CN116024310B - Detection method and system for amplifying electrical signals based on ferrous ions - Google Patents

Abstract

The invention belongs to the technical field of analytical chemistry and electrochemistry, and specifically relates to a detection method and system for amplifying electric signals based on ferrous ions, including: preparation of chips; hybridization of chips; initial detection of chip electric signals; detection of chip electric signals Amplification; and the secondary detection of the chip amplified electrical signal, when the electrochemical signal is detected by square wave voltammetry, the free ferrous ions close to the ferrocene marker can form with the ferrous core of ferrocene The conjugated system acts as an electron donor as a whole. Due to the increase of the electron donor, the current amplification effect is realized, which improves the electrical signal difference between the negative sample and the positive sample. Due to the signal amplification, the electrochemical detection method reduces the electrical signal of the instrument. The dependence on the sensitivity and accuracy of the detection element makes the low-cost electrochemical detection instrument possible.

Description

Detection method and system based on ferrous ion amplified electric signal

technical field

The invention belongs to the technical field of analytical chemistry and electrochemistry, and in particular relates to a detection method and system for amplifying electric signals based on ferric ions.

Background technique

DNA electrochemical probe detection is gradually becoming popular in the genetic detection market. It is based on the improvement of the PCR fluorescent probe method, and uses electrochemical markers with redox properties such as transition metals and inorganic semiconductor nanoparticles to replace traditional probes. Fluorescent markers. These DNA probes containing electrochemical markers can specifically bind nucleic acid amplification products, and then in a specific electric field, the electrochemical labeling material undergoes a redox reaction to generate a transient current, and the current signal is detected to report whether there is a Genes detected.

At present, this method still has certain limitations. The most common problem is the small current signal. It is difficult for a DNA probe containing a single electrochemical marker to generate a large current, which requires a professional electrochemical workstation produced by AUTOLAB to detect it. The price is expensive and the volume is huge, which is difficult to promote. Furthermore, currents generated by DNA probes containing a single electrochemical label tend to be in the range of 10-100 nA . However, due to the presence of background noise in the solution, the electrical signal of a positive sample is often covered by the background noise, making it difficult to distinguish.

The existing technology solves this problem by increasing the number of labeled molecules. For example, the electrochemical probe adopted by the American GENMARK company binds 8 ferrocene molecular labels to ensure signal detection, but this method will bring additional problems. . The first is that it is necessary to carry out difficult labeling on the target DNA . In DNA synthesis technology, the recovery rate of the product gradually decreases with the increase of the number of labeled molecules, so the cost gradually increases. A domestic company quotes 30,000 probes for similar products. Each DNA probe with a different sequence needs to be customized and it is difficult to guarantee the success rate. The second is that too many molecular labels will produce steric hindrance, which will affect the ability of the probe to bind and recognize specific nucleotide sequences.

Therefore, based on the above technical problems, it is necessary to design a new detection method and system based on the amplified electric signal of ferrous ions.

Contents of the invention

The purpose of the present invention is to provide a detection method and system for amplifying electrical signals based on ferrous ions.

In order to solve the above-mentioned technical problems, the present invention provides a detection method based on ferrous ion amplified electric signal, comprising:

Chip preparation;

Chip hybridization;

The initial detection of chip electrical signals;

Amplification of chip electrical signals; and

The chip amplifies the secondary detection of the electrical signal.

Further, the preparation of the chip includes:

The surface of the gold electrode is modified by chemical reagents to modify its surface conductivity, including binding DNA capture probes and sealing the insulating molecular layer. Further, the hybridization method of the chip includes: the hybridization of the target gene and the signal probe, and the hybridization of the target gene and the chip;

The hybridization method of described target gene and signaling probe comprises:

A mixed solution is configured according to the category of the target gene, and PBS buffer is added to the configured mixed solution so that the liquid volume of the hybridization solution reaches a preset volume.

Further, configure the target gene solution according to the first preset concentration;

configuring a signal probe solution according to a second preset concentration;

After mixing the target gene solution and the signal probe solution, PBS buffer is added, so that the liquid volume of the hybridization solution of PBS buffer, target gene solution and PBS buffer reaches a preset volume.

