 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. When an electric field is applied, a quantifiable fluorescent band is formed.

This embodiment takes the detection of lung cell carcinoma markers as an example, and the sequence information involved is shown in Table 1.

Table 1: Sequence information table of the present invention

(II) Preparation of multi-channel microfluidic chips

The structure of the multi-channel microfluidic chip of the present invention is shown in FIG2 . The chip has three parallel microchannels, each of which is 400 μm wide and 45 μm high. Each channel has a liquid reservoir, which can detect different targets respectively. In addition, each channel has a separate inlet, and the three channels share an outlet. When detecting lung cell carcinoma markers, the three inlets on the chip are filled with DNase I degradation supernatants of CEA, AFP and miR-21 respectively. The two sides of the three channels are buffer channels. After preparing a template with microchannels on a silicon wafer using SU-8 photoresist and a standard wet etching process, a PDMS microchannel chip is prepared by molding.

The method for preparing the multi-channel microfluidic chip of the present invention comprises the following steps:

(1) Place the silicon wafer template in a vacuum pump with 25 mL of trimethylsilyl chloride solution and silanize for 1 hour to prevent adhesion and make it easier to remove the PDMS glue.

(2) The silanized silicon wafer template was repeatedly cleaned with isopropanol and ultrapure water, and then blown dry with nitrogen.

(3) PDMS and curing agent were fully mixed in a volume ratio of 10:1, degassed in a vacuum pump until the gas disappeared, and then slowly poured onto a silicon wafer and cured at 95°C for 3 hours. The PDMS was then peeled off from the silicon wafer, so that the microstructure on the silicon wafer was transferred to the elastic PDMS.

(4) A Nafion membrane was fixed on a glass slide by patterning. The width of the Nafion membrane was 400 μm and the depth was 45 μm.

(5) Drill holes (1.5 mm in diameter) at the sample inlet and sample outlet, then place the glass slide with the PDMS chip and the Nafion membrane patterned and fixed in a plasma cleaner for surface modification, and then irreversibly bond the PDMS chip to the glass slide, with the chip channel and the Nafion membrane perpendicular to each other.

(III) Optimization of detection methods based on microfluidic chips

1. Optimization of DNase I concentration and incubation time

In order to obtain the best detection sensitivity, this section optimized the concentration and incubation time of DNase I. In this section, the (FF ₀ )/F ratio was selected as the sensitivity index. The specific optimization method is as follows:

(1) CEA (100 pg/mL) was added to a culture buffer (containing 20 mM Tris, 100 mM NaCl, 5 mM MgCl ₂ , 1 mM CaCl ₂ , pH 7.6) containing the corresponding FAM-Apt (10 nM) and rGO (20 μg/mL) and incubated at 37°C for 20 min.

(2) Different final concentrations of DNase I (1 U/mL, 2 U/mL, 5 U/mL, 10 U/mL, 20 U/mL and 30 U/mL) were added to the above mixture, and the final volume of the mixture was 100 μL. The mixed solution was incubated at 37°C for 5 min to 1.5 h, and then heated at 75°C for 15 min to terminate the enzymatic reaction.

(3) Centrifuge at 13000r/min for 10min to remove rGO, and take the supernatant for chip detection. Before detection, the microchannel was modified and passivated with 1% bovine serum albumin (BSA) for 10min to prevent nonspecific adhesion, and then the channel was rinsed three times with ultrapure water and filled with 1×PBS buffer for standby use. A 10μL pipette tip with the tip cut off was inserted into the chip hole as a reservoir, and then the electrode was inserted into the reservoir and connected to a DC power supply. A 30V DC voltage was applied in all experiments.

The test results are shown in A and B in Figure 3, indicating that increasing the DNase I concentration and prolonging the degradation time are beneficial to the cyclic dissociation of FAM-Apts. As the DNase I concentration increases to 20U/mL, (F-F0)/F0 gradually increases and then decreases at higher concentrations. This is because high concentrations of DNase I replace FAM-Apts on the rGO surface, resulting in a higher background signal. The (F-F0)/F0 value increases with the increase in DNase I degradation time and reaches a plateau after 60 minutes of incubation. Therefore, 20U/mL DNase I incubation for 1 hour was selected as the optimal condition after optimization.

