CN109507260A - Electrochemical Detection chip, electrochemical sensor and its preparation method and application - Google Patents
Electrochemical Detection chip, electrochemical sensor and its preparation method and application Download PDFInfo
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- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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- G—PHYSICS
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- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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Abstract
本发明公开了一种电化学检测芯片，包括电极层，所述电极层的工作电极包括依次层叠设置的导电层、纳米材料层和凝血反应层；所述凝血反应层产生用于检测凝血酶原时间的电信号，所述纳米材料层传递和放大所述电信号。利用纳米材料层的高导电性、大比表面积以及良好的生物相容性等，实现对凝血酶原时间检测过程中电信号的放大、增强，以提高芯片检测的灵敏度、缩短检测时间。本发明公开了一种电化学传感器，包括上述的电化学检测芯片，能够实现对凝血酶原时间的快速、灵敏检测，且具有较高的检测稳定性和重复性。本发明公开了一种电化学传感器的制备方法，适于制得上述灵敏度高、检测结果准确的电化学传感器。
The invention discloses an electrochemical detection chip, comprising an electrode layer, and a working electrode of the electrode layer includes a conductive layer, a nanomaterial layer and a coagulation reaction layer which are stacked in sequence; the coagulation reaction layer is used for detecting prothrombin. The electrical signal over time, the nanomaterial layer transmits and amplifies the electrical signal. The high conductivity, large specific surface area and good biocompatibility of the nanomaterial layer are used to amplify and enhance the electrical signal during the detection of prothrombin time, so as to improve the sensitivity of the chip detection and shorten the detection time. The invention discloses an electrochemical sensor, comprising the above electrochemical detection chip, which can realize rapid and sensitive detection of prothrombin time, and has high detection stability and repeatability. The invention discloses a preparation method of an electrochemical sensor, which is suitable for preparing the electrochemical sensor with high sensitivity and accurate detection result.
Description
Technical field
The present invention relates to hemostasis examination technical fields, and in particular to a kind of Electrochemical Detection chip, electrochemical sensor, electricity
The method of modifying of the working electrode of chemical detection chip and the preparation method of electrochemical sensor.
Background technique
Coagulation function normally whether be related to Human Physiology health status, the assessment of coagulation function is tested to a variety of diseases
Diagnosis, treatment and Index for diagnosis are significant.Clotting time is then the important indicator for measuring coagulation, wherein fibrin ferment
Former time (PT, ProthrombinTime) is the conventional index of current clinically extrinsic coagulation Function detection, is to judge body
Pathological change, the preoperative important indicator for carrying out disorders of blood coagulation screening occur for hemostasis and blood coagulation system, and clinically examine at present
The preferred index that disconnected and observation thrombosis, anticoagulant therapeutic effect monitor.
Prothrombin time PT depends on factor, fibrinogen and factor II in blood, V, VII, X etc.
Level, wherein coagulation factor is mainly synthesized by liver, thus PT hepatopaths many for cirrhosis, hepatitis etc. treatment tool
There is meaning of crucial importance, for some congenital or the type deficiency of coagulation factors patient day after tomorrow, thrombotic diseases patient and congenital FV
Increase disease patient etc. also requires the variation in monitor closely PT clotting time.Large-scale blood clotting since conventional PT detection at present is more
Instrument mostly uses the methods of thrombelastography, optical method to measure the clotting time, not only has to the operative skill of operator
High requirement, there is also it is complicated for operation, take a long time the problems such as.
Prothrombin time detection based on electrochemical method is not needed using large scale equipment, easy to operate, reduce inspection
Survey cost.A kind of prothrombin time test based on electrochemical method is disclosed in Chinese patent literature CN106680339A
Card: a certain amount of recombinant tissue factor (rTF) and phospholipid solution are mixed, tissue factor carboxylate is prepared.By tissue factor esterification
Object is dispersed on electrically-conductive backing plate and dries film forming, obtains a kind of prothrombin time detection card based on electrochemical method.
The above-mentioned prothrombin time based on electrochemical method detects card, and prothrombin time detection reagent is directly scattered in electrically-conductive backing plate
In, the electric signal generated during Coagulation test is directly transmitted to electrically-conductive backing plate.Due to Coagulation test process be related to multiple protein,
The participation of enzyme, the polypeptide chain in albumen, enzyme have the three-dimensional structure of bending fold, are influenced by its three-dimensional structure, coagulation process
The electronics of middle generation is difficult to transmit, and the electrical signal intensity generated in coagulation process is low, limits to prothrombin time detection
Sensitivity.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is that when overcoming in the prior art based on Electrochemical Detection factor
Between equipment have the defects that electronics be difficult to transmit, electrical signal intensity it is low.
For this purpose, the invention provides the following technical scheme:
In a first aspect, the present invention provides a kind of Electrochemical Detection chip, including electrode layer, the work electricity of the electrode layer
Pole includes the conductive layer being cascading, layer of nanomaterial and Coagulation test layer；The Coagulation test layer is generated for detecting
The electric signal is transmitted and amplified to the electric signal of prothrombin time, the layer of nanomaterial.
Preferably, above-mentioned Electrochemical Detection chip, the layer of nanomaterial is by carbon nanotube, graphene and zinc oxide
Any formation；Preferably, the layer of nanomaterial is formed by carbon nanotube；Preferably, the carbon nanotube is multi wall carbon
Nanotube.
Preferably, above-mentioned Electrochemical Detection chip, the Coagulation test layer include several Coagulation test units；Each institute
The electric signal area that Coagulation test unit includes fibrin ferment cog region, is connected with the fibrin ferment cog region is stated, and makes fibrin ferment
Original is converted into the zone of transformation of fibrin ferment；The fibrin ferment cog region separates under thrombin action with the electric signal area.
