Biosensor for exosome detection based on sulfide colloidal quantum dots as well as preparation and application of biosensor

Abstract

The invention belongs to the technical field of biochemical sensing, and particularly relates to a sulfide colloidal quantum dot-based biosensor for exosome detection as well as preparation and application thereof. Transferring the sulfide colloidal quantum dot solution to a metal gate electrode of a high electron mobility transistor or a working electrode of a screen printing three-electrode to form a sulfide colloidal quantum dot film; coating the sulfide colloidal quantum dot film with an antibody protein modification layer, and closing non-specific active sites on the antibody protein modification layer by adopting bovine serum albumin; the antibody protein can be specifically bound with transmembrane protein corresponding to the surface of an exosome to be detected, so that the exosome to be detected is captured on the surface of the electrode, and meanwhile, based on the unique physical and chemical effect of the sulfide colloidal quantum dots, the modified electrode has the capability of sensing the distribution change of external charges, so that the detection sensitivity of the exosome to be detected is improved. Therefore, rapid and quantitative detection of the exosome is realized.

Description

Technical Field

[0001] The present invention belongs to the field of biochemical sensing technology, and more specifically, relates to a biosensor for detecting exosomes based on sulfide colloidal quantum dots, and its preparation and application. Background Art

[0002] Exosomes are extracellular vesicles with a diameter of about 50 to 150 nm. Their outer layer is a lipid bilayer, containing proteins, RNA, DNA and other substances. As a type of small membrane vesicles actively released by most living cells in the human body, exosomes can circulate in a variety of body fluids such as blood, urine, and tears and are widely detected. More importantly, exosomes carry proteins, nucleic acids, and metabolic molecules related to the physiological / pathological state of parent cells. Molecules such as proteins and miRNAs protected by the lipid bilayer of exosomes can provide rich biological information for clinical diagnostic research and pathological mechanisms. Therefore, quantitative analysis and detection of exosomes in body fluids has gradually developed into an emerging in vitro diagnostic strategy for early diagnosis and prognosis of diseases.

[0003] In 1998, Escola et al. confirmed that certain transmembrane proteins (CD63, CD9, CD81, CD82, etc.) are highly enriched in exosomes, that is, they exist in large quantities on the surface of exosomes, which provides a certain basis for the identification and detection of exosomes. At the same time, as researchers further discovered, exosomes are membrane structures containing many proteins, and the content of exosome membrane proteins can reflect the proteomic information of parent cells. Therefore, the analysis and detection of exosomes can be transformed into quantitative detection of exosome membrane proteins, and the specific binding of receptors such as CD63 antibodies and CD9 antibodies to exosome membrane surface proteins can be used to capture and detect exosomes, thereby greatly improving the efficiency of exosome detection.

[0004] In recent years, various methods have been used to analyze and detect exosomes, such as transmission electron microscopy (TEM), scanning electron microscopy (SEM), nanoparticle tracking analysis (NTA), enzyme-linked immunosorbent assay (ELISA), and Western blotting (WB). These methods provide comprehensive and precise analysis of exosomes. NTA, SEM, and TEM are common techniques for the characterization of nanoscale particles, including exosomes. However, as purely physical techniques, these methods have no biochemical specificity for exosomes, and they are usually combined with other methods to specifically detect exosomes. At the same time, NTA has limitations in the detection of low-concentration exosomes, and SEM and TEM require large-scale, expensive equipment and time-consuming sample preparation processes. WB is a classic analytical technique in molecular biology and immunogenetics, which is usually used to detect specific proteins, but exosome detection also requires a complex sample preparation process. ELISA is one of the most commonly used labeled immunoassay techniques, which is only suitable for laboratory analysis and takes hours to days to detect.

[0005] In order to overcome the limitations of the above methods, advanced biosensors based on different principles have been proposed for exosome detection, such as optical biosensors, acoustic biosensors, electrochemical biosensors, and field effect transistor biosensors. However, they all have their own defects. For example, optical biosensors require expensive light sources and precise measuring instruments, which increases the cost of biomolecule detection; acoustic biosensors are difficult to ensure stability, and the results are easily interfered by the outside world; electrochemical biosensors have the advantages of low cost and simple principles, but how to achieve high-performance biomolecule detection to meet daily needs still requires more innovation and exploration; field effect transistor (FET) biosensors have the advantages of fast electrical response, high sensitivity, and label-free detection, so they show great potential in biomolecule detection. However, large-scale manufacturing of ultra-sensitive, highly reliable, and reproducible FET-based biosensors still has certain challenges, and innovative transformation of traditional FET technology is required.