Further, the hybridization solution is placed in an ep tube, and the hybridization is carried out at a preset temperature for a preset time, so that the hybridization of the hybridization solution is completed.

Further, the hybridization method of the target gene and the chip comprises:

Take out a preset amount of hybridization solution after hybridization, add the removed hybridization solution to the detection area of the modified electrode chip, and place it at the preset hybridization temperature for hybridization with a preset hybridization time to obtain a hybridized chip ;

If the preset hybridization temperature is different from the preset detection temperature, put the preset stabilization time before the detection and then perform the detection.

Further, the method for the initial detection of the chip electrical signal includes:

Perform electrochemical detection on the hybridized chip, connect the three electrodes with the leads of the electrochemical workstation, insert the hybridized chip so that the connection area is correctly in contact with the adapter;

Adjust each parameter according to the preset parameters after contact;

The first detection is performed after parameter adjustment.

Further, the method for amplifying the electrical signal of the chip includes: after the first detection, the PBS solution is gently washed away with clean water, and then a ferrous ion donor reagent of a preset concentration is added, and then the second detection is performed.

Further, the method for secondary detection of the chip amplified electrical signal includes:

Add ferrous ions to the hybridized chip and place it in the AUTOLAB electrochemical workstation for electrical signal detection;

Compare the electrochemical signal difference between the positive sample and the negative sample before and after adding the ferrous ion donor reagent.

In the second aspect, the present invention also provides a system using the above-mentioned detection method based on the amplified electric signal of ferrous ions, including:

Preparation of modules and chips;

Hybridization module, chip hybridization;

The initial detection module, the initial detection of the chip electrical signal;

Amplifying module, amplifying the electrical signal of the chip; and

The module is detected again, and the chip amplifies the electrical signal for secondary detection.

The beneficial effects of the present invention are that the present invention adopts the preparation of the chip; the hybridization of the chip; the primary detection of the chip electric signal; the amplification of the chip electric signal; When the signal is detected, the free ferrous ions close to the ferrocene marker can form a conjugated system with the ferrous core of ferrocene, which acts as an electron donor as a whole, and the current amplification effect is realized due to the increase of the electron donor. The electrical signal difference between the negative sample and the positive sample is improved. Due to the amplification of the signal, the dependence of the electrochemical detection method on the sensitivity and accuracy of the electrical signal detection components of the instrument is reduced, making low-cost electrochemical detection instruments possible.

Additional features and advantages of the invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and appended drawings.

In order to make the above-mentioned objects, features and advantages of the present invention more comprehensible, preferred embodiments will be described in detail below together with the accompanying drawings.

Description of drawings

In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the following will briefly introduce the accompanying drawings that need to be used in the specific implementation or description of the prior art. Obviously, the accompanying drawings in the following description The drawings show some implementations of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

Fig. 1 is the flow chart of the detection method based on ferrous ion amplification electric signal of the present invention;

Fig. 2 is the schematic diagram of the detection method based on ferrous ion amplified electric signal of the present invention;

Fig. 3 is a schematic diagram of the first detection result of the present invention;

Fig. 4 is a schematic diagram of the second detection result of the present invention.

Detailed ways

In order to make the purpose, 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 in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. the embodiment. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Example 1

As shown in Figures 1 to 4, the present embodiment 1 provides a detection method based on the amplified electric signal of ferrous ions, including: preparation of the chip; hybridization of the chip; initial detection of the electric signal of the chip; Amplification; and the secondary detection of the chip amplified electrical signal, when the electrochemical signal is detected by square wave voltammetry, the free ferrous ions close to the ferrocene marker can form with the ferrous core of ferrocene The conjugated system acts as an electron donor as a whole. Due to the increase of electron donors, the current amplification effect is realized, which improves the electrical signal difference between the negative sample and the positive sample. Due to the signal amplification, the electrochemical detection method reduces the electrical signal of the instrument. The dependence on the sensitivity and accuracy of detection components makes it possible to use low-cost electrochemical detection instruments, which avoids the small electrochemical signal of ferrocene-labeled DNA and is easily interfered by background signals.

