Starfish biosensor design method based on cascade signal amplification strategy

Abstract

The invention provides a cascading signal amplification strategy-based design method of a starfish biosensor, and belongs to the field of biosensor design, and the method comprises the following steps: designing a DNA probe, constructing an electrochemical biosensor, carrying out electrochemical test on the constructed electrochemical biosensor, and detecting the biosensor. Construction and feasibility analysis of the electrochemical biosensor are carried out, performance evaluation of the electrochemical biosensor is carried out, and design of the electrochemical biosensor is completed. The electrochemical biosensor combines a catalytic hairpin assembly (CHA) strategy and a hybrid chain reaction (HCR) strategy, signal amplification without depending on protease labeling is achieved, and the trace detection requirement is met. In the field detection of South China Sea, the applicability of the electrochemical biosensor is verified again. In addition, compared with ddPCR, the reliability and accuracy of the biosensor are further verified.

Description

Technical Field

[0001] The invention relates to the field of biosensor design, and in particular to a method for designing a long-spined sea star biosensor based on a cascade signal amplification strategy. Background Art

[0002] As a natural enemy of reef-building corals, repeated outbreaks of crown-of-thorns starfish (COTS) have become one of the most important causes of the coral reef crisis in the Indo-Pacific region. Repeated outbreaks of crown-of-thorns starfish have led to massive coral mortality and a continuous decline in coral coverage, which has seriously affected the sustainability and biodiversity of marine ecosystems. Surveys show that between 1985 and 2012, the live coral cover of Australia's Great Barrier Reef decreased by 50.7%, of which 42% of the corals died from outbreaks of crown-of-thorns starfish, and it is currently experiencing its fourth severe outbreak of crown-of-thorns starfish. In addition, in some waters of the South China Sea, the mortality rate of corals due to outbreaks of crown-of-thorns starfish is as high as 95%. However, due to the low visibility and hidden behavior of crown-of-thorns starfish larvae and adults, conventional ecological survey methods such as manta ray trawling can only detect 5-10% of crown-of-thorns starfish, which cannot accurately track the early stages of the disaster outbreak, thus hindering timely intervention. Therefore, there is still an urgent need for reliable and accurate early warning technologies for crown-of-thorns starfish outbreaks.

[0003] At present, environmental DNA (eDNA) molecular detection technology has shown great potential in biological monitoring due to its non-invasiveness, high efficiency and accuracy. Based on this, Uthicke and Kwong et al. used eDNA technology to detect low-density long-spined sea star populations through digital droplet PCR (ddPCR), and quantified the shedding rate and degradation rate of eDNA, providing support for early warning of long-spined sea star outbreaks. However, since eDNA is easily affected by various external factors and is difficult to store and transport for a long time, the above methods also require large instruments and well-trained personnel. Therefore, it is particularly important to develop an on-site, low-cost and rapid method for the detection of long-spined sea star eDNA to improve monitoring efficiency and accuracy.

[0004] In recent years, electrochemical biosensors have become an attractive means of molecular detection due to their portability, simplicity, economy and high sensitivity. The working principle of these biosensors is to convert the hybridization events of target molecules with probes fixed on the electrode surface into quantifiable signals (such as impedance, potential or current) and record them by electrochemical methods. It is worth noting that Wang et al. used electrochemical biosensors for the first time to realize the rapid on-site detection of long-spined sea star environmental DNA (eDNA), providing a new method for monitoring long-spined sea stars in the early stages of an outbreak. However, the dilution effect of the ocean results in quite low eDNA concentration levels in the ocean. Therefore, higher sensitivity and lower detection limits remain a huge challenge for accurate early warning of long-spined sea star disasters.

[0005] In order to solve the above problems, signal amplification strategies are needed to meet the requirements of high sensitivity and low detection limit. At present, enzyme-free DNA self-assembly amplification strategies, including catalytic hairpin assembly (CHA) or hybridization chain reaction (HCR), are widely used in the construction of biosensors. CHA is to start the assembly of hairpin probes under the catalysis of specific targets, produce a series of double-stranded DNA (dsDNA), with low background and target cyclic amplification. HCR is to trigger the programmed alternating hybridization of two hairpin DNAs through target recognition to generate long dsDNA, which can provide exponential amplification effect and achieve target amplification. However, the efficiency of a single amplification strategy (CHA or HCR) is still limited. Therefore, the combination of the above amplification techniques is conducive to providing more accurate analytical performance.

[0006] Electrochemical biosensors are widely used in the field of DNA detection because of their portability, ease of operation, economy and high sensitivity. Wang et al. constructed an electrochemical biosensor with a single-probe strategy and used it for the first time to rapidly detect long-spined sea star environmental DNA (eDNA) on site, providing a new method for monitoring long-spined sea stars in the early stages of an outbreak. However, due to the dilution effect of the ocean, the concentration level of eDNA in the ocean is quite low. Electrochemical biosensors based on a single-probe strategy are susceptible to strong background signals and are prone to false positives. At the same time, the working principle of the single-probe strategy is to directly hybridize with the target DNA without undergoing a signal amplification process, so it is impossible to meet the needs of on-site trace detection. Therefore, higher sensitivity and lower detection limits remain a huge challenge for accurate early warning of long-spined sea star disasters. Summary of the invention

[0007] The purpose of the present invention is to provide a method for designing a long-spined sea star biosensor based on a cascade signal amplification strategy to solve the technical problems mentioned in the background technology.