2. rGO concentration optimization

In order to obtain the best detection sensitivity, the concentration of rGO was optimized in this section. In this optimization, the (FF ₀ )/F ratio was selected as the sensitivity index. The specific optimization method is as follows:

(1) CEA (100 pg/mL) was added to culture buffer (containing 20 mM Tris, 100 mM NaCl, 5 mM MgCl ₂ , 1 mM CaCl ₂ , pH 7.6) containing the corresponding FAM-Apt (10 nM) and different concentrations of rGO (5 μg/mL, 10 μg/mL, 15 μg/mL, 20 μg/mL, 30 μg/mL) and incubated at 37°C for 20 min.

(2) DNase I (20 U/mL) was added to the above mixtures respectively, and the final volume of the mixture was 100 μL. The mixed solution was incubated at 37°C for 1 hour, and then heated at 75°C for 15 minutes to terminate the enzymatic reaction.

(3) Centrifuge at 13000r/min for 10min to remove rGO, and take the supernatant for chip detection. Before detection, the microchannel was modified and passivated with 1% bovine serum albumin (BSA) for 10min to prevent nonspecific adhesion, and then the channel was rinsed three times with ultrapure water and filled with 1×PBS buffer for standby use. A 10μL pipette tip with the tip cut off was inserted into the chip hole as a reservoir, and then the electrode was inserted into the reservoir and connected to a DC power supply. A DC voltage of 30V was applied in all experiments.

The test results are shown in Figure 4. The rGO concentration affects the background and the binding efficiency of the target and Apt. (F-F0)/F0 reaches the maximum value when the rGO concentration is 20 μg/mL. Therefore, 20 μg/mL rGO was selected as the optimal condition after optimization.

3. Optimization of PBS concentration

In order to obtain the best detection sensitivity, this section optimizes the concentration of the sample running buffer PBS. In this section, the (FF ₀ )/F ratio is selected as the sensitivity index. The specific optimization method is as follows:

(1) CEA (100 pg/mL) was added to the culture buffer (containing 20 mM Tris, 100 mM NaCl, 5 mM MgCl ₂ , 1 mM CaCl ₂ , pH 7.6) containing the corresponding FAM-Apt (10 nM) and rGO (20 μg/mL), and incubated at 37°C for 20 min.

(2) DNase I (20 U/mL) was added to the above mixtures respectively, and the final volume of the mixture was 100 μL. The mixed solution was incubated at 37°C for 1 hour, and then heated at 75°C for 15 minutes to terminate the enzymatic reaction.

(3) The rGO was removed by centrifugation at 13000 r/min for 10 min, and the supernatant was used for chip detection. Before detection, the microchannel was modified and passivated with 1% bovine serum albumin (BSA) for 10 min to prevent nonspecific adhesion. After that, the channel was rinsed three times with ultrapure water and filled with different concentrations of PBS buffer (0.1×, 0.5×, 1×, 1.5×, 2×) containing 4% (v/v) CH ₃ CN for use. A 10μL pipette tip with the tip cut off was inserted into the chip hole as a reservoir, and then the electrode was inserted into the reservoir and connected to a DC power supply. A DC voltage of 30V was applied in all experiments.

The results are shown in Figure 5A, which shows that the enrichment efficiency increases with the increase of PBS concentration, and 1×PBS reaches the maximum value. When it exceeds 1×PBS, the enrichment efficiency decreases. This is because too high or too low buffer ion strength is not conducive to sample enrichment, so 1×PBS was selected for further study.

4. Optimization of CH ₃ CN concentration in PBS buffer

CH3CN is often used as an additive for online sample pre-concentration, allowing rapid accumulation of nucleic acids by transient pseudo-isotachophoresis. In order to obtain the best detection sensitivity, this section optimizes the concentration of CH3CN in the running buffer PBS. In this section, the (FF ₀ )/F ratio is selected as the sensitivity index. The specific optimization method is as follows:

(1) CEA (100 pg/mL) was added to a culture buffer (containing 20 mM Tris, 100 mM NaCl, 5 mM MgCl ₂ , 1 mM CaCl ₂ , pH 7.6) containing the corresponding FAM-Apt (10 nM) and rGO (20 μg/mL) and incubated at 37°C for 20 min.