In this embodiment, the preparation of the chip includes: modifying the surface of the gold electrode with chemical reagents, modifying the conductivity of the surface, including binding DNA capture probes, and sealing with an insulating molecular layer.

In this embodiment, the hybridization method of the chip includes: the hybridization of the target gene and the signal probe, and the hybridization of the target gene and the chip; obtaining the hybridized chip is convenient for detection, so as to compare the positive sample and the negative sample. difference in electrochemical signals.

The method for the hybridization of the target gene and the signal probe comprises: configuring a mixed solution according to the category of the target gene, and adding PBS buffer in the configured mixed solution so that the liquid volume of the hybridization solution reaches a preset volume; the target gene can be It is a single-stranded synthetic DNA ( ssDNA ), or it can be a LAMP product; for example, if the target gene is a single-stranded synthetic DNA , configure a mixed solution according to Table 1 below, and finally add PBS buffer to make the liquid volume 50 μL .

Table 1: Solution configuration table

Different solutions can be configured according to requirements to obtain different concentrations for easy detection.

In this embodiment, the target gene solution is configured according to the first preset concentration; the signal probe solution is configured according to the second preset concentration; PBS buffer is added after the target gene solution and the signal probe solution are mixed, so that the PBS buffer, The liquid volume of the hybridization solution of the target gene solution and PBS buffer reaches a preset volume; for example, PBS buffer is finally added so that the liquid volume of the hybridization solution is 50 μL .

In this embodiment, the hybridization solution is placed in the ep tube, and the hybridization is carried out at the preset temperature for a preset time, so that the hybridization of the hybridization solution is completed; for example, the prepared hybridization solution is placed in the ep tube at 42°C Hybridize for 30 minutes.

In this embodiment, the hybridization method of the target gene and the chip includes: taking out a preset amount of hybridization solution after hybridization, adding the hybridization solution to the detection area of the modified electrode chip, and placing it in the pre-set Set the hybridization temperature for the preset hybridization time to obtain the hybridized chip; if the preset hybridization temperature is different from the preset detection temperature, set the preset stability time before detection before detection; for example, if the hybridization is completed Take out 50 μL of the hybridization solution and add it to the detection area of the modified electrode chip, and place it at 37°C for 30 minutes for hybridization. Do not wash after the hybridization is completed. Stand still for 10 minutes and wait for the temperature of the hybridization solution to remain stable before using the device for electrochemical detection.

In this embodiment, the method for the initial detection of the chip electrical signal includes: performing electrochemical detection on the hybridized chip, connecting the three electrodes to the leads of the electrochemical workstation, inserting the hybridized chip so that the connection area is correctly in contact with the adapter; Adjust the parameters according to the preset parameters after contact; perform the first detection after parameter adjustment; that is, directly place the hybridized chip (hybridized chip) in the AUTOLAB electrochemical workstation for electrical signal detection; for example, use Square-wave voltammetry is used to electrochemically detect the chip, using a three-electrode system. The three electrodes are composed of: working electrode, reference electrode, and counter electrode (auxiliary electrode); first, ensure that the three electrodes are connected correctly to the three leads of the electrochemical workstation. , insert the chip so that the connection area is correctly in contact with the adapter, adjust the detection interface parameters as shown in Table 2 below, and then start the first detection.

