An electrochemical biosensor based on ultrasonic bidirectional driving and a detection method

Abstract

The application provides an electrochemical biosensor based on ultrasonic bidirectional driving and a detection method, and belongs to the technical field of biosensing. The sensor comprises a porous membrane and an ultrasonic bidirectional driving unit. The pores of the porous membrane are fixed with probes capable of specifically capturing target objects. The ultrasonic bidirectional driving unit is composed of a first ultrasonic transducer and a second ultrasonic transducer symmetrically arranged on both sides of the porous membrane. By synchronously executing reverse power gradient changes, the ultrasonic bidirectional driving unit can form alternating pressure differences on both sides of the membrane, thereby driving the liquid to repeatedly cross the porous membrane. The detection method comprises the steps of injecting the liquid to be detected, ultrasonic bidirectional driving, ultrasonic auxiliary cleaning and electrochemical detection. The application uses bidirectional ultrasonic active driving force to overcome the problems of insufficient probe utilization, low mass transfer efficiency and non-specific adsorption existing in the traditional passive diffusion mechanism, significantly improves the detection sensitivity, speed and signal-to-noise ratio, and is suitable for rapid and high-sensitivity quantitative analysis of various analytes.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biosensing, and particularly relates to an electrochemical biosensor based on ultrasonic bidirectional driving and a detection method. BACKGROUND

[0002] The electrochemical biosensor, with the high efficient coupling characteristics of biological recognition unit and electrochemical signal conversion, has become one of the core technologies for realizing the rapid quantitative detection of biological molecules such as nucleic acid, protein and tumor marker, and has important application value in the fields of disease diagnosis and health monitoring. In the prior art, the electrochemical biosensor based on a porous membrane mainly relies on a passive diffusion mass transfer mechanism to realize the specific binding of a target object and a probe (such as an antibody or an aptamer) fixed in a membrane pore, that is, the target molecules in the sample are naturally diffused to the probe sites on the surface of a carrier (such as an electrode or a membrane pore) through static incubation. However, this technology has the following obvious limitations:

[0003] 1. Insufficient utilization of probes: Under the condition of static incubation, limited target molecules are difficult to effectively contact and bind to the probes in the deep part of the pore, resulting in low capture efficiency, insufficient detection sensitivity for low-concentration samples, and difficulty in meeting the demand for high-sensitivity early diagnosis.

[0004] 2. Low mass transfer efficiency: Under the passive diffusion mode, the transmission rate of the target molecules is limited by Brownian motion, and especially in the micro-nano pore, the diffusion mainly relies on the concentration gradient, resulting in a slow combination process of the probe and the marker, and a significant prolongation of the overall detection time.

[0005] 3. Non-specific adsorption interferes with signal-to-noise ratio: During the passive incubation process, the impurities, cell fragments and the like in the complex sample are easily adsorbed on the surface of the probe carrier through hydrophobic action or electrostatic action, forming background noise, covering the target binding signal, and seriously reducing the specificity and signal-to-noise ratio of the sensor.

[0006] Therefore, there is an urgent need for an electrochemical biosensor technology method that can actively regulate the mass transfer process, improve the utilization efficiency of the probe and effectively inhibit non-specific adsorption. SUMMARY

[0007] The present application proposes an electrochemical biosensor based on ultrasonic bidirectional driving and a detection method to solve the above problems in view of the low mass transfer efficiency, insufficient utilization of probes and non-specific adsorption interference with signal-to-noise ratio caused by the passive diffusion mechanism.

[0008] To achieve the above purpose, the present application adopts the following technical solutions:

[0009] The application discloses an electrochemical biosensor based on ultrasonic bidirectional driving, which comprises a porous membrane and an ultrasonic bidirectional driving unit. A probe for capturing a target object is combined in a channel of the porous membrane, the probe can specifically combine with the target object, and the concentration of the target object is converted into an electrical signal output, so that the detection of the target object in a to-be-detected liquid is realized. The ultrasonic bidirectional driving unit comprises a first ultrasonic transducer and a second ultrasonic transducer which are symmetrically arranged on opposite sides of the porous membrane. The two transducers can synchronously perform a power gradient change operation through the opposite arrangement mode, and through the alternately formed pressure difference with opposite directions on the two sides of the porous membrane, the liquid is actively driven to repeatedly cross the channel of the porous membrane, so that the mass transfer process is significantly enhanced.

