CN116004766A - Probe and method for analyzing target object based on deoxyribozyme micro-thermophoresis - Google Patents

Abstract

The invention provides a microthermophoresis method for analyzing DNAzyme cofactor target objects based on fluorescent dye-labeled RNA shearing DNAzyme probes, which can realize sensitive and rapid analysis and detection of lead ions and small molecules. When the target exists, the activity of deoxyribozyme cleavage substrate is activated, and the detection signal of microthermophoresis analysis changes significantly. The detection limit of the method of the present invention detects lead ion reaches 49pM or 6pM, while the detection of L-histidine The limit reached 3.9 μM. The method of the present invention has the following advantages: high sensitivity, rapid detection, small required sample volume, high throughput and can detect 16 samples at the same time without immobilization or separation of molecules, and only one-step incubation is required for detection. At the same time, the deoxyribozyme has good molecular stability, strong selectivity and high catalytic activity, which ensure the high selectivity and high sensitivity of the method.

Description

Probe and method for analysis of target based on deoxyribozyme microthermophoresis

technical field

The invention relates to the field of biological detection, and specifically relates to a probe and a method for analyzing target objects based on deoxyribozyme microthermophoresis.

Background technique

DNAzyme is a catalytically active DNA sequence that can catalyze various reactions, such as cleavage of RNA molecules, ligation of RNA molecules, ligation of DNA, cleavage of DNA, redox reactions, etc. RNA-cleaving DNAzymes can catalyze the cleavage of substrate molecules containing specific RNA sites in the presence of cofactors. Based on this feature, RNA-cleaving DNAzymes can be used to develop detection cofactors method. These cofactors are usually metal ions, small molecules, and the like. The RNA-cleaving DNAzyme targeting a certain cofactor can be obtained by in vitro screening. Since the RNA cleavage-type DNAzyme has the function of catalyzing the cleavage of the corresponding substrate sequence, the method for detecting cofactors by using the RNA-cleavage-type DNzyme can realize detection signal amplification and has high sensitivity. Currently, commonly used methods for detecting cofactors based on RNA-cleaving DNAzymes include electrochemical detection, fluorescence detection, and colorimetry. However, the electrochemical method needs to immobilize DNAzyme or substrate molecules on the electrode surface, the amount of sample is large, the electrochemical signal reproducibility is poor, and the detection throughput is relatively low, and only one sample or several samples can be analyzed at a time. The preparation of electrodes is time-consuming. Fluorescent detection methods often require simultaneous labeling of fluorescent dye molecules and quenching groups on DNAzymes or substrates (or the use of nanomaterials such as nano-gold, graphene, etc.), and the modification of multiple functional groups increases the cost. In addition, fluorescence The determination of the signal intensity (here refers to the fluorescence intensity signal) is easily affected by various factors, and the detection signal is prone to fluctuations.

Microthermophoresis analysis is a new technique for characterizing molecular interactions, which can sensitively measure the response of fluorescent molecules in capillary solution to infrared laser heating. This technique is simple, sensitive, rapid, requires less sample, has high throughput, and does not require separation and immobilization of molecules. Microthermophoresis analysis usually uses fluorescent dye-labeled affinity ligands and the interaction between molecules, etc., and the determination of affinity is realized according to the signal changes generated before and after the fluorescent dye-labeled affinity ligands bind to molecules. This technology has been used in There are a wide range of applications in many fields. Microthermophoresis analysis also shows potential in the detection of target substances, but it often requires the use of fluorescent dye-labeled affinity ligands that can bind to the analyte, and the lack of effective signal amplification means limits the improvement of detection sensitivity.

Contents of the invention

The invention provides a method for analyzing target objects based on deoxyribozyme microthermophoresis and corresponding fluorescent dye-labeled probes.

In order to achieve the above object, the present invention adopts the following technical solutions:

On the one hand, the present invention provides the probe that is used for the micro-thermophoresis analysis of target object, it is characterized in that, described probe is:

a. The sequence formed by coupling the RNA-cleaving DNAzyme and its substrate sequence together, and one end of the formed sequence is labeled with a fluorescent dye; or

b. comprising an RNA-cleaving DNAzyme and its substrate sequence, and one of the RNA-cleaving DNAzyme and its substrate sequence is labeled with a fluorescent dye.

In another aspect, the present invention provides a method for micro-thermophoresis analysis of a target, characterized in that it comprises the following steps:

a. Incubate the above-mentioned probe with the target object to be detected in a buffer;

b. Analyzing the signal changes generated by micro-thermophoresis, and obtaining the concentration of the target substance to be measured according to the signal changes.

In another aspect, the present invention provides a kit for microthermophoresis analysis of a target, characterized by comprising the above-mentioned probe and buffer.

In some embodiments, the fluorescent dye is a fluorescein molecule or tetramethylrhodamine.

