CN104212711B - Electronic sensor and the crto gene method based on electronic sensor - Google Patents
Electronic sensor and the crto gene method based on electronic sensor Download PDFInfo
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Abstract
The present invention relates to field of biological detection, a kind of electronic sensor and the crto gene method based on electronic sensor are disclosed.In invention, electronic sensor includes ISFET, etching forms the first groove in nanowire channel between the source electrode of the ISFET and drain electrode, the bottom of first groove is provided with nano-pore, or place chemical molecular, or open up nano-pore, and chemical molecular is placed at nano-pore, so that the side that DNA molecular is no longer chemically molecule or nano-pore unblockedly slides into opposite side without control at a high speed, only after single stranded DNA to be measured and detection base pairing success, a base could be moved forward by chemical molecular, it can accurately detect to detect base and the base-pairing events in single stranded DNA to be measured, so that crto gene result is more accurate, precision is higher, sensitivity is higher.
Description
Technical field
The present invention relates to field of biological detection, more particularly to a kind of electronic sensor and the gene based on electronic sensor visit
Survey method.
Background technology
With the development of science and technology, DNA (Deoxyribonucleic acid, be abbreviated as DNA) is sequenced
Turn into the conventional study project of whole world biological medicine research laboratory.The reason for bringing this phenomenon is probably due to sequencing
Developing rapidly for technology, causes the cost for possessing sequencing equipment to decline, it is also possible to be due to the bioid required for being sequenced
Product and other necessity costs decline.The cost of overall human genome sequencing is carried out based on so-called first generation sequencing technologies
Rate of descent at 2001 to 2007 meets the Moore's Law of microelectronics.As seen from the figure, large-scale parallel sequencing of future generation
Technology entered the application stage in 2005, caused cost rate of descent to exceed the several orders of magnitude of Moore's Law, but rate of descent is certainly
Remained basically stable since 2012.The technological revolution of a new round is to realize only to spend 1000 dollars of completions pair in the target set at that time
Whole human genome is sequenced.However, in order that gene order-checking walks out R＆D units, and penetrate into health care portion
Door, to the sequencing of each genome into should be well below 1000 dollars, therefore, further development sequencing technologies be definitely must
Want.
Up to the present, commercially available sequencing technologies are almost all based on optical fluorescence/chemical labeling method completely.It is heavy
Machine and tediously long operating time, and high cost, be the major defect of this technology.By ion torrent (Ion
Torrent) company shows revolutionary progress in the commercialized semiconductor sequencing technologies of in December, 2010 progress.Semiconductor is surveyed
Sequence technology is based entirely on silicon micromachining technology and microelectronics, and without mark and optical instrument.It is Ion as shown in Figure 1
Ion-sensitive field effect transistor (ISFET) schematic diagram of Torrent systems (111 be substrate, and 104 be reference electrode).In figure,
Carried on magnetic bead 101 it is substantial amounts of (quantity be more than 104To 106) identical single stranded DNA 102, the magnetic bead, which is placed on, is full of electrolysis
In the ISFET grooves 107 of matter.The groove is connected to the fluid for supplying four kinds of nucleotides (also referred to as " base ") in a predetermined sequence
Pond, four kinds of bases are respectively：Adenine (A), cytimidine (C), guanine (G), thymidine (T), not necessarily by A, C, G, T
Sequentially, each type occurs once in but substantially often flowing.In figure, it is not drawn into fluid pool, but depicts base entering ISFET
The direction 103 of groove 107.When pairing occurs in the base to be measured in the detection base in fluid and single stranded DNA, (i.e. A-T or C-G are tied
Close, form double-strand) when, substantial amounts of hydrogen ion will be discharged in a fluid.This will cause the soda acid (pH) of ISFET groove Inner electrolysis matter
It is worth instantaneous variation, and more importantly causes the hydrogen ion concentration moment of the upper surface of ISFET piled grids 109,110 to become
Change.And hydrogen ion concentration change in surface will cause the change of ISFET surface potentials, so as to cause ISFET source electrodes 105 and drain electrode
The change of channel current between 106.In order to detect this change to greatest extent, it can be applied on the surface of piled grids 109
Cover the metal or metal oxide 108 particularly sensitive to protonated/deprotonated (or pH value change).
The great advantage of Ion Torrent technologies is that its cost is extremely cheap.By decades develop rapidly and
Huge investment, the microchip of current semiconductor factory processes all great competitiveness in expense and complexity.Chip on piece
Ion Torrent sensor arrays and various electronic circuits are integrated with, is calibrated, noise processed, data management etc. so that this
One emerging technology reduce further holistic cost.
However, there is a defect in the chip (up to the present all products) of Ion Torrent companies, it is exactly in sequencing
In, it will usually produce the bit error rate higher than optical means.Specifically, the two big masters that Ion Torrent sequencing technologies are present
Wanting defect is：
(1) when occurring a large amount of repetition bases on single stranded DNA, it is impossible to determine the precise figures of its repeat length.For example,
GGGGG (repeat length is 5) and GGGGGG (repeat length is 6), when similar repeat occur, it is contemplated that there may be stronger electricity
Signal, because releasing more hydrogen ions in single fluid, result in bigger pH value change.But this change is difficult to
It is determined that it is also due to have used caused by substantial amounts of identical single-stranded DNA copy caused by long repetitive sequence to be due to.
(2) the reading length (i.e. the total bases of single stranded DNA) of Ion Torrent technologies is usually less than 400 bases
It is right, it is shorter than other sequence measurements.When the DNA to there is more repetition or have many structure variations is sequenced, longer reading
It is favourable to take length.Such as, it is particularly advantageous that assembling of the length to from the beginning genome is read in longer sequencing.
