Biosensor, kit and detection method for rapidly and highly sensitively detecting uranyl ions

Abstract

The invention relates to a biosensor, a kit and a detection method for rapidly and highly sensitively detecting uranyl ions. According to the biosensor, a four-leaf clover-shaped rolling loop mediated quadruple Walker is adopted as a signal amplifier to amplify a signal to detect UO2 < 2 + >; the four-leaf clover-shaped rolling loop mediated quadruple Walker comprises an L1 / L2 / L3 / L4 / Asist compound, a UO2 < 2 + > dependent DNAzyme substrate chain, a streptavidin modified magnetic bead and a buffer solution. In the presence of UO2 < 2 + >, the activated Walker can circularly cut a fluorescently-labeled substrate chain to generate an amplified fluorescence signal, so that high-sensitivity and high-specificity detection of UO2 < 2 + > is realized.

Description

Technical Field

[0001] This invention relates to the technical field of analytical detection, specifically to a biosensor, reagent kit, and detection method for rapid and highly sensitive detection of uranyl ions. Background Technology

[0002] Due to its high radioactivity, chemical toxicity, water solubility, and long half-life, uranium in contaminated soil can easily enter the food chain, ultimately posing a serious threat to human health. As a common stable form of uranium in phytoremediation, uranyl ions have become a key indicator for accurately assessing the enrichment level of uranium in vivo.

[0003] Currently UO2 2+ Conventional detection methods, such as inductively coupled plasma mass spectrometry (ICP-MS) and X-ray fluorescence spectroscopy (XRF), while possessing extremely high sensitivity and accuracy, require centralized laboratories, complex equipment, and specialized personnel. Novel methods, such as fluorescence spectroscopy and electrochemical sensors, are often affected by interference from coexisting ions, require the synthesis of complex probes, or rely on expensive and unstable enzymes.

[0004] To improve detection sensitivity, various signal amplification techniques have been widely adopted as efficient tools. Among them, DNAzyme biosensors have attracted attention due to their advantages such as flexible design and low cost; however, the sensitivity of existing technologies is often insufficient to meet the needs of trace detection. Therefore, there is an urgent need to develop an environmentally friendly, economical, and low-maintenance solution for uranium contamination remediation and detection.

[0005] Chinese patent document CN 112255173 A discloses a kit and method for the visual detection of uranyl ions. The kit typically includes a chromogenic agent, a buffer solution, and a colorimetric card. This technical solution utilizes UO2... 2+ Uranyl ions undergo a complexation reaction with a specific chromogenic reagent, and the color change is observed visually or the absorbance is measured using a portable spectrometer, then compared with a colorimetric card to achieve semi-quantitative or quantitative detection. However, such colorimetric methods based on traditional complexation reactions generally have low detection sensitivity, with detection limits typically only at the micromolar level. Furthermore, these methods suffer from poor selectivity and insufficient specificity.

[0006] Therefore, there is an urgent need to develop a UO2 with high sensitivity and excellent selectivity. 2+ The detection method makes the detection process highly sensitive, reduces costs, and is easy to promote. Summary of the Invention

[0007] To address the shortcomings of the existing technologies, this invention aims to provide a biosensor, reagent kit, and detection method for the rapid and highly sensitive detection of uranyl ions, solving the problem that existing technologies struggle to achieve ultra-trace UO2 at the picomolar (pM) level in environmental samples. 2+ It achieves precise detection, especially by raising the detection limit to the pM level, while also possessing higher sensitivity and excellent selectivity.

[0008] To solve the above problems, the present invention adopts the following technical solution: In a first aspect, the present invention provides a biosensor for rapid and highly sensitive detection of uranyl ions, wherein the biosensor employs a cloverleaf quadruple Walker amplifier as a signal amplifier to amplify and detect UO2. 2+ ; The four-leaf clover-shaped quadruple Walker includes an enzyme chain L1 / L2 / L3 / L4 / Assist complex and UO2. 2+ DNA-dependent substrate strand, streptavidin-modified magnetic beads, and buffer solution; the UO2 2+ The DNA-dependent substrate strand contains the fluorescent group CY5 and the fluorescence quenching group BH.