[0008] The problem of low sensitivity and poor specificity of the traditional single-probe strategy, which is difficult to meet the requirements of on-site trace detection of long-spined sea star eDNA, has been solved. The present invention has developed a new electrochemical biosensor that adopts a cascade amplification strategy to improve detection sensitivity. The electrochemical biosensor combines catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) strategies to achieve signal amplification without relying on protease labeling, meeting trace detection requirements. The applicability of the electrochemical biosensor has been verified again in on-site detection in the South China Sea. In addition, by comparing with ddPCR, the reliability and accuracy of the biosensor were further verified.

[0009] In order to solve the problem that the traditional single-probe strategy has low sensitivity and poor specificity, and is difficult to meet the requirements of on-site trace detection, a new type of electrochemical biosensor was successfully constructed, which adopted a cascade amplification strategy to improve the detection sensitivity. The electrochemical biosensor combines the catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) strategies to achieve signal amplification without relying on protease labeling, and achieves on-site trace level long-spined sea star eDNA tracing. Therefore, the electrochemical biosensor based on the cascade amplification strategy developed by the present invention has high sensitivity, strong applicability, and high accuracy, and provides a powerful tool for on-site long-spined sea star eDNA detection and long-spined sea star outbreak warning.

[0010] The CHA and HCR amplifications were combined for ultrasensitive detection of long-spined sea star eDNA. Due to the combination of CHA and HCR, the biosensor can achieve signal amplification without proteases and labels, solving the problems of low sensitivity and poor specificity of traditional single-probe strategies. In addition, the portable biosensor was used to detect long-spined sea star eDNA on-site at several typical epidemic sites in the South China Sea. Therefore, the present invention provides new insights and unique technical support for on-site warning of long-spined sea star outbreaks.

[0011] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0012] A method for designing a long-spined sea star biosensor based on a cascade signal amplification strategy, the method comprising the following steps:

[0013] Step 1: Design DNA probe;

[0014] Step 2: Construction of electrochemical biosensor;

[0015] Step 3: Electrochemical testing of the constructed electrochemical biosensor;

[0016] Step 4: Construction and feasibility analysis of electrochemical biosensor;

[0017] Step 5: Evaluate the performance of the electrochemical biosensor and complete the design of the electrochemical biosensor.

[0018] Further, in step 1, the sequence of the capture probe of the DNA probe is: TGG TTC TGT TTA TAG TGGATT TT(C6)-SH, the sequence of the hairpin probe 1 is: TAT AAA CAG AAC CAC GCT AGA TAA TGG AGG GTAGAC CAA CCA CCT CTA CCC TCC ATT AT, the sequence of the hairpin probe 2 is: CTT GGG TTT CAT GTG TGTAGA GGT GGT TGG TCT ACC CTC CAT TCC AAC CAC C, the sequence of the hairpin probe 3 is: ACA CAT GAAACC CAAGTG CTG ACT TGG GTT TC, the sequence of the hairpin probe 4 is: CTT GGG TTT CAT GTG TGA AACCCA AGT CAG CA, and the sequence of the target DNA is: TCT ACC CTC CAT TAT CTA GCG;

[0019] All hairpin probes were annealed at 95°C for 10 min before use and then cooled to room temperature at a rate of 1°C / min to ensure the formation of stable hairpins. All probes were stored at 4°C before use.

[0020] Furthermore, the specific process of step 2 is:

[0021] The gold electrode AuE was polished with 0.30 and 0.05 μm alumina powders, and then the residual alumina on the electrode surface was removed by alternating ultrasonic treatment with ethanol and ultrapure water for 30 seconds. Next, the AuE was electrochemically cleaned and activated in 0.5 MH2SO4. Finally, the electrode was cleaned with ultrapure water and dried in a nitrogen stream to obtain a bare gold electrode with a clean surface.

[0022] Before use, the capture probe CP needs to be treated with 10mM TCEP for 1 hour to reduce the disulfide bonds. Then, 10μLCP is dropped on the AuE surface and incubated at 4°C overnight. Finally, the electrode surface is treated with 1mM MCH at room temperature in the dark for 30 minutes to block the nonspecific binding sites on the surface. Finally, the constructed electrode is stored at 4°C before use, and the electrode is rinsed with washing buffer after each modification.