Caused by the sensitivity that these defects are on duty mainly due to plane ISFET phases, it has its source in plane ISFET's
Cone on raceway groove cuts body micro recessed structure (as shown in Figure 1), because its dominant sidewall areas is not effective detection
Area.Therefore, such design be able to should be improved, so as to obtain more preferable sensitivity.In addition, making on each magnetic bead
With substantial amounts of identical DNA copy so that the hydrogen ion of release is difficult in concert with change surface potential, so as to cause non-
Linear problem, and be difficult to differentiate between growing repetition.For intuitively, with a small amount of DNA copy, or even only use one, be sequenced,
It is very favorable, because it not only solves nonlinear problem, it also avoid the polymerase chain reaction (PCR) because of DNA cloning
Surveyed by mistake Deng caused by.It is in fact possible to remove time-consuming PCR steps.
Semiconductor sequencing technologies are referred to as " 2.5 generation ", and individual molecule sequencing is considered as the third generation.It is most representative
It may is that and use nano-pore, including the nano-pore based on graphene platelet, carry out electronic counting and determine base, so as to carry out straight
Sequencing is connect, without DNA cloning, fluorescence/chemical labeling or optical instrument.The structure and operation principle of the device are very simple, such as scheme
Shown in 2A and 2B.When single stranded DNA electrophoresis is by nano-pore, when particularly base is by nano-pore, upper and lower electrolyte liquid storage tank
Between gas current reduce.It can be changed by recording these gas currents, obtain the base number passed through, and by reference to
Distance between known base, obtains the length of single stranded DNA.
However, being so far mainly been used for measuring DNA length using nano-pore, length can be surveyed and reach several thousand bases, without
The order of sequence is to determine, because blocking the difference of electric current very small as the ion produced by four kinds of different bases.Moreover, from
Electron current is very low in itself, 10-11To 10-10Ampere (A) order of magnitude, accurate detection is carried out to it also extremely difficult.In addition, receiving
The shapes and sizes of metre hole, DNA base is relative to the position and direction of nano-pore, and the ionic nature in electrolyte liquid storage tank
And ionic strength, influence of these parameters to its result of detection be also very big.The nuance of electronic structure is used between base
(as shown in Figure 2 B, in figure, 201 be tunnel probe tunneling probe to transverse electric tunnelling current, and 202 be backgate back
Gate it is) also by no means easy to measure.
The content of the invention
It is an object of the invention to provide a kind of electronic sensor and the crto gene method based on electronic sensor, make base
Because result of detection is more accurate, precision is higher, and sensitivity is higher.
In order to solve the above technical problems, the invention provides a kind of electronic sensor, including ion sensitive field effect crystal
Etching forms first groove in pipe ISFET, the nanowire channel between the source electrode of the ISFET and drain electrode；
The bottom of first groove has one of following structure or combination：
The bottom of first groove is provided with nano-pore, the bottom of first groove and is placed with chemical molecular；
Wherein, the chemical molecular is used for the movement for controlling gene.
Present invention also offers a kind of crto gene method based on above-mentioned electronic sensor, comprise the steps of：
By the upper electrolyte liquid storage tank of first groove connection；
On described reference electrode is inserted in electrolyte liquid storage tank；
Single stranded DNA to be measured is delivered into the upper electrolyte liquid storage tank；
The single stranded DNA to be measured is moved to first bottom portion of groove under the bias voltage effect of the reference electrode；
To the upper electrolyte liquid storage tank supply detection base；Wherein, in a predetermined sequence supply four kinds of bases, every time to
The upper electrolyte liquid storage tank supplies a kind of base；Also, every time before replacing base, from the upper electrolyte liquid storage tank, institute
State in the first groove and to rinse away former base；
Detect base and after the base pairing success in the single stranded DNA, the single stranded DNA to be measured moves a base；
The electric current peaks or valleys that the gas current of the nano-pore is occurred by resistance are detected, the length of the single stranded DNA to be measured is determined
Degree or base number；Wherein, when the base is by the nano-pore, the gas current of the nano-pore is hindered；
Or, in base-pairing events, the release hydrogen ions into the nano bowl, detect the ISFET source electrode and
Curent change between drain electrode.
Compared with prior art, etching forms one in the nanowire channel in the present invention between ISFET source electrode and drain electrode
Individual first groove, the bottom of first groove or is provided with nano-pore, or places chemical molecular, or opens up nano-pore, and in nanometer
Chemical molecular is placed at hole so that DNA molecular is no longer the side of chemically molecule or nano-pore unblockedly at a high speed without control
Ground slides into opposite side, only after single stranded DNA to be measured and detection base pairing success, could move one forward by chemical molecular
Individual base, can accurately detect to detect base and the base-pairing events in single stranded DNA to be measured, so that crto gene result
More accurate, precision is higher, and sensitivity is higher.
In addition, first groove can be nano bowl, chemical molecular is placed with nanometer bowl bottom.
In addition, first groove can be nano bowl, the bottom of the nano bowl is provided with nano-pore, the ISFET's
Substrate is provided with the second groove centered on the nano-pore.
In addition, first groove can be nano bowl, the bottom of the nano bowl is provided with nano-pore, the ISFET's
Substrate is provided with the second groove centered on the nano-pore, and the nano-pore position is placed with chemical molecular；Wherein,
The chemical molecular only allows the double stranded section of gene from the nano-pore while being moved to another side.
In addition, wedge angle knife-edge is formed around the nano-pore on the bottom of the nano bowl, so as to more accurately
Detect the transverse electric tunnelling current of nano-pore
In addition, the height and diameter of the nano bowl are less than 200 nanometers；Or, the volume of the nano bowl is less than 10-17
Rise.Nano bowl there is sufficiently small volume can further make crto gene result is more accurate, precision is higher, and sensitivity is higher.