[0009] Furthermore, the UO2 2+ The sequence of the substrate strand of the DNA-dependent DNAzyme is shown in SEQ ID NO. 1.

[0010] Furthermore, the sequence of the L1 / Assist complex is shown in SEQ ID NO.2.

[0011] Furthermore, the sequence of the L2 / Assist complex is shown in SEQ ID NO.3.

[0012] Furthermore, the sequence of the L3 / Assist complex is shown in SEQ ID NO.4.

[0013] Furthermore, the sequence of the L4 / Assist complex is shown in SEQ ID NO.5.

[0014] Furthermore, in the sequence SEQ ID NO.1, rA is the cleavage site, and T(CY5) represents the T-base quenching group CY5; in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, T(BHQ2) all represent the T-base fluorophore BHQ2.

[0015] Furthermore, the buffer solution is a 50 mM MES buffer solution containing 300 mM NaCl, with a pH of 5.5.

[0016] Secondly, this invention provides a label-free fluorescent detection method for UO2. 2+ The kit includes: L1 / Assist complex, L2 / Assist complex, L3 / Assist complex, L4 / Assist complex, and UO2. 2+ The invention comprises a DNA-dependent substrate strand, streptavidin-modified magnetic beads, and a buffer solution, wherein the buffer solution includes a MES buffer solution at pH 5.5; the sequence of the DNA-dependent substrate strand is shown in SEQ ID NO.1; the sequence of the L1 / Assist complex is shown in SEQ ID NO.2; the sequence of the L2 / Assist complex is shown in SEQ ID NO.3; the sequence of the L3 / Assist complex is shown in SEQ ID NO.4; and the sequence of the L4 / Assist complex is shown in SEQ ID NO.5.

[0017] Thirdly, the present invention provides a label-free fluorescent detection of UO2 using the aforementioned kit. 2+ The method, characterized in that it includes: The L1 / Assist complex, L2 / Assist complex, L3 / Assist complex, and L4 / Assist complex were mixed in 50 mM MES buffer and pre-hybridized at 95°C for 5 minutes to form the enzyme chain L1 / L2 / L3 / L4 / Assist complex. The enzyme chain L1 / L2 / L3 / L4 / Assist complex and the substrate chain Substratestrand CY5-S labeled with fluorescein CY5 / BHQ2 were mixed with MBs and streptavidin magnetic beads at a molar ratio of 50:150:1 and shaken at 25°C for 24 h. Magnetic separation and washing were performed, the solution was centrifuged at 13000 rpm for 25 min and washed with sodium phosphate buffer. The solution was resuspended in 50 mM MES buffer to form a cloverleaf rolling ring mediated quadruple Walker. Add the sample to be tested and incubate at 25°C for 80 minutes. Detect the fluorescence signal at an excitation wavelength of 650 nm and an emission wavelength of 670 nm. Based on the linear relationship between the concentration of the enzyme-digested substrate strand and the fluorescence intensity, UO2 is detected. 2+ .

[0018] The beneficial effects of this invention are as follows: This invention calculates UO2 using fluorescence signals.2+ The concentration makes the detection process rapid and simple, with high sensitivity, specificity, and excellent selectivity; it also reduces costs, is easy to promote, and is effective against UO2 in the environment. 2 + Rapid detection is of great significance. Attached Figure Description

[0019] Figure 1 This invention provides a label-free fluorescent detection method for UO2. 2+ The schematic diagram of the method.

[0020] Figure 2 This invention provides a label-free fluorescent detection method for UO2. 2+ The results are shown in the figure below; where A is the real-time fluorescence kinetics of the DNase cleavage reaction; B is the PAGE electrophoresis analysis of four cloverleaf DNase Walker (lane 1: L1, lane 2: L1+L2, lane 3: L1+L2+L3, lane 4: L1+L2+L3+L4); C is the dynamic light scattering diagram of DNA-free (blue) and DNA-containing (orange) microspheres; D is the UV-Vis absorption curve of DNA-free and DNA-containing microspheres (the histogram shows the absorbance intensity at 260 nm, and the error bar represents the standard deviation of three independent measurements at each reaction temperature).

