PSA aptamer sensor based on screen-printed electrodes, preparation and detection method

Abstract

This invention discloses a PSA aptamer sensor based on a screen-printed electrode, its preparation, and detection method. In this invention, a composite material of AuNPs and graphene aerogel is used to modify the surface of a screen-printed electrode. The aptamer is modified onto the sensor by forming gold-sulfur (Au-S) bonds with a thiol-modified DNA single-stranded aptamer. Excess gold active sites are blocked with MCH to construct the PSA aptamer sensor. A certain concentration of PSA and UA is selected and incubated with the sensor. The EIS test method is used to detect PSA by recording its impedance changes, and the detection limit of the sensor is obtained. Utilizing the advantages of large-scale mass production and low cost of screen printing technology, combined with the excellent physical and electrical properties of nanomaterials, an electrochemical sensor for the detection of prostate-specific antigen (PSA) is prepared, enabling convenient, efficient, and low-cost detection, providing a new approach for prostate cancer patients to be detected outside of hospitals and laboratories.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of micro-nano biological detection, and particularly relates to a PSA aptamer sensor based on a screen-printed electrode, a sensor preparation method and a detection method. BACKGROUND

[0002] Prostate specific antigen (PSA) is secreted by prostate epithelial cells and has very high tissue organ specificity. The normal value of PSA concentration of adult men is not more than 4 ng / mL. The canceration and other lesions of the prostate can cause the increase of PSA concentration, and therefore PSA can be used as a tumor marker of prostate cancer.

[0003] According to the Chinese Prostate Cancer Screening and Early Treatment Early Diagnosis Guidelines (2022, Beijing), PSA detection is recommended as the preferred prostate cancer screening method in China.

[0004] At present, the detection of many tumor markers including PSA mainly concentrates on the laboratory of a hospital, and large biochemical analyzers are used to detect blood samples. The use of large biochemical analyzers to detect blood samples has the problems of low portability and poor instantaneity.

[0005] With the continuous expansion of demand, people have higher requirements for the convenience and instantaneity of biochemical detection, and instant detection has gradually become the research power and direction of biological sensors. SUMMARY

[0006] The present application aims to solve the defects of the prior art and provide a simple and portable PSA aptamer sensor based on a screen-printed electrode, which can realize instant monitoring and rapid detection, and a preparation method thereof.

[0007] The present application also aims to provide a PSA aptamer sensor detection method based on a screen-printed electrode.

[0008] The technical scheme provided by the present application is as follows: The present application discloses a PSA aptamer sensor preparation method based on a screen-printed electrode, which comprises modifying a composite material of AuNPs and graphene aerogel on the surface of a screen-printed electrode, forming a gold-sulfur bond between the composite material and thiol-modified PSA aptamer to realize modification on the surface of the screen-printed electrode, and constructing a PSA aptamer sensor.

[0009] Further, the screen-printed electrode comprises a substrate 1, a detection area 2 and a wiring area 7, and an insulating layer 6 arranged on the surface of the electrode, the detection area 2 comprises a counter electrode 3, a working electrode 4 and a reference electrode 5, the wiring area 7 is used for current transmission, and the insulating layer 6 separates the detection area 2 and the wiring area 7 of the electrode.

[0010] Furthermore, this includes the following steps:

[0011] S1. Rinse the screen-printed electrode with ultrapure water and let it dry. Cut it into a suitable size and place it in a culture dish. Then, add 4-6 μL of a mixture of AuNPs and graphene aerogel to the working electrode area. Add an appropriate amount of ultrapure water to the empty space in the culture dish. After sealing the dish, a humid environment is formed. Then, place the culture dish in a water bath and incubate at 40-50℃ for 10-12 h.

[0012] S2. Remove the petri dish, wipe off any condensation inside the lid, close the lid, and immediately freeze at -20~-30℃ overnight.

[0013] S3. Immediately after removing the petri dish, place it in a freeze dryer for 10-12 hours of freeze drying.

[0014] S4. Dilute the aptamer 1-3 times with pure water, take 5-10 μL and drop it onto the surface of the working electrode, then place it in a refrigerator at 0-4℃ overnight. After that, slowly rinse the working electrode area with pure water to clean the unbound aptamer, and blow it dry with nitrogen gas at a low flow rate.

[0015] S5. Add 5-10 μL of MCH to the surface of the working electrode, let it stand at room temperature for 1-2 h to block excess active sites, then slowly rinse the working electrode area with pure water to wash away excess MCH, and blow dry with nitrogen gas at a low flow rate to obtain the aptamer sensor.