[0006] Patent document CN104880557A discloses a GaAs-based HEMT biosensor for detecting tumor markers and a method for making the same. The sensor includes a GaAs-based HEMT, the source and drain electrodes are in ohmic contact with the source region and drain region of the GaAs-based HEMT respectively, the gate electrode is formed in the gate region of the GaAs-based HEMT, and the biosensitive film is formed on the gate electrode, which can specifically identify tumor markers, and is affected by the charge of the tumor marker itself, so that the charge distribution on the surface of the gate electrode changes, thereby affecting the concentration of the two-dimensional electron gas in the channel of the GaAs-based HEMT, so that the source-drain current changes. However, the response is small if the charge of the tumor marker itself on the gate causes the change of the source-drain current under the gate dielectric layer, resulting in low sensitivity in tumor marker detection.

[0007] Patent document CN112557473A relates to a preparation method and application of an electrochemical sensor based on a zirconium metal-organic framework. Malignant glioma is one of the most deadly tumors in the brain. Due to the complexity of carcinogenic causes and the obstruction of the blood-brain barrier, early diagnosis is still technically challenging. Specific exosomes secreted by malignant glioma can cross the blood-brain barrier and circulate in body fluids, with the potential for non-invasive early diagnosis. Therefore, a new label-free electrochemical biosensor based on a zirconium metal-organic framework (Zr-MOF) is proposed. In this method, the designed polypeptide chain can specifically capture the highly expressed human epidermal growth factor receptor (EGFR) and EGFR variant (v) III mutant (EGFRvIII) in malignant glioma exosomes. At the same time, Zr-MOF (MB@Zr-MOF) wrapped with methylene blue (MB) is used as a chemical selection marker to generate sensitive electrochemical signals by interacting with the phosphate groups outside the exosome phospholipid bilayer membrane. By monitoring the electroactive molecules in Z-MOF, the concentration of exosomes can be quantitatively detected, showing broad clinical diagnostic application value. The preparation steps of this invention are complicated. It not only requires the preparation of Zr-MOF with stable performance, but also the design of polypeptide chains that can specifically capture target molecules. It also requires the encapsulation of MB outside Zr-MOF. Each step requires a lot of time and technical costs, which increases the preparation cost of the sensor.

[0008] Patent CN113281514A discloses a SPRi biosensor for detecting exosomes of liver cancer cells and its preparation and application. The detection method is as follows: (1) Extraction of exosomes: extract exosomes from liver cancer cells; (2) Synthesis of gold nanoparticles-tyramide complex: synthesize gold nanoparticles first, and then combine gold nanoparticles with tyramide to generate gold nanoparticles-tyramide complex; (3) SPRi detection: first modify the chip surface with an aptamer hairpin capture probe with a G4 chain, and then load exosomes. After the exosomes are loaded, the aptamer specifically binds to the protein on the surface of the exosomes, opens the hairpin, releases the G4 sequence, and forms a G4-hemin enzyme. Finally, the gold nanoparticles-tyramide complex is loaded, and the gold nanoparticles are bound to the chip surface by tyramide signal amplification (TSA), and detected by SPR. The sensor is a label-free SPRi biosensor that can detect liver cancer cell-derived exosomes in real time, with high sensitivity and specificity. This method is an indirect method for measuring liver cancer cell exosomes, which relies on the interaction between the enzyme produced by the specific binding of the G4 chain aptamer to the exosome and the gold nano-tyramide complex. It has the problem of low sensitivity. It also requires SPR technology and corresponding optical equipment, which is costly. Summary of the invention

[0009] In view of the defects of the prior art, the purpose of the present invention is to provide a biosensor for detecting exosomes based on sulfide colloidal quantum dots, and its preparation and application, so as to solve the technical problems that the detection range of the prior art biosensors for detecting the presence of exosomes is not wide enough, the sensitivity is low, or the preparation method is complicated.

