CN101975810A - High-pass detection electrode of complex sample and preparation method thereof - Google Patents

Abstract

The invention provides an electrode capable of online simultaneous detection of analytes containing multiple components. The electrode can be combined with a potentiostat, a multiplexer, and data processing software to use the specificity of different target objects of different DNA sequence pairs for online detection of the analyte, which combines the intuitiveness of the array with the specificity of DNA. There are few reports on the combined sensing device. For the analytes containing multiple components, this device can also realize simultaneous online detection, and has strong operability and has the advantage of simple instruments.

Description

High-throughput detection electrode for complex samples and its preparation method

technical field

The invention belongs to the field of analytical chemistry, and more specifically relates to a high-flux detection electrode for complex samples and a preparation method thereof.

Background technique

In the detection of water samples, blood or human cells, these samples contain a variety of complex components at the same time, such as those contained in water samples (such as Pb ²⁺ , Hg ²⁺ , UO ²⁺ , microorganisms), proteins in blood ( Thrombin) or pathogenic cells in the human body, it is often necessary to detect one by one in general detection (for example, metal ions can be detected by atomic absorption method, inductively coupled plasma method, anodic dissolution method, capillary electrophoresis, etc.) The time to get the result greatly increase. Although these conventional methods have the advantage of high sensitivity, the instruments used in the detection are very precise, and complex samples need to be pre-treated, and well-trained personnel are required to perform skilled operations. People have developed a series of sensors by using some physical and chemical properties of substances, which can express the existence and content of these substances through signals that are easily obtained in the laboratory, such as light, electricity, etc., so that general laboratories can pass some simple sensors. However, these sensors also have shortcomings, that is, their selectivity is not satisfactory compared with conventional methods.

In the past ten years, DNA technology has been greatly developed. In addition to hybridizing with complementary sequences, DNA (or RNA) can also specifically combine with metal ions, small biological molecules, and even cells to form an aptamer-target complex structure. More than 100 aptamers have been discovered so far, but there are still a large number of aptamers that have not been screened out by people. People have developed a large number of sensors using existing aptamers. These sensors have the advantages of good selectivity, high sensitivity, and low detection limit. At the same time, they are easy to operate, and the cost of instruments and drugs is low, which overcomes the defects of conventional methods. It has broad application prospects. Due to the high sensitivity of electrochemistry and the easy operation of the instrument, there have been many reports on electrochemistry-based DNA sensors. The principle of these methods is mainly based on the introduction of the target substance to break or strip the DNA strand, which changes the DNA structure on the electrode surface, thereby causing changes in electrochemical signals (such as current value, electricity, and impedance signals, etc.). However, these methods can only detect one target object (at most two or three types), and the signal expression is not intuitive. If there are a large number of objects to be tested, the electrodes must be modified again, which requires a large number of electrodes and waiting samples. Analytes, therefore, the simultaneous detection of complex analytes containing multiple components with clearly identifiable results has become the focus of attention.

Contents of the invention

The object of the present invention is to provide an electrode capable of multi-channel detection, which can be combined with a potentiostat, a multiplexer, and a program for controlling and collecting data, so as to realize simultaneous detection of multiple components The test object is tested.

An electrode capable of realizing multi-channel detection is an array electrode, which is composed of transverse electrodes and longitudinal electrodes intersecting each other. The preparation method of the array electrode is as follows:

1) Cut the two pieces of glass into appropriate sizes, wash them, ultrasonically clean them with secondary water, ethanol and acetone respectively for 10min-30min, and place them in acetone for 8-12h;

2) After ashing the surface of the above-mentioned two glass sheets, evenly coat a layer of positive photoresist; place them flat on the platform of the exposure instrument, and place a pre-etched Cr template on the glass sheet, UV light is exposed through the Cr plate for 15 seconds, cleaned and cured with a developer;