[0016] Furthermore, the preparation method of the AuNPs / graphene aerogel composite material includes the following steps:

[0017] Sa1. Dilute the graphene oxide dispersion with ultrapure water 1-3 times and shake thoroughly. Weigh 20-30 mg of L-ascorbic acid and add it to 1-2 mL of graphene oxide dispersion. Shake well and then sonicate for 30-40 min.

[0018] Sa2. Take HAuCl4 and dilute it 1-3 times with pure water. Add 0.2-0.3 μL to the above solution, shake well, sonicate, and store in a refrigerator at 0-4℃.

[0019] Furthermore, in step S4, the PSA aptamer is a single-stranded DNA that folds into a secondary structure through different sequences and binds to the target.

[0020] Furthermore, the aptamer sensor is sealed and stored in an environment of 0-4℃.

[0021] Furthermore, the area ratio of the working electrode to the counter electrode is approximately 1:2.

[0022] Furthermore, conductive carbon paste is selected as the material for the working electrode and the counter electrode; silver / silver chloride paste is selected as the printing material for the reference electrode; conductive silver paste with good conductivity and relative stability is selected as the current transmission line layer material; and polyethylene terephthalate is selected as the substrate material.

[0023] The present invention also discloses a PSA aptamer sensor based on screen-printed electrodes, including an aptamer sensor prepared by the above-described method for preparing a PSA aptamer sensor based on screen-printed electrodes.

[0024] The present invention also discloses a detection method for a PSA aptamer sensor based on a screen-printed electrode, comprising using the above-mentioned PSA aptamer sensor based on a screen-printed electrode, preparing antigen test solutions of different concentrations with pure PBS, adding 10-15 μL to the working electrode area of ​​the aptamer sensor, incubating at room temperature for 2-3 h, then slowly rinsing the surface of the working electrode with PBS 3-5 times, gently drying with nitrogen gas at a low flow rate, and storing at 0-4℃.

[0025] Then, a test electrolyte solution containing potassium ferrocyanide, potassium ferrocyanide, and potassium chloride was prepared using PBS. The sensor to be tested was clamped onto the electrochemical workstation, and the working area of ​​the electrode was completely immersed in the electrolyte. PSA was detected and the impedance value was obtained. Different colored curves represented different PSA detection concentrations, and electrochemical impedance spectroscopy and linear fitting graphs were plotted.

[0026] The advantages of this invention compared to the prior art are:

[0027] 1. By utilizing the advantages of large-scale mass production and low cost of screen printing technology, combined with the excellent physical and electrical properties of nanomaterials, an electrochemical sensor for the detection of prostate-specific antigens was prepared, enabling convenient, efficient, and low-cost detection. This provides a new approach for prostate cancer patients to be tested outside of hospitals and laboratories, and also lays the theoretical and technical foundation for the future development of portable personal testing platforms.

[0028] 2. A one-step reduction method was used to prepare graphene aerogels containing gold nanoparticles. Au in chloroauric acid 3+ It has strong oxidizing properties and is unstable, easily reduced to Au by weak reducing agents. 0 Therefore, ascorbic acid can be used to reduce graphene oxide at the same time, and graphene oxide molecules can be used as nuclei to grow nanoparticles in situ and embed them in graphene, ultimately forming a composite material of AuNPs and graphene aerogel.

[0029] 3. Au-S bonds are readily formed spontaneously and release heat, and have been frequently used in recent years for biomolecular immobilization in nucleic acid sensors. To reduce modification steps and lower the difficulty of binding the biomodified layer, gold-sulfur bonds are formed between AuNPs on the modified electrode surface and thiol-modified aptamers, thereby achieving aptamer modification on the sensor. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the detection principle of an embodiment of the present invention;

[0031] Figure 2 This is a schematic diagram of the structure of the screen-printed electrode in an embodiment of the present invention;

[0032] Figure 3 This is an impedance variation diagram of different concentrations of HAuCl4 in the embodiments of the present invention;

[0033] Figure 4 These are SEM images of graphene aerogels with different concentrations of HAuCl4 in the embodiments of the present invention.

[0034] Figure 5 The diagram shows the detection response of the aptamer sensor under different processing conditions in the embodiments of the present invention.