Table 2: Parameter List

The method for amplifying the electrical signal of the chip includes: after the first detection, after the PBS solution is gently washed away by clear water, adding a ferrous ion donor reagent of a preset concentration (characterized by the formation of ferrous ions) compound, potassium ferrocyanide is used in this embodiment, FeSO ₄ , FeCl _{2 ,} etc. can also be used), and then the second detection is carried out; the method for the second detection of the amplified electrical signal of the chip includes: adding the chip after hybridization After the ferrous ion is placed in the AUTOLAB electrochemical workstation for electrical signal detection; compare the electrochemical signal difference between the positive sample and the negative sample before and after adding the ferrous ion donor reagent; after the first detection, rinse gently with water Remove the PBS solution, add potassium ferrocyanide solution at a concentration of 500 uM , and perform a secondary detection according to the above parameters again, and compare the electrochemical signal difference between the positive sample and the negative sample before and after adding the potassium ferrocyanide solution. Before the signal is amplified, the electrical signal value of the synthesized actin single-stranded DNA is the same as that of the synthesized GAPDH single-stranded DNA , which are all covered by the background noise generated by the ions of the hybridization solution itself. After adding 500 uM potassium ferrocyanide solution, only the signal value of the positive sample is amplified, the background noise signal of the negative sample remains unchanged, and the negative and positive samples can be clearly distinguished. When the electrochemical signal is detected by square wave voltammetry, the free ferrous ions close to the ferrocene marker can form a conjugated system with the ferrous iron core of ferrocene, and the whole acts as an electron donor. The increase of donors realizes the current amplification effect, and the DNA electrochemical probe is labeled with a single ferrocene molecule, and ferrous ions are added to the detection solution as an amplification reagent, so that the DNA electrochemical probe labeled with a single ferrocene molecule The current signal generated by the probe is amplified by a factor of 100.

In this embodiment, it is verified on the microarray biosensor by square wave voltammetry that the ferrous ions have an amplification effect on the electrical signal of the ferrocene-labeled DNA probe; the specific equipment involved may include: microarray biosensor Sensors, electrochemical workstation ( AUTOLAB ), incubator, various types of pipettes, sterile filter pipette tip (10 µL , 20 µL ), 1.5 ml microcentrifuge tube, 100 mm diameter Petri dish, signal probe solution S - probe , PBS buffer , samples to be tested ( positive samples : synthetic actin single - stranded DNA ; negative samples : synthetic GAPDH single - stranded DNA ) , ferrous ion donor reagent ( characterized by the formation of ferrous ions Ionic compound, potassium ferrocyanide is used in this embodiment, FeSO ₄ , FeCl _{2 ,} etc. can also be used); the hybridization of the chip can be performed through the above equipment and the electrochemical detection of the hybridized chip can be performed.

Example 2

On the basis of Example 1, this Example 2 also provides a system using the detection method based on ferrous ion amplified electrical signals in Example 1, including: preparation module, chip preparation; hybridization module, chip hybridization The initial detection module, the initial detection of the chip electrical signal; the amplification module, the amplification of the chip electrical signal; and the detection module again, the secondary detection of the chip amplified electrical signal; No longer.

In summary, the present invention adopts the preparation of the chip; the hybridization of the chip; the initial detection of the chip electrical signal; the amplification of the chip electrical signal; During detection, the free ferrous ions close to the ferrocene marker can form a conjugated system with the ferrous iron core of ferrocene, and act as an electron donor as a whole. Due to the increase of electron donors, the current amplification effect is realized, which improves the The electrical signal difference between negative samples and positive samples, due to signal amplification, reduces the dependence of electrochemical detection methods on the sensitivity and accuracy of instrument electrical signal detection components, making low-cost electrochemical detection instruments possible.

In the several embodiments provided in this application, it should be understood that the disclosed devices and methods may also be implemented in other ways. The device embodiments described above are only illustrative. For example, the flowcharts and block diagrams in the accompanying drawings show the architecture, functions and possible implementations of devices, methods and computer program products according to multiple embodiments of the present invention. operate. In this regard, each block in a flowchart or block diagram may represent a module, program segment, or part of code that includes one or more Executable instructions. It should also be noted that, in some alternative implementations, the functions noted in the block may occur out of the order noted in the figures. For example, two blocks in succession may, in fact, be executed substantially concurrently, or they may sometimes be executed in the reverse order, depending upon the functionality involved. It should also be noted that each block of the block diagrams and/or flowchart illustrations, and combinations of blocks in the block diagrams and/or flowchart illustrations, can be implemented by a dedicated hardware-based system that performs the specified function or action , or may be implemented by a combination of dedicated hardware and computer instructions.

