A non-labeled Cd based on SYBR Green I dye 2+ Rapid detection fluorescent aptamer sensor and its quantitative detection kit

Abstract

This invention belongs to the field of environmental and food safety testing technology, and specifically discloses a label-free Cd based on SYBR Green I dye. 2+ A rapid detection fluorescent aptamer sensor and its quantitative detection kit are designed to achieve highly sensitive and specific quantitative detection of cadmium ions in samples such as fruits, vegetables, and food products. The fluorescent aptamer sensor includes the nucleic acid aptamer Cd-(21) and its complementary strand CS. Cd‑(21) And SYBR Green I dye; this fluorescent aptamer sensor is used to detect Cd. 2+ This invention offers advantages such as simple operation, short processing time, high sensitivity, and good selectivity. It also provides a matching reagent kit containing the necessary aptamers, complementary strands, SGI, buffer solutions, and other reagents, facilitating operation and application. The kit is simple in design, suitable for various laboratory and field testing applications, and has broad application prospects. The label-free fluorescent aptamer sensor and its matching reagent kit of this invention are Cd... 2+ This provides a new solution for efficient detection, with good technical and practical application value.

Description

Technical Field

[0001] This invention relates to a label-free Cd based on SYBR Green I dye. 2+ Rapid detection of fluorescent aptamer sensors and their matching kits, specifically for the quantitative detection of Cd in fruits and vegetables. 2+ It belongs to the field of environmental and food safety testing technology. Background Technology

[0002] In the fields of environmental monitoring and food safety, heavy metal pollutants, especially cadmium ions (Cd), are monitored. 2+ The detection of cadmium is of great significance. Cadmium is a highly toxic heavy metal element, widely present in industrial emissions, agricultural production, and the natural environment. Long-term exposure to cadmium pollution can cause serious harm to human health, including kidney damage, bone diseases, and cancer risk. Therefore, establishing a sensitive, rapid, and highly specific method for detecting cadmium ions is crucial for environmental protection and public health.

[0003] Existing methods for cadmium ion detection mainly include atomic absorption spectrometry (AAS), inductively coupled plasma mass spectrometry (ICP-MS), fluorescence detection, and electrochemical detection. While these methods offer high accuracy and sensitivity, they also have some significant drawbacks. For example, AAS and ICP-MS instruments are expensive, complex to operate, and require specialized personnel; electrochemical detection methods are susceptible to sample matrix interference, leading to unstable results.

[0004] In recent years, biosensors based on fluorescent aptamers have gradually become a research hotspot for heavy metal ion detection due to their high sensitivity, high specificity, and convenience. Aptamers are a class of single-stranded nucleic acids that can specifically bind to target molecules and are obtained through in vitro screening techniques (SELEX). Fluorescent aptamer sensors typically use fluorophores to label aptamers, which generate a fluorescence signal change upon binding to the target molecule, thereby achieving detection. For example, Chinese patent CN116676310A discloses a nucleic acid aptamer that specifically recognizes cadmium ions and its application. The provided nucleic acid aptamer has one or more chemical modifications at its 5' or 3' end, including FAM, FITC, biotin, amino, and thiol groups. The nucleic acid aptamer sequence in this application contains 80 bases and includes a complex stem-loop structure. Furthermore, this application mainly involves the screening and identification of aptamers, not the design and development of a detection sensor method. Traditional labeled fluorescent aptamer sensors have some limitations, such as complex labeling processes, the influence of labeling position on aptamer function, and significant interference from other metal ions.

[0005] To overcome the aforementioned problems, label-free fluorescent aptamer sensors have emerged, which detect target molecules through exogenous fluorescent signal probes. SYBR Green I (SGI) is a commonly used fluorescent dye that can insert into the grooves of double-stranded DNA and emit a strong fluorescent signal. Therefore, SGI-based label-free fluorescent aptamer sensors not only avoid the influence of fluorophore labeling on aptamer affinity but also simplify the sensor construction process, significantly improving detection sensitivity and specificity.

[0006] Chinese patent CN117665286A discloses a Cr based on label-free fluorescent dyes and nucleic acid aptamers. 6+ The detection method utilizes a nucleic acid aptamer sequence containing 80 bases and a complex stem-loop structure, which binds to an SGI dye to form a self-assembled detection system; when Cr is present... 6+ When present, the stem-ring structure partially opens, the fluorescence intensity of the reaction system decreases, and the final fluorescence intensity change is related to Cr. 6+ The concentration is directly proportional to the concentration. However, this scheme targets Cr. 6+ However, it does not involve cadmium ions, and the signal probe is only the nucleic acid aptamer itself and does not involve the complementary strand. The nucleic acid aptamer has a long single-strand length, high cost, and poor performance.

[0007] Based on the above background, this invention proposes a label-free fluorescent aptamer sensor based on SGI dye and its matching kit for efficient, sensitive, and specific detection of Cd in solution. 2+ This sensor utilizes the aptamer Cd-(21) and Cd 2+ The sensor utilizes the specific binding of SGI dyes and their fluorescence properties to achieve quantitative detection of cadmium ions. Compared with existing technologies, the sensor of this invention has the advantages of simple operation, low cost, high detection sensitivity, and strong anti-interference ability. The developed kit is suitable for cadmium ion detection in environmental monitoring, food safety, and biomedicine. Summary of the Invention

[0008] Regarding Cd in existing technologies 2+ To address the shortcomings of existing detection technologies, this invention provides a label-free Cd based on SYBR Green I dye. 2+ A rapid detection fluorescent aptamer sensor and its quantitative detection kit are disclosed. The nucleic acid aptamer of this invention is low-cost, easy to synthesize, and has a broad target detection range. The label-free fluorescent sensor based on this nucleic acid aptamer is used for the detection of Cd. 2+ It has advantages such as simple operation, short operation time, high sensitivity and good selectivity.