(2) DNase I (20 U/mL) was added to the above mixtures respectively, and the final volume of the mixture was 100 μL. The mixed solution was incubated at 37°C for 1 hour, and then heated at 75°C for 15 minutes to terminate the enzymatic reaction.

(3) The rGO was removed by centrifugation at 13000 r/min for 10 min, and the supernatant was used for chip detection. Before detection, the microchannel was modified and passivated with 1% bovine serum albumin (BSA) for 10 min to prevent nonspecific adhesion. After that, the channel was rinsed three times with ultrapure water and filled with 1×PBS buffer containing different concentrations (0%, 1%, 2%, 4%, 6%, 8%, V/V) of CH ₃ CN for use. A 10μL pipette tip with a cut tip was inserted into the chip hole as a reservoir, and then the electrode was inserted into the reservoir and connected to a DC power supply. A DC voltage of 30 V was applied in all experiments. The optimal CH 3 CN concentration was determined by comparing the fluorescence intensity of the fluorescent strips on the chip.

The results are shown in FIG. 5B , which show that when the CH 3 CN concentration is 4% (v/v), the enrichment efficiency of the microfluidic chip is significantly improved, so 4% (v/v) CH ₃ CN is selected as the optimal concentration.

5. Optimization of Nafion membrane width and thickness

The FAM molecules generated by the DNase I cycle triggered by 100pg/mL CEA were used as optimization verification objects. The enrichment effect of the chips with different widths of Nafion membranes (100μm, 200μm, 400μm, 600μm and 700μm) and different thicknesses of Nafion membranes (20μm, 45μm, 100μm, 200μm) was investigated. The buffer contained 1×PBS, pH7.4, 4% (V/V) CH ₃ CN, and a DC voltage of 30V.

The results are shown in C and D in Figure 5, which show that the width and depth of the Nafion membrane have a significant effect on the sensitivity of the detection. As the size of the Nafion membrane increases, the chip enrichment efficiency first increases and then decreases. When the width of the Nafion membrane is 400μm and the depth is 45μm, the chip enrichment efficiency is the highest. Therefore, the Nafion membrane width of 400μm and depth of 45μm are used as the optimal detection conditions.

In summary, the optimized conditions were as follows: 20 U/mL DNase I incubation for 1 h, 20 μg/mL rGO, 1×PBS (pH 7.4) containing 4% (v/v) CH ₃ CN, and a Nafion membrane with a width of 400 μm and a depth of 45 μm.

(IV) Feasibility verification of the detection method based on DNase I/Apt/rGO combined with multi-channel microfluidic chip

1. Feasibility verification of DNase I cyclic dissociation and degradation of FAM-Apt

This section uses the above optimized conditions to verify the feasibility of DNase I cyclic dissociation and degradation of FAM-Apt. The specific method is as follows:

Using the microfluidic chip detection method optimized in Part (III), four groups of experiments were designed, including group a: rGO+FAM-Apt (without adding target CEA and DNase I), group b: rGO+FAM-Apt+DNase I, group c: rGO+FAM-Apt+CEA100pg/mL, and group d: rGO+FAM-Apt+CEA100pg/mL+DNase I.

The detection results are shown in A and B in Figure 6, showing that in the absence of target, the fluorescence bands a and b are negligible, indicating that FAM-Apts are adsorbed on the rGO surface and prevent the degradation of DNase I. As expected, in the presence of target (CEA as a model analyte), a fluorescence band appeared (as shown in band c), and when the target and DNase I appeared at the same time, a significant fluorescence band was detected (as shown in band d), indicating the feasibility of DNase I cyclic dissociation and degradation of FAM-Apt.

2. Feasibility verification of detection of CEA, AFP and miR-21

This section uses the above optimized conditions to verify the feasibility of simultaneous detection of CEA, AFP and miR-21. The specific method is as follows:

The microfluidic chip detection method optimized in part (III) was used. In step (1), high-concentration targets (1000pg/mL CEA, 2000pg/mL AFP, 5000fg/mL miR-21), medium-concentration targets (100pg/mL CEA, 500pg/mL AFP, 500fg/mL miR-21) and low-concentration targets (50pg/mL CEA, 150pg/mL AFP, 50fg/mL miR-21) were added for detection, and the feasibility of simultaneous detection of CEA, AFP and miR-21 was determined by their fluorescence intensity.