[0010] Further, the material of the porous membrane comprises a polyethylene terephthalate (PET) film, a polycarbonate (PC) film or a polyimide (PI) film, the pore size of the channel is 0.01-12 mu m, and the pore density is 1*10 3 ~1*10 15 / cm2, so that the fluid dynamics characteristics of the liquid crossing are considered while the sufficient specific surface area is ensured.

[0011] Further, the inner wall of the channel of the porous membrane is subjected to activation treatment to generate functional groups, so as to provide covalent combination sites for probe fixation.

[0012] Further, the two sides of the porous membrane are respectively provided with a first chamber and a second chamber to form independent fluid cavities. The first chamber is provided with an injection port, the second chamber is provided with a discharge port, a first valve body is arranged at the injection port, and a second valve body is arranged at the discharge port, so as to accurately control the fluid on-off. The structure design realizes the fully-closed operation of the detection process, and avoids external pollution.

[0013] Further, the sensor further comprises an electrochemical detection unit, which comprises a counter electrode arranged in the first chamber, a working electrode arranged in the second chamber, and an electrochemical detector electrically connected with the counter electrode and the working electrode. This design enables the sample processing and signal detection to be completed in the same device, and realizes the integration of the detection process.

[0014] Further, the working frequency of the first ultrasonic transducer and the second ultrasonic transducer is 0.5-5 MHz, and the first ultrasonic transducer and the second ultrasonic transducer are configured to synchronously perform opposite power changes with a period of 2-120 seconds, so that the output powers of the two ultrasonic transducers are synchronously and reversely alternately changed between a high pressure value P1 and a low pressure value P2, wherein P1>P2, the range of the high pressure value P1 is 0.1-1.2 W, and the range of the low pressure value P2 is 0.01-0.8 W.

[0015] The application further provides a detection method of the electrochemical biosensor based on ultrasonic bidirectional driving.

[0016] S1. The test liquid is introduced and driven by ultrasonic waves: the test liquid is injected into the first chamber and the second chamber through the injection port, after the test liquid fills the chambers, the first valve body and the second valve body are closed, the first ultrasonic transducer and the second ultrasonic transducer are started, the output power is alternately changed between the high pressure value P1 of 0.1-1.2 W and the low pressure value P2 of 0.01-0.8 W in a cycle of 2-120 seconds at a working frequency of 0.5-5 MHz, the test liquid is driven to reciprocally pass through the porous membrane, and the process lasts for 5-15 minutes, in which the ultrasonic action significantly improves the binding rate of the target and the probe.

[0017] Cleaning: the first valve body and the second valve body are opened, the cleaning liquid is continuously injected through the injection port, and the first ultrasonic transducer and the second ultrasonic transducer are started, the output power is alternately changed between the high pressure value P1 of 0.1-1.2 W and the low pressure value P2 of 0.01-0.8 W in a cycle of 2-120 seconds at a working frequency of 0.5-5 MHz, the cleaning liquid is driven to reciprocally pass through the porous membrane, and then is discharged from the discharge port, and the process lasts for 3-8 minutes, effectively removing non-specifically adsorbed impurities;

[0018] S3. Electrochemical detection: stop injecting the cleaning liquid, inject the electrolyte into the first chamber and the second chamber through the injection port until the chambers are filled, then close the first valve body and the second valve body and stop the ultrasonic driving, based on the electrochemical detection system constructed by the counter electrode and the working electrode, the electrochemical detector is used to collect the electrochemical response signal of the porous membrane, and the precise quantitative analysis of the target to be detected is realized.

[0019] Further, the electrolyte is 0.05-0.2 mol / L PBS buffer, and the electrochemical detection adopts the cyclic voltammetry or differential pulse voltammetry, and the detection voltage range is-2 V-2 V.