[0023] Further, in step 3, target DNA of different concentrations was incubated with hairpin probe 1 and hairpin probe 2 at 37°C for 2 hours to perform CHA amplification reaction, and then the CHA product was dropped onto the pre-prepared AuE surface and hybridized with the CP fixed on AuE for 0.5 hours. Next, 15 μL of a mixture of hairpin probe 3 and hairpin probe 4 was dropped onto the electrode surface and incubated at 37°C for 1.5 hours to perform HCR amplification reaction. Finally, 10 μL of methylene blue was dropped onto the electrode surface and incubated at room temperature for 15 minutes to allow MB to embed into the double-stranded DNA on the electrode surface. After completing the above steps, the electrode was connected to the workstation and the target DNA was electrochemically tested. The electrode was rinsed with washing buffer after each modification.

[0024] Electrochemical measurements were performed using a three-electrode system. DPV was performed by immersing the electrode in a working buffer solution with a pulse width of 0.05 s, a pulse period of 0.2 s, a pulse amplitude of 50 mV, and a potential scanning range of -0.5 V to 0.1 V. The cyclic voltammetry (CV) parameters were set to a potential range of -0.2 to 0.6 V and a scanning rate of 0.1 V / s. The frequency range of the electrochemical impedance spectroscopy (EIS) test was 10 KHz to 0.1 Hz, and the Coulomb curve test parameters were set to a potential range of 0.5 to -0.2 V, a pulse width of 0.25 s, and a sampling interval of 2 ms. The portable electrochemical biosensor was performed using an integrated chip electrode, and the DPV test parameters were set to a scanning voltage of -0.55 to -0.15 V, a pulse voltage of 0.05 V, a pulse width of 0.05 s, and a pulse period of 0.5 s.

[0025] Furthermore, in step 4, the stepwise assembly steps of the electrochemical biosensor are characterized by electrochemical impedance spectroscopy. The EIS curves of all electrodes are composed of high-frequency arcs and low-frequency linear curves, where the semicircle diameter of the high-frequency arc region determines the electron transfer kinetics at the electrode interface. The Randles equivalent circuit is used to fit the EIS data, where R S Represents the solution resistance, R ct represents the charge transfer resistance, Z W represents the Warburg diffusion element, C dl Represents the double-layer capacitance between the electrode and the electrolyte solution. The impedance curve of AuE is approximately a straight line. After fitting, R ct The capture probe is then anchored to the AuE surface via Au-S bonds, and the negatively charged DNA phosphate backbone is bonded to [Fe(CN)6] 3- / 4- The repulsive force between molecules makes R ct Increase, after fixing MCH on the electrode, R ctIn the presence of target DNA, the hairpin probe 1 / hairpin probe 1 complex generated by the CHA reaction will be captured by the CP on the electrode surface, forming a longer dsDNA chain on the electrode surface, allowing the DNA phosphate backbone to interact with [Fe(CN)6] 3- / 4- The repulsive force between molecules is enhanced, thereby increasing R ct By adding hairpin probes 3 and 4, HCR is carried out on the electrode surface, and negatively charged DNA chains are introduced, making the electrode surface and [Fe(CN)6] 3- / 4- The repulsive force between molecules is enhanced, increasing R ct , the results proved the successful construction of the long-spined sea star DNA biosensor;

[0026] In order to evaluate the feasibility of the constructed electrochemical biosensor for the detection of eDNA of long-spined sea star, differential pulse voltammetry tests were performed. In the absence of target DNA, only hairpin probe 1 could hybridize with CP and thus anchor on the electrode surface, generating a background signal lower than the set value. In contrast, in the presence of target DNA, the CHA reaction was triggered to form a stable hairpin probe 1 / hairpin probe 2 complex, and the current signal increased. Next, when the electrode was incubated with hairpin probes 3 and 4, the HCR reaction was triggered to generate dsDNA on the electrode surface, and the current signal increased. In the presence of target DNA, CHA and HCR were successfully carried out, resulting in longer dsDNA molecules and more MB accumulation, and a higher current signal was obtained.

[0027] Further, in step 5, under the experimental conditions of CP concentration of 1.25 μM, MB incubation time of 15 min, CHA reaction time of 120 min, and HCR reaction time of 90 min, the analytical performance of the constructed electrochemical biosensor based on the programmed CHA / HCR cycle amplification strategy was evaluated by DPV. As the target DNA concentration increased from 50 fM to 10 nM, the current signal gradually increased, which is consistent with the fact that the increase in target DNA concentration promotes the production of more dsDNA and further embeds more MB to display electrical signals. According to formula (1), there is a linear relationship between the current signal and the logarithm of the target DNA concentration, with a correlation coefficient of 0.982:

[0028] I=1.799lgC+28.983 (1)

[0029] The detection limit LOD of the constructed electrochemical biosensor was calculated based on 3 times the SD of the blank detection as the benchmark, and was 18.4 fM. The LOD of the constructed electrochemical biosensor based on the programmed CHA / HCR cycle amplification strategy was 10 lower than that of the existing electrochemical biosensor based on the single probe strategy. 6According to the 10-fold SD corresponding to the blank test, the LOQ of the constructed electrochemical biosensor was 41.1 fM, achieving ultrasensitive detection of long-spined sea star eDNA.