In some embodiments, the RNA-cleaving DNAzyme is shown in SEQ ID NO: 3, the substrate sequence is shown in SEQ ID NO: 2, and the target to be detected is lead ion.

In some embodiments, the RNA-cleaving DNAzyme is shown in SEQ ID NO: 6, the substrate sequence is shown in SEQ ID NO: 5, and the target to be detected is L-histidine.

In some embodiments, the probe is as shown in SEQ ID NO: 1, and the target to be detected is lead ion.

In some embodiments, the probe is shown in SEQ ID NO: 4, and the target to be detected is L-histidine.

In some embodiments, the RNA-cleaving DNAzyme and the substrate sequence are coupled together through multiple bases, preferably, the multiple bases are TTTTT.

In some embodiments, the buffer is a Tris-HCl buffer containing either magnesium ions and/or sodium ions and/or potassium ions.

In some embodiments, the magnesium ions are from MgCl ₂ , and the concentration of MgCl ₂ is 0-10 mM, preferably 2 mM.

In some embodiments, the sodium ions are from NaCl, and the NaCl concentration is 0-1000 mM, preferably 200-500 mM.

In some embodiments, the potassium ions are from KCl, and the concentration of KCl is 1-1000 mM, preferably 200-600 mM, more preferably 400 mM.

In some embodiments, the pH of the buffer is 6-9, such as 6.0, 6.5, 7.0, 7.5, 7.8, 8.0, 8.5, 9.0, preferably pH 7.5.

In some embodiments, the incubation temperature ranges from 4°C to 37°C, preferably the temperature is 20°C or 25°C.

In some embodiments, the incubation time is 15-90 minutes, preferably 15-60 minutes.

The analysis method of the present invention can be realized by the following Scheme 1 and Scheme 2.

Scheme 1: The RNA-cleaving DNAzyme and its substrate sequence are coupled together through several bases, and the fluorescent dye is labeled to the 3' end or 5' end of the DNA sequence. When the target to be detected exists, the activity of the deoxyribozyme is activated, the corresponding substrate sequence is catalyzed, and the molecular structure is changed. When the microthermophoresis analysis is performed, the signal of the microthermophoresis analysis produces obvious signal changes. The detection of cofactor target objects (such as lead ions, small molecule L-histidine, etc.) can be realized according to the change of detection signal by micro-thermophoresis analysis.

Scheme 2: Two separate DNA sequences are used, which are RNA-cleaving deoxyribozyme and its corresponding substrate sequence molecule, and one of the two sequences is end-labeled with a fluorescent dye molecule. When detecting the target substance, mix the two according to the ratio of 1:1, incubate with different concentrations of the target substance to be tested, the presence of the test substance activates the activity of the RNA-cleaving DNAzyme, cleaves the substrate, and then performs microcalorimetry Swimming analysis, based on the change of the microthermophoresis detection signal, the detection of the cofactor target (such as lead ion, small molecule L-histidine, etc.) is realized.

For the detection of lead ions in some embodiments, the corresponding DNA sequence is as follows.

SEQ ID NO:1-3'FAM, 5'- CTA T rA GGA AGA GAT GAT GTC TGT TTT TT A CAGACA TCA TCT CTG AAG TAG CGC CGC CGT ATA G-3'(SEQ ID NO:1), its 3' The end-labeled fluorescein molecule (FAM for short), in which the underlined part corresponds to the substrate molecule sequence of the deoxyribozyme, the rA in the sequence represents the RNA nucleoside (only rA is the RNA nucleoside), the italic part is the deoxyribozyme molecule, both There are several T bases in between, and the deoxyribozyme molecule and its substrate molecule are coupled together.

Abbreviated as SEQ ID NO:1-5'FAM, 5'- CTA T rA GGA AGA GAT GAT GTC TGT TTT TTA CAGACA TCA TCT CTG AAG TAG CGC CGC CGT ATA G-3'(SEQ ID NO:1), its 5' The terminal marker FAM, wherein the underlined part corresponds to the substrate molecular sequence of DNase, the rA in the sequence represents the RNA nucleoside, the part in italics is the DNase molecule, and there are several T bases between the two. The DNase molecule and The two substrate molecules are coupled together.

SEQ ID NO:1-3'TMR, 5'- CTA T rA GGA AGA GAT GAT GTC TGT TTT TTA CAG ACATCA TCT CTG AAG TAG CGC CGC CGT ATA G-3'(SEQ ID NO:1), its 3' end Mark Tetramethylrhodamine (TMR for short), where the underlined part corresponds to the substrate molecule sequence of DNAzyme, rA in the sequence represents RNA nucleoside, the italic part is the DNAzyme molecule, and there are several T bases in between , to couple the deoxyribozyme molecule and its substrate molecule together.