[0010] To achieve the above objectives, the present invention provides an ultra-sensitive biosensor based on sulfide colloidal quantum dots, which aims to further optimize the detection performance of exosome detection biosensors and promote the development of biosensors towards miniaturization and integration. Specifically, the present invention provides a method for preparing a biosensor for exosome detection based on sulfide colloidal quantum dots, comprising the following steps:

[0011] (1) transferring the sulfide colloidal quantum dot solution onto a metal gate electrode of a high electron mobility transistor or onto a working electrode of a screen-printed three-electrode to form a sulfide colloidal quantum dot film;

[0012] (2) coating an antibody protein modification layer on the sulfide colloidal quantum dot film in step (1), and blocking the non-specific active sites on the antibody protein modification layer with bovine serum albumin to prepare a biosensor for exosome detection based on sulfide colloidal quantum dots;

[0013] The surface of the sulfide colloidal quantum dots contains carboxyl groups, which can undergo a dehydration condensation reaction with the amino groups in the antibody protein to form a -CO-NH- chemical bond to achieve antibody fixation; in addition, the active sites on the surface of the lead sulfide colloidal quantum dots are conducive to the electrostatic adsorption of the antibody protein; and the S element in the sulfide colloidal quantum dots can form a chemical bond with the electrode, so that the antibody protein modification layer is further modified on the metal gate electrode of the transistor or on the working electrode of the screen-printed three-electrode, and a good charge transfer interface is formed;

[0014] The antibody protein can specifically bind to the transmembrane protein corresponding to the surface of the exosomes to be detected, thereby capturing the exosomes to be detected on the electrode surface. At the same time, based on the quantum effect of the sulfide colloidal quantum dots, the capacitance of the solution on the electrode surface increases, affecting the electrode surface potential and the output current, thereby achieving rapid and quantitative detection of exosomes.

[0015] Preferably, the sulfide colloidal quantum dots are lead sulfide colloidal quantum dots, bismuth sulfide colloidal quantum dots, stannous sulfide colloidal quantum dots or zinc sulfide colloidal quantum dots.

[0016] Preferably, the concentration of sulfide colloidal quantum dots in the sulfide colloidal quantum dot solution is 5-15 mg / mL.

[0017] Preferably, in step (1), the sulfide colloidal quantum dot solution is modified onto the metal gate electrode of a high electron mobility transistor or the working electrode of a screen-printed three-electrode by spin coating, drop coating or electrofluid printing; the modification amount is 0.5-1.5 μL.

[0018] Preferably, step (2) of coating the antibody protein modification layer on the sulfide colloidal quantum dot film in step (1) specifically comprises the following steps: dropping the antibody protein solution onto the surface of the sulfide colloidal quantum dot film, incubating at 37° C. for 1.5-2.5 hours, then rinsing with phosphate buffer solution to remove the unbound antibodies on the surface, and drying at room temperature for later use.

[0019] Further preferably, 0.5-1.5 μL of the antibody protein solution is dripped onto the surface of the sulfide colloidal quantum dot film.

[0020] Preferably, the antibody protein is CD63 antibody or CD9 antibody.

[0021] Preferably, the exosomes to be tested are exosome samples isolated from the culture medium of MCF-7 breast cancer cell line, MDA-MB-231 breast cancer cell line, MDA-MB-453 breast cancer cell line or BT-474 breast cancer cell line.

[0022] According to another aspect of the present invention, a biosensor for detecting exosomes prepared by the preparation method is provided.

[0023] According to another aspect of the present invention, there is provided an application of the biosensor for detecting exosomes secreted by breast cancer cell lines.

[0024] In general, the above technical solution conceived by the present invention has the following advantages compared with the prior art:

[0025] Beneficial effects:

[0026] (1) The present invention provides a new solution for the fixation of receptor molecules in the process of biosensor chip manufacturing. The CD63 antibody or CD9 antibody protein is stably modified on the HEMT gate or the working electrode of the screen-printed three-electrode by using the abundant active sites on the surface of lead sulfide colloidal quantum dots; and the problem of efficient transduction of biomolecular information is solved. With the advantage of the small size of quantum dots, the Debye shielding effect of the modified layer is weakened, and the charge distribution on the surface of the modified electrode changes, which leads to a change in the electric field, so that the biological signal of the specific binding of the transmembrane protein on the surface of the exosome and the antibody protein is efficiently transduced into an electrical signal output. The present invention has high sensitivity for exosome detection, a wide detection range, and greatly reduces sample consumption. At the same time, it is easy to operate, the detection time is shortened to 1 minute, and it has the advantages of integration and batch manufacturing, and has broad application prospects in the fields of disease diagnosis.