[0035] Figure 6 This is an impedance variation diagram of different concentrations of aptamers in an embodiment of the present invention;

[0036] Figure 7 These are electrochemical impedance spectroscopy spectra of different concentrations of PSA in the embodiments of the present invention;

[0037] Figure 8 This is a linear fitting graph of different concentrations of PSA in the embodiments of the present invention. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] The present application will now be described in detail with reference to specific embodiments. Figure 1 As shown, a PSA aptamer sensor based on a screen-printed electrode according to an embodiment of the present invention first requires the reduction of graphene oxide, and before it forms a gel, 0.25 μL of HAuCl4 with a concentration of 6 mg / mL is added, mixed and shaken, and stored at low temperature.

[0040] The above mixed solution was added dropwise to the working electrode area of ​​the screen printing electrode, and water bath, freezing and freeze-drying operations were performed respectively.

[0041] Next, 10 μL of the 10 μM aptamer was dropped onto the surface of the working electrode, incubated at low temperature overnight, and then cleaned and dried.

[0042] Then, 10 μL of 1 mM MCH was added to the surface of the working electrode to block the excess active sites, and the electrode was cleaned, dried, and stored at low temperature.

[0043] Finally, by combining it with a portable electrochemical workstation, the detection of PSA at different concentrations can be completed.

[0044] The above-mentioned fabrication principle enables the aptamer sensor to be low-cost, lightweight, sensitive, portable, reliable, and highly resistant to interference and real-time, which helps to improve the accuracy of test results.

[0045] A detection method for a PSA aptamer sensor based on screen-printed electrodes, comprising the following steps:

[0046] S1: Preparation of composite materials of AuNPs and graphene aerogel; specifically including

[0047] S11. Dilute the graphene oxide dispersion to 4 mg / mL with ultrapure water and shake thoroughly. Weigh 25 mg of L-ascorbic acid and add it to 1 mL of graphene oxide dispersion with a concentration of 4 mg / mL. Shake well and then sonicate for 30 minutes.

[0048] S12. Take HAuCl4 and dilute it with pure water to 6 mg / mL. Take 0.25 μL and add it to the above solution. Shake well and sonicate for 30 minutes. Store in a refrigerator at 0-4℃.

[0049] S2: Modified electrodes and immobilized biosensitive layers; specifically including...

[0050] S21. Rinse the screen-printed electrode with ultrapure water and let it dry. Cut it into a suitable size and place it in a culture dish. Then, add 5 μL of the mixed solution from step 1 to the working electrode area. Add an appropriate amount of ultrapure water to the empty space in the culture dish. After sealing the dish, a humid environment is formed. Then, place the culture dish in a water bath and incubate it at 40°C for 10 hours.

[0051] S22. Remove the petri dish, wipe off any condensation inside the lid, close the lid, and immediately freeze at -20°C overnight.

[0052] S23. Immediately after removing the petri dish, place it in a freeze dryer for 10 hours of freeze drying.

[0053] S24. Dilute the aptamer to 10 μM with pure water, take 10 μL and drop it onto the surface of the working electrode, then place it in a refrigerator at 0-4℃ overnight. After that, slowly rinse the working electrode area with pure water until the unbound aptamer is cleaned, and then blow it dry with nitrogen gas at a low flow rate.

[0054] S25. Add 10 μL of 1 mM MCH to the surface of the working electrode, let it stand at room temperature for 1 hour to block the excess active sites, then slowly rinse the working electrode area with pure water to wash away the excess MCH, and blow it dry with nitrogen gas at a low flow rate. Then seal the prepared sensor and store it at 0-4℃ for later use.

[0055] S3: Establish a detection model for the aptamer sensor, specifically including...

[0056] S31. Different concentrations of PSA were tested to determine whether the impedance value of the aptamer sensor increased continuously with the increase of PSA concentration.

[0057] S32. Using the logarithm of PSA concentration as the x-axis and the corresponding impedance value as the y-axis, a linear fitting line was obtained, and the two showed a good linear relationship.

[0058] S4: Sensor selectivity test, specifically including

[0059] The sensor was incubated with CEA, AFP, PSA, and Glucose, LA, and UA, respectively. The sensor showed good selectivity for PSA and had a large response.

[0060] The screen-printed electrode adopts a three-electrode system, including a substrate 1, a detection area 2, a counter electrode 3, a working electrode 4, a reference electrode 5, an insulating layer 6, and a wiring area 7. Its overall size is 34 mm, the working electrode diameter is 4 mm, and the area ratio of the working electrode to the counter electrode is approximately 1:2.