[0030] The present invention has the following beneficial effects due to the adoption of the above technical solution:

[0031] (1) The present invention solves the problem that the traditional single-probe strategy has low sensitivity and poor specificity, which makes it difficult to meet the requirements of on-site trace detection of long-spined sea star eDNA. A new electrochemical biosensor is developed, which adopts a cascade amplification strategy to improve the detection sensitivity. The electrochemical biosensor combines catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) strategies to achieve signal amplification without relying on protease labeling, meeting the requirements of trace detection. The applicability of the electrochemical biosensor was verified again in the on-site detection in the South China Sea. In addition, the reliability and accuracy of the biosensor were further verified by comparison with ddPCR.

[0032] (2) Through the cascade amplification strategy, i.e., the combination of catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR), a new electrochemical biosensor was successfully constructed for the detection of eDNA of long-spined sea star. The sensor constructed by this strategy can achieve signal amplification without relying on proteases and labels, thus realizing the tracing of long-spined sea star eDNA at trace levels on site. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 1 is the EIS and DPV response diagram of the electrochemical biosensor of different modified electrodes of the present invention;

[0034] Figure 2 is a graph of the DPV response of the present invention and the corresponding calibration curve of the target using the developed biosensor;

[0035] Figure 3 are the selective sensing results, reproducibility and sensing result graphs in species used for biosensor selectivity studies of the present invention;

[0036] Figure 4 It is a diagram of on-site detection results of the present invention. DETAILED DESCRIPTION

[0037] 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 with reference to the accompanying drawings and preferred embodiments. However, it should be noted that many details listed in the specification are only for the purpose of enabling the reader to have a thorough understanding of one or more aspects of the present invention, and these aspects of the present invention can be implemented even without these specific details.

[0038] A method for designing a long-spined sea star biosensor based on a cascade signal amplification strategy, the method comprising the following steps:

[0039] Step 1: DNA probe design

[0040] The oligonucleotides used in the present invention were synthesized and purified by Shanghai Sangon Biotechnology Co., Ltd. (China), and the oligonucleotide sequences are shown in Table 1.

[0041] Table 1 Oligonucleotide sequences used in this work

[0042]

[0043]

[0044] Hairpin probe 1 (H1) and hairpin probe 2 (H2) were designed based on the principle of enzyme-free strand displacement nucleic acid loop system. All hairpin probes were annealed at 95°C for 10 minutes before use, and then slowly cooled to room temperature at a rate of 1°C / min to ensure the formation of stable hairpins. Finally, all probes were stored at 4°C before use.

[0045] Step 2: Construction of electrochemical biosensor

[0046] First, the gold electrode (AuE) was polished with 0.30 and 0.05 μm alumina powder. Subsequently, ethanol and ultrapure water were alternately ultrasonicated for 30 seconds to remove the residual alumina on the electrode surface. Next, the AuE was electrochemically cleaned and activated in 0.5M H2SO4. Finally, the electrode was washed with ultrapure water and dried in a nitrogen stream. Through the above steps, a bare AuE with a clean surface was finally obtained.

[0047] Before use, the capture probe (CP) needs to be treated with 10mM TCEP for 1 hour to reduce the disulfide bonds. Then, 10μL CP (1.25μM) was dropped on the AuE surface and incubated at 4°C overnight. Finally, the electrode surface was treated with 1mM MCH at room temperature in the dark for 30 minutes to block the nonspecific binding sites on the surface. Finally, the constructed electrode was stored at 4°C before use. It should be noted that the electrode should be rinsed with washing buffer (PBS, pH=7.4) after each modification.

[0048] Step 3: Electrochemical Testing

[0049] First, different concentrations of target DNA were incubated with H1 (1 μM) and H2 (1 μM) at 37 °C for 2 h to perform CHA amplification reaction. Subsequently, the CHA product was added dropwise to the pre-prepared AuE surface and hybridized with the CP immobilized on AuE for 0.5 h. Next, 15 μL of a mixture of hairpin probe 3 (H3, 1 μM) and hairpin probe 4 (H4, 1 μM) was dropped on the electrode surface and incubated at 37 °C for 1.5 h for HCR amplification reaction. Finally, 10 μL of methylene blue (MB, 100 μM) was dropped on the electrode surface and incubated at room temperature for 15 min to allow MB to embed into the double-stranded DNA on the electrode surface. After completing the above steps, the electrode was connected to the workstation and the target DNA was electrochemically tested. It should be noted that the electrode should be rinsed with washing buffer (PBS, pH = 7.4) after each modification.

[0050] Electrochemical measurements were performed using a three-electrode system. DPV was performed by immersing the electrode in a working buffer solution, with a pulse width of 0.05s, a pulse period of 0.2s, a pulse amplitude of 50mV, and a potential scan range of -0.5V to 0.1V; the cyclic voltammetry (CV) parameters were set to a potential range of -0.2 to 0.6V, a scan rate of 0.1V / s; the frequency range of the electrochemical impedance spectroscopy (EIS) test was 10KHz to 0.1Hz; the Coulomb curve (CC) test parameters were set to a potential range of 0.5 to -0.2V, a pulse width of 0.25s, and a sampling interval of 2ms. The portable electrochemical biosensor was performed using an integrated chip electrode, and the DPV test parameters were set to a scan voltage of -0.55 to -0.15V, a pulse voltage of 0.05V, a pulse width of 0.05s, and a pulse period of 0.5s.