[0020] The detection principle of the application is as follows:

[0021] The electrochemical detection principle of the application is based on the change of the electrochemical signal caused by the specific binding of the target and the probe. When the target on both sides of the porous membrane binds with the probe fixed in the pore channel of the porous membrane, the effective pore size of the porous membrane is reduced or the flux is reduced, thereby changing the ion transmission impedance or charge distribution of the working electrode interface, and finally causing the regular change of the electrochemical response signal (such as current value). The change has a certain correlation with the concentration of the target. By establishing a standard curve with a target standard sample of a known concentration, the detected electrochemical signal can be converted into the actual concentration of the target to be detected, so that the quantitative detection of the target is realized.

[0022] Compared with the prior art, the application has the following beneficial effects:

[0023] 1. The method of driving liquid back and forth through the pores of a porous membrane by alternately forming pressure differences of opposite directions on both sides of the membrane with an ultrasonic bidirectional driving unit, thereby improving the mass transfer rate and the binding efficiency of the target and the probe.

[0024] 2. The method of ultrasonic bidirectional driving strengthens the use of probes in the deep part of the pores of the porous membrane, improves the capture efficiency of the target, and is especially suitable for high-sensitivity detection of low-concentration biomarkers, and can provide reliable technical means for early disease diagnosis.

[0025] 3. Through the designed ultrasonic cleaning step, non-specifically adsorbed impurities on the surface of the membrane and in the pores can be effectively removed, the background noise is reduced, and the signal-to-noise ratio and specificity of the detection are improved. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 A schematic diagram of an electrochemical biosensor based on ultrasonic bidirectional driving

[0027] Figure 2 A power timing diagram of ultrasonic driving

[0028] Figure 3 I-V curve diagram of responses of different concentrations of Mycoplasma pneumoniae under ultrasonic driving and non-ultrasonic driving conditions.

[0029] Figure 4 ΔI-lgC quantification curve diagram of the current change rate of responses of different concentrations of Mycoplasma pneumoniae under ultrasonic driving and non-ultrasonic driving conditions.

[0030] Figure 5 Curve diagram of the current change rate changing with time under ultrasonic driving and non-ultrasonic driving conditions.

[0031] Figure 6 Bar chart of specificity and non-specificity detection under ultrasonic driving and non-ultrasonic driving conditions.

[0032] REFERENCE NUMERALS:

[0033] 1. Porous membrane; 11. First chamber; 111. Inlet; 112. First valve body; 12. Second chamber; 121. Outlet; 122. Second valve body; 2. Ultrasonic bidirectional driving unit; 21. First ultrasonic transducer; 22. Second ultrasonic transducer; 3. Electrochemical detection unit; 31. Counter electrode; 32. Working electrode; 33. Electrochemical detector. DETAILED DESCRIPTION

[0034] The application will be further described below in conjunction with specific embodiments.

[0035] Example 1

[0036] This embodiment specifically provides an ultrasound-based bidirectional driven electrochemical biosensor for detecting Mycoplasma pneumoniae.

[0037] The schematic diagram of the ultrasonic bidirectional driven electrochemical biosensor is as follows: Figure 1 As shown, porous membrane 1 is a polyethylene terephthalate (PET) membrane with a pore size of 0.2 μm and a pore density of 5 × 10⁻⁶. 8 / cm². The probe immobilization process of the porous membrane 1 is as follows:

[0038] First, the PET membrane was placed in a mixed solution prepared with 0.1 M MES buffer (pH 6.0) containing 10 mg / mL EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and 1 mg / mL NHS (N-hydroxysuccinimide) and reacted at room temperature for 1 hour to activate the carboxyl functional groups on the inner wall of the porous membrane pores. Then, the activated porous membrane 1 was immersed in an 8 μM aminated modified capture probe solution for immobilization for 45 min. The capture probe was a specific complementary sequence of the Mycoplasma pneumoniae 23S-5S rDNA spacer sequence (ITS), with the sequence: 5'-GGAAGCCTTTGGTAGGAAATACGCAGG-3'. After immobilization, the membrane was washed with MES buffer to remove unbound capture probes and other impurities. Finally, the remaining active sites on the membrane were blocked using bovine serum albumin (BSA) solution to obtain the functionalized porous membrane 1.