[0027] (2) The present invention utilizes the abundant active sites and carboxylic acid groups on the surface of sulfide colloidal quantum dots to achieve dual effective fixation of CD63 antibodies or CD9 antibodies. Colloidal quantum dots are a zero-dimensional semiconductor material that can be stably dispersed in aqueous / organic solutions. They have a size diameter range of 2 to 20 nm, a large number of dangling bonds on the surface, and abundant active sites, which are conducive to the physical adsorption and fixation of antibodies. In addition, the colloidal quantum dots synthesized in the present invention have carboxyl groups on the surface, which will dehydrate and condense with the amino groups in the antibody protein to form a -CO-NH- chemical bond to achieve antibody fixation. No organic crosslinking agents such as PBASE, glutaraldehyde, EDC, NHS, etc. are required, which greatly reduces the complexity and processing time of the biosensor manufacturing process.

[0028] (3) The S element in the sulfide colloidal quantum dots of the present invention can form a gold-sulfur bond (Au-S) with the gold electrode, so that the PbSCQDs / antibody modified layer is further stably modified on the gold electrode and forms a good charge transfer interface, which lays the foundation for exosome molecular information transduction. In addition, the colloidal quantum dots have solution processability and are compatible with a variety of rigid / flexible substrates, which expands the application range of the modified layer.

[0029] (4) Different from the traditional quantum dot luminescence effect to detect biological molecules, colloidal quantum dots have a variety of unique quantum effects (such as small size effect, quantum confinement effect, quantum tunneling effect and Coulomb blockade effect), which can effectively regulate the carrier transport at the colloidal quantum dot-biomolecule interface under the action of an external electric field, promote the charge transfer at the solid-liquid interface on the biosensor surface, and improve the detection sensitivity of the sensor.

[0030] (5) The present invention combines colloidal quantum dots and the intrinsic signal amplification mechanism of HEMT to construct a fast and sensitive HEMT biosensor for exosome detection. When detecting exosome samples, CD63 antibody or CD9 antibody protein will specifically bind to the corresponding transmembrane protein on the surface of the exosome, thereby capturing the exosome on the gate surface and affecting the HEMT gate charge distribution; at the same time, based on the unique physical and chemical properties of sulfide colloidal quantum dots, the capacitance of the gate surface solution increases and the partial pressure of the gate dielectric layer changes. The above two points will affect the gate surface potential at the same time, thereby changing the drain-source current I of the transistor. DS , thus achieving rapid and quantitative detection of exosomes. Experiments have shown that compared with the existing biosensors for detecting exosomes, the HEMT-based biosensor of the present invention can detect exosomes with a detection range of up to 10 5 -10 9 particles / mL, and the minimum detectable amount is 20 particles / uL; the detection range of the three-electrode biosensor for exosomes can reach 10 2 -10 8 particles / mL, with a minimum detectable amount of 19 particles / mL, which is significantly better than the existing exosome detection technology NTA. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a schematic diagram of the principle of using the working electrode of the screen-printed three-electrode chemically modified with sulfide colloidal quantum dots of the present invention for exosome detection.

[0032] Figure 2 It is a schematic diagram of the process of chemically modifying the metal gate of HEMT with sulfide colloidal quantum dots of the present invention.

[0033] Figure 3 The results of lead sulfide quantum dots modifying three electrodes and detecting exosome samples in Example 1;

[0034] Figure 4 This is the result of testing the exosome samples using lead sulfide quantum dots to modify the HEMT chip in Example 2. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0036] The present invention provides a method for preparing a biosensor for exosome detection based on sulfide colloidal quantum dots, comprising the following steps:

[0037] (1) transferring the sulfide colloidal quantum dot solution onto a metal gate electrode of a high electron mobility transistor or onto a working electrode of a screen-printed three-electrode to form a sulfide colloidal quantum dot film;

[0038] (2) coating an antibody protein modification layer on the sulfide colloidal quantum dot film in step (1), and blocking the non-specific active sites on the antibody protein modification layer with bovine serum albumin to prepare a biosensor for exosome detection based on sulfide colloidal quantum dots.