[0051] Step 4: Sensor construction and feasibility analysis, characterize the step-by-step assembly steps of the electrochemical biosensor by electrochemical impedance spectroscopy (EIS). Figure 1 As shown in (A), the EIS curves of all electrodes are composed of high-frequency arcs and low-frequency linear curves, where the semicircular diameter of the high-frequency arc region determines the electron transfer kinetics at the electrode interface. The Randles equivalent circuit is used to fit the EIS data ( Figure 1 (A), inset), where R S Represents the solution resistance, R ct represents the charge transfer resistance, Z W represents the Warburg diffusion element, C dl Represents the double layer capacitance between the electrode and the electrolyte solution. The impedance curve of AuE is approximately a straight line. After fitting, R ctThe capture probe (CP) is then anchored to the AuE surface via Au-S bonds, and the negatively charged DNA phosphate backbone is bonded to [Fe(CN)6] 3- / 4- The repulsive force between molecules makes R ct Then, after fixing MCH on the electrode, R ct This is due to the poor conductivity of MCH, which increases R ct In addition, in the presence of target DNA, the H1 / H2 complex generated by the CHA reaction will be captured by the CP on the electrode surface, forming a long dsDNA chain on the electrode surface, allowing the DNA phosphate backbone to interact with [Fe(CN)6] 3- / 4- The repulsive force between molecules is enhanced, thereby increasing R ct (2625Ω). Subsequently, by adding H3 and H4, HCR was rapidly carried out on the electrode surface, introducing a large number of negatively charged DNA chains, making the electrode surface and [Fe(CN)6] 3- / 4- The repulsive force between molecules is enhanced, which significantly increases R ct (3670Ω). Therefore, the above results demonstrated the successful construction of the long-spined sea star DNA biosensor.

[0052] In order to evaluate the feasibility of the constructed electrochemical biosensor for detecting eDNA of long-spined sea star, differential pulse voltammetry (DPV) tests were performed ( Figure 1 (B)). In the absence of target DNA, only H1 can hybridize with CP and thus anchor on the electrode surface, generating a low background signal. In contrast, in the presence of target DNA, a CHA reaction is triggered, forming a stable H1 / H2 complex, and the current signal increases significantly. Next, when the electrode is incubated with H3 and H4, an HCR reaction is triggered, generating a large amount of dsDNA on the electrode surface, and the current signal increases significantly. Therefore, in the presence of target DNA, CHA and HCR proceed successfully, resulting in longer dsDNA molecules and more MB accumulation, resulting in a higher current signal.

[0053] Figure 1 (A) EIS of different modified electrodes. Among them, (a1) AuE; (a2) CP / AuE;

[0054] (a3)MCH / CP / AuE; (a4)target+H1+H2 / MCH / CP / AuE;

[0055] (a5) H3+H4 / target+H1+H2 / MCH / CP / AuE. (B) DPV response of electrochemical biosensor. Among them, (b1) H3+H4 / without target+H1+H2 / MCH / CP / AuE; (b2) 1nM target+H1+H2 / MCH / CP / AuE; (b3) H3+H4 / 1nM target+H1+H2 / MCH / CP / AuE.

[0056] Step 5: Performance evaluation. Under the optimal experimental conditions (CP concentration, 1.25 μM; MB incubation time, 15 min; CHA reaction time, 120 min; HCR reaction time, 90 min), the analytical performance of the constructed electrochemical biosensor based on the programmed CHA / HCR cycle amplification strategy was evaluated using DPV. First, as the target DNA concentration increased from 50 fM to 10 nM, the current signal gradually increased ( Figure 2 (A)), which is consistent with the fact that the increase in target DNA concentration promotes the production of more dsDNA and further embeds more MBs to show electrical signals. According to (1), there is an excellent linear relationship between the current signal and the logarithm of the target DNA concentration ( Figure 2 (B)), the correlation coefficient is 0.982:

[0057] I=1.799lgC+28.983 (1)

[0058] The detection limit (LOD) of the constructed electrochemical biosensor was calculated based on 3 times the SD of the blank detection as the benchmark to be 18.4 fM (0.125 fg / μL). It is worth noting that the LOD of the electrochemical biosensor based on the programmed CHA / HCR cycle amplification strategy constructed in this work is 10 lower than the LOD of the previously reported electrochemical biosensor based on the single probe strategy. 6 In addition, according to the 10-fold SD corresponding to the blank test, the limit of quantification (LOQ) of the constructed electrochemical biosensor is 41.1 fM. Therefore, the electrochemical biosensor constructed by the present invention has a wide linear detection range, low LOD and low LOQ, which is helpful to achieve ultrasensitive detection of eDNA of long-spined sea star.

[0059] Figure 2 (A) DPV responses and (B) corresponding calibration curves for 50 fM, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM, and 10 nM targets using the developed biosensor.