[0039] The first chamber 11 and the second chamber 12 on both sides of the porous membrane 1 are made of polydimethylsiloxane (PDMS). The inlet 111 and outlet 121 are connected to an external injection pump via a silicone tube, forming a controllable fluid path. This allows for the directional injection and discharge of the Mycoplasma pneumoniae test solution inside the sensor, ensuring that the detection is performed in a controlled fluid environment. Both the first valve body 112 and the second valve body 122 employ a one-way valve structure to control the flow path. In the ultrasonic bidirectional drive unit 2, both the first ultrasonic transducer 21 and the second ultrasonic transducer 22 are piezoelectric ceramic transducers. Their operating frequency is set to 1MHz, and they synchronously perform reverse power changes with a 30-second cycle: the high-pressure value P1 is 0.8 W, and the low-pressure value P2 is 0.1 W. During operation, the power changes over time as follows: Figure 2 (The ultrasonic drive power timing diagram is shown). In the electrochemical detection unit 3, the counter electrode 31 is a platinum electrode, the working electrode 32 is a silver chloride electrode, and the electrochemical detector 33 performs electrochemical detection using an electrochemical workstation.

[0040] Example 2

[0041] This embodiment provides a detection method for Mycoplasma pneumoniae based on an ultrasound-driven bidirectional electrochemical biosensor, comprising the following steps:

[0042] (1) Sequentially test samples of different concentrations: Inject a specific concentration of Mycoplasma pneumoniae test solution (e.g., concentrations of 0 and 10) into the first chamber 11 and the second chamber 12 through injection port 111. 2 10 3 10 4 10 5 10 6 10 7 After the test solution fills the first chamber 11 and the second chamber 12, the first valve body 112 and the second valve body 122 are closed. The first ultrasonic transducer 21 and the second ultrasonic transducer 22 are started, with a working frequency of 1MHz and a cycle of 30 seconds, so that the output power alternates synchronously and in opposite directions between a high pressure value P1 of 0.8W and a low pressure value P2 of 0.1W, driving the Mycoplasma pneumoniae test solution to reciprocate through the functionalized porous membrane 1 prepared as in Example 1. This process lasts for 10 minutes.

[0043] (2) Open the first valve body 112 and the second valve body 122, and continuously inject cleaning fluid through the injection port 111. At the same time, start the first ultrasonic transducer 21 and the second ultrasonic transducer 22. With a working frequency of 1MHz and a cycle of 30 seconds, the output power alternates synchronously and in opposite directions between the high pressure value P1 of 0.8W and the low pressure value P2 of 0.1W, driving the cleaning fluid to repeatedly pass through the functionalized porous membrane 1 prepared in Example 1, and then discharge from the outlet. This process lasts for 5 minutes.

[0044] (3) Stop injecting the cleaning solution, and inject 0.1 mol / L PBS buffer through injection port 111 until the first chamber 11 and the second chamber 12 are filled. Close the first valve body 112 and the second valve body 122 and stop the ultrasonic drive. In the completed electrochemical detection unit 3, apply a scanning voltage from -2 V to 2 V, and record the current-voltage (IV) response curve using differential pulse voltammetry. Figure 3 A), the current value measured at a blank control with a Mycoplasma pneumoniae concentration of 0 is recorded as I0, and the current values ​​measured at other concentrations are recorded as I. Quantitative analysis of the target analyte is achieved by calculating the rate of change of current (I0-I) / I0, and a standard curve is plotted with the Mycoplasma pneumoniae concentration on the x-axis and the rate of change of current on the y-axis. Figure 4 (Red curve).

[0045] Example 3

[0046] To verify the beneficial effects achieved by the present invention, three control experiments without ultrasound were set up.