It is further preferred that above-mentioned Electrochemical Detection chip, the fibrin ferment cog region is formed by polypeptide material, described
Electric signal area is formed by electric signal material, and the conversion zone is formed by the organization material containing thromboplastin.
It is further preferred that above-mentioned Electrochemical Detection chip, the electric signal material is p-phenylenediamine.
Preferably, above-mentioned Electrochemical Detection chip, the working electrode are screen printing electrode.
Preferably, above-mentioned Electrochemical Detection chip, the electrode layer further include reference electrode and to electrode；Preferably,
The reference electrode be screen printing electrode and/or it is described be screen printing electrode to electrode.
Second aspect, the present invention provides a kind of electrochemical sensors, comprising:
The electrode layer of above-mentioned Electrochemical Detection chip, the Electrochemical Detection chip is set on basal layer；
Encapsulated layer is set on one side of the electrode layer far from the basal layer.
Preferably, above-mentioned electrochemical sensor is additionally provided with insulating layer, institute between the electrode layer and the encapsulated layer
Stating insulating layer has opening, and the electrode layer exposes the reaction zone to form the electrochemical sensor in the position of the opening.
The third aspect, the present invention provides above-mentioned Electrochemical Detection chips or above-mentioned electrochemical sensor in blood coagulation
Application in zymogen time detection.
Fourth aspect, the present invention provides a kind of method of modifying of the working electrode of Electrochemical Detection chip, including it is following
Step:
Nano material powder is dispersed in perfluor sulfoacid resin solution, nano material dispersion liquid is obtained；By nanometer
Material dispersion forms layer of nanomaterial in conductive layer surface in the surface of the conductive layer of working electrode, drying；
Thrombin substrate solution is coated in layer of nanomaterial, thrombin substrate solution includes containing thrombin cleavage site
Polypeptide, the electric signal molecule being connected with the polypeptide, and the tissue factor containing thromboplastin；It is dry, in nano material
Coagulation test layer is formed on layer, the working electrode of surface modification is made；
Preferably, the nano material is the carbon nano-tube material of purifying.
5th aspect, the present invention provides a kind of preparation methods of electrochemical sensor, comprising the following steps:
On the base layer the conductive layer of preparation work electrode, to electrode and reference electrode；
With the method for modifying of above-mentioned working electrode, the working electrode of surface modification is obtained；
Encapsulated layer is set on one side of the electrode layer far from basal layer to get the electrochemical test sensors.
Technical solution of the present invention has the advantages that
1. a kind of Electrochemical Detection chip, including electrode layer, the working electrode of the electrode layer includes being cascading
Conductive layer, layer of nanomaterial and Coagulation test layer；The Coagulation test layer generates the telecommunications for detecting prothrombin time
Number, the electric signal is transmitted and amplified to the layer of nanomaterial.
Above-mentioned Electrochemical Detection chip, when being used to detect prothrombin time, shortage blood platelet and calcium to be detected
The blood of ion is contacted with Coagulation test layer, makes conversion of prothrombin fibrin ferment, the blood coagulation of triggering prothrombin time detection
Process.Meanwhile Coagulation test layer generates the electric signal for detecting prothrombin time, by acquiring the variation of electric signal, energy
It is enough that coagulation process under thrombin action is monitored, realize the purpose of detection prothrombin time.Utilize nanometer
The advantages that electric conductivity height of material, large specific surface area, load capacity be high and good biocompatibility, nano material can accelerate electronics
Transmitting, improve current responsing signal, realize to electric signal molecule generate electric signal transmitting and amplification.Pass through nano material pair
The transmitting and amplification of electric signal, the sensitivity raising for detecting Electrochemical Detection chip to prothrombin time, detection time contracting
Sample size needed for short, detection is reduced.Electrochemical Detection chip is to the detection of prothrombin time independent of fiber egg in blood
The combination of white equal substances and chip surface, therefore the requirement to chip surface environment consistency is low, and there is good detection to repeat
Property.
2. Electrochemical Detection chip provided by the invention, the layer of nanomaterial is by carbon nanotube, graphene and zinc oxide
Any one of formed；Preferably, the layer of nanomaterial is formed by carbon nanotube.Carbon nanotube good, machine with mechanical characteristic
Tool intensity is high, electric conductivity is high, and large specific surface area, electrochemical stability height, higher aspect ratio and pore size are in a certain range
The advantages that interior controllable, provides possibility to support transmitting and the transmitting of Coagulation test layer and the effective electric signal of generation.Carbon
The chemical stability of nanotube is good, advantageously reduces the signal fluctuation as caused by complicated ingredient in blood, reduces foreign matter
Interference, improve detection repeatability and stability.
3. Electrochemical Detection chip provided by the invention, institute's Coagulation test layer includes several Coagulation test units；Often
The electric signal area that a Coagulation test unit includes fibrin ferment cog region, is connected with the fibrin ferment cog region, and make to coagulate
Hemase original is converted into the zone of transformation of fibrin ferment；The fibrin ferment cog region separates under thrombin action with the electric signal area.
After the contacting blood of Coagulation test layer and shortage blood platelet and calcium ion, the factor in blood is converted into solidifying on zone of transformation
Hemase, thrombin action separate the electric signal area being connected with fibrin ferment cog region in fibrin ferment cog region.Electric signal area exists
Under external voltage effect, the electric current that electronics is mobile, and formation can be recorded is generated, it is real by the variation of record current response signal
Now to the detection of prothrombin time.
4. Electrochemical Detection chip provided by the invention, the fibrin ferment cog region is formed by polypeptide material, the telecommunications
Number area is formed by electric signal material, and the conversion zone is formed by the organization material containing thromboplastin.