[0060] Selectivity is an important parameter for evaluating sensor performance. In this work, we selected some typical species living in the coral reef area of ​​the South China Sea as interfering species to evaluate the selectivity of biosensors ( Figure 3 (A)), including Favia speciosa, Chaetodon speculum, Choriaster granulatus, Pocilloporamadamicornis and Sargocentron caudimaculatum. Among them, the DNA concentration of the interfering species used for detection is more than 10 times that of the long-spined sea star DNA. Figure 3 As shown in (B), the difference between the current response of the interfering species and the current response of the blank sample is negligible. In the presence and absence of interfering species, the response current to the long-spined sea star DNA produced similar and significantly enhanced current responses. Therefore, the above results show that the electrochemical biosensor we constructed based on the cascade amplification strategy has excellent selectivity.

[0061] In addition, five identically prepared electrodes were used to perform parallel assays with different target DNA concentrations (100 fM, 10 pM, and 1 nM) to evaluate the reproducibility of the electrochemical biosensor. Figure 3 As shown in (C), the relative standard deviations of the three concentrations were 4.71%, 4.28%, and 3.33%, respectively, indicating that the constructed electrochemical biosensor achieved satisfactory reproducibility. In addition, the constructed biosensor electrode was stored at 4°C for several days, and then 1 nM target DNA was detected to evaluate the stability of the biosensor ( Figure 3 (D)). The results show that the electrode still maintained 91% of the initial response current on the 4th day. As the storage time increases, the DNA chain will partially degrade, but the response current of the electrode remains at 83% after 6 days, which meets the requirements of practical applications. Therefore, the above results show that the electrochemical biosensor we constructed has excellent reproducibility and stability.

[0062] Figure 3 (A) Species used for biosensor selectivity study. (B) Selective sensing results of the developed biosensor. (C) Reproducibility of the biosensor (0.0001, 0.01 and 1 nM target, respectively). (D) Sensing results of the developed biosensor after storage for 1-7 days. Error bars represent standard deviation (n=3). ***P≤0.001, ***P≤0.01, ns>0.05.

[0063] Step 6: On-site detection. To further study the applicability of the electrochemical biosensor based on the cascade amplification strategy we developed, we conducted on-site detection of long-spined sea star eDNA at six sampling sites in the Xisha Islands in the South China Sea. A portable biosensor based on screen-printed carbon electrodes (SPCE) was used throughout the on-site analysis. The response current of the portable biosensor directly reflects the concentration level of long-spined sea star eDNA at each sampling site (histogram). Strong current signals were detected at sites Q1, Q3, Q4, Q5, and Q6, indicating that the concentration of long-spined sea star eDNA was high; on the contrary, at site Q2, the biosensor could only detect background signals. In addition, we also used ddPCR to verify the accuracy of the on-site detection results. The detection results of the portable biosensor were completely consistent with those of the ddPCR detection, and there was a strong correlation between the two (r=0.992; P<0.001). This shows that the portable electrochemical biosensor has completely reliable accuracy and specificity for on-site detection.

[0064] Portable electrochemical biosensing device. Map of sampling sites in the Xisha Islands of the South China Sea and histogram of COTS eDNA concentrations from field tests. Inset shows the correlation between COTS eDNA quantification results provided by the portable biosensor and COTS eDNA quantification results obtained by ddPCR.

[0065] The present invention successfully constructed a new electrochemical biosensor for the detection of eDNA of long-spined sea star through a cascade amplification strategy, i.e., a strategy combining catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR). The sensor constructed by this strategy can achieve signal amplification without relying on proteases and labels, and realizes the tracing of long-spined sea star eDNA at trace levels on site.

[0066] In order to solve the problem that the traditional single-probe strategy has low sensitivity and poor specificity, and is difficult to meet the requirements of on-site trace detection, a new type of electrochemical biosensor was successfully constructed, which adopted a cascade amplification strategy to improve the detection sensitivity. The electrochemical biosensor combines the catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) strategies to achieve signal amplification without relying on protease labeling, and achieves on-site trace level long-spined sea star eDNA tracing. Therefore, the electrochemical biosensor based on the cascade amplification strategy developed by the present invention has high sensitivity, strong applicability, and high accuracy, and provides a powerful tool for on-site long-spined sea star eDNA detection and long-spined sea star outbreak warning.

[0067] The probe sequence used in the present invention is designed for the specific DNA fragment of long-thorn sea star, but probes designed with other specific DNA fragments of long-thorn sea star can also realize the present invention. Therefore, the cascade amplification strategy using other specific fragments of long-thorn sea star as target recognition sites is an alternative solution.

[0068] In the present invention, we use a gold electrode. However, the present invention can also be achieved by electrodepositing gold nanoparticles on a glassy carbon electrode, so using a glassy carbon electrode treated with gold nanoparticles is an alternative.