[0039] The surface of the sulfide colloidal quantum dots contains carboxyl groups, which can undergo a dehydration condensation reaction with the amino groups in the antibody protein to form a -CO-NH- chemical bond to achieve antibody fixation; in addition, the active sites on the surface of the lead sulfide colloidal quantum dots are conducive to the electrostatic adsorption of the antibody protein; and the S element in the sulfide colloidal quantum dots can form a chemical bond with the electrode (when the metal gate electrode or the working electrode of the screen-printed three-electrode is a gold electrode, the S element forms a gold-sulfur bond with it; when the working electrode of the screen-printed three-electrode is a carbon electrode, the S element forms a carbon-sulfur bond with it), so that the antibody protein modification layer is further stably modified on the metal gate electrode of the transistor or on the working electrode of the screen-printed three-electrode, and a good charge transfer interface is formed;

[0040] The antibody protein can specifically bind to the transmembrane protein corresponding to the surface of the exosomes to be detected, thereby capturing the exosomes to be detected on the electrode surface. At the same time, based on the quantum effect of the sulfide colloidal quantum dots, the capacitance of the solution on the electrode surface increases, affecting the electrode surface potential and the output current, thereby achieving rapid and quantitative detection of exosomes.

[0041] Figure 1 This is a schematic diagram of the principle of using the working electrode of the screen-printed three-electrode chemically modified with sulfide colloidal quantum dots for exosome detection, wherein 1 is sulfide colloidal quantum dots, 2 is CD63 antibody protein, and 3 is an exosome sample obtained by ultracentrifugation separation, whose surface contains CD63 transmembrane protein. Figure 2It is a schematic diagram of the process of chemically modifying the metal gate of HEMT with sulfide colloidal quantum dots of the present invention. A uniform and dense sulfide colloidal quantum dot film is formed on the metal gate electrode of the transistor chip by spin coating / drip coating / electrofluidic printing technology, and then the device is functionalized with CD63 antibody, and non-specific active sites are blocked with bovine serum albumin.

[0042] The high electron mobility transistor (HEMT) used in the present invention is manufactured by a series of semiconductor processes such as molecular beam epitaxy, photolithography, plasma enhanced chemical vapor deposition, electron beam evaporation, and etching.

[0043] In some embodiments of the present invention, the sulfide colloidal quantum dots, taking lead sulfide colloidal quantum dots as an example, the preparation method thereof comprises the following steps:

[0044] (1) First, 1-octadecene (ODE) was stirred under vacuum at 80°C for 8 hours to extract water and oxygen. Then, 1.8 g of lead oxide (PbO), 6 ml of oleic acid (OA) and 20 ml of ODE were mixed in a three-necked flask and stirred under vacuum at 120°C for 8 hours to obtain a lead precursor.

[0045] (2) In order to obtain the sulfur precursor, 280 μL C6H 18 SSi2 was dissolved in 10 ml ODE.

[0046] (3) In a nitrogen environment, the sulfur precursor is quickly injected into the three-necked flask to react with the lead precursor. It can be observed that the solution changes from transparent to black, that is, PbS crystal nuclei are instantly formed in the solution. After the reaction continues for 30 seconds, the heater is turned off and the three-necked flask is transferred to a cold water bath for rapid cooling.

[0047] (4) After cooling to room temperature, the precipitate was collected and washed several times by centrifugation with toluene and acetone, and then dispersed in n-octane after vacuum drying to finally prepare a PbS CQDs solution with a concentration of 10 mg / mL.

[0048] In some embodiments, the sulfide colloidal quantum dots are lead sulfide colloidal quantum dots, bismuth sulfide colloidal quantum dots, stannous sulfide colloidal quantum dots or zinc sulfide colloidal quantum dots; and the concentration of the sulfide colloidal quantum dots in the sulfide colloidal quantum dot solution is 5-15 mg / mL.

[0049] In some embodiments, step (1) modifies the sulfide colloidal quantum dot solution onto the metal gate electrode of a high electron mobility transistor or the working electrode of a screen-printed three-electrode by spin coating, drop coating or electrofluidic printing; the modification amount is 0.5-1.5 μL.

[0050] In some embodiments, step (2) of coating the antibody protein modification layer on the sulfide colloidal quantum dot film in step (1) specifically comprises the following steps: dropping 0.5-1.5 μL of antibody protein solution onto the surface of the sulfide colloidal quantum dot film, incubating at 37° C. for 1.5-2.5 hours, then washing with phosphate buffer solution to remove unbound antibodies on the surface, and drying at room temperature for later use. The concentration of the antibody protein solution is 0.2-0.5 mg mL -1 .

[0051] In some embodiments, the antibody protein is CD63 antibody or CD9 antibody.