[0009] The objective of this invention can be achieved through the following technical solutions:

[0010] In a first aspect, the present invention provides a method for specifically identifying Cd. 2+ The nucleic acid aptamer Cd-(21) has the nucleotide sequence shown in SEQ ID No. 1. Specifically, it is 5'-ATTGGTTACGGCTACCGTGAA-3', and the dissociation constant Kd of the nucleic acid aptamer is 12.9 μM, indicating good affinity.

[0011] Among all heavy metal ion biosensors, one nucleic acid aptamer is used as a probe to recognize target molecules, and the unlabeled fluorescent dye SYBR Green I is used as a signal amplification reagent. It is not only simple to operate, fast to analyze, highly sensitive, specific, and has a low detection limit, but also easy to miniaturize and port.

[0012] The present invention also provides a method for specifically identifying Cd 2+ Variants of the nucleic acid aptamer that specifically recognize Cd 2+ The variant of the nucleic acid aptamer is obtained by deleting, adding, or substituting nucleotides of the sequence as described in SEQ ID No. 1, and is associated with the specific recognition of Cd. 2+ The nucleic acid aptamers are single-stranded DNA molecules with the same specific recognition function.

[0013] The present invention also provides a method for specifically identifying Cd 2+ Derivatives of nucleic acid aptamers, for the specific recognition of Cd 2 + The nucleic acid aptamer is modified by amination, thiolation or isotopy to obtain the specific Cd-recognizing aptamer. 2+ Derivatives of nucleic acid aptamers that have the same function as nucleic acid aptamers.

[0014] The present invention also provides the specific identification of Cd 2+ Nucleic acid aptamers or specific recognition of Cd 2+ Variants or derivatives of nucleic acid aptamers in Cd 2+ Applications in detection.

[0015] The present invention also provides a method for specifically identifying Cd 2+ Nucleic acid aptamers or specific recognition of Cd 2+ Nucleic acid aptamer derivatives in the preparation, detection, or specific recognition of Cd 2+ Applications in products that specifically identify Cd, said products. 2+ Identification probe or Cd 2+ The fluorescent aptamer sensor was detected.

[0016] Secondly, the present invention provides a label-free Cd based on SYBR Green I dye. 2+A rapid detection fluorescent aptamer sensor, comprising the nucleic acid aptamer Cd-(21) and its complementary strand CS Cd-(21) And SYBR Green I dye, the complementary chain CS Cd-(21) The nucleotide sequence is shown in SEQ ID No. 2. Specifically, it is: 5'-TTCACAACAGCCGCGGCCAAT-3'.

[0017] The complementary chain CS Cd-(21) In the absence of Cd 2+ At this time, it forms a stable double-stranded DNA structure with the aptamer Cd-(21) through base pairing; using SGI as a fluorescent probe, when the aptamer Cd-(21) and the complementary strand CS Cd-(21) When double-stranded DNA is formed, SGI inserts into the small grooves in the double-stranded DNA, emitting a strong fluorescent signal; in Cd... 2+ When it exists, Cd 2+ By binding the aptamer Cd-(21), its conformation is altered so that it cannot bind to the complementary chain CS. Cd-(21) When double-stranded DNA is formed, SGI exhibits only a weak fluorescent signal; Cd is detected by measuring changes in fluorescence intensity. 2+ Quantitative analysis.

[0018] This invention also provides label-free Cd based on SYBR Green I dye. 2+ Rapid detection of fluorescent aptamer sensors in Cd 2+ Applications in detection.

[0019] This invention also provides label-free Cd based on SYBR Green I dye. 2+ Rapid detection of fluorescent aptamer sensors in the preparation of Cd detection 2+ Application in quantitative detection kits.

[0020] Thirdly, the present invention provides a label-free Cd based on SYBR Green I dye. 2+ Rapid quantitative detection kit. This kit is suitable for Cd detection in real samples. 2+ Testing includes, but is not limited to, drinking water, food extracts, and biological samples.

[0021] Specifically, the kit includes: a solution of nucleic acid aptamer Cd-(21); complementary strand CS Cd-(21) Solution; SYBR Green I (SGI) dye; 10 mM HEPES buffer, pH 7.6; standard Cd 2+Solutions are used for calibration and standard curve plotting; other auxiliary reagents, including MgCl2 solution, are used to optimize reaction conditions. The SGI dye is a 5× stock solution, diluted to the required working concentration before use.

[0022] Fourthly, the present invention provides a method for detecting Cd using the aforementioned fluorescent aptamer sensor or kit. 2+ The detection method specifically includes the following steps:

[0023] S1. Prepare the nucleic acid aptamer Cd-(21) and its complementary strand CS. Cd-(21) , change Cd 2+ The specific nucleic acid aptamer Cd-(21) was reacted with different concentrations of Cd 2+ After the solution is mixed well, the first incubation is carried out.