SEQ ID NO:1-5'TMR, 5'- CTA TrA GGA AGA GAT GAT GTC TGT TTT TTA CAG ACATCA TCT CTG AAG TAG CGC CGC CGT ATA G-3'(SEQ ID NO:1), its 5' end tag TMR, where the underlined part corresponds to the substrate molecule sequence of DNAzyme, the rA in the sequence represents RNA nucleoside, the italic part is the DNAzyme molecule, and there are several T bases between the two, and the DNAzyme molecule and its substrate The two molecules are coupled together.

SEQ ID NO: 2: Corresponding to the substrate molecular sequence of lead ion deoxyribozyme, its sequence is 5'-CTA T rA GGAAGA GAT GAT GTC TGT-3', wherein rA represents RNA nucleoside.

SEQ ID NO:2-5'FAM, corresponding to the substrate molecular sequence of lead ion deoxyribozyme, 5'-CTA T rAGGA AGAGAT GAT GTC TGT-3'(SEQ ID NO:2), where rA represents RNA nucleoside , whose 5' end is labeled with FAM.

SEQ ID NO:3-3'FAM, corresponding to the lead ion deoxyribozyme molecule, 5'-ACA GAC ATC ATC TCT GAAGTA GCG CCG CCG TAT AG-3'(SEQ ID NO:3), its 3' end is marked with FAM.

SEQ ID NO: 3: corresponding to lead ion deoxyribozyme, 5'-ACA GAC ATC ATC TCT GAA GTA GCG CCGCCG TAT AG-3'.

SEQ ID NO:4-3'FAM, 5'- CTA T rA GGA AGA GCC G TT TTT CGG CTC TTA ACG GGGCTG TGC GGC TAG GAA GTA ATA G-3'(SEQ ID NO:4), its 3' end tag FAM, wherein the underlined part is the substrate molecular sequence of L-type histidine deoxyribozyme, the italic part is the sequence of L-type histidine deoxyribozyme, and there are several T bases between the two, and the deoxyribozyme molecule and its Both substrate molecules are coupled together.

SEQ ID NO:4-5'FAM, 5'- CTAT rAGGAAGAGCCG TT TTT CGG CTC TTA ACG GGG CTGTGC GGC TAG GAA GTA ATA G-3'(SEQ ID NO:4), its 5' end is labeled FAM, wherein the underlined part is The substrate molecular sequence of L-type histidine deoxyribozyme, the italic part is the sequence of L-type histidine deoxyribozyme, and there are several T bases between the two, and the deoxyribozyme molecule and its substrate molecule are coupled linked together.

SEQ ID NO:7-3'FAM, 5'- CTATrAGGAAGAGATGATGTCTGT TTT TT A CAG ACA TCATCT CTG AAG TTA TAC CGC CGT ATA G-3'(SEQ ID NO:7), wherein 3' marks FAM, and the underlined part corresponds to the deoxygenated nucleus The substrate molecular sequence of the enzyme, the rA in the sequence represents the RNA nucleoside, the italic part is the deoxyribozyme molecule, and there are several T bases between the two, which couple the deoxyribozyme molecule and its substrate molecule together , but cannot be used for the detection of lead ions.

Other similar nucleic acid sequences with appropriate modifications to the above sequences (such as base substitution or sequence extension) can also be used. Fluorescent dyes Other types of fluorescent dye molecules can also be used.

The present invention has following advantage and effect:

In the present invention, by rationally designing the RNA splicing type deoxyribozyme sequence and the corresponding substrate sequence, using the corresponding DNA sequence of the fluorescent dye molecule at the end of the sequence, under optimized experimental conditions, using a specific RNA splicing type deoxyribozyme Enzyme has developed a microthermophoresis assay that can sensitively detect the cofactor lead ion or the small molecule histidine. This approach has the following significant advantages:

Simple operation, only one-step incubation, no need for separation and immobilization;

The detection is fast, and the detection of the sample only takes less than 20 seconds or less;

High sensitivity, RNA cleavage DNAzyme has the activity of catalyzing the cleavage of the substrate sequence, resulting in signal amplification, and the designed scheme is also conducive to the generation of significant detection signal changes;

The signal stability is good. The signal recorded by micro-thermophoresis analysis is the ratio of the fluorescence intensity of the sample before and after infrared laser heating, and the signal deviation is small;

High selectivity, the RNA-cleaving deoxyribozyme used selectively binds to its cofactor, and can only have the activity of cutting the substrate when the cofactor exists, resulting in a signal change;

High detection throughput, microthermophoresis analysis can analyze 16 samples at the same time, and other instrument models can be used to detect more samples, such as 96 samples or more;

The amount of sample used is small, and each sample detection only needs less than 4 microliters.