The detection results are shown in Figure 7, which show that the three different concentrations of the targets induced a regular increase in the FF ₀ value, indicating that it is feasible to simultaneously detect CEA, AFP and miR-21 on the chip.

3. Repeatability test

This section uses the above optimized conditions to test the repeatability of simultaneous detection of CEA, AFP and miR-21. The specific method is as follows:

The optimized microfluidic chip detection method in part (III) was used. In step (1), the same concentration of targets (100 pg/mL CEA, 300 pg/mL AFP, and 100 fg/mL miR-21) were added for repeated detection, and the intra-day (n=6) and inter-day (n=15) repeatability of the F-F0 values of CEA, AFP, and miR-21 within 4 days were analyzed.

The test results are shown in Figure 8, showing that the relative standard deviations (RSDs) of CEA within days (n=6) and between days (n=15) were 3.6% and 5.1%, AFP were 5.2% and 5.4%, and miR-21 were 3.5% and 4.4%, indicating that the method has high repeatability (precision).

4. Specificity detection

This section uses the above optimized conditions to detect the specificity of CEA, AFP and miR-21 respectively. The specific methods are as follows:

The optimized microfluidic chip detection method in part (III) was used. Different targets (100pg/mL CEA, 300pg/mL AFP, 100fg/mL miR-21) were added in step (1). Under the same detection conditions, HSA, AFP, CRP, IL-6 and Her-2 were used as interfering molecules for detecting CEA; HSA, CEA, CRP, IL-6 and Her-2 were used as interfering molecules for detecting AFP; miR-141, miR-375, single base mismatch (mis-1) and random RNA sequences were used as interfering molecules for miR-21. The FI values measured for the three targets were compared with the FI values of common interfering molecules to verify the specificity of this method.

The specific detection results are shown in Figure 9, showing that the FI values of CEA (100pg/mL), AFP (300pg/mL) and miR-21 (100fM) were significantly higher than those of the interfering substances. The FI values of the above interfering substances were all lower than that of blank + 3SD (n = 11), indicating that the method has high selectivity and there is a good linear relationship between different concentrations (x) of the targets (CEA, AFP and miR-21) and FF ₀ (y).

5. Accuracy test

This section uses the above optimized conditions to test the accuracy of CEA, AFP and miR-21 respectively. The specific methods are as follows:

Using the optimized microfluidic chip detection method in part (III), standard stock solutions of CEA (100 μg/mL), AFP (100 μg/mL) and miR-21 (100 μM) were prepared in deionized water. The stock solutions were appropriately diluted with deionized water to prepare standard solutions of different concentrations. Standard solutions of CEA, AFP and miR-21 of different concentrations were added to the incubation buffer (20 mM Tris, 100 mM NaCl, 5 mM MgCl ₂ , 1 mM CaCl ₂ , pH 7.6) including the corresponding FAM-apt (10 nM) and rGO (20 μg/mL), respectively, and incubated at 37°C for 20 min, and then DNase I (20 U/mL) was added to the above mixture. After the mixed solution was incubated at 37°C for 1 h, it was heated at 75°C for 15 min, and finally centrifuged at 13000 r/min for 10 min to remove rGO. The supernatant fluorescence induced by different concentrations of targets was detected using the chip.

The calibration curve results are shown in Figure 10 and Table 2.

Table 2: Regression equations and related parameters for CEA, AFP, and miR-21 detection

The results showed that the relative coefficients (r) of CEA, AFP and miR-21 were 0.9945, 0.9981 and 0.9937, respectively, indicating good linear correlation. The detection limits (S/N=3) of CEA, AFP and miR-21 were 4.5 pg/mL, 37.0 pg/mL and 1.3 fM, respectively.