The blood to be detected for lacking blood platelet and calcium ion organizes material in the presence of forming the organization material of zone of transformation
Conversion of prothrombin in blood is fibrin ferment by thromboplastin in material, under thrombin action, soluble fibre in blood
Fibrillarin original is converted into insoluble fibrin, and blood is made to be changed into not flowable gel by the flow regime of liquid
Coagulation test occurs for state, and the time of above-mentioned Coagulation test is prothrombin time (PT), is the blood coagulations such as detection II, V, VII, X
The index of the factor and fibrinogen activity.
Contain thrombin cleavage site in polypeptide material, peptide residue, release and polypeptide are cut under thrombin action
The electric signal material of connection.Under external voltage effect, electric signal material generates the electric current that electronics is mobile, and formation can be recorded.
Electric signal material is supported in layer of nanomaterial, and nano material can promote the oxidation/reduction reaction of electric signal molecule, accelerates electricity
The transmitting of son improves current responsing signal, realizes the transmitting and amplification that electric signal is generated to electric signal material.The life of nano material
Object compatibility is good, suitable for supporting the biomaterials such as polypeptide, albumen, enzyme；Its large specific surface area can be improved the load to biomaterial
Carrying capacity expands the electric signal generated after fibrin ferment cutting polypeptide, realizes transmitting and amplification of the layer of nanomaterial to electric signal, make
Sample size needed for sensitivity raising that Electrochemical Detection chip detects prothrombin time, detection time are shortened, detected is reduced,
And there is good detection stability and repeatability.
5. Electrochemical Detection chip provided by the invention, the electric signal material is p-phenylenediamine.Know when forming fibrin ferment
After the polypeptide material in other area is cut by fibrin ferment, peptide residue is generated, while discharging p-phenylenediamine, applies electricity on the working electrode (s
Pressure, p-phenylenediamine is oxidized, and loses electronics, and electronics movement forms the electric current that can be recorded.
6. Electrochemical Detection chip provided by the invention, the working electrode is screen printing electrode, silk-screen printing preparation
Electrode have that preparation is simple, detection sensitivity is high and the advantages such as detection time is short.
Electrode layer further includes reference electrode and to electrode, and Electrochemical Detection chip uses three-electrode system, utilizes reference electricity
The reference signal of pole is to reduce the disturbing factor from instrument and equipment, detection environment and test sample, since blood constituent is multiple
It is miscellaneous, the interference of foreign matter is advantageously reduced using three-electrode system, improves detection stability and testing result accuracy.Into one
Step, reference electrode and/or to electrode be screen printing electrode, when further increasing Electrochemical Detection chip to factor
Between detect sensitivity, shorten detection time.
7. the present invention provides a kind of electrochemical sensor, including above-mentioned Electrochemical Detection chip.Pass through Electrochemical Detection
Chip effectively enhances current-responsive during detecting blood coagulation zymogen time, enhances the signal strength of electrode, to reach
High detection sensitivity, short response time and few detection sample size, can be realized electrochemical sensor to factor
Quick, the highly sensitive and specific detection of time improves the accuracy of prothrombin time detection.Prothrombin time
As judge body hemostasis and blood coagulation system disease, diagnosis and observation thrombosis, and prompt body whether suffer from hepatitis, liver
The diagnosis of the related diseases such as hardening, liver cancer and prognostic indicator, using electrochemical sensor realize to prothrombin time it is quick,
Sensitive Detection, the diagnosis, treatment and curative effect of medication monitoring for being suitable for clinical disease provide and effectively instruct foundation.
In addition, utilizing the technical level easy to operate, to operator of electrochemical sensor detection prothrombin time
Extensive universal, application of less demanding, suitable for factor detection.
6. the preparation method of electrochemical sensor provided by the invention, preparation process is simple, and operability is high, can prepare
Obtain above-mentioned having that amplification current responsing signal, high sensitivity, stability be good, the quick Electrochemical Detection chip of detection.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the structural schematic diagram for the Electrochemical Detection chip that the embodiment of the present invention 1 provides；
Fig. 2 is the working principle that the Electrochemical Detection chip that the embodiment of the present invention 1 provides is used for prothrombin time detection
Figure；
Fig. 3 is a kind of structural schematic diagram for electrochemical sensor that the embodiment of the present invention 2 provides；
Fig. 4 is a kind of structural schematic diagram for electrochemical sensor that the embodiment of the present invention 2 provides；
Fig. 5 is the top view of electrochemical sensor shown in Fig. 4；
Fig. 6 is the structural schematic diagram for the Electrochemical Detection chip that the embodiment of the present invention 3 provides；
Fig. 7 is the structural schematic diagram for the electrochemical sensor that the embodiment of the present invention 4 provides；
Fig. 8 shows the flow chart of the preparation method of the electrochemical sensor provided in embodiment 5；
Fig. 9 shows the flow chart of the preparation method of the electrochemical sensor provided in embodiment 5；
Figure 10 shows the modification figure being surface modified to working electrode；
Figure 11 shows the result figure of the prothrombin time of three groups of blood samples of electrochemical sensor detection in experimental example 1；
Figure 12 shows that electrochemical sensor and clinic SYSMEXCS5100 optics Blood coagulation instrument detect blood coagulation protoenzyme in experimental example 1
The matched curve figure of time；
Figure 13 shows that the electrochemical sensor of MWCNTs type electrochemical sensor and unmodified MWCNTs are to solidifying in experimental example 2
The comparison result figure of blood zymogen time detection；
Figure 14 shows the repeated testing result figure of 5 groups of electrochemical sensors in experimental example 3；
Description of symbols:
1- encapsulated layer, 11- sample introduction zone, 12- sample detection area, 13- reaction zone, 14- is into Man Qu, 15- interface area；
2- electrode layer, 21- working electrode, 22- is to electrode, 23- reference electrode, 24- strip conductor；
3- basal layer；
4- insulating layer.