[0024] S2. Add the aptamer complementary chain CS to the mixture after the first incubation. Cd-(21) The SGI dye is used to induce base pairing between the complementary strand of the aptamer and the nucleic acid aptamer to form a double strand, with the SGI fluorescent dye embedded in the formed double strand. After mixing, a second incubation is performed. The fluorescence intensity F is measured at the excitation and emission wavelengths using a multi-functional microplate reader, without the addition of Cd. 2+ The buffer solution was used as a control group, and the fluorescence intensity F0 was measured. Based on the fluorescence intensity, the concentrations of Cd at different known concentrations were calculated. 2+ The difference in fluorescence intensity of the solution is ΔF = F0 - F;

[0025] S3. Based on Cd 2+ The difference between concentration and fluorescence intensity ΔF was fitted to obtain a linear equation;

[0026] S4. Using an unknown concentration of Cd-containing... 2+ Replace the standard with the test sample and proceed according to steps S1-S2 to detect the fluorescence intensity difference ΔF between the test samples; then, based on the curve fitted to the standard in step S3, calculate the Cd concentration in the test sample. 2+ concentration.

[0027] In one embodiment of the present invention, the buffer solution used in the reaction is HEPES with a concentration of 10 mM, the reaction temperature is 25°C, and the pH is 7.6.

[0028] In one embodiment of the present invention, the nucleic acid aptamer and the aptamer complementary strand are dissolved in HEPES buffer, wherein the concentration of the nucleic acid aptamer is 6.25-100 nmol / L and the concentration of the aptamer complementary strand is 6.25-100 nmol / L.

[0029] In one embodiment of the present invention, the concentration of the SGI dye is 0.1-0.3×.

[0030] In one embodiment of the present invention, the concentration of MgCl2 added to the reaction is 200 μM.

[0031] In one embodiment of the present invention, the nucleic acid aptamer and Cd 2+ The first incubation time is 5-25 min, and the second incubation time is 5-25 min after adding the aptamer complementary strand and SGI fluorescent dye.

[0032] In one embodiment of the present invention, the excitation wavelength is 480 nm and the emission wavelength is 525 nm.

[0033] In one embodiment of the present invention, the linear fitting line equation is y = 8.03x + 513.23, where x represents the Cd in the detected sample. 2+ The concentration is in nmol / L, and y represents the difference in fluorescence intensity of the samples, ΔF.

[0034] This invention reveals that optimal detection conditions were obtained by gradient optimization of the final concentrations of the nucleic acid aptamer and complementary strand, as well as the interaction time between the aptamer and complementary strand in the detection system: the optimal final concentration of SGI was 0.2×, the optimal final concentration of the nucleic acid aptamer was 75 nM, the optimal final concentration of the complementary strand was 75 nM, and the optimal final concentration of Cd was... 2+ The optimal interaction time for nucleic acid aptamers is 15 min, and the optimal interaction time for complementary strands with SGI fluorescent dye is 10 min.

[0035] This invention surprisingly discovered that when the aptamer is Cd... 2+ When the nucleotide sequence of the aptamer (SEQ ID No. 1) is obtained, it is associated with Cd. 2+ It has good specificity and high sensitivity.

[0036] The principle of this invention is: when only SGI or Cd exists in the system 2+ With SGI, there is almost no fluorescence signal. When there is no Cd in the system... 2+ And there exist Cd-(21) and CS Cd-(21) At that time, due to Cd-(21) and CS Cd-(21) During incubation, complementary double strands are formed through self-stacking, thus SGI insertion into the double-helix grooves of the double-stranded structure generates a strong fluorescent signal. At this point, Cd is introduced... 2+ After that, Cd 2+ The specific binding of Cd-(21) prevents hybridization between the nucleic acid aptamer and its complementary strand, hindering the formation of complementary double strands and thus significantly reducing fluorescence intensity. Therefore, the fluorescence intensity recovery value ΔF at the emission wavelength of 520 nm after excitation at a wavelength of 480 nm can be used to indicate the presence of Cd in the test solution. 2+ The content of [the substance / method]. The principle of this invention can be referred to [reference needed].Figure 4 .

[0037] In existing technologies, labeled fluorescent aptamer sensors suffer from problems such as narrow linear detection range and susceptibility to interference from other metal ions. This invention constructs a aptamer comprising Cd-(21) and its complementary chain CS... Cd-(21) Label-free fluorescent aptamer sensors, including those using SGI dye, introduce SGI as an exogenous fluorescent signal probe, avoiding the influence of fluorophore labeling on aptamer binding affinity, thereby improving detection specificity and sensitivity. This invention also optimizes experimental parameters, including SGI concentration, aptamer and complementary strand concentration, incubation time, MgCl2 concentration, buffer type, and system pH. Under optimized conditions, the novel sensor exhibits a wide linear detection range and good anti-interference capability, enabling the detection of Cd in real samples. 2+ Perform accurate testing.

[0038] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0039] The nucleic acid aptamer sequence used in this invention contains only 21 bases and has a simple stem-loop structure. Its base composition is similar to that of existing Cd... 2+ The specific nucleic acid aptamers are significantly different.