[0069] The signal amplification strategy used by this biosensor is a cascade combination signal amplification strategy of catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR). First, CP is fixed to the electrode surface through Au-S bonds. Subsequently, MCH acts as a blocking agent to block the nonspecific binding sites and active sites on the AuE surface to prevent nonspecific connection of the probe. In the presence of target DNA, H1 and target DNA are opened through a chain-mediated reaction and form a Target / H1 complex, exposing the trigger chain of H2. Then, the Target / H1 complex undergoes a chain displacement reaction with H2 to form a more stable H1 / H2 complex. The released target DNA participates in another CHA reaction, thereby generating a large number of H1 / H2 complexes to achieve cyclic amplification of the target DNA. The H1 / H2 complex can hybridize with CP and be fixed to the electrode surface. Next, the HCR amplification process of H3 and H4 will be triggered by the H1 / H2 complex on the electrode surface, thereby generating a longer double-stranded DNA (dsDNA) polymer on the electrode surface. Finally, the electroactive indicator MB molecule will be inserted into the dsDNA groove to achieve signal amplification. In contrast, when there is no target DNA, the progress of the above-mentioned programmed CHA / HCR cycle amplification strategy will be hindered. H1 and H2 will maintain a hairpin structure, the CHA reaction will not occur, and only H2 will hybridize with CP and be fixed to the electrode surface. Similarly, the HCR reaction cannot proceed. Ultimately, compared with when the target DNA is present, only a small amount of MB is inserted into the dsDNA groove. Therefore, the above strategy is a reliable solution for ultrasensitive detection of eDNA of long-spined sea star.

[0070] The CHA and HCR amplifications are cleverly combined for ultrasensitive detection of long-spined sea star eDNA. Due to the combination of CHA and HCR, the biosensor can achieve signal amplification without proteases and labels, solving the problems of low sensitivity and poor specificity of traditional single-probe strategies. In addition, the portable biosensor was used to detect long-spined sea star eDNA on-site at several typical epidemic sites in the South China Sea. Therefore, the present invention provides new insights and unique technical support for on-site warning of long-spined sea star outbreaks.

[0071] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for designing a long-spined sea star biosensor based on a cascade signal amplification strategy, characterized in that: The method comprises the following steps: Step 1: Design DNA probe; Step 2: Construction of electrochemical biosensor; Step 3: Electrochemical testing of the constructed electrochemical biosensor; Step 4: Construction and feasibility analysis of electrochemical biosensor; Step 5: Evaluate the performance of the electrochemical biosensor and complete the design of the electrochemical biosensor.

2. The method for designing a long-spined sea star biosensor based on a cascade signal amplification strategy according to claim 1, characterized in that: In step 1, the sequence of the capture probe of the DNA probe is: TGG TTC TGT TTATAG TGG ATT TT(C6)-SH, the sequence of the hairpin probe 1 is: TAT AAA CAG AAC CAC GCT AGA TAA TGG AGG GTAGACCAACCA CCT CTA CCC TCC ATT AT, the sequence of the hairpin probe 2 is: CTT GGG TTT CAT GTG TGT AGAGGT GGT TGG TCT ACC CTC CAT TCC AAC CAC C, the sequence of the hairpin probe 3 is: ACACAT GAAACCCAAGTG CTG ACT TGG GTT TC, the sequence of the hairpin probe 4 is: CTT GGG TTT CAT GTG TGA AAC CCAAGT CAG CA, and the sequence of the target DNA is: TCT ACC CTC CAT TAT CTA GCG; All hairpin probes were annealed at 95°C for 10 min before use and then cooled to room temperature at a rate of 1°C / min to ensure the formation of stable hairpins. All probes were stored at 4°C before use.

3. The method for designing a long-spined sea star biosensor based on a cascade signal amplification strategy according to claim 1, characterized in that: The specific process of step 2 is: The gold electrode AuE was polished with 0.30 and 0.05 μm alumina powders, and then the residual alumina on the electrode surface was removed by alternating ultrasonic treatment with ethanol and ultrapure water for 30 seconds. Next, the AuE was electrochemically cleaned and activated in 0.5 M H2SO4. Finally, the electrode was cleaned with ultrapure water and dried in a nitrogen flow to obtain a bare gold electrode with a clean surface. Before use, the capture probe CP needs to be treated with 10mM TCEP for 1 hour to reduce the disulfide bonds. Then, 10μL CP is dropped on the AuE surface and incubated at 4°C overnight. Finally, the electrode surface is treated with 1mM MCH at room temperature in the dark for 30 minutes to block the surface nonspecific binding sites. Finally, the constructed electrode is stored at 4°C before use, and the electrode is rinsed with washing buffer after each modification.