[0021] Figure 3 For biosensors subjected to different concentrations of UO2 2+ The results after processing are shown in the figure; where A is a typical fluorescence spectrum; and B is the relationship between target concentration and fluorescence response (four clover-shaped carriers: 100 nM; orbital: 300 nM).

[0022] Figure 4 Label-free fluorescent detection of UO2 2+ The method for different concentrations of UO2 2+ The specific detection experimental results are shown in the figure; where A represents different concentrations of strontium ions (Sr). 2+ ), cesium ions (Cs 2+ ), cadmium ions (Cd) 2+ ), manganese ions (Mn 2+ ), copper ions (Cu 2+ ), zinc ions (Zn 2+ ), chromium ions (Cr 3+ ), lead ions (Pb 2+ Fluorescence standard curves for thiamethoxam (TMX), atrazine (AT), and penicillin (PCN); B is... Figure 4 The fluorescence value of A at 670 nm. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to specific embodiments.

[0024] It should be noted that these embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Simple improvements to the method under the premise of the present invention are all within the scope of protection claimed by the present invention.

[0025] Example 1 The uranyl ion detection biosensor uses a cloverleaf rolling ring mediated quadruple Walker amplifier as a signal amplifier to amplify and detect UO2. 2+ The clover-shaped rolling circle-mediated tetra-Walke complex includes the enzyme chain L1 / L2 / L3 / L4 / Assist complex and UO2. 2+ The enzyme consisted of a DNA-dependent substrate strand, streptavidin-modified magnetic beads, and a buffer solution; the L1 / L2 / L3 / L4 / Assist complex contained UO2. 2+ The enzyme strands enzyme strands (L1, L2, L3, L4) and the assist strand of the DNA-dependent DNA enzyme work together to produce UO2. 2+ The DNA-dependent substrate chain Substratestrand contains the fluorescent group CY5, the fluorescence quenching group BHQ2, and rA bases for co-modification, while the enzyme chains L1 / L2 / L3 / L4 / Assist contain the fluorescence quenching group BHQ2 for co-modification.

[0026] UO2 2+ The sequence of the DNA-dependent substrate strand is shown in SEQ ID NO.1; SEQ ID NO.1 is: 5'-Biotin-ACTCACTAT(CY5)rAGGAAGAGAT(BHQ2)GGACGTG-3'; The sequence of the L1 / Assist complex is shown in SEQ ID NO.2, which is: 5'-Biotin-ATAGGTTCATAGGTTCATCCCCCACGTCCATCTCTGCAGTCGGGTAGTTAAACCGACCTTCAGACAT(BHQ2)AGTGAGTCCCCCGAAACTTCGAAAC-3'; The sequence of the L2 / Assist complex is shown in SEQ ID NO.3, which is: 5'-Biotin-GTTTCGAAGTTTCGCCCCCACGTCCATCTCTGCAGTCGGGTAGTTAAACCGACCTTCAGACAT(BHQ2)AGTGAGTCCCCCTTGAATGCTTGAATGCT-3'; The sequence of the L3 / Assist complex is shown in SEQ ID NO.4, which is: 5'-Biotin-AGCATTCAAGCATTCAAGCCCCCACGTCCATCTCTGCAGTCGGGTAGTTAAACCGACCTTCAGACAT(BHQ2)AGTGAGTCCCCGCTTTGAAGCTTTG-3'; The sequence of the L4 / Assist complex is shown in SEQ ID NO.5, which is: 5'-Biotin-CAAAGCTTCAAAGCCCCCCACGTCCATCTCTGCAGTCGGGTAGTTAAACCGACCTTCAGACAT(BHQ2)AGTGAGTCCCCATGAACCTATGAACCTAT-3'.

[0027] In sequence SEQ ID NO.1, rA is the cleavage site, and T(CY5) represents the T-base quenching group CY5; in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, T(BHQ2) all represent the T-base fluorophore BHQ2.