3) Ash the glass surface after 2) development, and evenly sputter Cd and Au on the glass surface in a metal sputterer. The thicknesses of Cd and Au are 0.5-1.5nm and 5-8nm respectively; The glass is cleaned with acetone, and the previously unexposed part is peeled off from the glass surface with the cleaning of acetone, and copper wires are connected to the solder joints of the metal layer, and one of them is designated as a horizontal electrode;

4) After ashing the other electrode obtained in 3) again, coat the surface with a layer of negative photoresist, place it flat on the platform of the exposure instrument, and place another Cr plate above it to allow ultraviolet light to pass through the Cr plate , exposing and etching the electrode, cleaning the developer with ethanol, peeling off the surface of the unexposed part, forming n×n micropores of 100×100×5 μm, length×width×height on the electrode, and obtaining a longitudinal electrode;

5) The horizontal electrode and the vertical electrode obtained in 3) and 4) are placed vertically across each other, so that the micropores of the vertical electrode are aligned with the electrode lines of the horizontal electrode; the two electrodes are separated by double-sided adhesive tape.

The positive photoresist used is commonly used positive photoresist, which can be S1818 positive photoresist; the negative photoresist used in step 4) is commonly used negative photoresist, which can be SU-8 negative photoresist.

The array electrode is combined with a potentiostat, a multiplexer, and a program for controlling and collecting data to realize the simultaneous online monitoring of multiple components. The steps are:

Step 1), immobilization of the capture probe: the capture probe is immobilized in the microwell of the longitudinal electrode through a gold sulfhydryl bond;

Step 2), hybridize the target aptamer with the capture probe, inject potassium ferricyanide solution between the two electrodes, and apply 0 and 0.6V voltages on the two array electrodes using a potentiostat, The obtained current is converted into a current topography map, and the depth of the color on the topography map reflects the strength of the current;

Step 3), adding the target substance to the microwell, the target substance can bind to the aptamer, causing the part of the aptamer to break or peel off the surface of the microwell; after that, the surface is cleaned to remove the fallen DNA fragments, and then Apply voltages of 0 and 0.6V to the two electrodes respectively, and the obtained current value is converted into a current intensity topography map. By comparing with step 2), it is possible to judge whether certain substances exist by changing the color of the topography map.

Significant advantage of the present invention is:

The invention designs a novel multi-channel detection electrode device. The DNA is first fixed in the microwell of the longitudinal electrode. When the target substance is introduced, the amount of DNA on the microwell changes, which causes the change of the negatively charged phospholipid skeleton and the change of the current signal. Since one micropore can immobilize one kind of DNA, and different DNA can respond to specific targets, this method changes the situation that the previous sensor can only detect a single target, and can analyze multiple different components at the same time. Moreover, the electronegativity of the DNA phosphate backbone is used to achieve label-free detection without changing the characteristics of the DNA. At the same time, the detected current signal can be converted into a topography map of the current, and the strength of the current can be visually identified through the depth of the color on the topography map. Due to the good specificity of DNA, when the target coexists with other substances, the interference caused by other substances can be avoided, so this method can well realize the simultaneous online monitoring of multiple components.

Description of drawings

Fig. 1 is array electrode of the present invention and potentiostat, multiplexer, is used for the schematic diagram of the device that the program of collecting data is combined with high-throughput detection, and wherein 1 is multiplexer; 2 is constant Potentiometer; 3 is a program for controlling and collecting data; 4 is an array electrode.

Fig. 2 is a schematic diagram of an array electrode of the present invention, wherein (a) is a top view of the electrode, and (b) is a front view of the electrode. 1 is the horizontal electrode; 2 is the vertical electrode; 3' and 3'' are the glass substrates of the horizontal electrode and the vertical electrode respectively; 4 is the hardened photoresist; 5 is the double-sided adhesive.

