CN101915799A - An Extended Gate Field Effect Transistor Sensor Chip for DNA Molecular Detection - Google Patents

Abstract

The invention discloses an extended grid field effect transistor sensing chip for detecting DNA molecules, which is characterized in that: a grid electrode of a field effect transistor (FET) has an extended structure. The extended grid structure is characterized in that: an aluminum wire serving as a lead is connected with an extended gold plane electrode (the length of a side is 0.2 to 1.0 mm). The gold electrode structure is characterized in that: a silicon dioxide (SiO2) layer, a chromium layer, a nickel-copper alloy layer and a gold layer are plated by a sputtering coating method from bottom to top in turn. The sensing chip for detecting the DNA molecules is prepared by self-assembling DNA segments which are complemented with sulfydryl group-containing molecules on the extended gold electrode through the bridge connection of thioalcohol molecules. The sensing chip can detect 1 to 10 DNA molecules per 100 nm<2> on a gold substrate. The sensing chip has the advantages of simple structure, simple and convenient operation and high generality.

Description

A kind of prolongation grid field effect transistor sensing chip that is used for the dna molecular detection

Technical field

The invention belongs to the detection of nucleic acids field, particularly relate to a kind of small detection sensing chip that can detect dna molecular, this sensing chip utilization has field effect transistor (FET) technology that prolongs grid, can detect target dna molecule quickly and easily.

Background technology

Entered since 21 century, be accompanied by developing rapidly of biotechnology, the electronic technology biochip that has been born that combines with biotechnology, this is a kind of revolutionary achievement.Use biochip technology to carry out the DNA detection analysis at present and in molecular biology, medical science, clinical practice, become increasingly mature.The gordian technique of biochip is its detection and quantification to crossover process between the dna molecular.The preparation of most of DNA chips is based on the optical detecting method that fluorescently-labeled nucleic acid molecules and the probe that is fixed are hybridized, and utilizes the method for the electrochemical sensing detection crossover process that electric signal combines with bio signal also to become important.By with the combining of semiconductor technology, field effect transistor (FET) sensor has been widely used in the biological detection analysis.Use fluorescently-labeled method relatively with comparatively traditional DNA chip, FET gate sensing chip does not need the reagent and the shading box of usage flag, has simplified operation steps, has saved the detection cost.

For this reason, the present invention prolongs certain distance with the grid of FET, proposed a kind of brand-new serve as the FET sensing chip that prolongs grid with golden plate electrode, the Electrochemical Detection that is used for dna molecular all has very important application prospect in all fields relevant with vital movement such as biomedicine, pharmaceutical engineering, agricultural and environmental science.

Summary of the invention

The purpose of this invention is to provide a kind of brand-new serve as the FET sensing chip that prolongs grid with golden plate electrode, this sensing chip is that a kind of energy is simple, the small pick-up unit of fast detecting target dna molecule.

In order to achieve the above object, the technical solution used in the present invention is: the molecule self assembly that utilizes chemical reaction will contain mercapto groups is prolonging as field effect transistor on the golden plate electrode of grid, for detecting certain specific dna molecular, to contain the dna fragmentation complementary and pass through the bridging effect self assembly of thiol molecule on the plate that prolongs with it, finish the fixing of dna probe with this, thereby finish the preparation that prolongs grid field effect transistor sensing chip.If contain target dna molecule in the test sample, target dna molecule just can combine by the dna fragmentation of hybridization with complementation, can capture the crossover process that prolongs on the plate this moment by the detection of electric signal, and calculate the dna molecular number that hybridization takes place with this on the unit area of sensor.

The concrete device that the present invention relates to comprises: one has prolongation grid (grid prolongation 0.1-40mm, this is golden plate electrode, specification: grow and the long 0.2-1.0mm of being of broadside) field effect transistor, the Ag/AgCl contrast electrode of a built-in saturated KCl solution, and detect the electronic equipment that each parameter of transistor is used.Described prolongation grid field effect transistor comprises two parts: a prolongation grid gold plate electrode that is used for fixing dna probe; Be exactly in addition occurring in the FET part that crossover process on the gold electrode changes into electric signal.Wherein the auri matter that prolongs on the grid sensitive zones at FET is at SiO₂The chromium (20-200nm) of sputter one adhesion layer is followed by one deck monel layer (100-1000nm) on the basalis, is that the order of one deck gold (30-300nm, and gold surface is very smooth) constitutes at last.Gate insulator in the FET part is by by the SiO in the wet oxidation₂Layer and top thereof are by the Si in the chemical vapor deposition₃N₄Layer is formed.Gate terminal then is connected by the metal aluminum steel with the gold electrode part.FET part by silica gel sealing to reach the effect of shielding and insulating effect.

In the invention design of device, it is that grid is prolonged certain distance (0.1-40mm) that its innovation part is different with traditional F ET, makes up to prolong the grid gold electrode, utilizes to prolong the grid gold electrode and survey dna molecular, and can arrive every 100nm²Detect the level of 1-10 dna molecular.By on the prolongation grid, carrying out chemical modification, arrive the purpose of fixing DNA probe.And use FET to monitor the change in electric that transmission comes on the prolongation grid, it comprises the process of thiol molecule self assembly and the process of dna molecule hybridize.But also can calculate the dna molecular number that hybridization takes place by electric signal parameter.Use fluorescently-labeled method relatively with most of DNA chips, FET prolongs solvent and the shading box that the grid sensing chip does not need usage flag, has crucial clinical value as a kind of microbiosensor.

The invention has the beneficial effects as follows that device is simple, and is easy and simple to handle, can determine the lip-deep dna molecular number of auri matter exactly, highly versatile.