Specific embodiment
Technical solution of the present invention is clearly and completely described below in conjunction with attached drawing, it is clear that described implementation
Example is a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, ordinary skill
Personnel's every other embodiment obtained without making creative work, shall fall within the protection scope of the present invention.
In the description of the present invention, it should be noted that term " center ", "upper", "lower", "left", "right", "vertical",
The orientation or positional relationship of the instructions such as "horizontal", "inner", "outside" be based on the orientation or positional relationship shown in the drawings, merely to
Convenient for description the present invention and simplify description, rather than the device or element of indication or suggestion meaning must have a particular orientation,
It is constructed and operated in a specific orientation, therefore is not considered as limiting the invention.In addition, term " first ", " second ",
" third " is used for descriptive purposes only and cannot be understood as indicating or suggesting relative importance.
In the description of the present invention, it should be noted that unless otherwise clearly defined and limited, term " installation ", " phase
Even ", " connection " shall be understood in a broad sense, for example, it may be being fixedly connected, may be a detachable connection, or be integrally connected；It can
To be mechanical connection, it is also possible to be electrically connected；It can be directly connected, can also can be indirectly connected through an intermediary
Connection inside two elements.For the ordinary skill in the art, above-mentioned term can be understood at this with concrete condition
Concrete meaning in invention.
As long as in addition, the non-structure each other of technical characteristic involved in invention described below different embodiments
It can be combined with each other at conflict.
Embodiment 1
The present embodiment provides a kind of Electrochemical Detection chips, including basal layer 3 and take shape in the electrode layer 2 on basal layer 3,
As shown in Figure 1,2 working electrode 21 of electrode layer, to electrode 22 and reference electrode 23.Wherein, working electrode 21 includes stacking gradually
Conductive layer, layer of nanomaterial and the Coagulation test layer of setting.Coagulation test layer generates the telecommunications for detecting prothrombin time
Number, layer of nanomaterial transmitting and amplification electric signal.Electrochemical Detection chip lacks blood platelet for detecting prothrombin time
It is contacted with the blood of calcium ion with Coagulation test layer, makes conversion of prothrombin fibrin ferment, triggering prothrombin time detection
Coagulation process.Meanwhile Coagulation test layer generates the electric signal for detecting prothrombin time, electric signal through layer of nanomaterial to
Conductive layer transmitting, since advantages, the layer of nanomaterial such as the conductive height of layer of nanomaterial, large specific surface area can accelerate electronics
The amplification to electric signal is realized in transmitting, improves the detection sensitivity of Electrochemical Detection chip, detection time shortens.
Coagulation test layer includes several Coagulation test units, and Coagulation test unit includes fibrin ferment cog region and fibrin ferment
The electric signal area that cog region is connected, and make the zone of transformation of conversion of prothrombin fibrin ferment, fibrin ferment cog region is in fibrin ferment
Effect is lower to be separated with electric signal area.Specifically, fibrin ferment cog region uses the polypeptide (sequence: Tos- of thrombin cleavage site
Gly-Pro-Arg) material is formed, and electric signal area is formed by p-phenylenediamine, and polypeptide is connected with p-phenylenediamine with amido bond, is formed
Polypeptide-p-phenylenediamine (Tos-Gly-Pro-Arg-p-Phenylenediamine).Zone of transformation is by containing thromboplastin
Organization material formed, organization material selects prothrombin time detection reagent, containing needed for extrinsic coagulation in detection reagent
Tissue factor and the substances such as calcium ion.Layer of nanomaterial is by multi-walled carbon nanotube (Multi-Walled
CarbonNanotubes, MWCNTs) material formed.The material of conductive layer selects conductive metallic materials or the stones such as gold paste, silver paste
The conductive carbon pastes material such as black alkene, carbon nanotube, with realize Electrochemical Detection chip and detection device (such as: bioelectric interface molecule
Analyzer) electric signal connection.
Working principle of the Electrochemical Detection chip for factor detection is as shown in Figure 2: the blood coagulation of working electrode 21 is anti-
Answer layer with without containing blood platelet, calcium ion contacting blood after, in the presence of the tissue factor of zone of transformation, zone of transformation organization material
In thromboplastin by the conversion of prothrombin in blood be fibrin ferment (Fig. 2 a → Fig. 2 b).Under thrombin action, in blood
Soluble fibrinogen is converted into insoluble fibrin, is changed into blood by the flow regime of liquid not flowable
Gel state, occur Coagulation test.At the same time, after generating fibrin ferment, fibrin ferment cuts the polypeptide of fibrin ferment cog region,
Peptide residue is generated, the p-phenylenediamine being connected with polypeptide is released (Fig. 2 b → Fig. 2 c), following reaction occurs:
Apply voltage on working electrode 21, the p-phenylenediamine in electric signal area is oxidized, and loses electronics, the mobile shape of electronics
At the current responsing signal that can be recorded.On Coagulation test layer is set in the layer of nanomaterial of multi-walled carbon nanotube formation, benefit
It can accelerate electron transmission, strengthening electric current response with the high electric conductivity of carbon nanotube.The good biocompatibility of carbon nanotube is suitable for negative
Carry polypeptide, the biomaterials such as albumen, large specific surface area, load capacity are high, can increase to polypeptide material, organization material but
Amount amplifies current responsing signal to promote the generation and transmitting of electronics.Current signal is acquired by bioelectric interface analysis of molecules instrument
Variation, the coagulation process under thrombin action can be monitored, realize detection prothrombin time purpose.