In addition, the diameter of the nano-pore is less than 10 nanometers, matched with the size of gene, effectively to be detected.
In addition, thickness of the bottom of the nano bowl around the nano-pore is less than 5 nanometers.
In addition, Coating Ions sensitive membrane on the madial wall of the nano bowl.The ion-sensitive membrane material can be metal
Or metal oxide, these ion-sensitive membrane materials are more sensitive to hydrogen ion and suction-operated is stronger, and adsorption concentration is higher,
Sensitivity is good, thus makes measurement result effective much sooner.
In addition, Organic ionic groups can be combined on the ion sensitive membrane.It can be made by organic ion with activated surface
The electronic sensor of present embodiment is more sensitive to hydrogen ion.
In addition, the chemical molecular is Phi29DNAP.
In addition, first groove can be infundibulate；The funnel-shaped bottom portion is provided with nano-pore；And described first is recessed
Trench bottom forms wedge angle knife-edge around the nano-pore.
In addition, the wedge angle position is placed with chemical molecular；Wherein, the chemical molecular only allows the double-strand of gene
Part is from the nano-pore while being moved to another side.
In addition, when detecting the curent change between the source electrode of the ISFET and drain electrode, recording when electric current changes
The base type supplied.
In addition, when the gas current for detecting the nano-pore is hindered the electric current peaks or valleys occurred, record is in electric current hair
The base type supplied during changing.
In addition, after the step of detecting the curent change between the source electrode of the ISFET and drain electrode, also comprising following step
Suddenly：
According to basepairing rule, the base type of record is found out into its complementary base in order, the list to be measured is obtained
The base sequence of chain DNA；
Wherein, the basepairing rule is：Adenine A and thymidine T is matched, and guanine G and cytimidine C is matched.
In addition, after the step of obtaining the base sequence of the single stranded DNA to be measured, also comprising the steps of：
With reference to distance between known base, according to the base sequence of the single stranded DNA to be measured, obtain described to be measured single-stranded
DNA length.
In addition, in the step of detecting the curent change between the source electrode of the ISFET and drain electrode, also comprising following sub-step
Suddenly：
The number of times that record current changes；
After treating that crto gene is finished, the total degree that the electric current recorded is changed is used as the single stranded DNA to be measured
Base number.
In addition, in the step of delivering single stranded DNA to be measured into electrolyte liquid storage tank on described, only delivering one every time and treating
Survey single stranded DNA；
In the step of supplying base to electrolyte liquid storage tank on described, every time to the upper electrolyte liquid storage tank supply one
Individual detection base.
It is sequenced by supplying a detection single single stranded DNA of base-pair every time, its sequencing result is more accurate.Also,
Due to every time only for a detection base is answered, if the curent change between the ISFET detected source electrode and drain electrode, then,
Its result is only possible to be the detection base of supply and the base pairing success in single stranded DNA；And pass through Phi29 DNAP control
System, is repeated even if there is base in single stranded DNA, every time also can only with a base pairing after double stranded section, it is therefore, right
It is long to repeat base, it can also detect exactly.
In addition, in the step of delivering single stranded DNA to be measured into electrolyte liquid storage tank on described, delivering some identical to be measured
The copy of single stranded DNA；
Before the step of supplying base to electrolyte liquid storage tank on described, copying for the identical single stranded DNA to be measured is judged
Whether shellfish is moved to the bottom of first groove, if it is not, waiting until that the copy of the identical single stranded DNA to be measured is moved
After the bottom for moving first groove, then to the upper electrolyte liquid storage tank supply base；
In the step of supplying base to electrolyte liquid storage tank on described, if being supplied every time to the upper electrolyte liquid storage tank
Dry detection base；Wherein, the base number of the supply is more than or equal to the copy number of the identical single stranded DNA to be measured.
By multiple detection bases and the base pairing in the copy of multiple identical single stranded DNAs to be measured, it can make ISFET's
Curent change increase between source electrode and drain electrode, or the curent change duration growth between ISFET source electrode and drain electrode,
So as to further make gene sequencing result more accurate.
Brief description of the drawings
Fig. 1 is the ISFET schematic diagrames of Ion Torrent systems in the prior art；
Fig. 2A is the structural representation of the nano-pore direct gene sequencing based on graphene platelet in the prior art；
Fig. 2 B are the operation principle schematic diagrams of the nano-pore direct gene sequencing based on graphene platelet in the prior art；
Fig. 3 is the electronic sensor schematic diagram in first embodiment of the invention；
Fig. 4 is the system schematic of the electronic sensor progress crto gene in first embodiment of the invention；
Fig. 5 A are the electronic sensor diagrammatic cross-sections in second embodiment of the invention；
Fig. 5 B are the electronic sensor top views in second embodiment of the invention；
Fig. 6 be electronic sensor in second embodiment of the invention carry out mrna length and the detection of base number be
System schematic diagram；
Fig. 7 is the system schematic of the electronic sensor progress gene sequencing in second embodiment of the invention；
Fig. 8 is a kind of structural representation of the electronic sensor in third embodiment of the invention；
Fig. 9 is another structural representation of the electronic sensor in third embodiment of the invention；
Figure 10 is the system schematic of the electronic sensor progress gene sequencing in third embodiment of the invention；
Figure 11 is the crto gene system signal that the crto gene method in four embodiment of the invention is used
Figure；
Figure 12 is the flow chart of the crto gene method in four embodiment of the invention；
Figure 13 is the crto gene system signal that the crto gene method in fifth embodiment of the invention is used
Figure；
Figure 14 is the flow chart of the crto gene method in fifth embodiment of the invention.