[0061] Conductive carbon paste was chosen as the material for both the working and counter electrodes, and silver / silver chloride paste was selected as the printing material for the reference electrode. Highly conductive and relatively stable conductive silver paste was chosen as the material for the current transmission line layer, and polyethylene terephthalate (PET) was chosen as the base material. The interface between the different inks was located outside the working area, and the silver paste layer was directly connected to the instrument to reduce contact resistance. An insulating layer was used on the outermost layer of the conductive layer to separate the detection area and the wiring area of ​​the electrode sheet.

[0062] The working electrode is the main electrode where the electrochemical reaction occurs and is also the electrode for subsequent modification. The counter electrode and the working electrode together form a circuit, where electron gain and loss occur. In the electrochemical system, the electrode forming the test current circuit has a large current flowing through it, making it easily polarized and difficult to measure the accurate potential. Therefore, an electrode that is not easily polarized is introduced as a reference electrode to provide a more stable potential reference value.

[0063] This three-electrode system constitutes two current loops: one is a measurement loop consisting of a reference electrode and a working electrode, used to measure the relative potential of the working electrode. The current in this loop is very small and will not cause polarization of the reference electrode.

[0064] Another polarization circuit consists of a working electrode and a counter electrode, which forms the main electrochemical reaction circuit. Polarization occurs at the electrodes in this circuit. Therefore, the screen-printed electrode must include these three independent electrodes.

[0065] The aptamer is a single-stranded DNA with the sequence 5'-TTT AAT TAAAGC TCG CCA TCAAAT AGC TTT-3', with end modification 5'SH C6. It is purified by HPLC-CE and folds into a secondary structure through different sequences to bind to the target.

[0066] Among them, the PSA concentration was 5×10 -8 mg / mL to 5×10 -5 Within the range of mg / mL, the logarithm of PSA concentration exhibits a strong linear relationship with the impedance value (R0). 2 =0.992).

[0067] The sensor's sensitivity is 241.52 Ω / [Log(mg / mL)], and its detection limit (3σ / m) is 0.0306 ng / mL.

[0068] The concentrations of CEA, AFP, and PSA were 50 ng / mL, while the concentrations of glucose, LA, and UA were 10 mM. The sensor's response resistance to PSA reached nearly 900 Ω.

[0069] In this embodiment, the gold nanoparticles play an important role in fixing the aptamer in the material, and can be reduced to gold nanoparticles together with the help of excess ascorbic acid.

[0070] The specific procedure involves adding a certain concentration of HAuCl4 after reducing graphene oxide but before it gels. At this point, the HAuCl4 can be rapidly reduced by excess ascorbic acid, and gold nanoparticles grow from the graphene oxide as nuclei, attaching to its reduced surface. Aggregation of gold nanoparticles is detrimental to their properties and can lead to aptamer aggregation; therefore, the concentration of doped HAuCl4 should not exceed 6 mg / mL. Figure 3 , 4 As shown.

[0071] In this embodiment, the aptamer is directly incubated without further treatment after thawing.

[0072] like Figure 5 As shown, corresponding sensors were prepared by selecting three different temperatures (95℃, 75℃, and no heating) as activation conditions. The initial response of the electrode and its response to PSA at a concentration of 10 ng / mL were recorded. It was found that the initial response of the sensor incubated with the aptamer was between 180 Ω and 200 Ω, while a difference was observed after incubation with PSA.

[0073] The impedance of the untreated electrode increased significantly after incubation with PSA, while the impedance of the two annealed groups decreased slightly after incubation with aptamers, and the deviation was large.

[0074] The similar initial responses indicate that different treatment conditions have little effect on the incubation of aptamers, and all of them can bind to the electrode via gold-sulfur bonds; however, the difference in the PSA detection response reflects that different treatment conditions can affect the recognition and binding of aptamers to antigens.

[0075] like Figure 6 As shown, when the aptamer concentration increased from 1 μM to 10 μM, the impedance increased by 150 Ω to 200 Ω. However, when the concentration increased from 10 μM to 20 μM, the impedance increase was less than 50 Ω. This indicates that the 10 μM aptamer is close to saturation in capturing PSA at a concentration of 50 ng / mL. Further increasing the aptamer concentration will not capture more PSA and will only increase costs. Therefore, 10 μM was chosen as the optimized aptamer incubation concentration.

[0076] The detection performance of the aptamer sensor in the above embodiments, such as Figure 7 As shown.

[0077] Curves of different color depths represent different PSA detection concentrations, and their impedance values ​​increase continuously with increasing PSA concentration. For example... Figure 8 As shown, the aptamer sensor in this embodiment has a wider linear range, a lower detection limit, and higher sensitivity than the immunosensor, resulting in a significant improvement in overall detection capability.