Under the optimized sequence design and experimental conditions, the developed detection method shows that the signal of microthermophoresis analysis changes significantly with the increase of the target concentration. When the RNA-cleaving DNAzyme of lead ions is used to construct the corresponding microthermophoresis analysis method, the detection limit of lead ions can be as low as 49pM or lower, and the method can be used for the sensitive detection of lead ions in actual water samples.

Description of drawings

Figure 1 is the result of detecting lead ions using SEQ ID NO:1-3'FAM. Figure 1A shows the curves of the fluorescence signal of the micro-thermophoresis analysis corresponding to different concentrations of lead ions as a function of time; Figure 1B shows the relationship between the corresponding Fnorm value and the concentration of lead ions when the selected time is 5 seconds; Figure 1C shows the relationship between 6nM lead ions and other metal ions including Zn ²⁺ , Cu ²⁺ , Ni ²⁺ , Mn ²⁺ , Hg ²⁺ , Ca ²⁺ , Cd ²⁺ (concentration is 6nM), Fnorm compared with blank sample solution Value (corresponding value at 5 seconds) change; Figure 1D shows the change of Fnorm value (corresponding value at 5 seconds) when detecting lead ions added in 20-fold diluted lake water and tap water samples.

Fig. 2 is the result that adopts SEQ ID NO:1-5'FAM to detect lead ion. Figure 2A shows the time-varying curves of the fluorescence signal of the microthermophoresis analysis corresponding to different concentrations of lead ions; Figure 2B shows the relationship between the corresponding Fnorm value and the lead ion concentration when the selected time is 5 seconds.

Figure 3 is the result of detecting lead ions using SEQ ID NO:2 and SEQ ID NO:3-3'FAM. Figure 3A shows the curve of the fluorescent signal changing with time as the lead ion concentration increases; Figure 3B shows the relationship between the corresponding Fnorm value and the lead ion concentration when the selected time is 5 seconds; Figure 3C shows the relationship between 6nM lead ion and other In the presence of metal ions including Zn ²⁺ , Cu ²⁺ , Ni ²⁺ , Mn ²⁺ , Hg ²⁺ , Ca ²⁺ , and Cd ²⁺ (the concentration is 6nM ⁾ , the signal change compared with the signal of the blank sample solution ( The corresponding Fnorm value at 5 seconds); Figure 3D shows the changes in the fluorescence signal (corresponding to the Fnorm value at 5 seconds) when detecting lead ions added in 20-fold diluted lake water and tap water samples.

Fig. 4 is the result that adopts SEQ ID NO:2-5'FAM and SEQ ID NO:3 to detect lead ion. Figure 4A shows the curve of the fluorescence signal changing with time as the lead ion concentration increases; Figure 4B shows the relationship between the corresponding Fnorm value and the lead ion concentration when the selected time is 5 seconds.

Figure 5 is the result of detecting lead ions using SEQ ID NO:1-3'TMR (corresponding to Figures 5A and 5B) or SEQ ID NO:1-5'TMR (corresponding to Figures 5C and 5D). Figure 5A shows the curve of SEQ ID NO: 1-3' TMR as the concentration of lead ions increases, the fluorescence signal changes with time, and Figure 5B shows the relationship between the corresponding Fnorm value and the concentration of lead ions when the selected time is 5 seconds; Figure 5C shows the curve of the fluorescence signal of SEQ ID NO:1-5'TMR as the concentration of lead ions increases with time, and Figure 5D shows the relationship between the corresponding Fnorm value and the concentration of lead ions when the selected time is 5 seconds.

Fig. 6 is the result that adopts SEQ ID NO:4-3'FAM to detect L-type histidine. Figure 6A shows the curves of the fluorescence signal of different concentrations of L-histidine corresponding to the micro-thermophoresis analysis as a function of time; Figure 6B shows the corresponding Fnorm value and the concentration of L-histidine when the selected time is 5 seconds Relationship; Figure 6C shows the changes in the signal (the corresponding Fnorm value at 5 seconds) produced when L-histidine and other types of amino acid molecules exist; Figure 6D shows the detection of serum samples and urine diluted 50 times with buffer solution The fluorescence signal (the corresponding Fnorm value at 5 seconds) changes when L-histidine is added to the sample.

Fig. 7 is the result that adopts SEQ ID NO:4-5'FAM to detect L-type histidine. Figure 7A shows the time-dependent curves of the fluorescent signal of the micro-thermophoresis analysis corresponding to different concentrations of L-histidine; Figure 7B shows the relationship between the corresponding Fnorm value and the concentration of L-histidine when the selected time is 5 seconds .

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials and reagents used in the following examples were purchased from conventional reagent companies unless otherwise specified.

The DNA sequences used were synthesized, prepared and purified by Sangon Bioengineering (Shanghai) Co., Ltd.