Enhancing and amplification due to carbon nanotube to current responsing signal, Electrochemical Detection chip for prothrombin time detection when
Between shorten, detection sensitivity improve, detection needed for blood sample amount reduce.Electrochemical Detection chip is to prothrombin time
Detect the combination independent of the substances such as fibrin in blood and chip surface, the requirement to chip surface environment consistency
It is low.Simultaneously as the good electrochemical stability of carbon nanotube, can reduce unrelated ingredient complicated in blood to factor
The interference of time detection, makes Electrochemical Detection chip have good detection stability and repeatability.
Further, as shown in Figure 1, electrode layer 2 further includes the strip conductor 24 for being layed in 3 surface of basal layer, for passing
It is defeated by working electrode 21, the detection signal generated to electrode 22 and reference electrode 23.Working electrode 21, to electrode 22 and reference electricity
Pole 23 can be prepared using the techniques such as vacuum deposition, silk-screen printing, plating, the preparation based on silk-screen printing technique is simple,
Detection sensitivity height and the short advantage of detection time, working electrode 21 specifically use silk screen to electrode 22 and reference electrode 23
The method of printing is formed.It is conductive that preparation can choose gold paste, silver paste etc. to the electrode slurry of electrode 22 and reference electrode 23 respectively
The conductive carbon pastes material such as metal material or graphene, carbon nanotube, to realize Electrochemical Detection chip and bioelectric interface molecule point
The electric signal of analyzer connects.
Embodiment 2
The present embodiment also provides a kind of electrochemical sensor, as shown in figure 3, electrochemical sensor includes the above embodiments
The 1 Electrochemical Detection chip provided, and electrode layer 2 is set to far from the encapsulated layer 1 on 3 one side of basal layer.1 material of encapsulated layer
Select polymethyl methacrylate (PMMA), poly terephthalic acid class plastics (PET), polyether-ether-ketone (PEEK), polytetrafluoroethylene (PTFE)
(PTFE), polyvinyl chloride (PVC), polypropylene (PP) polymer etc., make encapsulated layer 1 have both stability, inoxidizability, anticorrosion
Property, bio-compatibility, and have certain hydrophily.Encapsulated layer 1 using epoxy AB glue substance, single layers of two-sided band, etc. from
The methods of daughter, ultraviolet light and thermocompression bonding are covered on electrode layer 2, are made to fit closely between encapsulated layer 1 and electrode layer 2, be subtracted
Few influence of the external environment to electrochemical sensor detection performance.To reinforce the stickiness between encapsulated layer 1 and electrode layer 2, also
A layer insulating 4 (Fig. 4) can be printed between encapsulated layer 1 and electrode layer 2, insulating layer 4 is certain hydrophilic exhausted using having
Edge ink is formed.Insulating layer 4 is set between encapsulated layer 1 and electrode layer 2, to improve the packaging effect of encapsulated layer 1, while exhausted
The working region that electrode layer 2 is reserved in edge layer 4 makes electrode layer 2 and blood complete haptoreaction.
After packaging is accomplished, the surface texture schematic diagram of Electrochemical Detection chip as shown in Figure 5 is obtained: in encapsulated layer 1
Upper formation sample introduction zone 11, sample detection area 12, reaction zone 13, into full area 14 and interface area 15.Sample introduction is offered in sample introduction zone 11
Hole is offering sample outlet hole into full area 14, and blood sample to be detected is flowed by the sample holes of sample introduction zone 11, in Electrochemical Detection
Under the capillarity of the water wetted material of chip, the sample outlet hole into full area 14 is flowed to.When flowing through sample detection area 12, sample introduction inspection
The reference electrode 23 for surveying area 12 generates sample introduction signal, and instruction sample flows into.Then be left to reaction zone 13, blood sample to be measured with
Coagulation test layer contacts, reacts in working electrode 21, generates electric signal, and electric signal is subsequent via the amplification of layer of nanomaterial
Continue and transmitted to the conductive layer of working electrode 21, by recording the variation of electric signal, is completed to sample to be tested prothrombin time
Detection.After blood sample to be measured flows through reaction zone 13, sample to be tested is flow into full area 14, and sample detection is prompted to complete.Interface
Area 15 can be realized to be connect with the electric signal of current signal acquisition instrument (such as: biomolecule interface analysis instrument), passes through interface
Realize the electric current generated from electrochemical sensor into current signal acquisition instrument output prothrombin time detection process in area 15
Signal.
By the way that the above-mentioned Electrochemical Detection chip with amplification current responsing signal function is arranged in electrochemical sensor,
Make electrochemical sensor that there are the advantages such as high sensitivity, response time are short, required sample size is few, can the time to factor when
Between quick, Sensitive Detection, and the advantages that have that reproducible, stability is high etc. are diagnosis, the treatment of clinical disease, and
Curative effect of medication monitoring, which provides, effectively instructs foundation.In addition, utilizing the operation letter of electrochemical sensor detection prothrombin time
Single, of less demanding to the technical level of operator, suitable for factor detection extensive universal, application.
Embodiment 3
The present embodiment provides a kind of Electrochemical Detection chip, the structure of electrode layer 2 is as shown in fig. 6, include being set to base
Strip conductor 24 on bottom 3, strip conductor 24 are specifically formed by conductive silver layer, and the starting point of strip conductor 24 is close to substrate
A side in width direction for layer 3 is arranged, and is extended along the length direction of basal layer 3, and conductor rail is ultimately formed
Clearing end of the road 24 on basal layer 3.The clearing end contact connectio of strip conductor 24 has the conductive layer of working electrode 21 and to electricity
The conductive layer of pole 22, working electrode 21 is specifically in the form of annular discs, and the conductive layer of working electrode 21 is conductive carbon ink electrode.To electrode
22 are spaced the ring structure for being arranged and surrounding working electrode 21 in working electrode 21, are conductive carbon ink electrode to electrode 22.