[0040] This invention can accurately and sensitively detect Cd in food within 25 minutes. 2+ The detection limit for the content is 0.64 nmol / L, and the detection concentration range is 1-1400 nmol / L. The technical solution provided by this invention avoids the disadvantages of traditional instrumental methods, such as complex operation, long processing time, and inability to monitor online in real time. The detection method provided by this invention does not require large instruments, is simple to operate, has high sensitivity, good selectivity, low cost, and high efficiency. It can quickly, easily, and accurately monitor food on-site and has good application prospects. Attached Figure Description

[0041] Figure 1 For Cd 2+ Simulated secondary structure diagram of nucleic acid aptamer Cd-(21);

[0042] Figure 2 For Cd 2+ Nucleic acid aptamer complementary strand CS Cd-(21) Simulated secondary structure diagram;

[0043] Figure 3 For nucleic acid aptamers Cd-(21) and Cd 2+ Combined isothermal titration thermograms;

[0044] Figure 4 For label-free Cd based on SYBR Green I dye2+ Schematic diagram of a rapid detection sensor for fluorescent aptamers;

[0045] Figure 5 For the detection of Cd by the aptamer sensor in Example 1 2+ The feasibility verification results diagram;

[0046] Figure 6 This is a graph showing the optimization results of SGI dye concentration in Example 2;

[0047] Figure 7 The nucleic acid aptamer Cd-(21) and complementary strand CS in Example 3 Cd-(21) Concentration optimization results diagram;

[0048] Figure 8 In Example 4, Cd-(21)+Cd 2+ and CS Cd-(21) Figure showing the results of optimized incubation time;

[0049] Figure 9 This is a schematic diagram showing the results of buffer solution and pH optimization in the detection system of Example 5;

[0050] Figure 10 The graph shows the results of the MgCl2 addition concentration optimization in Example 6;

[0051] Figure 11 Cd in Example 7 2+ The sensitivity results of the detection sensor are shown in the graph.

[0052] Figure 12 Cd in Example 7 2+ The sensitivity results of the detection sensor are shown in the figure. Detailed Implementation

[0053] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0054] In the following examples, the nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID No. 1. Specifically, it is: 5'-ATTGGTTACGGCTACCGTGAA-3'. Cd 2+ The simulated secondary structure diagram of the nucleic acid aptamer Cd-(21) is shown below. Figure 1 As shown in the figure, the structure of Cd-(21) is a typical stem-loop structure, indicating that it has a potential strong target binding ability.

[0055] The nucleotide sequence of the complementary strand of the aptamer is shown in SEQ ID No. 2. Specifically, it is: 5'-TTCACAACAGCCGCGGCCAAT-3'. Cd 2+ Nucleic acid aptamer complementary strand CS Cd-(21) The simulated secondary structure diagram is as follows Figure 2 As shown, it also exhibits stem-loop structure characteristics, but has fewer complementary base pairs, indicating that it is easy to unfold, thus allowing it to interact with the aptamer Cd. 2+ Nucleic acid aptamer Cd-(21) binds.

[0056] Nucleic acid aptamers Cd-(21) and Cd 2+ Combined isothermal titration thermograms, as shown Figure 3 As shown in the figure, it can be seen that as Cd... 2+ As the amount of added material increases, the peak area of ​​the exothermic peak in the system gradually decreases, indicating that Cd-(21) reacts with Cd... 2+ There is a bond between them. Based on the exothermic enthalpy change (ΔH), the dissociation constant Kd of Cd-(21) is fitted to be 12.9 μM, which shows good affinity.

[0057] Example 1

[0058] This embodiment involves the use of nucleic acid aptamer Cd-(21) and complementary strand CS. Cd-(21) Cd composed of SGI dyes 2+ Feasibility verification of rapid detection of fluorescent aptamer sensors.

[0059] The nucleic acid aptamer Cd-(21) and complementary strand CS were mixed in 10mM HEPES buffer at pH 7.4. Cd-(21) All concentrations were prepared to 2 μM. The detection principle was then validated.

[0060] (1) 1400 nM Cd 2+ Mix with 10 μL of 1 μM Cd-(21) and incubate for 10 min, then add 10 μL of 1 μM CS. Cd-(21) The mixture was incubated with 8 μL of 5×SGI dye for 10 min, with a total reaction volume of 200 μL. Simultaneously, SGI dye and SGI dye + Cd were added. 2+ SGI+CS Cd-(21) ,SGI+Cd-(21),SGI+Cd-(21)+CS Cd-(21) As a control group, samples were taken from black 96-well plates and fluorescence intensity was scanned using an Infinite M200 Pro microplate reader at an excitation wavelength of 480 nm and an emission wavelength of 510-700 nm. Figure 5 Figure A shows the initial SGI dye and SGI dye + Cd. 2+The fluorescence intensity of the group was extremely low. With the complete addition of the aptamer and complementary strand, the fluorescence intensity of the system reached its highest level.