Embodiment
To make the object, technical solutions and advantages of the present invention clearer, below in conjunction with each reality of the accompanying drawing to the present invention
The mode of applying is explained in detail.However, it will be understood by those skilled in the art that in each embodiment of the invention,
In order that reader more fully understands the application and proposes many ins and outs.But, even if without these ins and outs and base
Many variations and modification in following embodiment, can also realize each claim of the application technical side claimed
Case.
The first embodiment of the present invention is related to a kind of electronic sensor, as shown in figure 3, the electronic sensor includes ion
Etching forms one the in sensitive field effect transistor (ISFET), the nanowire channel 304 between its source electrode 302 and drain electrode 303
One groove, the bottom of first groove has one of following structure or combination：The bottom of first groove is provided with nano-pore, first recessed
The bottom of groove is placed with chemical molecular；Wherein, chemical molecular is used for the movement for controlling gene.That is, first groove
Bottom is provided with nano-pore, or places chemical molecular, or opens up nano-pore, and the placement chemical molecular at nano-pore so that DNA
Molecule is no longer that the side of chemically molecule or nano-pore unblockedly slides into opposite side without control at a high speed, only in list to be measured
After chain DNA and detection base pairing success, a base could be moved forward by chemical molecular, detection can be accurately detected
Base and the base-pairing events in single stranded DNA to be measured, so that crto gene result is more accurate, precision is higher, and sensitivity is more
It is high.
In the present embodiment, the first groove can be nano bowl, and the bottom of the nano bowl is provided with nano-pore 306, ISFET
Substrate 301 be provided with groove centered on nano-pore 306.
The volume of nano bowl is made sufficiently small, can be with the concentration of ion in relative increase electrolyte.Such as, the height of nano bowl
Spend and diameter is less than 200 nanometers, or, the volume of nano bowl is less than 10-17Rise.Specifically, the normal blood pH value of people is
7.4, corresponding at a fairly low proton concentration, about per cubic centimeter 24 × 1012, i.e., in the nano cubic body of the 360nm length of sides only
1 proton can be found.It is less than 100 nanometers with the height and diameter of nano bowl, or its volume is less than 10-18Rise (attolitre, i.e.,
106Cubic nanometer) exemplified by for, although the only one of which proton in the nano bowl of such small volume, its corresponding concentration is several
Than increasing by 50 times in the case of proton mean dispersion in the normal blood of people, correspondingly, one is produced in so small volume
Proton will correspondingly make pH value be changed to 5.7 from 1.7, or make 110 millivolts of potential shift (mV), considerably beyond typical ISFET's
Resolution limit (0.1-1mV).Therefore, there is nano bowl sufficiently small volume can further make crto gene result more accurate
Really, precision is higher, and sensitivity is higher.
The electronic sensor of present embodiment can be described as " 3NANO sensors " (abbreviation 3NANO), general comprising three nanoscales
Read：Nano wire, nano bowl and nano-pore.The SOI substrate specially designed has SiO2/Si3N4/SiO2Storehouse as insulating barrier (such as
In Fig. 3 301), it is that the follow-up nano-pore that makes is ready.The thickness of surface silicon layer can be 200 nanometers, first make and use base
In the nano wire ISFET of current ISFET preparation technologies, it would however also be possible to employ new beamwriter lithography (EBL) technology is received to make
Rice structure.Wide 50 to 300 nanometers of nano-wire array can be used for forming optimal nano bowl, and thickness (the i.e. nanometer of nano bowl
The height of bowl) easily it can be obtained from the thickness 200nm adjustment of surface silicon layer.The doping type and concentration of nano wire are also adjusted
It is whole to arrive under optimum polarity and bias condition.By suitably setting the gate bias voltage of electrolyte, it can match somebody with somebody base as far as possible
The hydrogen ion discharged to during is mobile to sensing surface (i.e. ion sensitive membrane 305), rather than to the direction of remote nano bowl
It is mobile.
When seen from above, nano bowl profile can be the combination of circular, oval or several different basic configurations, can
To select the accurate shape that sensitivity is optimal, machinability is optimal by emulation.The inner surface of nano bowl needs to be designed to mirror
Face, to eliminate corrosion and damage.Can be using SiO of the thermal oxidation technology formation with most stiff stability2/ silicon interfaces, its interface
Form and heat endurance are preferable, and sensitivity is high, and acoustic noise control capability is stronger.The top that ion sensitive membrane will be deposited
It is one layer, as detailed below.In addition, the 3NANO sensors of present embodiment have the source/drain of optimization, insulating barrier, passivation and gold
Belong to contact zone, to reduce external disturbance as far as possible.
Nano-pore positioned at nanometer bowl bottom can be by the silicon nitride film of several nanometer thickness, using beamwriter lithography
(EBL) mode top-down approach formation diameter reaches 20 nanometers of nano-pore, it would however also be possible to employ hard mask and conformal thin-film are heavy
Product (such as ald ALD) combines to form the nano-pore that diameter is less than 10 nanometers.The pore size of nano-pore can be with
Further reduced by ALD, but effective film thickness around nano-pore is also to need what is controlled, that is to say, that the bottom of nano bowl
Portion is less than 5 nanometers in the thickness of nanometer bore edges.Therefore, ALD number of times can be controlled as needed, made the aperture of nano-pore and received
Film thickness around metre hole is adjusted to suitable value.
Can Coating Ions sensitive membrane on the madial wall of nano bowl.The nano bowl etched in nano-wire transistor channel can be noted
Enter electrolyte, such design causes the inner side of almost whole nano bowl, in addition to nano-pore, including upright side walls, is
Sense surface.It is of course also possible to which the only Coating Ions sensitive membrane in upright side walls, can reach the same sensing effect.Using this
When the electronic sensor of embodiment carries out crto gene, due to Coating Ions sensitive membrane on the madial wall of nano bowl, so whole
The madial wall of individual nano bowl is used equally for detecting hydrogen ion concentration, makes detection base and the base pairing in single stranded DNA to be measured
Journey can be accurately detected, so that crto gene result is more accurate, precision is higher, and sensitivity is higher.