It is not connected with covering one layer of AgCl on working electrode 21 and termination part to electrode 22 in strip conductor 24, forms reference electrode
23.Working electrode 21 conductive layer along far from basal layer 3 direction along be cascading conductive layer, layer of nanomaterial and
Coagulation test layer.Layer of nanomaterial is formed by nano material, for example, carbon nanotube.The Coagulation test being arranged in layer of nanomaterial
Layer includes several Coagulation test units, and each Coagulation test unit includes fibrin ferment cog region, is connected with fibrin ferment cog region
Electric signal area, and make the zone of transformation of conversion of prothrombin fibrin ferment.Wherein, fibrin ferment cog region is by containing thromboplastin
Organization material is formed, and fibrin ferment cog region is formed by the polypeptide material containing thrombin cleavage site, electric signal Qu Youyu polypeptide
Electric signal material (for example, p-phenylenediamine) formation being connected.In coagulation process, is transmitted and amplified by right by carbon nanotube
The current signal that phenylenediamine generates, high sensitivity, reproducible quick detection with realization to prothrombin time.
As the first alternative embodiments of the present embodiment, working electrode 21 in electrode layer 2, to electrode 22 and reference
The structure of electrode 23 is also an option that any other structure, as long as passing through the nano material of 21 surface modification of working electrode, realizes
Transmitting and amplification to electric signal, to improve the detection effect to prothrombin time.
As second of alternative embodiments of the present embodiment, the conductive layer of working electrode 21, to electrode 22 and reference
The material of electrode 23 can also select the material of other conductive matter respectively, for example, Ag, Au metal material etc..
As the third alternative embodiments of the present embodiment, the layer of nanomaterial of working electrode 21 is also an option that it
The high nano material of his electric conductivity height, large specific surface area, bio-compatibility, for example, graphene, ZnO etc. are able to achieve to electricity
The transmitting and amplification of signal.
As the 4th kind of alternative embodiments of the present embodiment, electric signal material is also an option that other can generate electricity
Son is mobile, the material for being recorded electric current is formed, for example, o-phenylenediamine etc..
Embodiment 4
The present embodiment also provides a kind of electrochemical sensor, the Electrochemical Detection chip provided including embodiment 3.Such as Fig. 7
It is shown, it is covered with a layer insulating 4 on the surface of the electrode layer 2 of Electrochemical Detection chip, insulating layer 4 is formed by dielectric ink.
Opening is provided on insulating layer 4, electrode layer 2 is leaked out by the opening of insulating layer 4, forms the reaction zone of electrochemical sensor
13。
One layer of hydrophilic film is covered in the top of insulating layer 4, as the encapsulated layer 1 of electrochemical sensor, encapsulated layer 1 is covered instead
The electrode layer 2 that area 13 is exposed is answered, sample holes and sample outlet hole is offered in the position of the corresponding reaction zone 13 of encapsulated layer 1, makes blood to be measured
Liquid sample is flowing through the inflow electrode layer 2 of reaction zone 13, and Coagulation test occurs, is coagulated by the current signal that electrode layer 2 exports
The detection of blood zymogen time.
Embodiment 5
The present embodiment provides a kind of preparation method of electrochemical sensor in embodiment 4, the preparations of electrochemical sensor
Journey is as shown in Figure 8 and Figure 9, preparation method specifically includes the following steps:
S1 prepares strip conductor 24:
S11 selects polyethylene terephthalate (PET) as 3 material of basal layer, before printing, PET base material is used
Deionized water washes off surface impurity, and with being dried with nitrogen；
S12, first printing conductive silver layer, to improve the electric conductivity of screen printing electrode and leading as screen printing electrode
Electric rail 24；
S2, on basal layer 3 conductive layer of preparation work electrode 21, to electrode 22 and reference electrode 23
S21 prints chlorination silver paste in the end of conductive silver layer, forms reference electrode 23；
S22 continues to print conductive carbon ink slurry on basal layer 3, is respectively formed the conductive layer of working electrode 21 and to electricity
Pole 22.Wherein the conductive layer of working electrode 21 is discoid, is in be spaced to be arranged and around work with working electrode 21 to electrode 22
Make the ring structure of electrode 21；
S3 covers insulating layer 4 on one side of the electrode layer 2 far from basal layer 3:
Insulating layer 4 is fixed on electrode layer 2 using dielectric ink, is left for fixating reagent and the workspace to react
Domain, and being toasted again 30 minutes in 75 DEG C of dry insulating boxs, the Electrochemical Detection chip prepared aging 2d at room temperature, phase
Between be stored in and be protected from light in dry environment；
S4 is surface modified working electrode 21, and modification is as shown in Figure 8:
S41 dissolves the purifying of 0.8-1.2mg with Nafion membrane (perfluorinated sulfonic resin) solution of 0.8-1.2mL
MWCNTs (multi-walled carbon nanotube) powder, and be placed in KQ-400KDE type high power numerical control ultrasonic cleaner (city of Kunshan is super
Sound Instrument Ltd.) in oscillation 20 minutes or so it is evenly dispersed to MWCNTs, the MWCNTs that 0.8-1.2mg/mL is made is uniform
Dispersion liquid.Before modifying MWCNTs dispersion liquid, the Electrochemical Detection chip to complete is cleaned using PBS buffer solution, is used in combination
Drying machine drying 5 minutes.Then the coating modification of 4-6 μ LMWCNTs uniform dispersion is weighed in the work for the screen printed electrode made
Make 21 surface of electrode, and be placed in insulating box at 70 DEG C and dry 30 minutes, is made after dry and is set to working electrode 21 and leads
Layer of nanomaterial in electric layer；
S42 prepares 0.5% BSA solution with the PBS buffer solution and bovine serum albumin(BSA) powder of 0.1mol/L, then uses
BSA solution dissolves polypeptide-p-phenylenediamine (Tos-Gly-Pro-Arg-p-Phenylenediamine) freeze-dried powder
Polypeptide-p-phenylenediamine substrate solution of 300 μm of ol/L is made in (P14993, Shanghai Sheng Gong bioengineering limited liability company)；
By spare PT reagent (prothrombin time determination reagent) and 0.9% physiological saline progress 1:4 dilution, stirring
It mixes in machine and stirs 2 minutes, be placed in after evenly mixing to thrombin reagent in 37 DEG C of water-baths 3 minutes.PT reagent is for detecting people's blood
The prothrombin time of slurry is the mix reagent containing tissue factor etc. needed for extrinsic coagulation, so that blood solidifies
Reaction generates fibrin ferment.