Detailed ways

An electrode capable of multi-channel detection is an array electrode, which is composed of horizontal electrodes and longitudinal electrodes intersecting each other. The preparation method of the array electrode is:

1) Cut the two pieces of glass into appropriate sizes, wash them, ultrasonically clean them with secondary water, ethanol and acetone for 10min-30min respectively, and place them in acetone for 8-12h;

2) After ashing the surface of the above-mentioned two glass sheets, evenly coat a layer of positive photoresist; place them on the platform of the exposure instrument, place a pre-etched Cr template on the glass sheet, UV light is exposed through the Cr plate for 15 seconds, cleaned and cured with a developer;

3) Ash the glass surface after 2) development, and evenly sputter Cd and Au on the glass surface in a metal sputterer. The thicknesses of Cd and Au are 0.5-1.5nm and 5-8nm respectively; The glass is cleaned with acetone, and the previously unexposed part is peeled off from the glass surface with the cleaning of acetone, and copper wires are connected to the solder joints of the metal layer, and one of them is designated as a horizontal electrode;

4) After ashing the other electrode obtained in 3) again, coat the surface with a layer of negative photoresist, place it flat on the platform of the exposure instrument, and place another Cr plate above it to allow ultraviolet light to pass through the Cr plate , exposing and etching the electrode, cleaning the developer with ethanol, peeling off the surface of the unexposed part, forming n×n micropores of 100×100×5 μm, length×width×height on the electrode, and obtaining a longitudinal electrode;

5) The horizontal electrode and the vertical electrode obtained in 3) and 4) are placed vertically across each other, so that the micropores of the vertical electrode are aligned with the electrode lines of the horizontal electrode; the two electrodes are separated by double-sided adhesive tape.

 Using the array electrode of the present invention, potentiostat, multiplexer, program for controlling and collecting data in conjunction with a device with high-throughput detection to detect the sample to be tested includes the following steps:

Step 1, immobilization of the capture probe: the capture probe is immobilized in the micropore of the longitudinal electrode through a gold sulfhydryl bond.

Step 2: hybridize a series of target aptamers with the capture probes, apply a voltage on the two electrodes of the sensor using a potentiostat, and convert the obtained current into a current topography map, and the color on the topography map The depth reflects the strength of the current.

Step 3: Add the target substance into the microwell, and the target substance can bind to the aptamer, causing the part of the aptamer to break or peel off the surface of the microwell. Afterwards, clean the surface to remove the DNA fragments that fell off, and then apply a voltage on the two array electrodes, and the obtained current value is converted into a current topography map, which can be judged by the change in the color of the topography map by comparing with step 2. the existence of certain substances.

The following examples describe the specific working principles of the present invention.

Example 1

The production method of the electrode is as follows: 1) Cut two pieces of glass into appropriate sizes, wash them, ultrasonically clean them with secondary water, ethanol and acetone for 10 minutes, and place them in acetone for 8 hours. After the surface of the glass sheet is ashed, it is evenly coated with a layer of positive photoresist; it is placed flat on the platform of the exposure instrument, and a pre-etched Cr template is placed on the glass sheet, and the ultraviolet light is exposed through the Cr plate for 15 seconds. , cleaned and cured with a developer; 3) Ash the developed glass surface in 2), and uniformly sputter Cd and Au on the glass surface in a metal sputterer, the thickness of Cd and Au are 0.5nm and 5nm respectively; the rest The steps are the same as the specific implementation.

The water sample is used as the detection object for analysis. The water sample contains Pb ²⁺ , Zn ²⁺ , Cu ²⁺ , and UO ²⁺ . The number of micropores used is 4×4:

Step 1: Four kinds of probe DNAs (mainly metal-dependent DNAzyme for metals, so the probe DNA is the complementary strand of DNAzyme) are immobilized in four mutually independent microwells through gold-sulfhydryl bonds.

Step 2: add corresponding complementary strand substrates to the above four microwells respectively, and age for hybridization. Potassium ferricyanide solution is injected between the two electrodes, and voltages of 0 and 0.6V are applied respectively to allow ferricyanide ions to undergo oxidation and reduction between the electrodes to generate a current signal, and the current conversion results in a reflected current shape with 4×4 grids. The topography map, the depth of each grid color in the topography map reflects the strength of the current.