The material of ion sensitive membrane in the present embodiment can be metal or metal oxide, and metal more typical at present has
Gold, platinum, palladium etc., more typical metal oxide have hafnium oxide (HfO2), titanium dioxide (TiO2), aluminum oxide (Al2O3) or five
Tantalum oxide (Ta2O5) etc., these ion-sensitive membrane materials are more sensitive to hydrogen ion and suction-operated is stronger, adsorption concentration compared with
Height, sensitivity is good, thus makes measurement result effective much sooner.The ion sensitive membrane can pass through thermal oxide, chemical gaseous phase
Film is made in above-mentioned material by deposition or the mode of ald, and these modes are comparatively all relatively simple, into
Film device requires also not high, can reduce the preparation cost of whole device.
In addition, it is noted that can be combined with organic ionic group on ion sensitive membrane, such as hydroxyl OH- from
Son, hydrosulphuric acid hydrogen radical SH- ions.These organic ions with activated surface, can make the electronic sensor of present embodiment to hydrogen from
Son is more sensitive.
In addition, what deserves to be explained is, in the present embodiment, Semiconductor substrate can be p-type or N-type, if semiconductor is served as a contrast
Bottom is p-type, then what is formed by adulterating is exactly source electrode and the drain electrode of N-type；If Semiconductor substrate is N-type, formed by adulterating
Be exactly p-type source electrode and drain electrode.In figure 3, it is P-type semiconductor substrate, source electrode and the leakage of N-type is formed by doping
Pole, and form p-type heavy doping (P+ regions in figure) in source electrode and drain electrode.Because the mobility of electronics is much larger than the migration in hole
Rate, thus with the n-type doping that electronics is majority carrier do source electrode and drain electrode if, it is much better than by current capacity；In addition,
For control angle, N-type ISFET can be opened with positive voltage, be used more convenient.
The system for carrying out crto gene using the electronic sensor of present embodiment, its structure is as shown in figure 4, nano-pore institute
It is in place to be equipped with chemical molecular 401, the upper electrolyte liquid storage tank 402 of nano bowl connection, the lower electrolyte liquid storage of the second groove connection
Pond 403.
Some technologies have been occurred in that at present, by measuring electron stream by being integrated with the nano-wire fet of nano-pore, have been used
The basecount associated with displacement improves reliability.In the art, it is crucial that how to control DNA to move by nano-pore
The speed of position.Using light or it is named as " Phi29 archaeal dna polymerase " (referred to as Phi29DNAP) and can be proved to speed control
It is feasible.Phi29DNAP is also used in other sequencings, and most famous is in zero mould fiber waveguide (ZMW) technology, successfully
Using this magical enzyme, the single stranded DNA in single base level is controlled to move, each successful base pairing causes single-stranded
DNA base movement.That is, add Phi29DNAP in nano-pore porch or similar chemical molecular etc.,
Phi29DNAP presence cause DNA molecular be no longer unblockedly from the side of nano-pore (in figure above nano-pore, upper electricity
The side of solution matter liquid storage tank 402) opposite side is slided into without control at a high speed (in figure below nano-pore, the lower side of electrolyte liquid storage tank 403).
Under reasonable terms, Phi29DNAP only allows DNA double stranded section to pass through, that is to say, that only in single stranded DNA to be measured and inflow
Detection base A, G, C, T base pairing success after, DNA toward lower section could move a base by Phi29DNAP.
Using above-mentioned principle, when carrying out gene sequencing, single stranded DNA is delivered in upward electrolyte liquid storage tank, from fluid pool
Supply detection base (being indicated with five-pointed star) in upward electrolyte liquid storage tank, direction as shown in figure 404, detect base A, C, G,
T and the base pairing in single stranded DNA, after successful matching, single stranded DNA originally turns into double-strand, in Phi29DNAP control
Under, downward electrolyte liquid storage tank moves a base.In each base-pairing events in single stranded DNA, hydrogen ion is produced
406, it is discharged into nano bowl, the pH value of electrolyte in nano bowl is occurred transient change.In the bias voltage of reference electrode 405
Under effect, hydrogen ion moves (direction shown in 407 in figure) to the inwall of nano bowl, makes the ditch between ISFET source electrode and drain electrode
Transient change also occurs therewith for road electric current, can be accurate by detecting that the channel current between ISFET source electrode and drain electrode changes
At the time of ground obtains base pairing success.In short, (just some base is led to when the gas current for passing through nano-pore with surveying is hindered
When crossing nano-pore) change (electric current peaks or valleys occur) determine DNA length or base number；Or, can be by ISFET electricity
Electron current changes and flows into the synchronization process of detection base sequential, to determine DNA order；Also can be by nano-pore ion-conductance
Rheologyization and inflow detect the synchronization process of base sequential to determine DNA order；Both also may be used in combination afterwards, can improve
The accuracy rate (cross-check) of sequencing.
In addition, being worth noting is, ribonucleic acid (RNA) forms long chain molecule by ribonucleotide through the condensation of phosphide key.
One ribonucleic acid molecule is made up of phosphoric acid, ribose and base.RNA base mainly has 4 kinds, i.e. adenine (A), guanine
(G), cytimidine (C), uracil (U), wherein, U instead of the T in DNA.With being set forth above class is detected on single stranded DNA
Seemingly, the electronic sensor of present embodiment can also be used for detecting RNA, difference be to detect base type be A, G,
C, U, the chemical molecular similar with Phi29DNAP is placed in nano-pore porch, and RNA can be controlled lateral another from nano-pore one
The movement of side, the principle to RNA progress length, base number, subsequent detection is similar with DNA, does not repeat one by one herein.