[0061] (2) Prepare different concentrations (10, 50, 100, 200, 400, 800 nM) of Cd 2+ The solution was mixed with 10 μL of 1 μM Cd-(21) and incubated for 10 min, followed by the addition of 10 μL of 1 μM CS. Cd-(21) Mix with 8 μL of 5×SGI dye and incubate for 10 min; the total reaction volume is 200 μL. Samples are taken into black 96-well plates and fluorescence intensity is scanned using an Infinite M200 Pro microplate reader at 480 nm excitation and 525 nm emission wavelengths. Simultaneously, without Cd... 2+ As a blank control, the average fluorescence intensity value F of the system calculated after three repeated experiments is used as the ordinate, and Cd is used as the ordinate. 2+ Plotting solution concentration on the x-axis, as shown below. Figure 5 As shown in Figure B, it can be seen from the figure that as Cd... 2+ As the concentration increased, the fluorescence intensity of the system gradually decreased, further confirming the feasibility of the principle.

[0062] Example 2

[0063] Based on the method of Example 1, this example optimizes the SGI dye concentration.

[0064] For optimization of SGI dye concentration, different SGI dye concentrations (0.1×, 0.15×, 0.2×, 0.25×, 0.3×) were set at 1400 nM Cd. 2+ The experimental group (F) and the Cd-free group 2+ For the HEPES blank group (F0), 10 μL of 1 μM nucleic acid aptamer Cd-(21) solution was added to a 1.5 mL brown centrifuge tube and incubated for 10 min, followed by the addition of 10 μL of 1 μM CS. Cd-(21) The mixture was co-incubated with different concentrations of SGI dye for 10 min, maintaining a total reaction volume of 200 μL. Fluorescence intensity was measured in black 96-well plates using an Infinite M200 Pro microplate reader, obtaining F and F0, and calculating ΔF. The average difference in fluorescence intensity ΔF obtained after three repetitions was plotted on the ordinate, with the SGI dye concentration on the abscissa. Figure 6 As shown, the fluorescence recovery value of the system gradually increases with the increase of SGI dye concentration, reaching the highest value at 0.2×. Therefore, the concentration value (0.2×) corresponding to the maximum ΔF value is selected as the optimal concentration of SGI dye solution.

[0065] Example 3

[0066] Based on the method of Example 1, this example optimizes Cd-(21) and CS. Cd-(21) concentration.

[0067] For optimization of Cd-(21) concentration, a concentration of 1400 nM Cd was set. 2+ The experimental group (F) and the Cd-free group 2+ The HEPES blank group (F0) was incubated with 6.25, 12.5, 25, 50, 75, and 100 nM Cd-(21) aptamer solutions in 1.5 mL brown centrifuge tubes for 10 min, followed by the addition of 10 μL of 1 μM CS. Cd-(21) Incubate with 8 μL of 5×SGI dye for 10 min, maintaining a total reaction volume of 200 μL. Samples are taken from black 96-well plates and fluorescence intensity is measured using an Infinite M200 Pro microplate reader. F and F0 are obtained, and ΔF is calculated. The average of the fluorescence intensity differences ΔF obtained after three repetitions is plotted on the ordinate, and the concentration of the nucleic acid aptamer FAM-Cd-(21) solution is plotted on the abscissa. Figure 7 As shown in Figure A, the fluorescence recovery value of the system gradually increases with the increase of Cd-(21) concentration, reaching the highest value at 75 nM. Therefore, the concentration value (75 nM) corresponding to the maximum ΔF value is selected as the optimal concentration of the nucleic acid aptamer Cd-(21) solution.

[0068] For CS Cd-(21) Concentration optimization was performed, with a setting of 1400 nM Cd. 2+ The experimental group (F) and the Cd-free group 2+ The HEPES blank group (F0) was incubated with 75 nM Cd-(21) in a 1.5 mL brown centrifuge tube for 10 min, followed by the addition of 6.25, 12.5, 25, 50, 75, and 100 nM complementary strand CS, respectively. Cd-(21) Add 8 μL of 5×SGI dye to the solution and incubate for another 10 min, maintaining a total reaction volume of 200 μL. Samples are taken from black 96-well plates and fluorescence intensity is measured using an Infinite M200 Pro microplate reader. F and F0 are obtained, and ΔF is calculated. The average of the fluorescence intensity differences ΔF obtained after three repetitions is used as the ordinate, with the complementary strand CS... Cd-(21) Plot the solution concentration on the x-axis. For example... Figure 7 As shown in B, with CS Cd-(21) With increasing concentration, ΔF initially increases and then decreases, reaching a peak at 75 nM. Therefore, the concentration value corresponding to the maximum ΔF (75 nM) is selected as the complementary chain CS. Cd-(21) The optimal concentration of the solution.

[0069] Example 4

[0070] Based on the methods of Examples 1-3, this example optimizes Cd-(21) and CS. Cd-(21) Incubation time.

[0071] For optimization of the incubation time of aptamer Cd-(21), a setting of 1400 nM Cd was used. 2+ The experimental group (F) and the Cd-free group 2+ The HEPES blank group (F0) was incubated with 75 nM of nucleic acid aptamer Cd-(21) solution in a 1.5 mL brown centrifuge tube for 5, 10, 15, 20 and 25 min, respectively. Then, 75 nM of complementary strand CS was added. Cd-(21) Add 0.2×SGI dye and continue incubation for 10 min, maintaining a total reaction volume of 200 μL. Samples were taken from black 96-well plates and fluorescence intensity was measured using an Infinite M200 Pro microplate reader. F and F0 were obtained, and ΔF was calculated. The average of the fluorescence intensity differences ΔF obtained after three repetitions was used as the ordinate. Cd-(21)+Cd 2+ Plot the incubation time on the x-axis. Figure 8 As can be seen from A, with Cd-(21)+Cd 2+ With increasing incubation time, Cd-(21) and Cd 2+ The binding between them becomes more complete, reaching saturation at 15 minutes, at which point ΔF reaches its peak. Beyond this time, the fluorescence of the SGI dye naturally quenches due to prolonged excitation, leading to a decrease in ΔF. Therefore, the Cd-(21) incubation time (15 min) corresponding to the maximum ΔF value is selected as the optimal incubation time.