Step 3: Clean the electrodes carefully, add the solution to be tested that may contain the above four metal ions into the micropores, react for a period of time, and carefully clean the micropores. Then, repeat the detection step in step 2 of the experimental example to obtain a current topography map of 4×4 grids. By comparing with the figure obtained in step 2, identify the type of ions contained in the liquid to be tested according to the change in color depth.

Example 2

The production method of the electrode is as follows: 1) Cut two pieces of glass into appropriate sizes, wash them, ultrasonically clean them with secondary water, ethanol and acetone for 30 minutes, and place them in acetone for 10 hours. After the surface of the glass sheet is ashed, it is evenly coated with a layer of positive photoresist; it is placed flat on the platform of the exposure instrument, and a pre-etched Cr template is placed on the glass sheet, and the ultraviolet light is exposed through the Cr plate for 15 seconds. , cleaned and cured with a developer; 3) Ash the developed glass surface in 2), and uniformly sputter Cd and Au on the glass surface in a metal sputterer, the thickness of Cd and Au are 1.5nm and 8nm respectively; the rest The steps are the same as the specific implementation.

The blood samples of drug addicts were used as the research object for analysis. The blood samples contained cocaine, adenosine triphosphate, and thrombin. The number of microwells used was 4×4:

Step 1: Immobilize the three probe DNAs (since the aptamer can specifically bind small molecules and proteins, the probe DNA is the complementary strand of the corresponding aptamer) in three independent microwells through gold-sulfhydryl bonds middle.

Step 2: adding corresponding complementary strand substrates to the above three microwells respectively, and aging for hybridization. Potassium ferricyanide solution is injected between the two array electrodes, and voltages of 0 and 0.6V are applied respectively, so that ferricyanide ions undergo oxidation and reduction between the electrodes to generate current signals, and the current conversion results in a reflected current intensity with 4×4 grids Weak topography map, the depth of each grid color reflects the strength of the current.

Step 3: Clean the electrode carefully, add the treated blood sample to be tested into the microwell, react for a period of time, and carefully clean the microwell. Then, repeat the detection step in step 2 of the experimental example to obtain a current topography map of 4×4 grids. By comparing with the image obtained in step 2, the small molecules and protein types contained in the blood sample liquid to be tested are identified according to the change in color depth.

Example 3

The production method of the electrode is as follows: 1) Cut two pieces of glass into appropriate sizes, wash them, ultrasonically clean them with secondary water, ethanol and acetone respectively for 20 minutes, and place them in acetone for 9 hours. After the surface of the glass sheet is ashed, it is evenly coated with a layer of positive photoresist; it is placed flat on the platform of the exposure instrument, and a pre-etched Cr template is placed on the glass sheet, and the ultraviolet light is exposed through the Cr plate for 15 seconds. , cleaning and curing with a developer; 3) Ashing the developed glass surface in 2), uniformly sputtering Cd and Au on the glass surface in a metal sputterer, the thickness of Cd and Au are 1nm and 7nm respectively; the rest of the steps Same as the specific implementation.

 Using human cell samples as the research object for analysis, the samples include human B lymphoma cells and prostate cancer cells, and the number of microwells used is still 4×4:

In step 1, the two probe DNAs (since the aptamer can specifically bind to the surface of the corresponding cell, the probe DNA is the complementary strand of the corresponding aptamer) are immobilized on two independent microstructures through gold-sulfhydryl bonds. in the hole.

Step 2: adding corresponding complementary strand substrates to the above two microwells respectively, and aging for hybridization. Potassium ferricyanide solution is injected between the two electrodes, and voltages of 0 and 0.6V are applied respectively to allow ferricyanide ions to undergo oxidation and reduction between the electrodes to generate current signals, and the current conversion results in a reflected current morphology with 4×4 grids In the figure, the depth of each grid color in the topographical figure reflects the strength of the current.