Description of drawings

The present invention is further described below in conjunction with accompanying drawing.

Fig. 1 is the planar structure synoptic diagram that prolongs grid FET sensor.

Fig. 2 is the planing surface effect synoptic diagram that prolongs the substrate of grid gold plate electrode.

Among Fig. 2: 1-gold layer; 2-monel layer; 3-chromium layer; 4-SiO₂Layer.

Embodiment

As shown in Figure 1, the golden plate electrode part that prolongs grid with FET prepares described sensing chip as working surface, and the FET part then is used to detect the change in electric of dna molecule hybridize process.

The voltage that applies that the present invention uses is the 20-200mV DC voltage, and contrast electrode is the Ag/AgCl contrast electrode of built-in saturated KCl solution, the characteristic electron parameter of FET such as grid voltage (V_G), leakage current (I_D) will after on the gold substrate, measure at thiol molecule and dna molecular self assembly.

Concrete implementation step is as follows:

At first prepare following solution system and oligonucleotide molecule (standby):

1.Na₂SO₄Solution: 0.10mol/L;

2.TE buffer solution: add the EDTA solution preparation of 1.0mmol/L, pH=7.4 by the Tris-HCl of 10mmol/L;

The EDTA solution preparation of 3.TE-NaCl buffer solution: Tris-HCl, the 1.0mmol/L of NaCl, the 10mmol/L of usefulness 1.0mol/L, pH=7.4;

4.DNA probe is the structure of the HS-ssDNA of sulfhydrylation, its base sequence is: contain HS-(CH₂)₂5 ' of-group-CACGACGTTGTAAAACGACGGCCAG (P1); The strand base sequence of target dna is: 5 '-CTGGCCGTCGTTTTACAACGTCGTG (P2).

Embodiment 1

Gold electrode is immersed in the TE buffer solution that contains 1.0-100 μ mol/L P1 (HS-ssDNA) molecule reacts 1-12h, finish the preparation process of probe sensing chip, obtain gold-single strand dna structure (Au-ssDNA), and the sensing chip for preparing is immersed in 0.10mol/L Na₂SO₄Carry out electronic surveying in the solution.

Embodiment 2

Gold electrode after P1 modified is immersed in the TE-NaCl buffer solution that contains 1.0-100 μ mol/L P2 molecule reaction 1-4h in the time of 50-120 ℃, the double chain DNA molecule structure (Au-dsDNA) that to finish hybridization reaction then is cooled to room temperature, clean with TE-NaCl buffer solution and deionized water rinsing, use nitrogen drying at last.The same withembodiment 1, the sensing chip of finishing hybridization is immersed in 0.10mol/L Na₂SO₄Carry out electronic surveying in the solution.

Embodiment 3

Data analysis:

By the rapid electric signal measurement of first two steps as can be known, when leakage current during at 3600 μ A, the threshold voltage (V of the Au-ssDNA behind the assembling P1_T) be offset 69mV to positive X-direction.Finish the threshold voltage (V of the Au-dsDNA behind the hybridization reaction_T) be offset 47mv to positive X-direction.Can calculate the V that causes by hybridization reaction thus_TThe change value is 22mv.This is by due to the minimizing of gate surface negative charge behind the generation hybridization reaction, and the reason that causes negative charge to reduce is when should be P1 and the target molecule P2 complementary with it hybridization reaction takes place, and neutralization has taken place the positive charge on the negative charge of phosphate group and the oligonucleotide chain footing.

By electrochemical measurement, the electric capacity that draws the sensing chip of the Au-dsDNA after hybridizing is 7.52 * 10^-6F/cm², because the Δ V of hybridization reaction front and back takes place_T=22mV, the quantity of electric charge that can calculate the double chain DNA molecule after the hybridization thus is 1.65 * 10^-7C/cm², the double chain DNA molecule density that can derive on the FET prolongation grid gold electrode surfaces is 1.714 * 10¹²Molecule/cm², be equivalent to every 100nm on gold substrate²Can detect the level of 1.714 dna moleculars, show that prolonging grid type field effect transistor can well be applied to the detection of dna molecular, and be applicable to and make bio-sensing chip.

Claims (6)

1. one kind is used for the prolongation grid field effect transistor sensing chip that dna molecular detects, and it is characterized in that the gate design of field effect transistor (FET) is become the structure of prolongation, utilizes prolongation grid gold electrode to survey dna molecular.

2. sensing chip according to claim 1 is characterized in that grid is prolonged the distance of 0.1-40mm, makes up to prolong grid gold plate electrode structure.

3. prolongation grid structure according to claim 2 is characterized in that connecting the gold electrode that prolongs with conventional wires (being aluminum steel in the diagram, can also be copper cash, silver-colored line etc.), and long and broadside is long to be 0.2-1.0mm.

4. gold electrode structure according to claim 3, it is characterized in that method by sputter coating by from the bottom up order respectively with SiO₂Layer, chromium layer, monel layer, gold layer plate, wherein SiO₂Layer is substrate, and other layer thickness is respectively: chromium layer 20-200nm, and monel layer 100-1000nm, gold layer 30-300nm, and gold surface is very smooth.

5. sensing chip according to claim 1, it is characterized in that utilizing chemical reaction the dna probe molecule self assembly of sulfhydrylation on the golden plate electrode that prolongs grid, only need reaction 1-12h, get final product probe molecule in the self assembly, thereby finish the preparation of sensing chip.

6. sensing chip according to claim 1 is characterized in that at the every 100nm of golden plate electrode substrate²Can detect 1-10 dna molecular on the area.

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