[0047] (1) This control experiment uses the detection method described in Example 2, but in step (1), the first ultrasonic transducer 21 and the second ultrasonic transducer 22 are not activated. Instead, the functionalized porous membrane 1 is incubated in different concentrations of Mycoplasma pneumoniae test solutions for 60 minutes. The remaining experimental steps are the same as in Example 2, and the current-voltage (IV) response curves without ultrasonic action are measured. Figure 3 B), and plotted the corresponding standard curve ( Figure 4 (Black curve).

[0048] pass Figure 3 and Figure 4 Data comparison shows that the current-voltage (IV) under ultrasonic driving is significantly different from that under static incubation, indicating that this invention breaks the limitation of static diffusion, enabling Mycoplasma pneumoniae molecules in the test solution to rapidly transport into the porous membrane channels, greatly improving the "spatial accessibility" of molecules, thereby significantly improving the probe utilization rate in the functionalized porous membrane and enhancing the probe capture efficiency. Simultaneously, both standard curves (with and without ultrasonic driving) show a good linear relationship, indicating the reliability of the detection method proposed in this invention. Furthermore, the curve with ultrasonic driving shifts upwards overall, indicating that at the same concentration, ultrasonic treatment significantly increases the current change rate (I0-I) / I0, i.e., enhances the detection signal. It also improves the detection sensitivity at low concentrations.

[0049] (2) This control experimental system compared the relative change rate of current (I0-I) / I0 over time under ultrasonic driving conditions and static incubation conditions. Electrochemical measurements were performed at time points of 1, 2, 3, 4, 5, 10, 20, 40, 60, 90 and 120 minutes. Figure 5 The results showed that the current change response was more rapid and significant in the ultrasonic-driven group, indicating that the present invention significantly accelerated the binding process between Mycoplasma pneumoniae molecules and functionalized membrane probes, and greatly shortened the time for the reaction to reach stability. In contrast, the response change under static incubation conditions was relatively slow. These results further validate the significant advantages of the ultrasonic-driven strategy in improving detection mass transfer efficiency and response kinetics.

[0050] (3) This control experiment was conducted to investigate the effect of ultrasound-driven conditions on the specificity and signal-to-noise ratio of the target. Standard solutions of Chlamydia pneumoniae, respiratory syncytial virus, metapneumovirus, and rhinovirus were used as controls. Detection was performed with and without ultrasound-driven conditions according to the detection method described in Example 2. The results are as follows: Figure 6As shown, the current change rates induced by Mycoplasma pneumoniae, Chlamydia pneumoniae, respiratory syncytial virus, metapneumovirus, and rhinovirus without ultrasound driving were (23.45±1.3)%, (5.95±1.28)%, (8.74±1.5)%, (4.61±1.8)%, and (3.38±1.43)%, respectively; while the current change rates corresponding to each pathogen under ultrasound driving were (30.32±2.11)%, (4.39±1.02)%, (6.51±2.14)%, (3.66±1.9)%, and (2.87±1.1)%, respectively. These data demonstrate that the present invention significantly improves the detection signal of Mycoplasma pneumoniae, with its current change rate being significantly higher than that of other pathogens, showing good specificity. Simultaneously, the response changes of non-target pathogens under ultrasound conditions were small or even slightly inhibited, indicating that the method has strong anti-interference ability and a high signal-to-noise ratio. While enhancing the target signal, it did not induce significant non-specific binding, further demonstrating the application potential of this detection strategy in complex samples.

[0051] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An electrochemical biosensor based on bidirectional ultrasonic actuation, characterized in that, It includes a porous membrane (1) and an ultrasonic bidirectional drive unit (2); the porous membrane (1) has probes for capturing target objects attached to its pores. The probes can specifically bind to the target objects and convert the concentration of the target objects into an electrical signal output to realize the detection of the target objects in the test liquid; the ultrasonic bidirectional drive unit (2) includes a first ultrasonic transducer (21) and a second ultrasonic transducer (22) respectively disposed on opposite sides of the porous membrane (1). The first ultrasonic transducer (21) and the second ultrasonic transducer (22) are arranged opposite to each other and are configured to synchronously perform power gradient change operation to alternately form pressure differences in opposite directions on both sides of the porous membrane (1) to drive the liquid to repeatedly pass through the porous membrane (1).