The step of micro-thermophoresis analysis of the target object: incubate the corresponding DNA probes and cofactors in a preferred buffer solution, and after incubation for a certain period of time, perform micro-thermophoresis analysis on the sample solution. Micro-thermophoresis analysis was performed using a micro-thermophoresis analyzer (Monolith NT.115, NanoTemper Technologies). According to the type of fluorescent dye, the corresponding LED excitation light channel was selected for fluorescence detection. Select the blue LED channel for fluorescein (FAM) dye-labeled probes and the green LED channel for tetramethylrhodamine (TMR) dye-labeled probes. If there is no special instruction, the LED excitation intensity is selected as the automatic mode, the infrared laser intensity is selected as the intermediate level, and the measurement temperature is 25°C. During the determination, the sample solution is transferred to the standard capillary used in the instrument, and the micro-thermophoresis analyzer is used to measure according to the selected parameters, and the normalized fluorescent signal Fnorm (Fnorm=F _hot / F _cold expressed in per thousand). F _cold is the fluorescence signal of the sample solution before the MST infrared laser is turned on, and F _hot is the fluorescence signal of the sample solution after the MST infrared laser is turned on). Select the Fnorm signal corresponding to a specific time (such as 5 seconds) within 20 seconds, draw the relationship between it and the concentration of the target substance, and detect the target substance according to the change of the Fnorm signal.

When detecting lead ions, use the following solution, 50mM Tris-HCl (pH 7.5)+2mM MgCl ₂ . In some embodiments, the reaction buffer solution contains sodium ions or magnesium ions (such as NaCl, its concentration is 0-1000mM, preferably 200-500mM; such as MgCl ₂ , its concentration is 0-10mM, preferably 2mM ) in Tris-HCl buffer solution, pH 6-9 (eg 6.0, 6.5, 7.0, 7.5, 7.8, 8.0, 8.5, 9.0), preferably pH 7.5. In some embodiments, the detected temperature ranges from a specific temperature ranging from 4°C to 37°C, preferably 20°C or 25°C. In some embodiments, the incubation time is 15-90 minutes, wherein the preferred incubation time is 15-60 minutes.

When detecting L-histidine, the following solution is used, 20mM Tris-HCl (pH 7.5)+400mM KCl+10mMMgCl ₂ . In some embodiments, the reaction buffer solution is a Tris-HCl buffer solution with a pH of 6-9 (eg, 6.0, 6.5, 7.0, 7.5, 7.8, 8.0, 8.5, 9.0), preferably a pH of 7.5. In some embodiments, the detected temperature ranges from a specific temperature ranging from 4°C to 37°C, preferably 20°C or 25°C. In some embodiments, the incubation time is 15-90 minutes, wherein the preferred incubation time is 15-60 minutes.

Embodiment 1: detect lead ion based on SEQ ID NO:1-3'FAM

Incubate 20nM SEQ ID NO:1-3'FAM with different concentrations of lead ions in buffer solution 50mM Tris-HCl (pH7.5)+2mM MgCl ₂ at 25 degrees for 60 minutes, then transfer the sample solution to a micropipette In the capillary used for thermophoresis analysis, micro-thermophoresis analysis was performed.

Figure 1A shows the time-varying curves of the fluorescent signal of the micro-thermophoresis analysis corresponding to different concentrations of lead ions. With the increase of the concentration of lead ions, the curve of the fluorescent signal changing with time gradually moves down, and the curves correspond from top to bottom. The lead ion concentrations were 0, 6pM, 12pM, 24pM, 49pM, 98pM, 195pM, 390pM, 781pM, 1.56nM, 3.13nM, 6.25nM, 12.5nM, 25nM, 50nM, 100nM. Figure 1B shows the relationship between the Fnorm value and the lead ion concentration when the selected time is 5 seconds. As the lead ion concentration increases, Fnorm decreases, and the corresponding fitting curve is drawn. The detection of lead ions can be realized according to the change of Fnorm. The detectable concentration range of lead ion is from 49pM to 100nM, the concentration detection limit is 49pM, and the maximum change of Fnorm signal is about 251‰.

The method has good selectivity, as shown in Figure 1C, other metal ions include Zn ²⁺ , Cu ²⁺ , Ni ²⁺ , Mn ²⁺ , Hg ²⁺ , Ca ²⁺ , Cd ²⁺ (concentration average When 6nM) exists, there is no significant change in signal compared with the blank sample solution signal, ΔFnorm is the Fnorm signal corresponding to the sample minus the Fnorm signal of the blank sample solution, and when 6nM lead ions exist, compared with the blank sample solution signal, Fnorm The signal (Fnorm signal corresponding to 5s) was significantly reduced by about 232‰.

The method can also be used to detect lead ions added in 20-fold diluted lake water and tap water samples, the results are shown in Figure 1D, and the detection limit can reach 49pM, which can be used to monitor whether the lead ion content exceeds the standard, and lead ions are harmful substances .