Step 3, carefully clean the electrode, add the cell sample into the microwell, react for a period of time, and carefully clean the microwell. Then, repeat the detection step in step 2 of the experimental example to obtain a current topography map of 4×4 grids. By comparing with the image obtained in step 2, the type of cancer cells contained in the cell sample is identified according to the change in color depth.

The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

Claims (5)

1. the high throughput testing electrode of complex sample, it is characterized in that: described electrode is an array electrode, is intersected mutually to place by transverse electrode and longitudinal electrode to constitute.

2. the preparation method of the high throughput testing electrode of complex sample, it is characterized in that: the preparation method of described array electrode is:

1) two blocks of glass sheet is cut into appropriate size, clean,, be placed on 8-12h in the acetone with secondary water, ethanol and acetone difference ultrasonic cleaning 10min-30min;

2) above-mentioned two blocks of glass sheet handling well are carried out surperficial ashing after, apply the positive photoresist of last layer equably; Lie on the platform of exposure instrument, place a textured Cr template of etching in advance on the glass sheet, ultraviolet light sees through Cr plate exposure 15 seconds, cleans with developer and solidifies;

3) with 2) glass surface ashing after developing, in the metal sputtering device with glass surface Cd and Au in the even sputter, the thickness of Cd and Au is respectively 0.5-1.5nm and 5-8nm; The glass that plates metal cleans with acetone, and unexposed before part connects copper conductor along with the cleaning of acetone is peeled off from glass surface at the solder joint place of metal level, will be wherein one be decided to be transverse electrode;

4) with 3) another cube electrode of obtaining once more after the ashing at the negative photoresist of surface-coated last layer, lie on the platform of exposure instrument, another piece Cr plate is placed in the top, allow ultraviolet light see through this Cr plate, to the electrode etching of exposing, developer cleans with ethanol, unexposed part stripper surface, form n * n 100 * 100 * 5 μ m on electrode, length * wide * high micropore obtains longitudinal electrode;

5) with 3) with 4) transverse electrode and the longitudinal electrode that obtain intersect vertical placement mutually, and the micropore of longitudinal electrode is alignd with the electrode wires of transverse electrode; Separate by double faced adhesive tape between two plate electrodes.

3. the preparation method of an array electrode as claimed in claim 2 is primarily characterized in that: step 2) in the positive photoresist that adopted for positive photoresist commonly used, can be the positive photoresist of S1818; The negative photoresist that is adopted in the step 4) is negative photoresist commonly used, can be the negative photoresist of SU-8.

4. array electrode as claimed in claim 1 or 2 is primarily characterized in that: this electrode can with potentiostat, multiplexer is used to control the program associating with image data, has realized multi-component while on-line monitoring.

5. array electrode as claimed in claim 4 is primarily characterized in that: described array electrode and potentiostat, and multiplexer is used to control the program associating with image data, has realized multi-component while on-line monitoring, and its detection method is:

Step 1), capture probe fixing: in the micropore of longitudinal electrode by golden mercapto key fixing on capture probe;

Step 2), the aptamers and the capture probe ageing of object are hybridized, between two plate electrodes, inject potassium ferricyanide solution, utilize potentiostat on two chip arrays electrodes, to apply 0 and 0.6V voltage respectively, the electric current that obtains is converted into the electric current shape appearance figure, and the depth of color is represented the power of electric current on the shape appearance figure;

Step 3) is added to object in the micropore, and object can combine with aptamers, allows aptamers partly take place to rupture or separate micropore surface; Afterwards surface clean is removed the dna segment that splits away off, on two chip arrays electrodes, apply 0 and 0.6V voltage more respectively, the current value that obtains is converted into the electric current shape appearance figure, by with step 2) compare, can judge whether some material exists by the change of shape appearance figure color.

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