In addition, each functional module in each embodiment of the present invention can be integrated together to form an independent part, or each module can exist independently, or two or more modules can be integrated to form an independent part.

If the functions are implemented in the form of software function modules and sold or used as independent products, they can be stored in a computer-readable storage medium. Based on this understanding, the essence of the technical solution of the present invention or the part that contributes to the prior art or the part of the technical solution can be embodied in the form of a software product, and the computer software product is stored in a storage medium, including Several instructions are used to make a computer device (which may be a personal computer, a server, or a network device, etc.) execute all or part of the steps of the methods described in various embodiments of the present invention. The aforementioned storage media include: U disk, mobile hard disk, read-only memory ( ROM , Read - Only Memory ), random access memory ( RAM , Random Access Memory ), magnetic disk or optical disk, and other media that can store program codes .

Inspired by the above-mentioned ideal embodiment according to the present invention, through the above-mentioned description content, relevant workers can make various changes and modifications within the scope of not departing from the technical idea of the present invention. The technical scope of the present invention is not limited to the content in the specification, but must be determined according to the scope of the claims.

Claims (5)

1. A detection method based on ferrous ion amplified electric signal, is characterized in that, comprises: Chip preparation; Chip hybridization; The initial detection of chip electrical signals; Amplification of chip electrical signals; and Secondary detection of chip amplified electrical signal; The preparation of the chip includes: Modify the surface of the gold electrode with chemical reagents to modify its surface conductivity, including binding DNA capture probes and sealing the insulating molecular layer; The hybridization method of the chip comprises: the hybridization of the target gene and the signal probe, and the hybridization of the target gene and the chip, and the signal probe is a DNA electrochemical probe labeled by a single ferrocene molecule; The method for the initial detection of the chip electrical signal comprises: Perform electrochemical detection on the hybridized chip, connect the three electrodes with the leads of the electrochemical workstation, insert the hybridized chip so that the connection area is correctly in contact with the adapter; Adjust each parameter according to the preset parameters after contact; Perform the first detection after parameter adjustment; The method for amplifying the electrical signal of the chip includes: after the first detection, after the PBS solution is gently rinsed off with clear water, a ferrous ion donor reagent of a preset concentration is added, and then the second detection is performed; The method for secondary detection of the chip amplified electrical signal includes: Add ferrous ions to the hybridized chip and place it in the AUTOLAB electrochemical workstation for electrical signal detection; Compare the electrochemical signal difference between the positive sample and the negative sample before and after adding the ferrous ion donor reagent. 2. the detection method based on ferrous ion amplification electric signal as claimed in claim 1, is characterized in that, The hybridization method of described target gene and signaling probe comprises: A mixed solution is configured according to the category of the target gene, and PBS buffer is added to the configured mixed solution so that the liquid volume of the hybridization solution reaches a preset volume. 3. the detection method based on ferrous ion amplified electrical signal as claimed in claim 2, is characterized in that, configuring the target gene solution according to the first preset concentration; configuring a signal probe solution according to a second preset concentration; After mixing the target gene solution and the signal probe solution, PBS buffer is added, so that the liquid volume of the hybridization solution of PBS buffer, target gene solution and PBS buffer reaches a preset volume. 4. the detection method based on ferrous ion amplified electric signal as claimed in claim 3, is characterized in that, The hybridization solution is placed in an ep tube, and the hybridization is performed at a preset temperature for a preset time, so that the hybridization of the hybridization solution is completed. 5. the detection method based on ferrous ion amplified electric signal as claimed in claim 4, is characterized in that, The hybridization method of described target gene and chip comprises: Take out a preset amount of hybridization solution after hybridization, add the removed hybridization solution to the detection area of the modified electrode chip, and place it at the preset hybridization temperature for hybridization with a preset hybridization time to obtain a hybridized chip ; If the preset hybridization temperature is different from the preset detection temperature, put the preset stabilization time before the detection and then perform the detection.

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