[0052] In some embodiments, in step (2), the bovine serum albumin solution is coated on the antibody-modified electrode surface and blocked at 37° C. for 0.5-1.5 hours to block the nonspecific active sites on the surface, and then the film surface is rinsed with phosphate buffer and dried.

[0053] In some embodiments, the exosomes to be tested are exosome samples isolated from the culture medium of MCF-7 breast cancer cell line, MDA-MB-231 breast cancer cell line, MDA-MB-453 breast cancer cell line or BT-474 breast cancer cell line.

[0054] The exosome detection biosensor prepared by the present invention can be used to detect exosomes secreted by various breast cancer cell lines, including but not limited to MCF-7 breast cancer cell line, MDA-MB-231 breast cancer cell line, MDA-MB-453 breast cancer cell line or BT-474 breast cancer cell line.

[0055] In some embodiments, the prepared biosensor (based on screen-printed glassy carbon three electrodes) was connected to an electrochemical workstation, and phosphate buffer (blank control sample) or exosome test solution with different concentration gradients was added dropwise. The test mode was selected as DPV detection mode, and the starting voltage was set to -0.6V, the end voltage was set to 1V, the pulse width was set to 50ms, the pulse amplitude was set to 50mV, and the potential increment was set to 4mV.

[0056] In other embodiments, the prepared biosensor (based on gold-gate HEMT) was connected to a vacuum probe station, and phosphate buffer (blank control sample) or exosome test solution with different concentration gradients was added dropwise, and the gate voltage V was set by a semiconductor parameter instrument (B1500A). G and drain-source voltage V DS , testing the source-drain current I of the HEMT biosensor DS , the test time remains the same

[0057] The present invention is based on HEMT transistor devices and screen-printed three electrodes, and uses semiconductor nanomaterial sulfide colloidal quantum dots (such as lead sulfide colloidal quantum dots, bismuth sulfide colloidal quantum dots, etc.) as sensitive electrode modification materials to stably fix specific CD63 antibodies or CD9 antibodies on the surface of the biosensor, thereby preparing a biosensor that can quickly capture and quantitatively and sensitively detect exosomes. According to the electrical response curves of the biosensor to exosome samples from healthy people and patients, it is expected to achieve early diagnosis of breast cancer.

[0058] The following are examples:

[0059] Example 1

[0060] (1) Synthesis of lead sulfide colloidal quantum dots. The specific process is as follows: 1.8 g of lead oxide (PbO), 6 ml of oleic acid (OA), and 20 ml of octadecene (ODE, which is obtained by vacuum stirring and extracting water and oxygen at 80°C for 8 hours) are stirred at high speed. After vacuum extraction, the precursor temperature is raised to 120°C and vacuum stirred for 8 hours to obtain a lead precursor. In a glove box, 280 μL of C6H 18 SSi2 was dissolved in 10 mL ODE to prepare the sulfur precursor. The sulfur precursor was quickly injected into the lead precursor in a nitrogen environment and then cooled in a cooling bath for 30 seconds. The collected precipitate was washed with toluene and acetone several times, centrifuged and vacuum dried, and dispersed in n-octane. Finally, a precipitate with a size of about 3.5 nm and a concentration of 10 mg mL -1 PbS colloidal quantum dot solution;

[0061] (2) The lead sulfide quantum dot material (1 μL) synthesized above was modified by drop coating onto the working electrode of the screen-printed glassy carbon three-electrode so that it was evenly distributed on the surface of the working electrode;

[0062] (3) Then use a pipette to add 1 μL of CD63 antibody solution (CD63 antibody is dispersed in phosphate buffered saline (PBS) with a concentration of 0.36 mg mL -1 ) on the surface of the working electrode modified with lead sulfide quantum dots, incubated at 37°C for 2 hours, rinsed with phosphate buffer (1×PBS, 0.067M) to remove the remaining substances on the surface, and dried at room temperature;

[0063] (4) To reduce nonspecific adsorption, 10 mg mL -1 The serum albumin solution (BSA dispersed in phosphate buffer solution (PBS)) blocked the nonspecific active sites on the surface and made them evenly wrapped on the surface of the modified working electrode. After blocking at 37°C for 1 hour, the film surface was rinsed with phosphate buffer and dried.

[0064] (5) Exosome samples were extracted from the culture medium of MCF-7 breast cancer cell line by ultracentrifugation (UC) and diluted into different concentration gradients (10 2 ~10 8 partilcels mL -1 ).