Furthermore, the accuracy of the method was evaluated by spike recovery. Three different target concentrations (CEA: 20pg/mL, 250pg/mL, 850pg/mL, AFP: 60pg/mL, 600pg/mL, 1800pg/mL, miR-21: 10fM, 250fM, 2500fM) were added to 1% serum samples to analyze their recovery rates. The serum sample was prepared by taking 1ml of anticoagulant-free whole blood from the elbow vein of a volunteer, placing it at room temperature for 2 hours, then centrifuging it at 3000rpm for 15 minutes, taking the upper serum and storing it at -20°C for later use. The results are shown in Figure 11 and Table 3.

Table 3: Spike results of CEA, AFP and miRNA-21 in human serum

The results showed that the average recoveries of CEA, AFP and miR-21 were 106.5%, 108.2% and 109.4%, respectively, indicating that the detection method had excellent accuracy.

In summary, the present invention provides a detection method and application based on DNase I/Apt/rGO combined with a multi-channel microfluidic chip. This method develops a new strategy for the simultaneous detection of multiple biomarkers by combining FAM-Apt/rGO/DNase I with a multi-channel microfluidic enrichment chip. This method is highly efficient, and it only takes 30 minutes to simultaneously detect proteins and nucleic acids on the same chip. And under the dual signal amplification strategy, the limits of detection (LOD) of AFP, CEA and miR-21 are 37.0pg/mL, 4.5pg/mL and 1.3fM, respectively, which can meet the detection of low-concentration serum markers in HCC patients.

The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims (10)

1. A detection method based on DNase I/Apt/rGO combined with multi-channel microfluidic chip, which is characterized by including the following steps: S1. Incubate the sample to be tested by adding nucleic acid aptamers containing fluorescent groups and a buffer with a final concentration of 15 to 30 μg/mL of bioreduced graphene oxide, and then adding deoxyribonucleic acid with a final concentration of 5 to 40 U/mL. Enzyme reaction, centrifugation, and collection of supernatant; S2. Use a microfluidic chip to detect the supernatant, and then quantify the concentration of the target detection substance in the sample to be tested according to the fluorescence signal intensity; The detection method is used for non-disease diagnostic purposes. 2. The detection method according to claim 1, characterized in that the nucleic acid aptamer includes a nucleic acid aptamer for detecting proteins and/or a nucleic acid aptamer for detecting nucleic acids; Preferably, the nucleic acid aptamer includes at least one of Apt-CEA, Apt-AFP and Apt-miR 21; Preferably, the nucleotide sequence information of Apt-CEA is shown in SEQ ID NO.1; Preferably, the nucleotide sequence information of the Apt-AFP is shown in SEQ ID NO. 2; Preferably, the nucleotide sequence information of Apt-miR 21 is shown in SEQ ID NO.3. 3. The detection method according to claim 1, characterized in that the concentration of the fluorescent group-containing nucleic acid aptamer is 5-50 nM. 4. The detection method according to claim 1, characterized in that the buffer further contains 15-25mM Tris, 80-120mM NaCl, 3-8mM _MgCl2 and 0.5-1.5mM _CaCl2 . 5. The detection method according to claim 1, characterized in that the deoxyribonuclease comprises deoxyribonuclease I. 6. The detection method according to claim 1, characterized in that the incubation time is 10 to 30 minutes; Preferably, the reaction time is 10 to 120 minutes; Preferably, the centrifugal speed is 12000-13500r/min; Preferably, the centrifugation time is 5 to 15 minutes. 7. The detection method according to claim 1, characterized in that the microfluidic chip contains 1 to 4 parallel microchannels; Preferably, the width of the microchannel is 100-700 μm, and the height is 20-200 μm; Preferably, both sides of the microchannel are buffer channels. 8. The detection method according to claim 1, wherein the use of a microfluidic chip to detect the supernatant specifically includes: first modifying the microfluidic chip microchannel with bovine serum albumin, Then detect in PBS buffer containing CH ₃ CN; Preferably, the concentration of CH ₃ CN in the PBS buffer is 1 to 10% (v/v); Preferably, the pH value of the PBS buffer is 7.2 to 7.6; Preferably, the detected voltage is 25-35V. 9. Application of the detection method based on DNase I/Apt/rGO combined with multi-channel microfluidic chip according to any one of claims 1 to 8 in detecting biomarkers. 10. The application according to claim 9, characterized in that the biomarkers comprise liver cancer biomarkers.