Embodiment 2: adopt SEQ ID NO:1-5' FAM probe to detect lead ion

Incubate 20nM SEQ ID NO:1-5'FAM with different concentrations of lead ions in buffer solution 50mM Tris-HCl (pH7.5)+2mM MgCl ₂ at 25 degrees for 60 minutes, then transfer the sample solution to a micropipette In the capillary used for the thermophoresis analysis, the parameters are set, and the micro-thermophoresis analysis is performed.

Figure 2A shows the time-varying curves of the fluorescent signal of the micro-thermophoresis analysis corresponding to different concentrations of lead ions. With the increase of the concentration of lead ions, the curve of the fluorescent signal changing with time gradually moves down, and the curve from top to bottom corresponds to The lead ion concentrations were 0, 6pM, 12pM, 24pM, 49pM, 98pM, 195pM, 390pM, 781pM, 1.56nM, 3.13nM, 6.25nM, 12.5nM, 25nM, 50nM, 100nM. Figure 2B shows the relationship between the corresponding Fnorm value and the lead ion concentration when the selected time is 5 seconds. As the lead ion concentration increases, Fnorm decreases, and the detection of lead ions can be realized according to the change of Fnorm. The detectable concentration range of lead ion is 195pM to 100nM, and the concentration detection limit is 195pM. Compared with the method using SEQ ID NO:1-3'FAM for detection, when using SEQ ID NO:1-5'FAM probe to detect lead ions, the change of Fnorm signal is smaller, and the maximum change of Fnorm signal is about 38‰ .

Embodiment 3: adopt SEQ ID NO:2 and SEQ ID NO:3-3'FAM detects lead ion

Incubate SEQ ID NO:2 and SEQ ID NO:3-3'FAM at a ratio of 1:1 in buffer solution 50mM Tris-HCl (pH7.5)+2mM MgCl ₂ at 25°C for 15 minutes, and then add different Concentration of lead ions, the final concentration of SEQ ID NO: 2 and SEQ ID NO: 3-3'FAM is 20nM, incubated at 25°C for 60 minutes, after incubation, the sample solution is transferred to the capillary used for microthermophoresis analysis , set parameters, and perform micro-thermophoresis analysis.

Figure 3A shows that as the concentration of lead ions increases, the curve of the fluorescence signal changes with time gradually moves down. Figure 3B shows the relationship between the corresponding Fnorm value and the concentration of lead ions when the selected time is 5 seconds. As the concentration of lead ions increases, Fnorm Reduced, the detection of lead ions can be realized according to the change of Fnorm. The detection range is 49pM to 100nM, the corresponding detection limit is 49pM, and the maximum Fnorm signal (the signal corresponding to 5s) varies by about 376‰. Figure 3C shows that the corresponding method is selective. When other metal ions including Zn ²⁺ , Cu ²⁺ , Ni ²⁺ , Mn ²⁺ , Hg ²⁺ , Ca ²⁺ , and Cd ²⁺ (concentrations are all 6nM) exist, Compared with the blank sample solution signal, the signal has no significant change. ΔFnorm is the Fnorm signal corresponding to the sample minus the Fnorm signal of the blank sample solution, and when 6nM lead ions exist, compared with the blank sample solution signal, the Fnorm signal (the Fnorm signal corresponding to 5s signal) was significantly reduced. The method can also be used to detect lead ions added in 20 times diluted lake water and tap water samples, the results are shown in Figure 3D, and the detection limit can reach 98pM.

Embodiment 4: adopt SEQ ID NO:2-5'FAM and SEQ ID NO:3 to detect lead ion

Incubate SEQ ID NO:2-5'FAM and SEQ ID NO:3 in a 1:1 ratio in buffer solution 50mM Tris-HCl (pH7.5)+2mM MgCl ₂ at 25°C for 15 minutes, and then add different Concentration of lead ions, the final concentration of SEQ ID NO:2-5'FAM and SEQ ID NO:3 is 20nM and incubated at 25°C for 60 minutes, then the sample solution is transferred to the capillary used for micro-thermophoresis analysis, set The parameters were determined for microthermophoresis analysis.

Figure 4A shows that as the concentration of lead ions increases, the curve of the fluorescence signal changes with time gradually moves down. Figure 4B shows the relationship between the corresponding Fnorm value and the concentration of lead ions when the selected time is 5 seconds. As the concentration of lead ions increases, Fnorm Reduced, the detection of lead ions can be realized according to the change of Fnorm. The detection range is 98pM to 100nM, the corresponding detection limit is 98pM, and the maximum Fnorm signal (the signal corresponding to 5s) varies by about 61‰.