[0072] For complementary chain CS Cd-(21) Incubation time optimization, set to 1400 nM Cd 2+ The experimental group (F) and the Cd-free group 2+ For the HEPES blank group (F0), 75 nM of nucleic acid aptamer Cd-(21) solution was added to a 1.5 mL brown centrifuge tube and incubated for 15 min, followed by the addition of 75 nM of complementary strand CS. Cd-(21) The sample was incubated with 0.2×SGI dye for 5, 10, 15, 20, and 25 min, respectively, maintaining a total reaction volume of 200 μL. Fluorescence intensity was measured in black 96-well plates using an Infinite M200 Pro microplate reader, obtaining F and F0, and calculating ΔF. The average of the fluorescence intensity differences ΔF obtained after three repetitions was used as the ordinate, and CS... Cd-(21) Plot the incubation time on the x-axis. For example... Figure 8 As shown in B, with CS Cd-(21)The fluorescence difference ΔF of the system increases with incubation time, reaching a peak at 10 min. Afterward, the SGI fluorescence signal gradually decreases over time, thus reducing the system's ΔF. Therefore, the CS value corresponding to the maximum ΔF value was selected. Cd-(21) The incubation time (10 min) is considered the optimal incubation time.

[0073] Example 5

[0074] Based on the methods of Examples 1-4, this example optimizes the type of buffer solution and the pH of the system.

[0075] For buffer type optimization, a 1400nM Cd buffer was set. 2+ The experimental group (F) and the Cd-free group 2+ The corresponding buffer blank group (F0) was prepared. The reagents were dissolved in 10 mM pH 7.4 MOPS, Tris-HAC, PBS, HEPES, and Tris-HCl buffer. 75 nM nucleic acid aptamer Cd-(21) solution and 1400 nM Cd were added to a 1.5 mL brown centrifuge tube. 2+ Incubate for 15 minutes, then add 75 nM complementary strand CS Cd-(21) Add 0.2×SGI dye and incubate for another 10 min, maintaining a total reaction volume of 200 μL. Samples are taken from black 96-well plates and fluorescence intensity is measured using an Infinite M200 Pro microplate reader. F and F0 are obtained, and ΔF is calculated. The average of the fluorescence intensity differences ΔF obtained after three repetitions is plotted on the ordinate, and the buffer type on the x-axis. (Example:...) Figure 9 As shown in Figure A, the largest ΔF change was observed when HEPES was used as the buffer, indicating that different buffer types may affect the performance of the aptamer sensor. Therefore, the buffer corresponding to the maximum ΔF value (HEPES buffer) was selected as the optimal buffer type for the system reaction.

[0076] For pH optimization of the system, a concentration of 1400 nM Cd was set. 2+ The experimental group (F) and the Cd-free group 2+ The corresponding buffer blank group (F0) was prepared. Reagents were dissolved in 10 mM HEPES buffer at different pH values ​​(7.2, 7.4, 7.6, 7.8, 8.0, 8.2). 75 nM nucleic acid aptamer Cd-(21) solution and 1400 nM Cd were added to a 1.5 mL brown centrifuge tube. 2+ Incubate for 15 minutes, then add 75 nM complementary strand CS Cd-(21)Add 0.2×SGI dye and incubate for another 10 min, maintaining a total reaction volume of 200 μL. Samples are taken from black 96-well plates and fluorescence intensity is measured using an Infinite M200 Pro microplate reader. F and F0 are obtained, and ΔF is calculated. The average of the fluorescence intensity differences ΔF obtained after three repetitions is plotted on the ordinate, and the pH of the reaction system is plotted on the abscissa. Figure 9 As shown in Figure B, the ΔF value gradually increases in the pH range of 7.2 to 7.6, reaching a maximum at 7.6, and then decreases as the pH continues to rise. Changes in pH generally affect Cd⁻(2¹) and Cd⁻(2¹). 2+ The binding state between the molecules alters the generation and stability of the fluorescence signal, thus affecting the ΔF value. Therefore, the pH corresponding to the maximum ΔF value (7.6) was selected as the optimal pH for the system reaction.

[0077] Example 6

[0078] Based on the methods of Examples 1-5, this example optimizes the concentration of MgCl2 added.

