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Method for detecting concentration of target molecule in mixed systemand kit

A target and molecular technology, applied in the field of a method and kit for detecting the concentration of target molecules in a mixed system, can solve the problems of large limitations and achieve the effect of short time consumption and simple operation

Active Publication Date: 2021-06-18
北京聚树生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method can only detect small molecules with corresponding allosteric proteins, which has great limitations.

Method used

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  • Method for detecting concentration of target molecule in mixed systemand kit
  • Method for detecting concentration of target molecule in mixed systemand kit
  • Method for detecting concentration of target molecule in mixed systemand kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 Feasibility Verification Experiment

[0073]In order to verify the feasibility of the method, we chose ATP's adapter sequence (SEQ ID NO.1), and designed CrRNA-ATP sequences according to this sequence, combined with the characteristics and properties of Cas12a nuclease and its crrna ( SEQ IDNO.2). Positive control (PC, curve with a circular point) By forming a Cas12A-CrNA-adapter composite, CAS12A is activated, and the FQ Probe is cut by inverted active, and the other is added to the other group. The molecule acts as an experimental group (curve of the ribbon), due to the specific binding of the adapter, resulting in the effective adapter of the excitation, and the fluorescence value decreases (see Figure 2A ).

[0074] Considering the decline in the addition of ATP, it is possible to have such a target molecule that has an effect on the activity of the protein in the reaction system, thereby reducing the fluorescent signal, is not an inhibitory effect that the co...

Embodiment 2

[0098] Example 2. Optimization of the reaction system

[0099] 1 Optimization of the reaction buffer system:

[0100] Cas12a is a nuclease, and its cutting activity will definitely be affected by the surrounding environment. In the previous report, some people have used NEB buffer 2.1, NEB buffer 3.1, NEB buffer 4, Neb cutsmart buffer, or buffer itself. Therefore, we have studied the effects of buffer of different ingredients for CAS12A cutting activity (see Figure 3A with Figure 3B ). It can be seen that important factors affecting Cas12a lysis activity is the divalent cation mg in the test solution. 2+ concentration. Because the Cas12a RUVC domain clears SSDNA, MG by bimetallic ion mechanism 2+ Ions induce the conformational coordination of the RUVC domain and SSDNA by changing the spatial distribution around the SSDNA in the RUVC active cutting center. This result is consistent with the previous report. But when MG 2+ When the concentration is constant, different buffers also e...

Embodiment 3

[0114] Example 3 Sensitivity and specific experiment

[0115] Based on the above optimization conditions, we evaluate the sensitivity of the method to detect the ATP target molecule. We will use the adapter as a SSDNA-activated sub-activator as a positive control. After adding different concentrations of ATP, due to the specific binding of ATP and adapter, it is reduced by the adapter of activation, and the fluorescent signal has different degrees of decline. Figure 8A ). Quantitative analysis can be accomplished by monitoring the inhibition rate after 13 min after the start. We IR% of ATP y-axis, the logarithm of the ATP concentration is plotted as the x-axis, can be obtained by fitting a linear response, R2 = 0.9496, its dynamic range (dynamicrange) is 0.1mM-5mM (see Figure 8B ). According to the formula detection (LOD) = 3 * SD / slope. The LOD of this method can reach 2.66 μm. This sensitivity and dynamic range are better than the adapter-based color sensor, but may not reach ...

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Abstract

The invention discloses a method for detecting the concentration of a target molecule in a mixed system and a kit. The invention relates to a method for quantitatively detecting the concentration of a target molecule X. The method comprises the steps of adding a reporter molecule A into a reaction system containing the target molecule X for at least first given time, wherein specific binding activity exists between the reporter molecule A and the target molecule X; adding a competitive molecule B into the reaction system, wherein specific binding activity exists between the competitive molecule B and the reporter molecule A, the competitive molecule B and the target molecule X do not have any binding activity, and when the target molecule X does not exist, fluorescence intensity L0 with given intensity is emitted when the competitive molecule B is combined with the reporter molecule A; and uniformly mixing the system, and detecting the fluorescence intensity L of the system after waiting for at least a second given time, wherein in the ideal concentration range, a linear relation between the concentration of the target molecule X and the L0-L value can be established, and the established linear relation is used as a standard curve, and the concentration of the target molecule X can be calculated according to the actually measured fluorescence intensity change value L0-L. The invention also relates to a kit matched with the method.

Description

Technical field [0001] The present invention relates to the field of chemical analysis, and more particularly to a method and kit for detecting a target molecular concentration within a mixing system. Background technique [0002] The detection of material concentration is widely used in chemical, biomedicine, and medicine. The study of emerging and developing nucleic acid suitable ligands in the 1990s has even prompted the development of this area. The nucleic acid fit is a small sequence of oligonucleotide sequences or short polypeptides that are screened by vitro, which can provide high affinity and strong specificity with the corresponding ligand, which is provided as the chemical biological and biomedical community. A new research platform for highly efficient and rapid identification and shows a good application prospect in many ways. [0003] Conventional nucleic acid fittings The detection method of target molecular concentration is mostly involved in G4 integer color, fl...

Claims

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Application Information

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IPC IPC(8): G01N33/53
CPCG01N33/5308Y02A50/30
Inventor 张翀牛晨启邢新会
Owner 北京聚树生物科技有限公司
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