Embodiment 5: Adopt SEQ ID NO:1-3'TMR and SEQ ID NO:1-5'TMR to detect lead ion

Incubate 20nM SEQ ID NO:1-3'TMR with different concentrations of lead ions in buffer solution 50mM Tris-HCl (pH7.5)+2mM MgCl ₂ at 25 degrees for 60 minutes, then transfer the sample solution to a micropipette In the capillary used for thermophoresis analysis, micro-thermophoresis analysis was performed. Figure 5A shows that as the concentration of lead ions increases, the curve of the fluorescence signal changes with time gradually moves down. Figure 5B shows the relationship between the Fnorm value and the concentration of lead ions when the selected time is 5 seconds. As the concentration of lead ions increases, Fnorm Reduced, the detection of lead ions can be realized according to the change of Fnorm. The detection range is 49pM to 100nM, the corresponding detection limit is 49pM, and the maximum Fnorm signal (the signal corresponding to 5s) varies by about 229‰.

Incubate 20nM SEQ ID NO:1-5'TMR with different concentrations of lead ions in buffer solution 50mM Tris-HCl (pH7.5)+2mM MgCl ₂ at 25 degrees for 60 minutes, then transfer the sample solution to a micropipette In the capillary used for thermophoresis analysis, micro-thermophoresis analysis was performed. Figure 5C shows that as the concentration of lead ions increases, the curve of the fluorescence signal changes with time gradually moves down. Figure 5D shows the relationship between the Fnorm value and the concentration of lead ions when the selected time is 5 seconds. As the concentration of lead ions increases, Fnorm Reduced, the detection of lead ions can be realized according to the change of Fnorm. The detection range is 6pM to 100nM, and the corresponding detection limit is 6pM (this result corresponds to SEQ ID NO:1-5'TMR, which can detect lower concentrations of lead ions), and the maximum Fnorm signal (the signal corresponding to 5s) changes by about 244‰ .

Embodiment 6: detect L-type histidine with SEQ ID NO:4-3'FAM

20nM SEQ ID NO:4-3'FAM was incubated with different concentrations of L-histidine in buffer solution 20mM Tris-HCl (pH 7.5)+400mM KCl+10mM MgCl ₂ at 25 degrees for 60 minutes, and then The sample solution is transferred to a capillary for microthermophoresis analysis, and microthermophoresis analysis is performed.

Figure 6A shows the time-varying curves of the fluorescent signal of the micro-thermophoresis analysis corresponding to different concentrations of L-histidine. As the concentration of L-histidine increases, the curve of the fluorescent signal changing with time gradually shifts down , the concentration of L-histidine corresponding to the curve from top to bottom is 0 μM, 0.488 μM, 0.977 μM, 1.95 μM, 3.91 μM, 7.81 μM, 15.6 μM, 31.3 μM, 62.5 μM, 125 μM, 250 μM, 500 μM, 1000 μM , 2000 μM, 4000 μM, 8000 μM. Figure 6B shows the relationship between the Fnorm value and the concentration of L-histidine when the selected time is 5 seconds. As the concentration of L-histidine increases, Fnorm decreases. According to the change of Fnorm, the concentration of L-histidine can be realized. detection. The detectable concentration range of L-histidine is 3.9 μM to 8 mM, and the detection limit is 3.9 μM.

Fig. 6C shows the signal changes in the presence of other types of amino acid molecules, and the corresponding concentrations of the amino acids investigated are all 125 μM. Compared with the blank sample solution, when only L-type histidine exists, it can produce obvious signal changes (ΔFnorm is the Fnorm signal corresponding to the sample minus the Fnorm signal of the blank sample solution), and L-type phenylalanine (L-Phe ), L-aspartic acid (L-Asp), L-arginine (L-Arg) and L-tyrosine (L-Tyr) samples, the signal is close to that of the blank sample. The results show that the detection method has good selectivity, and other amino acids cannot produce obvious signal changes. When D-type histidine (D-His) exists, the corresponding signal is the same as that of the blank sample solution, indicating that the corresponding method has high selective.

The described method can also be used for the detection of L-histidine added in serum samples and urine samples diluted 50 times with buffer solution, and the results are shown in Figure 6D, indicating that the detection method can be used for L-histidine in complex samples For acid detection, the detection limit reached 3.9 μM.

Embodiment 7: detect L-type histidine with SEQ ID NO:4-5'FAM

20nM SEQ ID NO:4-5'FAM was incubated with different concentrations of L-histidine in buffer solution 20mM Tris-HCl (pH 7.5)+400mM KCl+10mM MgCl ₂ at 25 degrees for 60 minutes, and then The sample solution is transferred to a capillary for microthermophoresis analysis, and microthermophoresis analysis is performed.