Second embodiment of the invention is related to a kind of electronic sensor, second embodiment and first embodiment substantially phase
Together, it is in place of main difference：In the first embodiment, the first groove is nano bowl, and in this second embodiment, first is recessed
Groove is infundibulate, and funnel-shaped bottom portion is provided with nano-pore, and wedge angle (knife- is formed around nano-pore on the bottom of the first groove
Edge), its structure as shown in Figure 5 A and 5B, wherein, Fig. 5 A are the ISFET of present embodiment diagrammatic cross-sections, and Fig. 5 B are these
The ISFET of embodiment top view.
Specifically, the ISFET of present embodiment is by the Si in Fig. 2A3N4/SiO2Nano-pore be substituted for one it is only several
The wedge angle nano-pore ISFET of nanometer thickness, more accurately to detect the transverse electric tunnelling current of nano-pore.Using shown in Fig. 6
System, the change when gas current that can pass through nano-pore with surveying is hindered (when just some base is by nano-pore) (occurs
Electric current peaks or valleys) determine DNA length or base number.Using system shown in Figure 7, chemistry point is placed with wedge angle position
Son；The chemical molecular only allow the double stranded section of gene from nano-pore on one side (upside shown in figure) to another side (shown in figure
Downside) it is mobile, can be by the synchronization process to the change of ISFET electronic currents and inflow detection base sequential, to determine that DNA's is suitable
Sequence；Also DNA order can be determined by changing and flowing into the synchronization process of detection base sequential to nano-pore gas current；Afterwards
Both also may be used in combination, and can improve the accuracy rate (cross-check) of sequencing.Its general principle and first embodiment class
Seemingly, this is no longer going to repeat them.
Second embodiment of the invention is related to a kind of electronic sensor, second embodiment and first embodiment substantially phase
Together, it is in place of main difference：In the first embodiment, the first groove is nano bowl, and nanometer bowl bottom is provided with nano-pore；
In second embodiment, the first groove is nano bowl, but nanometer bowl bottom does not open nano-pore, directly places chemical molecular, and it is tied
Structure is as shown in Figure 8.Specifically, the bottom of nano bowl is stacked structure, has stacked gradually insulating barrier 801, the and of Si dielectric layers 802
Ion sensitive membrane 803；Chemical molecular 804 is placed on ion sensitive membrane.
Or, the first groove is nano bowl, and nanometer bowl bottom opens nano-pore, and being sitting in position in nano-pore places chemistry point
Son, its structure is as shown in Figure 9.In figure, 301 be substrate, and 304 be the nano-channel between ISFET source electrode 302 and drain electrode 303,
401 be chemical molecular.In the present embodiment, can be in the whole inwall Coating Ions sensitive membrane of nano bowl, can also be only perpendicular
Straight sidewall Coating Ions sensitive membrane 305.
The system for carrying out crto gene using the electronic sensor of present embodiment is as shown in Figure 10, in reference electrode 405
Under effect, deliver and moved to the single stranded DNA to be measured in upper electrolyte liquid storage tank to nanometer bowl bottom, in upward electrolyte liquid storage tank
When base pairing in injection detection base (being represented with five-pointed star), detection base and single stranded DNA to be measured is successful, into nano bowl
Release hydrogen ions, make the pH value of electrolyte in nano bowl occur transient change.Reference electrode bias voltage effect under, hydrogen from
Son is moved to the inwall of nano bowl, is made the channel current between ISFET source electrode and drain electrode that transient change also occur therewith, is passed through
The channel current change between ISFET source electrode and drain electrode is detected, at the time of base pairing success can be obtained exactly.Its base
Present principles are similar with first embodiment, and this is no longer going to repeat them.
Third embodiment of the invention is related to a kind of crto gene method, is the electronic sensor based on first embodiment
Crto gene method, specifically carry out single single stranded DNA detection, its specific detection principle as shown in figure 11, detection flow as scheme
Shown in 12.
Specifically, nano bowl is first connected into upper electrolyte liquid storage tank, the lower electrolyte liquid storage tank of groove connection；In upper electrolysis
Add dispensing in bias voltage, upward electrolyte liquid storage tank to be measured single-stranded between the electrolyte of matter liquid storage tank and lower electrolyte liquid storage tank
DNA, only delivers a single stranded DNA to be measured every time；The single stranded DNA to be measured is in the presence of bias voltage, by nano-pore, downwards
Electrolyte liquid storage tank is quickly moved, at nano-pore, is stopped by chemical molecular.
Upward electrolyte liquid storage tank supply detection base, supplies a detection base every time；Wherein, supply in a predetermined sequence
Four kinds of bases are answered, electrolyte liquid storage tank supplies a kind of base upwards every time, also, every time before replacing base, from upper electrolyte
Former base is rinsed away in liquid storage tank, nano bowl, groove, lower electrolyte liquid storage tank.
Detect base and after the base pairing success in single stranded DNA, the downward electrolyte liquid storage tank of single stranded DNA to be measured moves one
Individual base；
The electric current peaks or valleys that the gas current of nano-pore is occurred by resistance are detected, the length or base of single stranded DNA to be measured is determined
Number；Wherein, when base is by nano-pore, the gas current of nano-pore is hindered.
In base-pairing events, meeting release hydrogen ions into nano bowl therefore, it can detect ISFET source electrode and leakage
Curent change between pole, and record the base type supplied when electric current changes.Or, detecting nano-pore
During the electric current peaks or valleys that gas current is occurred by resistance, the base type supplied when electric current changes is recorded.Then, according to
Basepairing rule, finds out its complementary base by the base type of record, obtains the base sequence of single stranded DNA to be measured in order；
Wherein, basepairing rule is：Adenine A and thymidine T is matched, and guanine G and cytimidine C is matched.