[0079] For optimization of MgCl2 concentration, a concentration of 1400 nM Cd was set. 2+ The experimental group (F) and the Cd-free group 2+ The corresponding buffer blank group (F0) was prepared. Each reagent was dissolved in 10 mM HEPES buffer (pH 7.6) and different concentrations of MgCl2 (200, 400, 600, 800, 1000 μM) were added. Then, 75 nM Cd-(21) aptamer solution and 1400 nM Cd were added to a 1.5 mL brown centrifuge tube. 2+ Incubate for 15 minutes, then add 75 nM complementary strand CS Cd-(21) Add 0.2×SGI dye and incubate for another 10 min, maintaining a total reaction volume of 200 μL. Samples are taken from black 96-well plates and fluorescence intensity is measured using an Infinite M200 Pro microplate reader. F and F0 are obtained, and ΔF is calculated. The average of the fluorescence intensity differences ΔF obtained after three repetitions is plotted on the ordinate, and the MgCl2 concentration is plotted on the abscissa. Figure 10 As shown, the ΔF value reaches its maximum at 200 μM MgCl2, and then decreases as the concentration of MgCl2 continues to increase. Excessively high MgCl2 concentrations can easily generate a strong ion shielding effect, causing Cd-(21) to interact with Cd... 2+ The weakening of ionic interactions between the ions leads to a decrease in the ΔF value of SGI. Therefore, the MgCl2 concentration (200 μM) corresponding to the maximum ΔF value was selected as the optimal MgCl2 addition concentration for the system reaction.

[0080] Example 7

[0081] This embodiment provides a label-free Cd based on SYBR Green I dye. 2+ A rapid detection kit and its detection application, the kit comprising: pH 7.6 10mM HEPES buffer, 10mM MgCl2 solution, 10μM aptamer Cd-(21), 5μM Cd 2+ Standard solution, 10 μM aptamer complementary chain CS Cd-(21) 5×SGI dye (dilute to 0.2×working concentration before use), application method is as follows:

[0082] (1) First, dissolve each reagent in 10 mM pH 7.6 HEPES buffer. Add 75 nM of nucleic acid aptamer Cd-(21) solution to a 1.5 mL brown centrifuge tube. Then, add 0.5, 1, 5, 10, 20, 30, 50, 100, 200, 500, 800, 1100, 1400, and 1700 nM of Cd to the system, respectively. 2+ Incubate in solution for 15 min, then add 75 nM complementary chain CS Cd-(21) Add 0.2×SGI dye and incubate for another 10 min, maintaining a total reaction volume of 200 μL. Samples are taken from black 96-well plates and fluorescence intensity is measured using an Infinite M200 Pro microplate reader to obtain F and F0, and ΔF is calculated.

[0083] (2) Using different concentrations of Cd 2+ Plot the curve against the corresponding enhancement ΔF to obtain a standard curve, as shown below. Figure 11 As shown.

[0084] (3) Preparation of sample detection system: Take 0.2g of the sample to be tested and digest it according to the food-based sample in the national standard GB5009.15-2014. Then adjust the pH of the sample system to 7.6 and make up to 25mL with 10mM pH=7.6 HEPES buffer. Add it to the centrifuge tube of the detection system prepared in step (1) to replace the Cd dissolved in HEPES in step (1). 2+ Solution. After processing according to the above steps, determine ΔF.

[0085] (4) Substituting the calculated ΔF value into the standard curve, the Cd in the sample can be calculated. 2+ content.

[0086] (5) Verification: The method of this invention was used to determine the Cd content. 2+ One food sample (cabbage, broccoli, and grape) with concentrations of 5, 700, and 1400 nM was obtained, and the average recovery rate ranged from 95.23% to 111.32%, thus proving the reliability of this method. The results are shown in Table 1.

[0087] The method of the present invention is used to determine Cd in food samples. 2+ The concentration range is 1-1400 nM. In this embodiment, the linear fitting equation is y = 8.03x + 513.23, and the limit of detection is 0.64 nM. In the linear equation, x represents the concentration of Cd in the sample. 2+ The concentration (nM) is given by y, which represents the sample ΔF difference.

[0088] Table 1 Spike recovery in different food samples

[0089]

[0090]

[0091] This embodiment also includes Cd. 2+ The sensitivity test is detailed below:

[0092] For the study of system sensitivity, this embodiment used different heavy metal cation interfering agents. A 1400 nM Cd atmosphere was used. 2+ + Test groups (F) for each interfering heavy metal cation and Cd-free 2+ The HEPES blank group (F0) was prepared by adding 70 nM of nucleic acid aptamer Cd-(21) solution and 1400 nM of Cd to a 1.5 mL brown centrifuge tube. 2+ Incubate with different interfering heavy metal cations for 15 min, then add 75 nM complementary chain CS Cd-(21) Incubate with 0.2×SGI dye for 10 min, maintaining a total reaction volume of 200 μL. Samples are taken from black 96-well plates and fluorescence intensity is measured using an Infinite M200 Pro microplate reader. F and F0 are obtained, and ΔF is calculated. The average difference in fluorescence intensity ΔF obtained after three repetitions is plotted on the ordinate, and the names of each interfering substance are plotted on the abscissa. The specificity of the detection system is determined by comparing the magnitude of each ΔF value. Figure 12 As shown in the figure, under optimal experimental conditions, the presence of various interfering cations does not cause significant changes in the fluorescence response of the system, indicating that this sensor can detect Cd. 2+ It exhibits good specificity, which is crucial for the accuracy and reliability of this aptamer sensor in detecting complex food samples.

[0093] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A non-labeled Cd based on SYBR Green I dye 2+ A rapid detection fluorescent aptamer sensor characterized by, comprising a nucleic acid aptamer Cd-(21), a complementary strand CS of the nucleic acid aptamer Cd-(21) Cd-(21) and a SYBR Green I dye, the nucleotide sequences of the nucleic acid aptamer Cd-(21) and the complementary strand CS Cd-(21) being shown as SEQ ID No. 1~ SEQ ID No.