4. The method for designing a long-spined sea star biosensor based on a cascade signal amplification strategy according to claim 1, characterized in that: In step 3, target DNA of different concentrations was incubated with hairpin probe 1 and hairpin probe 2 at 37°C for 2 hours to perform CHA amplification reaction. Subsequently, CHA product was added dropwise to the pre-prepared AuE surface and hybridized with CP fixed on AuE for 0.5 hours. Next, 15 μL of a mixture of hairpin probe 3 and hairpin probe 4 was added dropwise to the electrode surface and incubated at 37°C for 1.5 hours to perform HCR amplification reaction. Finally, 10 μL of methylene blue was added dropwise to the electrode surface and incubated at room temperature for 15 minutes to allow MB to embed into the double-stranded DNA on the electrode surface. After completing the above steps, the electrode was connected to the workstation and the target DNA was electrochemically tested. The electrode was rinsed with washing buffer after each modification. Electrochemical measurements were performed using a three-electrode system. DPV was performed by immersing the electrode in a working buffer solution with a pulse width of 0.05 s, a pulse period of 0.2 s, a pulse amplitude of 50 mV, and a potential scanning range of -0.5 V to 0.1 V. The cyclic voltammetry (CV) parameters were set to a potential range of -0.2 to 0.6 V and a scanning rate of 0.1 V / s. The frequency range of the electrochemical impedance spectroscopy (EIS) test was 10 KHz to 0.1 Hz, and the Coulomb curve test parameters were set to a potential range of 0.5 to -0.2 V, a pulse width of 0.25 s, and a sampling interval of 2 ms. The portable electrochemical biosensor was performed using an integrated chip electrode, and the DPV test parameters were set to a scanning voltage of -0.55 to -0.15 V, a pulse voltage of 0.05 V, a pulse width of 0.05 s, and a pulse period of 0.5 s.

5. The method for designing a long-spined sea star biosensor based on a cascade signal amplification strategy according to claim 1, characterized in that: In step 4, the stepwise assembly steps of the electrochemical biosensor are characterized by electrochemical impedance spectroscopy. The EIS curves of all electrodes are composed of high-frequency arcs and low-frequency linear curves. The semicircle diameter of the high-frequency arc region determines the electron transfer kinetics at the electrode interface. The Randles equivalent circuit is used to fit the EIS data, where R S Represents the solution resistance, R ct represents the charge transfer resistance, Z W represents the Warburg diffusion element, C dl Represents the double-layer capacitance between the electrode and the electrolyte solution. The impedance curve of AuE is approximately a straight line. After fitting, R ct The capture probe is then anchored to the AuE surface via Au-S bonds, and the negatively charged DNA phosphate backbone is bonded to [Fe(CN)6] 3- / 4- The repulsive force between molecules makes R ct Increase, after fixing MCH on the electrode, R ct In the presence of target DNA, the hairpin probe 1 / hairpin probe 1 complex generated by the CHA reaction will be captured by the CP on the electrode surface, forming a longer dsDNA chain on the electrode surface, allowing the DNA phosphate backbone to interact with [Fe(CN)6] 3- / 4- The repulsive force between molecules is enhanced, thereby increasing R ct By adding hairpin probes 3 and 4, HCR is carried out on the electrode surface, and negatively charged DNA chains are introduced, making the electrode surface and [Fe(CN)6] 3- / 4- The repulsive force between molecules is enhanced, increasing R ct , the results proved the successful construction of the long-spined sea star DNA biosensor; In order to evaluate the feasibility of the constructed electrochemical biosensor for the detection of eDNA of long-spined sea star, differential pulse voltammetry tests were performed. In the absence of target DNA, only hairpin probe 1 could hybridize with CP and thus anchor on the electrode surface, generating a background signal lower than the set value. In contrast, in the presence of target DNA, the CHA reaction was triggered to form a stable hairpin probe 1 / hairpin probe 2 complex, and the current signal increased. Next, when the electrode was incubated with hairpin probes 3 and 4, the HCR reaction was triggered to generate dsDNA on the electrode surface, and the current signal increased. In the presence of target DNA, CHA and HCR were successfully carried out, resulting in longer dsDNA molecules and more MB accumulation, and a higher current signal was obtained.

6. The method for designing a long-spined sea star biosensor based on a cascade signal amplification strategy according to claim 1, characterized in that: In step 5, under the experimental conditions of CP concentration of 1.25 μM, MB incubation time of 15 min, CHA reaction time of 120 min, and HCR reaction time of 90 min, the analytical performance of the constructed electrochemical biosensor based on the programmed CHA / HCR cycle amplification strategy was evaluated by DPV. As the target DNA concentration increased from 50 fM to 10 nM, the current signal gradually increased, which is consistent with the fact that the increase in target DNA concentration promotes the production of more dsDNA and further embeds more MB to display electrical signals. According to formula (1), there is a linear relationship between the current signal and the logarithm of the target DNA concentration, with a correlation coefficient of 0.982: I=1.799lgC+28.983 (1) The detection limit LOD of the constructed electrochemical biosensor was calculated based on 3 times the SD of the blank detection as the benchmark, and was 18.4 fM. The LOD of the constructed electrochemical biosensor based on the programmed CHA / HCR cycle amplification strategy was 10 lower than that of the existing electrochemical biosensor based on the single probe strategy. 6 According to the 10-fold SD corresponding to the blank test, the LOQ of the constructed electrochemical biosensor was 41.1 fM, achieving ultrasensitive detection of long-spined sea star eDNA.

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