[0078] On the one hand, the introduction of gold nanoparticles makes the binding of aptamers to sensors more stable than that of antibodies; on the other hand, aptamers have better antigen capture performance and stability.

[0079] Aptamers are short oligonucleotide sequences that can specifically bind to ligands and are also known as "chemical antibodies." Compared to antibodies, aptamers have several advantages, including better stability and the ability to be synthesized in vitro.

[0080] In this specific embodiment, a commonly used PSA aptamer sequence was selected. The structure is a single-stranded DNA with the sequence 5'-TTTAAT TAAAGC TCG CCA TCAAAT AGC TTT-3', with an end modification of 5'SH C6. Purified by HPLC-CE, the sequence folds into a secondary structure to bind to the target. However, the PSA aptamers applicable to this invention are not limited to this; various PSA aptamer sequences can be selected.

[0081] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one specific embodiment of the present invention. The actual software and hardware structure is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without inventiveness, they should all fall within the protection scope of this patent.

Claims

1. A method for fabricating a PSA aptamer sensor based on screen-printed electrodes, characterized in that: A composite material of AuNPs and graphene aerogel is used to modify the surface of a screen-printed electrode. The modification on the screen-printed electrode surface is achieved by forming gold-sulfur bonds between the composite material and the thiol-modified PSA aptamer, thus constructing a PSA aptamer sensor. The screen-printed electrode includes a substrate 1, a detection area 2 and a wiring area 7, and an insulating layer 6 disposed on the electrode surface. The detection area 2 includes a counter electrode 3, a working electrode 4 and a reference electrode 5. The wiring area 7 is used for current transmission. The insulating layer 6 separates the detection area 2 and the wiring area 7 of the electrode. The area ratio of the working electrode 4 to the counter electrode 3 is 1:

2. Includes the following steps: S1. Rinse the screen-printed electrode with ultrapure water and let it dry. Cut it into a suitable size and place it in a culture dish. Then, add 4-6 μL of a mixture of AuNPs and graphene aerogel to the working electrode area. Add an appropriate amount of ultrapure water to the empty space in the culture dish. After sealing the dish, a humid environment is formed. Then, place the culture dish in a water bath and incubate at 40-50℃ for 10-12 h. The preparation method of the AuNPs-graphene aerogel composite material includes the following steps: Sa1. Dilute the graphene oxide dispersion with ultrapure water 1-3 times and shake thoroughly. Weigh 20-30 mg of L-ascorbic acid and add it to 1-2 mL of the graphene oxide dispersion. Shake well and then sonicate for 30-40 min. Sa2. Take HAuCl4 and dilute it 1-3 times with pure water. Take 0.2-0.3 μL and add it to the above solution. Shake well and then sonicate. Store in a refrigerator at 0-4℃. S2. Remove the petri dish, wipe off any condensation inside the lid, close the lid, and immediately freeze at -20~-30℃ overnight. S3. Immediately after removing the petri dish, place it in a freeze dryer for 10-12 hours of freeze drying. S4. Dilute the aptamer 1-3 times with pure water, add 5-10 μL to the surface of the working electrode, and then place it in a refrigerator at 0-4℃ overnight. Afterwards, gently rinse the working electrode area with pure water to remove unbound aptamers, and then dry it with nitrogen gas at a low flow rate. The PSA aptamer is a single-stranded DNA that folds into a secondary structure through different sequences and binds to the target. The sequence of the PSA aptamer is 5'-TTT AAT TAAAGC TCG CCA TCAAAT AGC TTT-3'. S5. Add 5-10 μL of MCH to the surface of the working electrode, let it stand at room temperature for 1-2 h to block excess active sites, then slowly rinse the working electrode area with pure water to wash away excess MCH, and blow dry with nitrogen gas at a low flow rate to obtain the aptamer sensor.

2. The method for fabricating a PSA aptamer sensor based on screen-printed electrodes according to claim 1, characterized in that: The aptamer sensor is sealed and stored at 0-4℃.

3. The method for fabricating a PSA aptamer sensor based on screen-printed electrodes according to claim 1, characterized in that: Conductive carbon paste is selected as the material for the working electrode and the counter electrode; silver / silver chloride paste is selected as the printing material for the reference electrode; conductive silver paste with good conductivity and relative stability is selected as the material for the current transmission line layer; and polyethylene terephthalate is selected as the substrate material.

4. A PSA aptor sensor based on screen-printed electrodes, characterized in that: It is prepared based on the PSA aptamer sensor fabrication method based on screen-printed electrodes as described in any one of claims 1-3.

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