In addition, after the base sequence of single stranded DNA to be measured is obtained, with reference to distance between known base, according to list to be measured
The base sequence of chain DNA, can obtain the length of single stranded DNA to be measured.Also, between detection ISFET source electrode and drain electrode
During curent change, the number of times that can be changed with record current；After treating that crto gene is finished, by the electricity recorded
The total degree that stream changes as single stranded DNA to be measured base number.
Present embodiment is sequenced by supplying a detection single single stranded DNA of base-pair every time, and its sequencing result is more
Accurately.Also, due to every time only for a detection base is answered, if the electric current between the ISFET detected source electrode and drain electrode
Change, then, its result is only possible to be the detection base of supply and the base pairing success in single stranded DNA；And pass through
Phi29DNAP control, every time also can only be with an alkali after double stranded section even if the base occurred in single stranded DNA is repeated
Basigamy pair, therefore, repeats base to long, can also detect exactly.
Four embodiment of the invention is related to a kind of crto gene method, is the electronic sensor based on first embodiment
Crto gene method, specifically carry out multiple single stranded DNA detections, its specific detection principle as shown in figure 13, detection flow as scheme
Shown in 14.
Specifically, nano bowl is first connected into upper electrolyte liquid storage tank, the lower electrolyte liquid storage tank of groove connection, in upper electrolysis
Add dispensing in bias voltage, upward electrolyte liquid storage tank to be measured single-stranded between the electrolyte of matter liquid storage tank and lower electrolyte liquid storage tank
DNA, delivers the copy of some identical single stranded DNAs to be measured；The single stranded DNA to be measured is in the presence of bias voltage, by nano-pore,
Downward electrolyte liquid storage tank is quickly moved, and at nano-pore, is stopped by chemical molecular.
Before upward electrolyte liquid storage tank supply base, judge whether the copy of identical single stranded DNA to be measured is moved to
Nano-pore, if it is not, wait until that the copy of identical single stranded DNA to be measured is moved to after nano-pore, then upward electrolyte liquid storage
Pond supplies base.
During upward electrolyte liquid storage tank supply detection base, some detection bases are supplied every time；Wherein, in a predetermined sequence
Four kinds of bases are supplied, electrolyte liquid storage tank supplies a kind of base upwards every time, and the base number of supply is more than or equal to identical to be measured
The copy number of single stranded DNA.Also, change before base, stored up from upper electrolyte liquid storage tank, nano bowl, groove, lower electrolyte every time
Former base is rinsed away in liquid pool.
Detect base and after the base pairing success in single stranded DNA, the downward electrolyte liquid storage tank of single stranded DNA to be measured moves one
Individual base；The electric current peaks or valleys that the gas current of nano-pore is occurred by resistance are detected, the length or base of single stranded DNA to be measured is determined
Number；Wherein, when base is by nano-pore, the gas current of nano-pore is hindered.
In addition, it is similar with the 3rd embodiment, single stranded DNA to be measured can also be carried out the detection such as to be sequenced, its operation principle
It is similar, to avoid repeating, it will not be repeated here.
In the present embodiment, because multiple detection bases are matched somebody with somebody with the base in the copy of multiple identical single stranded DNAs to be measured
Right, accordingly, there exist two kinds of situations：
(1) it is multiple detection bases simultaneously with the base pairing in single stranded DNA, if now successful matching, its produce hydrogen from
The hydrogen ion produced when son is by than single single stranded DNA is more, then the curent change between ISFET source electrode and drain electrode can also increase
Greatly, consequently facilitating detecting this curent change, it can further make gene sequencing result more accurate.
(2), can be certain if now successful matching with the base pairing in single stranded DNA when multiple detection bases are different
In time, hydrogen ion is persistently produced, so that the curent change duration between ISFET source electrode and drain electrode is also longer,
It is also beneficial to detect this curent change, can further makes gene sequencing result more accurate.
The step of various methods are divided above, be intended merely to description it is clear, can be merged into when realizing a step or
Some steps are split, multiple steps are decomposed into, as long as including identical logical relation, all protection domain in this patent
It is interior；To adding inessential modification in algorithm or in flow or introducing inessential design, but its algorithm is not changed
Core design with flow is all in the protection domain of the patent.
It will be understood by those skilled in the art that the respective embodiments described above are to realize the specific embodiment of the present invention,
And in actual applications, can to it, various changes can be made in the form and details, without departing from the spirit and scope of the present invention.
Claims (24)
1. a kind of electronic sensor, including ion-sensitive field effect transistor ISFET, it is characterised in that the source electrode of the ISFET
Etching forms first groove in nanowire channel between drain electrode；
The bottom of first groove has one of following structure or combination：
The bottom of first groove is provided with nano-pore, the bottom of first groove and is placed with chemical molecular；
Wherein, the chemical molecular is used for the movement for controlling gene.
2. electronic sensor according to claim 1, it is characterised in that first groove is nano bowl.
3. electronic sensor according to claim 2, it is characterised in that the nanometer bowl bottom is placed with the chemistry point
Son.
4. electronic sensor according to claim 2, it is characterised in that the bottom of the nano bowl is provided with nano-pore, institute
The substrate for stating ISFET is provided with groove centered on the nano-pore.
5. electronic sensor according to claim 4, it is characterised in that the nano-pore position is placed with chemistry point
Son；Wherein, the chemical molecular only allows the double stranded section of gene from the nano-pore while being moved to another side.
6. electronic sensor according to claim 2, it is characterised in that the height and diameter of the nano bowl are less than 200
Nanometer；
Or, the volume of the nano bowl is less than 10-17Rise.
7. electronic sensor according to claim 4, it is characterised in that the diameter of the nano-pore is less than 10 nanometers.