Figure 7A shows the time-varying curves of the fluorescent signal of the micro-thermophoresis analysis corresponding to different concentrations of L-histidine. As the concentration of L-histidine increases, the curve of the fluorescent signal changing with time gradually shifts down , the concentration of L-histidine corresponding to the curve from top to bottom is 0 μM, 0.488 μM, 0.977 μM, 1.95 μM, 3.91 μM, 7.81 μM, 15.6 μM, 31.3 μM, 62.5 μM, 125 μM, 250 μM, 500 μM, 1000 μM , 2000 μM, 4000 μM, 8000 μM. Figure 7B shows the relationship between the corresponding Fnorm value and the concentration of L-histidine when the selected time is 5 seconds. As the concentration of L-histidine increases, Fnorm decreases. According to the change of Fnorm, the concentration of L-histidine can be realized. detection. The detectable concentration range of L-histidine is 7.8 μM to 8 mM, and the detection limit is 7.8 μM. The maximum Fnorm (signal corresponding to 5s) signal change is about 95‰, which is smaller than the corresponding maximum Fnorm (signal corresponding to 5s) signal change when using SEQ ID NO:4-3'FAM for detection.

sequence

SEQ ID NO: 1: 5'-CTAT rA GGA AGAGAT GAT GTC TGT TTT TT ACAG ACATCATCTCTG AAG TAG CGC CGC CGT ATAG

SEQ ID NO:2: 5'-CTAT rAGGAAGAGAT GAT GTC TGT-3'

SEQ ID NO: 3: 5'-ACAGAC ATC ATC TCT GAAGTAGCG CCG CCG TAT AG-3'

SEQ ID NO: 4: 5'-CTAT rAGGAAGAGCC GTT TTT CGG CTC TTA ACG GGG CTG TGCGGC TAG GAA GTA ATA G-3'

SEQ ID NO:5: 5'-CTAT rA GGAAGA GCC G-3'

SEQ ID NO:6: 5'-CGG CTC TTAACG GGG CTG TGC GGC TAG GAA GTA ATA G-3'

SEQ ID NO: 7: 5'- CTAT rA GGA AGA GAT GAT GTC TGT TTT TT ACAG ACA TCA TCT CTG AAG TTA TAC CGC CGT ATA G -3'

The specific embodiments described above have further described the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above descriptions are only specific embodiments of the present invention, and are not intended to limit the present invention. Within the spirit and principles of the present invention, any modifications, equivalent replacements, improvements, etc., shall be included in the protection scope of the present invention.

Claims (10)

1. the probe that is used for the micro-thermophoresis analysis of object, it is characterized in that, described probe is: a. The sequence formed by coupling the RNA-cleaving DNAzyme and its substrate sequence together, and one end of the formed sequence is labeled with a fluorescent dye; or b. comprising an RNA-cleaving DNAzyme and its substrate sequence, and one of the RNA-cleaving DNAzyme and its substrate sequence is labeled with a fluorescent dye. 2. The micro-thermophoresis analysis method of target object, it is characterized in that, comprises the following steps: a. incubating the probe according to claim 1 with the target object to be tested in a buffer; b. Analyzing the signal changes generated by micro-thermophoresis, and obtaining the concentration of the target substance to be measured according to the signal changes. 3. A kit for microthermophoresis analysis of a target, comprising the probe and buffer according to claim 1. 4. The probe according to claim 1, the method according to claim 2 or the kit according to claim 3, wherein the fluorescent dye is a fluorescein molecule or tetramethylrhodamine. 5. probe according to claim 1, method according to claim 2 or kit according to claim 3, is characterized in that, described RNA cuts DNAzyme as shown in SEQ IDNO:3 , the substrate sequence is shown in SEQ ID NO: 2, and the target to be detected is a lead ion. 6. probe according to claim 1, method according to claim 2 or kit according to claim 3, is characterized in that, described RNA cuts DNAzyme as shown in SEQ IDNO:6 , the substrate sequence is shown in SEQ ID NO: 5, and the target to be detected is L-histidine. 7. probe according to claim 1, method according to claim 2 or kit according to claim 3, is characterized in that, described probe is as shown in SEQ ID NO:1, and described The target to be detected is lead ion; or the probe is shown in SEQ ID NO: 4, and the target to be detected is L-histidine. 8. probe according to claim 1, method according to claim 2 or test kit according to claim 3, is characterized in that, described RNA cuts DNAzyme and described substrate sequence by Multiple bases are coupled together, preferably, the multiple bases are TTTTT. 9. The method according to claim 2 or the kit according to claim 3, wherein the buffer is a Tris-HCl buffer containing magnesium ions and/or sodium ions/and or potassium ions. 10. The method according to claim 2, characterized in that, the incubation temperature ranges from 4°C to 37°C, preferably the temperature is 20°C or 25°C, and the incubation time is 15-90 minutes, preferably The incubation time is 15-60 minutes.

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