[0028] The buffer solution comprises a 50 mM MES buffer containing 300 mM NaCl, with a pH of 5.5. This simulates physiological conditions for base pairing. The composition, type, and pH of the buffer solution may vary slightly.

[0029] like Figure 1 As shown, the reaction principle of this technical solution is as follows: the clover-shaped walking device is constructed by pre-hybridizing BHQ2-labeled enzyme chains (L1, L2, L3, and L4), and uses the CY5 / BHQ2-modified substrate chain S as the walking track. Without the introduction of UO2... 2+ At this time, the clover-mediated tetrapod reaction is inactive. The fluorescence signal is quenched due to the close proximity of BHQ2 and CY5, resulting in a low background signal. When UO2 is introduced... 2+Subsequently, it specifically binds to DNase and activates the four-leaf clover-mediated four-walking reaction. The activated walker then simultaneously cleaves four adjacent CY5 / BHQ2-labeled orbitals, releasing BHQ2-labeled DNA fragments. The free four-leaf clover-mediated walker can then bind to new orbitals, initiating multiple walker cycles. Finally, the amplified fluorescence signal generated by the microarray platform can be used to detect trace amounts of UO2. 2+ .

[0030] The method exhibits a linear range from 10 pM to 1000 nM and a detection limit of 1.9 pM. It also demonstrates significant selectivity for other possible analogues. Actual UO2 from environmental water samples. 2+ Analysis shows that this method has good precision and accuracy.

[0031] Label-free fluorescence detection of UO2 2+ The test kit contains the following components: L1 / Assist complex, L2 / Assist complex, L3 / Assist complex, L4 / Assist complex, and UO2. 2+ The product consists of a DNA-dependent substrate strand, streptavidin-modified magnetic beads, and a buffer solution, including a MES buffer solution at pH 5.5.

[0032] Label-free fluorescence detection of UO2 2+ The method, specifically the steps, are as follows: Formation of the S1 L1 / L2 / L3 / L4 / Assist complex: The L1 / Assist, L2 / Assist, L3 / Assist, and L4 / Assist complexes were mixed in 50 mM MES buffer (300 mM NaCl, pH 5.5). Pre-hybridization was performed at 95°C for 5 minutes, followed by slow cooling to room temperature to form the enzyme chain L1 / L2 / L3 / L4 / Assist complex. S2 Cloverleaf Rolling Circle-Mediated Quadruple Walker Formation: The prepared enzyme chain L1 / L2 / L3 / L4 / Assist complex, CY5 / BHQ2-labeled substrate strand, and streptavidin magnetic beads were mixed at a molar ratio of 50:150:1 and incubated with shaking at 25°C for 24 hours to immobilize the substrate strand on the magnetic beads via biotin-streptavidin interaction. Subsequently, the mixture was magnetically separated and washed, and the supernatant was discarded. The solution was then centrifuged at 13000 rpm for 25 minutes. It was washed three times with sodium phosphate buffer (PBS, 5 M NaCl, 200 mM Na2HPO4 / NaH2PO4, 0.05% Tween 20, pH 7.4) to remove unbound DNA probes. Finally, it was resuspended in 50 mM MES buffer.

[0033] S3 UO2 2+ Detection: Four-leaf clover-shaped rolling ring mediated quadruple Walker enzyme was incubated with streptavidin magnetic beads at room temperature for 60–120 minutes, followed by measurement of fluorescence signal values ​​(observation blank group). The test sample was added to the product from step S2 and incubated at 25°C for 80 minutes, then fluorescence signals were detected at an excitation wavelength of 650 nm and an emission wavelength of 670 nm. Based on the linear relationship between the concentration of the enzyme-digested substrate strand and the fluorescence intensity, UO2 was detected. 2+ The purpose.