8. electronic sensor according to claim 4, it is characterised in that the bottom of the nano bowl is on the nano-pore side
The thickness of edge is less than 5 nanometers.
9. electronic sensor according to claim 2, it is characterised in that Coating Ions are quick on the madial wall of the nano bowl
Feel film.
10. electronic sensor according to claim 9, it is characterised in that the material of the ion sensitive membrane be metal or
Metal oxide.
11. electronic sensor according to claim 10, it is characterised in that organic ion is combined on the ion sensitive membrane
Group.
12. electronic sensor according to claim 10, it is characterised in that the bottom of the nano bowl is stacked structure,
Insulating barrier, Si dielectric layers and ion sensitive membrane are stacked gradually；
The chemical molecular is placed on the ion sensitive membrane.
13. electronic sensor according to claim 1, it is characterised in that the chemical molecular is Phi29DNAP.
14. electronic sensor according to claim 1, it is characterised in that first groove is infundibulate；The funnel
Shape bottom is provided with nano-pore；
First bottom portion of groove forms wedge angle knife-edge around the nano-pore.
15. electronic sensor according to claim 14, it is characterised in that the wedge angle position is placed with chemistry point
Son；Wherein, the chemical molecular only allows the double stranded section of gene from the nano-pore while being moved to another side.
16. electronic sensor according to claim 1, it is characterised in that the material of the nano-pore for it is following it is any it
One：
Silicon Si, silicon nitride SiNx, silicon oxide sio 2, diamond, graphene graphene.
17. a kind of crto gene method of the electronic sensor based on as described in any one of claim 1 to 16, its feature exists
In comprising the steps of：
By the upper electrolyte liquid storage tank of first groove connection；
On described reference electrode is inserted in electrolyte liquid storage tank；
Single stranded DNA to be measured is delivered into the upper electrolyte liquid storage tank；
The single stranded DNA to be measured is moved to the bottom of first groove under the bias voltage effect of the reference electrode；
To the upper electrolyte liquid storage tank supply detection base；Wherein, four kinds of bases are supplied in a predetermined sequence, every time to described
Upper electrolyte liquid storage tank supplies a kind of base；Also, before changing base every time, from the upper electrolyte liquid storage tank, described the
Former base is rinsed away in one groove；
Detect base and after the base pairing success in the single stranded DNA, the single stranded DNA to be measured moves a base；
Detect the electric current peaks or valleys that the gas current of the nano-pore is occurred by resistance, determine the single stranded DNA to be measured length or
Base number；Wherein, when the base is by the nano-pore, the gas current of the nano-pore is hindered；
Or, in base-pairing events, the release hydrogen ions into the nano bowl detect source electrode and the drain electrode of the ISFET
Between curent change.
18. crto gene method according to claim 17, it is characterised in that detecting the source electrode of the ISFET and leakage
During curent change between pole, the base type supplied when electric current changes is recorded.
19. crto gene method according to claim 17, it is characterised in that detecting the ion-conductance of the nano-pore
When flowing the electric current peaks or valleys by resistance appearance, the base type supplied when electric current changes is recorded.
20. the crto gene method according to claim 18 or 19, it is characterised in that detecting the source electrode of the ISFET
After the step of curent change between drain electrode, also comprise the steps of：
According to basepairing rule, the base type of record is found out into its complementary base in order, the single stranded DNA to be measured is obtained
Base sequence；
Wherein, the basepairing rule is：Adenine A and thymidine T is matched, and guanine G and cytimidine C is matched.
21. crto gene method according to claim 20, it is characterised in that obtaining the alkali of the single stranded DNA to be measured
After the step of base sequence, also comprise the steps of：
With reference to distance between known base, according to the base sequence of the single stranded DNA to be measured, the single stranded DNA to be measured is obtained
Length.
22. crto gene method according to claim 17, it is characterised in that detecting the source electrode of the ISFET and leakage
In the step of curent change between pole, also comprising following sub-step：
The number of times that record current changes；
After treating that crto gene is finished, alkali of the total degree that the electric current recorded is changed as the single stranded DNA to be measured
Radix.
23. crto gene method according to claim 17, it is characterised in that thrown into electrolyte liquid storage tank on described
In the step of putting single stranded DNA to be measured, a single stranded DNA to be measured is only delivered every time；
In the step of supplying base to electrolyte liquid storage tank on described, a spy is supplied to the upper electrolyte liquid storage tank every time
Survey base.
24. crto gene method according to claim 17, it is characterised in that thrown into electrolyte liquid storage tank on described
In the step of putting single stranded DNA to be measured, the copy of some identical single stranded DNAs to be measured is delivered；
Before the step of supplying base to electrolyte liquid storage tank on described, judging the copy of the identical single stranded DNA to be measured is
It is no to be moved to the bottom of first groove, if it is not, waiting until that the copy of the identical single stranded DNA to be measured is moved to
After the bottom of first groove, then to the upper electrolyte liquid storage tank supply base；
In the step of supplying base to electrolyte liquid storage tank on described, visited every time to the upper electrolyte liquid storage tank supply is some
Survey base；Wherein, the base number of the supply is more than or equal to the copy number of the identical single stranded DNA to be measured.
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| AU2016270887B2 (en) | 2015-06-03 | 2019-09-19 | Illumina, Inc. | Compositions, systems, and methods for sequencing polynucleotides using tethers anchored to polymerases adjacent to nanopores |
| CN105092647A (en) * | 2015-08-20 | 2015-11-25 | 中国科学院重庆绿色智能技术研究院 | Molecular conformation change-based sequence measuring method |
| CN106501340B (en) * | 2016-09-23 | 2019-07-09 | 上海小海龟科技有限公司 | The detection method of electrode, ion-sensitive sensor, capacitor and ionic activity |
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