2. The electrochemical biosensor based on bidirectional ultrasonic drive according to claim 1, characterized in that, The porous membrane (1) is a polyethylene terephthalate (PET) membrane, a polycarbonate (PC) membrane, or a polyimide (PI) membrane, with a pore size of 0.01~12μm and a pore density of 1×10⁻⁶. 3 ~1×10 15 / cm².

3. The electrochemical biosensor based on bidirectional ultrasonic drive according to claim 2, characterized in that, The porous membrane (1) has its pores inner walls activated to generate functional groups, and the probe is bound to the functional groups via covalent bonds.

4. The electrochemical biosensor based on bidirectional ultrasonic drive according to claim 1, characterized in that, The porous membrane (1) has a first chamber (11) and a second chamber (12) on both sides respectively. The first chamber (11) has an injection port (111) and the second chamber (12) has an outlet port (121). A first valve body (112) is provided at the injection port (111) and a second valve body (122) is provided at the outlet port (121) to control its opening and closing.

5. The electrochemical biosensor based on bidirectional ultrasonic drive according to claim 4, characterized in that, It also includes an electrochemical detection unit (3), which includes a counter electrode (31) disposed in the first chamber (11), a working electrode (32) disposed in the second chamber (12), and an electrochemical detector (33) electrically connected to the counter electrode (31) and the working electrode (32).

6. The electrochemical biosensor based on bidirectional ultrasonic drive according to claim 1, characterized in that, The first ultrasonic transducer (21) and the second ultrasonic transducer (22) operate at a frequency of 0.5~5MHz and are configured to synchronously perform opposite power changes with a period of 2~120 seconds, so that the output power of the two changes synchronously and in opposite directions between a high voltage value P1 and a low voltage value P2, wherein P1 > P2, the high voltage value P1 ranges from 0.1~1.2 W, and the low voltage value P2 ranges from 0.01~0.8 W.

7. A detection method based on an ultrasonic bidirectional driven electrochemical biosensor according to any one of claims 1-6, characterized in that, Includes the following steps: S1. Test liquid introduction and ultrasonic bidirectional drive: The test liquid is injected into the first chamber (11) and the second chamber (12) through the injection port (111). After the test liquid is filled, the first valve body (112) and the second valve body (122) are closed, and the first ultrasonic transducer (21) and the second ultrasonic transducer (22) are started. The output power is synchronously and in reverse alternating between the high pressure value P1 of 0.1~1.2 W and the low pressure value P2 of 0.01~0.8 W at a working frequency of 0.5~5MHz and a period of 2~120 seconds, driving the test liquid to reciprocate through the porous membrane (1) for 5~15 minutes. S2. Cleaning: Open the first valve body (112) and the second valve body (122), and continuously inject cleaning fluid through the injection port (111). At the same time, start the first ultrasonic transducer (21) and the second ultrasonic transducer (22) with a working frequency of 0.5~5MHz and a cycle of 2~120 seconds, so that the output power alternates between a high pressure value P1 of 0.1~1.2 W and a low pressure value P2 of 0.01~0.8 W synchronously and in reverse, driving the cleaning fluid to reciprocate through the porous membrane (1), and then discharge from the outlet, for 3~8 minutes. S3. Electrochemical detection: Stop injecting cleaning solution, inject electrolyte through injection port (111) until the first chamber (11) and the second chamber (12) are filled, close the first valve body (112) and the second valve body (122) and stop ultrasonic driving. Based on the electrochemical detection system constructed by the counter electrode (31) and the working electrode (32), the electrochemical response signal of the porous membrane (1) is collected by the electrochemical detector (33) to realize the accurate quantitative analysis of the target analyte.

8. The detection method according to claim 7, characterized in that, The electrolyte is 0.05~0.2 mol / L PBS buffer, and the electrochemical detection is performed using cyclic voltammetry or differential pulse voltammetry, with a detection voltage range of -2V~2V.

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