[0065] (6) After connecting the sensor prepared above to an electrochemical workstation (Shanghai Chenhua, CHI660E), phosphate buffer (blank control sample) or exosome test solution with different concentration gradients was added dropwise. The test mode was selected as DPV detection mode, and the pulse starting voltage was set to -0.6V, the end voltage was set to 1V, the pulse width was set to 50ms, the pulse amplitude was set to 50mV, and the potential increment was set to 4mV, so that a single test could be completed within one minute.

[0066] The detection result is drawn as follows Figure 3 As shown in content (a) and content (b), it can be seen that with the increase of exosome concentration, the current peak gradually increases. 2 ~10 8 The logarithmic concentration range of particles / mL showed a linear correlation, and the theoretical detection limit was deduced to be 19 particles / mL (Slope / 3SDblank).

[0067] This example demonstrates the effective fixation of CD63 antibodies by lead sulfide quantum dots, the practical feasibility of using lead sulfide quantum dots to detect exosomes, and the constructed sensor can obtain excellent detection performance.

[0068] Example 2

[0069] (PbS CQDs modified on HEMT chip and detected exosome samples)

[0070] (1) Synthesize lead sulfide quantum dots. For the specific process, refer to step (1) of Example 1.

[0071] (2) The HEMT transistor substrate is cleaned with n-octane and oleic acid, and finally the surface material is removed by cleaning with alcohol. The lead sulfide quantum dot material synthesized above is modified onto the transistor gate by electrofluidic printing so that it is evenly distributed on the metal gate electrode surface of the transistor.

[0072] (3) Then use a pipette to add 1 μL of CD63 antibody solution (CD63 antibody is dispersed in phosphate buffered saline (PBS) with a concentration of 0.36 mg mL -1) on the HEMT gate surface, and after incubation at room temperature for 3 hours, the film surface was rinsed with phosphate buffer (1×PBS, 0.067M) to remove the remaining surface substances and dried at room temperature.

[0073] (4) To reduce nonspecific adsorption, 10 mg mL -1 Serum albumin solution (BSA dispersed in phosphate buffer solution PBS) blocks the nonspecific active sites on the surface, making them evenly wrapped on the surface of the HEMT gate. After blocking for half an hour at room temperature, the film surface is rinsed with phosphate buffer and dried.

[0074] (5) Exosome samples were extracted from the culture medium of MCF-7 breast cancer cell line by ultracentrifugation (UC) and diluted into different concentration gradients (10 5 ~10 9 partilcels mL -1 );

[0075] (6) After the sensor prepared above is connected to the vacuum probe station, phosphate buffer (blank control sample) or exosome test solution with different concentration gradients is added dropwise, and the gate voltage V is set by semiconductor parameter instrument (B1500A). G and drain-source voltage V DS , testing the source-drain current I of the HEMT biosensor DS , the test time remains consistent.

[0076] Draw the detection results as an image Figure 4 As shown in content (a) and content (b), it can be seen that as the concentration of exosomes increases, the current value gradually increases. 5 -10 9 particles / mL showed a linear correlation in the logarithmic concentration range, and the theoretical detection limit was deduced to be 2.0×10 4 particles / mL(Slope / 3SD blank ).

[0077] Based on Example 1, Example 2 fixed lead sulfide quantum dots on the HEMT gate, and also achieved quantitative detection of exosomes. However, from the test results, the detection limit increased, which may be due to the small gate area of ​​the HEMT, and the number of exosomes that can be effectively captured is reduced compared to the three-electrode. However, compared with the three-electrode, HEMT has the advantages of miniaturization and easy integration, which better meets the development trend of future biosensors.

[0078] Table 1 compares different exosome detection technologies in recent years, and compares them with Examples 1 and 2 of the present invention. It can be clearly seen that the exosome biosensors of Examples 1 and 2 of the present invention have a lower detection limit and a wider detection range, which is expected to meet the clinical detection needs of exosomes.