It will mix, and stirring by the volume ratio of 1:2 with PT reagent containing polypeptide-p-phenylenediamine substrate solution
3 minutes are stirred on machine to being uniformly mixed, and are configured to thrombin substrate solution.
The solidifying of 6 μ L is modified in the conductive layer surface coating of the working electrode 21 of the Electrochemical Detection chip of step S41 preparation
Hemase substrate solution.It is put in insulating box later at 36 DEG C 20 minutes dry, supports thrombin substrate in carbon nanotube, shape
Working electrode 21 at the Coagulation test layer being located in layer of nanomaterial, after being modified.
Encapsulated layer 1 is arranged in S5 on one side of the electrode layer 2 far from basal layer 3:
One layer of hydrophilic film is sticked as encapsulated layer 1 in electrode surface, is formed the reaction zone 13 of a 5 μ L, is opened on encapsulated layer 1
Equipped with sample holes and sample outlet hole, the electrochemical sensor for PT detection is made, and be placed in vacuum seal bag wait test
It is required.
As the first alternative embodiments of the present embodiment, Electrochemical Detection chip is also an option that in embodiment 1
Any chip provided equally can obtain current signal enhancing, the electricity that detection sensitivity improves using above-mentioned preparation method
Chemical sensor.
Experimental example 1
1, experiment purpose: electrochemical sensor is examined to be used to detect the validity of blood coagulation raw coal time.
2, experimental method:
(1) blood sample of 3 patients is had chosen from Grade A hospital clinical laboratory, the electrochemistry electrification provided with embodiment 2
The prothrombin time of sensor detection plasma sample is detected, and test is all using the new of hospital same day acquisition patient every time
Blood liquid.Three electrodes of electrochemical sensor are connected with electrochemical workstation, and electrode is placed on stable experimental bench,
Before carrying out clotting assay, a certain amount of blood plasma is first extracted from blood sample, plasma sample is put into 37 DEG C of water-baths 3 points
5 μ L samples are injected reaction zone 13 by the sample holes of electrochemical sensor using pipettor by clock, due to the effect of hydrophilic film,
Sample can fill reaction zone 13 in 0.1s or so, using the reaction process of chronoamperometry record sample solidification.Nothing is used simultaneously
The electrochemical sensor of modification thrombin substrate heals slurry and carries out negative control experiment.
(2) blood sample of 10 donors is had chosen from hospital, with the electrochemical sensor that embodiment 4 provides, is used
Above-mentioned detection method detects the prothrombin time of blood sample, while using SYSMEXCS5100 optics Blood coagulation instrument
Test prothrombin time is verified.
3, experimental result:
Figure 11 show respectively electrochemical sensor detect three groups of blood samples prothrombin time be respectively 42.75s,
58.94s,82.86s.After blood sample injects the sample holes of electrochemical sensor, first with to be formed in 21 blood coagulation of working electrode anti-
The zone of transformation material in layer is answered to be dissolved, Coagulation test is activated in course of dissolution, the blood coagulation substrate of fibrin ferment cutting is generated,
Current signal is generated under the oxidation of electrode.When blood coagulation production of enzyme reaches maximum value during this, fibrin ferment cleavage reaction
Also most rapidly, and then kinetic current is made also to reach peak value；With the progress of cleavage reaction, thrombin amount is gradually decreased, electric current
On be also gradually reduced and tend towards stability therewith.As shown in Figure 11, clear, the different blood of 3 groups of collected signal curves of sample institute
Liquid sample generates the speed and dosage difference of fibrin ferment, so that the time for making electrode surface collect current peak is also different,
Therefore achieve the purpose that detect the clotting time, start to be reacted to electric current using sensor feed liquor in this experimental example and reach peak value
PT value of the time as Coagulation test.Figure 11 also shows the feminine gender that blood plasma is added dropwise on the sensor without modification thrombin substrate
Control experiment results, it can be seen that after addition blood plasma, the sample introduction peak only generated by sample introduction, rear rapid decrease is simultaneously attached in zero
It closely gradually tends towards stability, blood plasma Coagulation test is not activated in experiment, reflects the principle conscientiously and can be used for PT detection.
Figure 12 shows that the electrochemical sensor provided with embodiment 4 and the detection of clinic SYSMEXCS5100 optics Blood coagulation instrument are solidifying
The matched curve of blood protoenzyme time.The linear fit curve of measurement result, coefficient of determination R are shown by straight line in Figure 122For
0.991.The results show that electrochemical sensor provided by the invention can preferably be applied to clinical detection coagulation parameters PT.
Experimental example 2
1, experiment purpose: detection electrochemical sensor is used to detect the sensitivity of blood coagulation raw coal time.
2, experimental method: with the detection method in experimental example 1, to the electrochemical sensor that embodiment 4 provides, and not into
The current-responsive intensity of the carbon nano tube modified electrochemical sensor of row is detected.