[0034] Example 2: Feasibility of using kinetics to determine uranyl ion concentration, the specific steps are as follows: To validate the detection of UO2 by a fluorescent biosensor 2+ We verified its feasibility through solution tests under different conditions. For example... Figure 2 As shown in A, the real-time kinetic curves illustrate two types of UO2. 2+ Fluorescence response from 0 to 140 minutes at concentrations (0 nM blank control and 1000 nM). The blank control signal remained consistently low, while the signal after adding 1000 nM UO2... 2+ Subsequently, the fluorescence signal rapidly increased and stabilized after 100 minutes, indicating that the DNase walker achieved continuous activation and continuous cleavage. When UO2 was added... 2+ At that time, the solution signal significantly increased, indicating that the tetrapod walking mechanism mediated by the four-leaf clover structure was activated, forming a circular cleavage track on the magnetic bead surface, releasing CY5-labeled DNA fragments, thereby generating a high fluorescence signal for UO2. 2+ Testing.

[0035] The assembly steps of the clover-shaped walking machine were also verified by native polyacrylamide gel electrophoresis (PAGE). Figure 2 (B in the original text). Adding L1, L2, L3, and L4 successfully formed a four-leaf clover-shaped walker. These electrophoresis results not only verified the stepwise assembly process of the probe but also confirmed the formation of the four-leaf clover-shaped structure in the sensing system. Dynamic light scattering (DLS) detection results were further validated by comparing the particle size changes of microspheres (MBs) before and after the biotinylated DNA reaction. Figure 2 As shown in C, after biotinylated DNA modification, the average hydrodynamic diameter of MBs increased significantly from 14.92 nm to 68.69 nm, which fully demonstrates the success of DNA modification. Figure 2 Data from D showed that the DNA fixation efficiency reached 56.65%, indicating that most DNA molecules had been successfully anchored on the surface of the microspheres.

[0036] Example 3: Label-free fluorescence detection of UO2 2+ The method for different concentrations of UO2 2+ The specific steps for conducting the test are as follows: First, prepare UO2 2+ Standard solutions with concentrations of 0, 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1000 nM, and 2000 nM were stored at 4 °C; different concentrations of UO2 were then used. 2+ The solutions were tested using the detection method of Example 1. After the reaction was complete, the fluorescence intensity was observed. Figure 3 ( Figure 3 A in the figure: Under the condition of λex = 650 nm, different UO2 2+ The emission spectrum corresponding to the concentration; Figure 3 B in the equation: F under the condition of λex = 650 nm λem The fluorescence standard curve obtained at 670 nm is shown in the figure. UO2 at 1.9 pM... 2+ A noticeable fluorescence change was observed, indicating a detection limit of 1.9 pM. With UO2... 2+ As the concentration increases, the fluorescence intensity also increases, and gradually approaches saturation.

[0037] Example 4: Label-free fluorescence detection of UO2 2+ The method for different concentrations of UO2 2+ The specific detection steps are as follows: Prepare 0, 100 nM, 200 nM, and 300 nM standard solutions of other metal ions that may coexist in the aqueous environment, namely strontium ions (Sr... 2+ ), cesium ions (Cs 2+ ), cadmium ions (Cd) 2+), manganese ions (Mn 2+ ), copper ions (Cu 2+ ), zinc ions (Zn 2+ ), chromium ions (Cr 3+ ), lead ions (Pb 2+ Thiamethoxam (TMX), atrazine (AT), and penicillin (PCN); Different concentrations of interfering standard solutions (0, 100 nM, 200 nM, and 300 nM) and UO2 were respectively used. 2+ The solutions were reacted using the method described in Example 1, and the changes in fluorescence intensity were observed after complete reaction. The changes in fluorescence intensity are as follows: Figure 4 As shown, where Figure 4 In the figure, A represents the standard fluorescence curve. Figure 4 B in Figure 4 The fluorescence value of A at 670 nm in the image is from... Figure 4 Both A and B in the diagram show different concentrations of strontium ions (Sr). 2+ ), cesium ions (Cs 2+ ), cadmium ions (Cd) 2+ ), manganese ions (Mn 2+ ), copper ions (Cu 2+ ), zinc ions (Zn 2 + ), chromium ions (Cr 3+ ), lead ions (Pb 2+ The fluorescence intensities of thiamethoxam (TMX), atrazine (AT), and penicillin (PCN) were all significantly lower than those of UO2. 2+ This proves that the method is effective for UO2. 2+ The detection has good specificity.