[0079] Table 1. Comparison of different exosome detection technologies

[0080]

[0081] References mentioned in Table 1:

[0082] [1]Precise Capture and Direct Quantification of Tumor Exosomes via aHighly Efficient Dual-Aptamer Recognition-Assisted RatiometricImmobilization-Free Electrochemical Strategy;

[0083] [2]Miniaturized Biomedical Sensors for Enumeration of ExtracellularVesicles;

[0084] [3]In Situ Formation of Gold Nanoparticles Decorated Ti3C2 MXenesNanoprobe for Highly Sensitive Electrogenerated Chemiluminescence Detection of Exosomes and Their Surface Proteins;

[0085] [4]A homogeneous electrochemical sensing platform based on DNAzymewalker for accurate detection of breast cancer exosomes;

[0086] [5]Aptasensor with Expanded Nucleotide Using DNANanotetrahedra for Electrochemical Detection of Cancerous Exosomes;

[0087] [6]Hierarchical Au nanoarrays functionalized 2D Ti2CT x MXene membranes for the detection of exosomes isolated from human lung carcinoma cells;

[0088] [7]Integrated magneto-electrochemical sensor for exosome analysis;

[0089] [8]An electrochemical method for the detection of disease-specific exosomes.

[0090] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a biosensor for exosome detection based on sulfide colloidal quantum dots, characterized in that: The steps include: (1) transferring the sulfide colloidal quantum dot solution onto a metal gate electrode of a high electron mobility transistor or onto a working electrode of a screen-printed three-electrode to form a sulfide colloidal quantum dot film; (2) coating an antibody protein modification layer on the sulfide colloidal quantum dot film in step (1), and blocking the non-specific active sites on the antibody protein modification layer with bovine serum albumin to prepare a biosensor for exosome detection based on sulfide colloidal quantum dots; The surface of the sulfide colloidal quantum dots contains carboxyl groups, which can undergo a dehydration condensation reaction with the amino groups in the antibody protein to form a -CO-NH- chemical bond to achieve antibody fixation; in addition, the active sites on the surface of the lead sulfide colloidal quantum dots are conducive to the electrostatic adsorption of the antibody protein; and the S element in the sulfide colloidal quantum dots can form a chemical bond with the electrode, so that the antibody protein modification layer is further modified on the metal gate electrode of the transistor or on the working electrode of the screen-printed three-electrode, and a good charge transfer interface is formed; The antibody protein can specifically bind to the transmembrane protein corresponding to the surface of the exosomes to be detected, thereby capturing the exosomes to be detected on the electrode surface. At the same time, based on the quantum effect of the sulfide colloidal quantum dots, the capacitance of the solution on the electrode surface increases, affecting the electrode surface potential and the output current, thereby achieving rapid and quantitative detection of exosomes.

2. The preparation method according to claim 1, characterized in that The sulfide colloidal quantum dots are lead sulfide colloidal quantum dots, bismuth sulfide colloidal quantum dots, stannous sulfide colloidal quantum dots or zinc sulfide colloidal quantum dots; and / or, The concentration of the sulfide colloidal quantum dots in the sulfide colloidal quantum dot solution is 5-15 mg / mL.

3. The preparation method according to claim 1, characterized in that: Step (1) modifying the sulfide colloidal quantum dot solution onto the metal gate electrode of a high electron mobility transistor or the working electrode of a screen-printed three-electrode by spin coating, drop coating or electrofluidic printing; the modification amount is 0.5-1.5 μL.

4. The preparation method according to claim 1, characterized in that: Step (2) coating the antibody protein modification layer on the sulfide colloidal quantum dot film in step (1) specifically comprises the following steps: dropping the antibody protein solution onto the surface of the sulfide colloidal quantum dot film, incubating at 37° C. for 1.5-2.5 hours, then rinsing with phosphate buffer solution to remove the unbound antibodies on the surface, and drying at room temperature for later use.

5. The preparation method according to claim 4, characterized in that: 0.5-1.5 μL of antibody protein solution is dropped onto the surface of the sulfide colloidal quantum dot film.

6. The preparation method according to claim 1, characterized in that: The antibody protein is CD63 antibody or CD9 antibody.

7. The preparation method according to claim 1, characterized in that: Step (2) wrapping the bovine serum albumin solution on the antibody-modified electrode surface, blocking at 37° C. for 0.5-1.5 hours to block the non-specific active sites on the surface, and then rinsing the film surface with phosphate buffer and drying.

8. The preparation method according to claim 1, characterized in that: The exosomes to be tested are exosome samples separated from the culture medium of MCF-7 breast cancer cell line, MDA-MB-231 breast cancer cell line, MDA-MB-453 breast cancer cell line or BT-474 breast cancer cell line.

9. A biosensor for detecting exosomes prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the biosensor according to claim 9, characterized in that: Used to detect exosomes secreted by breast cancer cell lines.

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