3, experimental result:
Figure 13 shows the MWCNTs type electrochemical sensor that embodiment 4 provides and the electrochemical sensor of unmodified MWCNTs
To the comparison result of prothrombin time detection.As shown in Figure 13, compared with the electrochemical sensor of unmodified MWCNTs,
The current-responsive intensity of MWCNTs type electrochemical sensor increases the detection spirit of 36 ± 1%, MWCNTs type electrochemical sensor
Sensitivity is promoted.The negative right of the MWCNTs type sensor addition thrombin substrate for being not provided with Coagulation test layer is also shown in Figure 13
It according to the facts tests, it can be seen that after feed liquor, caused sample introduction peak, sample introduction when the reaction peak, only feed liquor of thrombin signal do not occur
Peak caused sensor immediate current variation when being to sensor sample introduction, declines and tends towards stability rapidly later.
Experimental example 3
1, experiment purpose: the repeatability of electrochemical sensor is detected.
2, detection method: the blood coagulation protoenzyme test method in test experience example 1 carries out following reperformance test:
(1) 3 groups of electrochemical sensors (1th, 2th, 3th) that embodiment 2 provides and 2 groups that embodiment 4 provides are chosen
Electrochemical sensor (4th, 5th) carries out same blood sample to repeat test.
(2) the electrochemistry tradition device that embodiment 2 provides is chosen, 5 duplicate measurements have been made respectively to 3 groups of plasma samples.
3, experimental result:
Figure 14 shows the repeated testing result of 5 groups of electrochemical sensors, by being clearly visible plots changes phase in Fig. 5
Together, there is preferable detection stability；Its appearance time CV value (coefficient of variation) is 1.36%, and peak point current CV value is
3.02%, therefore there is more outstanding repeatability.
Table 1
Table 1 shows the repeated testing result of 3 groups of blood samples, the CV value of 3 groups of experimental results is respectively 2.26%,
2.07% and 1.29%, it is possible thereby to judge sensor provided by the invention to measure PT with good uniformity and repetition
Property.This experimental method has recorded the transformation period that thrombin action in coagulation process generates electric current in Coagulation test layer, in other
Independent of in the substances such as fibrin in blood and the combination of sensor surface unlike measurement method, therefore PT test pair
The consistency etc. of MWCNTs electrochemical sensor surface environment has more loose limitation.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
Claims (12)
1. a kind of Electrochemical Detection chip, including electrode layer, which is characterized in that the working electrode of the electrode layer includes successively layer
Conductive layer, layer of nanomaterial and the Coagulation test layer of folded setting；The Coagulation test layer is generated for detecting prothrombin time
Electric signal, layer of nanomaterial transmitting and amplify the electric signal.
2. Electrochemical Detection chip according to claim 1, which is characterized in that the layer of nanomaterial by carbon nanotube,
The formation of any one of graphene and zinc oxide；Preferably, the layer of nanomaterial is formed by carbon nanotube；Preferably, described
Carbon nanotube is multi-walled carbon nanotube.
3. Electrochemical Detection chip according to claim 1 or 2, which is characterized in that the Coagulation test layer includes several
Coagulation test unit；Each Coagulation test unit includes fibrin ferment cog region, is connected with the fibrin ferment cog region
Electric signal area, and make the zone of transformation of conversion of prothrombin fibrin ferment；The fibrin ferment cog region under thrombin action with institute
State the separation of electric signal area.
4. Electrochemical Detection chip according to claim 3, which is characterized in that the fibrin ferment cog region is by polypeptide material
It is formed, the electric signal area is formed by electric signal material, and the conversion zone is formed by the organization material containing thromboplastin.
5. Electrochemical Detection chip according to claim 4, which is characterized in that the electric signal material is p-phenylenediamine.
6. Electrochemical Detection chip according to claim 1-5, which is characterized in that the working electrode is silk
Wire mark brush electrode.
7. Electrochemical Detection chip according to claim 1-6, which is characterized in that the electrode layer further includes
Reference electrode and to electrode；Preferably, the reference electrode be screen printing electrode and/or it is described be silk-screen printing to electrode
Electrode.
8. a kind of electrochemical sensor characterized by comprising
The described in any item Electrochemical Detection chips of claim 1-7, the electrode layer of the Electrochemical Detection chip are set to base
On bottom；
Encapsulated layer is set on one side of the electrode layer far from the basal layer.
9. electrochemical sensor according to claim 8, which is characterized in that between the electrode layer and the encapsulated layer also
It is provided with insulating layer, the insulating layer has opening, and the electrode layer exposes to form the electrochemistry in the position of the opening
The reaction zone of sensor.
10. the described in any item Electrochemical Detection chips of claim 1-7 or the described in any item electrifications of claim 8-9
Learn application of the sensor in prothrombin time detection.
11. a kind of method of modifying of the working electrode of Electrochemical Detection chip, which comprises the following steps:
Nano material powder is dispersed in perfluor sulfoacid resin solution, nano material dispersion liquid is obtained；By nano material
Dispersion forms layer of nanomaterial in conductive layer surface in the surface of the conductive layer of working electrode, drying；
Thrombin substrate solution is coated in layer of nanomaterial, thrombin substrate solution includes containing the more of thrombin cleavage site
Peptide, the electric signal molecule being connected with the polypeptide, and the tissue factor containing thromboplastin；It is dry, in layer of nanomaterial
Coagulation test layer is formed, the working electrode of surface modification is made；
Preferably, the nano material is the carbon nano-tube material of purifying.
12. a kind of preparation method of electrochemical sensor, which comprises the following steps:
On the base layer the conductive layer of preparation work electrode, to electrode and reference electrode；
With the method for modifying of the working electrode described in claim 11, the working electrode of surface modification is obtained；
Encapsulated layer is set on one side of the electrode layer far from basal layer to get the electrochemical test sensors.
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