[0038] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described with reference to preferred embodiments, those skilled in the art should understand that various changes in form and detail can be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A biosensor for rapid and high sensitive detection of uranyl ion, characterized in that, The biosensor adopts a clover-shaped quadruple Walker as a signal amplifier to detect UO2 2+ ; The clover shaped quadruple Walker comprises enzyme strands L1 / L2 / L3 / L4 / Assist complex, UO2 2+ Dependent DNAzyme substrate strand, streptavidin modified magnetic beads and buffer solution; the UO2 2+ Dependent DNAzyme substrate strand contains a fluorescent group CY5 and a fluorescence quenching group BH.

2. The biosensor of claim 1, wherein, The UO2 2+ The sequence of the substrate strand of the dependent DNAzyme is shown as SEQ ID NO.

1.

3. The biosensor of claim 1, wherein, The sequence of the L1 / Assist complex is shown as SEQ ID NO.

2.

4. The biosensor of claim 1, wherein, The sequence of the L2 / Assist complex is shown as SEQ ID NO.

3.

5. The biosensor of claim 1, wherein, The sequence of the L3 / Assist complex is shown as SEQ ID NO.

4.

6. The biosensor of claim 1, wherein, The sequence of the L4 / Assist complex is shown as SEQ ID NO.

5.

7. The biosensor of claim 1, wherein, The rA in the sequence of SEQ ID NO. 1 is a cleavage site, and T(CY5) represents a T base label quencher group CY5; the T(BHQ2) in the sequences of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5 all represent a T base label fluorescent group BHQ2.

8. The uranium ion rapid detection biosensor according to claim 1, wherein, The buffer solution is a 50 mM MES buffer solution containing 300 mM NaCl, and the pH is 5.

5.

9. A kit for the label-free fluorescent detection of UO2 2+ characterized in that, Comprising: L1 / Assist complex, L2 / Assist complex, L3 / Assist complex, L4 / Assist complex, UO2 2+ Dependent DNAzyme substrate strand, streptavidin-modified magnetic beads, and buffer solution, the buffer solution including MES buffer solution, pH = 5.5; the sequence of the dependent DNAzyme substrate strand is shown in SEQ ID NO. 1; the sequence of the L1 / Assist complex is shown in SEQ ID NO. 2; the sequence of the L2 / Assist complex is shown in SEQ ID NO. 3; the sequence of the L3 / Assist complex is shown in SEQ ID NO. 4; the sequence of the L4 / Assist complex is shown in SEQ ID NO.

5.

10. A method for label-free fluorescent detection of UO2 using the kit of claim 9, characterized in that, 2+ Comprising: ​ The L1 / Assist complex, the L2 / Assist complex, the L3 / Assist complex and the L4 / Assist complex are mixed in a 50 mM MES buffer solution, pre-hybridized at 95°C for 5 minutes to form an enzyme chain L1 / L2 / L3 / L4 / Assist complex; The enzyme chain L1 / L2 / L3 / L4 / Assist complex and the fluorescein CY5 / BHQ2 labeled substrate strand Substrate strand CY5-S are mixed with MBs and streptavidin magnetic beads at a molar ratio of 50:150:1, and oscillated at 25°C for 24 hours; magnetic separation and washing are performed, the solution is centrifuged at 13000 rpm for 25 minutes, and washed with a sodium phosphate buffer solution; the solution is resuspended in a 50 mM MES buffer solution to form a four-leaf clover-shaped rolling circle mediated four-way Walker; Add the sample to be tested, incubate at 25°C for 80 minutes, detect the fluorescence signal at excitation wavelength 650 nm, emission wavelength 670 nm; according to the linear relationship between the concentration of the enzyme-cleaved substrate strand and the fluorescence intensity, detect UO2 2+ .

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