2.

2. The non-labeled SYBR Green I dye-based Cd of claim 1 2+ A rapid detection fluorescent aptamer sensor, characterized in that, The complementary strand CS Cd-(21) In the absence of Cd 2+ forms a stable double-stranded DNA structure with the aptamer Cd-(21) by base pairing; Using SGI as a fluorescent probe, when the aptamer Cd-(21) and the complementary strand CS Cd-(21) When double-stranded DNA is formed, SGI inserts into the small groove of double-stranded DNA, emitting a strong fluorescence signal; In Cd 2+ Cd 2+ Combined with aptamer Cd-(21), change its conformation, so that it can't with complementary chain CS Cd-(21) Form double-stranded DNA, SGI only shows weak fluorescence signal; Quantitative analysis of Cd 2+ was achieved by measuring the change in fluorescence intensity.

3. The non-labeled Cd based on SYBR Green I dye according to claim 1 2+ Application of the rapid detection fluorescent aptamer sensor in detecting Cd 2+ .

4. The non-labeled Cd based on SYBR Green I dye according to claim 1 2+ Use of the rapid detection fluorescent aptamer sensor in the preparation of a kit for detecting Cd 2+ .

5. A non-labeled SYBR Green I dye-based Cd 2+ A rapid quantitative detection kit characterized in that, The kit comprises: The fluorescent aptamer sensor of claim 1; 10 mM HEPES buffer, pH 7.6; Standard Cd 2+ Solutions for calibration and standard curve preparation; Other auxiliary reagents, including MgCl2 solution, are used to optimize the reaction conditions.

6. The non-labeled SYBR Green I dye-based Cd of claim 5 2+ A rapid quantitative detection kit, characterized in that, The SYBR Green I dye in the fluorescent aptamer sensor is a 5x stock solution, which is diluted to a working concentration of 0.2x before use.

7. The non-labeled SYBR Green I dye-based Cd of claim 5 2+ A rapid quantitative detection kit, characterized by, The kits are suitable for Cd 2+ detection of real samples, including drinking water, food extracts and biological samples.

8. A method for detecting Cd2+ using the fluorescent aptamer sensor of claim 1 or the kit of claim 5. 2+ A method for detecting a target substance, characterized by, The kit comprises the following steps: S1. Preparation of nucleic acid aptamer Cd-(21) and aptamer complementary strand CS Cd-(21) The nucleic acid aptamer Cd-(21) was mixed with different concentrations of Cd 2+ After mixing, the solution was incubated for the first time; S2. Adding the aptamer complementary strand CS to the mixed solution after the first incubation Cd-(21) and SGI dye, so that the aptamer complementary strand base pairs with the nucleic acid aptamer to form double strands and the SGI fluorescent dye is embedded in the formed double strands. After mixing, the second incubation is carried out. The fluorescence intensity F is measured at the excitation wavelength and the emission wavelength by using a multifunctional enzyme marker. The buffer without Cd 2+ is used as a control group to measure the fluorescence intensity F0. The fluorescence intensity difference ΔF=F0-F of the Cd 2+ solution with different known concentrations is calculated according to the fluorescence intensity. S3. Based on Cd 2+ Concentration and fluorescence intensity difference ΔF, linear equation is fitted S4. Replace the standard with an unknown concentration of a sample containing Cd 2+ containing Cd, and perform steps S1-S2 to detect the fluorescence intensity difference ΔF of the sample. Then, according to the curve of the standard fitted in step S3, calculate the concentration of Cd 2+ in the sample.

9. The method of claim 8, wherein the Cd is performed. 2+ The method of claim 1, wherein the detecting is performed. The buffer used for the reaction is HEPES at a concentration of 10 mM, the reaction temperature is 25°C, and the pH is 7.6; The nucleic acid aptamer Cd-(21) and the complementary strand CS Cd-(21) The SGI dye was dissolved in HEPES buffer at a concentration of 6.25-100 nM, the concentration of the SGI dye was 0.1x-0.3x, and the concentration of MgCl2 added in the reaction was 200 μM. The nucleic acid aptamer binds to Cd 2+ The time for the first incubation is 5-25 min and the time for the second incubation after adding the aptamer complementary strand and SGI dye is 5-25 min. The measurement of the fluorescence intensity is carried out in a black 96-well microplate, and a fluorescence microplate reader is used for detection, with an excitation wavelength of 480 nm and an emission wavelength of 525 nm; The results of the assay were compared to a standard curve of fluorescence intensity change versus Cd 2+ concentration to enable quantitative analysis of Cd 2+ content in unknown samples.

10. The method of claim 9, wherein the performing comprises performing Cd 2+ The method of claim 1, wherein the detecting comprises detecting the presence of the at least one biomarker in the sample. The aptamer Cd-(21) and the complementary strand CS Cd-(21) at a concentration of 75 nM, respectively, and the SGI dye at a concentration of 0.2x; the nucleic acid aptamer was incubated with Cd 2+ The time of the first incubation was 15 min, and the time of the second incubation after addition of the aptamer complementary strand and the SGI dye was 10 min.

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