CN107064116B - A kind of electrochemical luminescence biochemical detection system and method based on mobile phone USB-OTG interface - Google Patents
A kind of electrochemical luminescence biochemical detection system and method based on mobile phone USB-OTG interface Download PDFInfo
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- CN107064116B CN107064116B CN201710116895.9A CN201710116895A CN107064116B CN 107064116 B CN107064116 B CN 107064116B CN 201710116895 A CN201710116895 A CN 201710116895A CN 107064116 B CN107064116 B CN 107064116B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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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- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
Abstract
The invention discloses a kind of electrochemical luminescence biochemical detection system and method based on mobile phone USB-OTG interface, the system devise the adjustable driving source system of electrochemical luminescence based on mobile phone USB-OTG interface, the generation for electrochemical luminescence；Then electrochemiluminescence analysis unit is devised, the quantitative analysis for electrochemical luminescence；Next explores concentration selection of influence and tris (bipyridine) ruthenium of the different voltages to electrochemical luminescence during electrochemical luminescence, the optimal value of voltage and tris (bipyridine) ruthenium concentration is obtained, for the subsequent electrochemical luminescence biochemistry detection based on mobile phone；Finally by different electrode modifications, the electrochemiluminescence analysis of BSA and fibrin ferment is realized on mobile phone.The present invention realizes excitation and analyzes integrated mobile phone electrochemiluminescence analysis platform, and be applied to the technology in the electrochemical luminescence detection process of BSA and fibrin ferment, have device portable general, stability is good, the advantages that convenient is operated, can adapt to the requirement of instant biochemistry detection.
Description
Technical field
The present invention relates to a kind of electrochemical luminescence biochemical detection system and method based on mobile phone USB-OTG interface, especially
Realize voltage drive and the integrated electrochemiluminescence analysis of image analysis on cell phone platform.
Background technique
USB has the property of hot plug, and is convenient for carrying and uses, while USB standard is also an open standard, it
It supports the distinct device of different vendor that can use on the same device, has and be more widely applied scene, difference is supported to set
It is standby.Based on the above feature, USB interface has obtained rapidly universal.But the USB of standard is using host-guest architecture, i.e., one
Main equipment connection one or more is from equipment, and in a transmission system, data transmission can only occur in main equipment and from equipment
Between, it cannot occur from equipment and between equipment.OTG agreement as may be implemented after the complementary agreement of USB from equipment with
From the connection between equipment.By utilizing OTG agreement, OTG peripheral hardware is connected to mobile phone, may be implemented two-way between peripheral hardware and mobile phone
Communication.Electrochemiluminescence analysis on traditional cell phone platform, the main image analysis function for utilizing mobile phone, voltage drive also need
External electrochemical workstation provides, and cannot be known as the electrochemical luminescence based on mobile phone in complete meaning, therefore cell phone platform powers on
Pressure excitation and the research of the integrated electrochemiluminescence analysis of image analysis have far-reaching significance.The present invention proposes that one kind is based on
The electrochemical luminescence biochemical detection system and method for mobile phone USB-OTG interface, and the technology is applied to BSA and fibrin ferment
In electrochemical luminescence detection process, have device portable general, stability is good, operates the advantages that convenient, can adapt to instant life
Change the requirement of detection.
Summary of the invention
In view of the above-mentioned deficiencies in the prior art, it is an object of the present invention to provide a kind of electrification based on mobile phone USB-OTG interface
Shine biochemical detection system and method are learned, voltage drive and the integrated electrochemistry of image analysis on cell phone platform are especially realized
Luminesceence analysis.
The purpose of the present invention is achieved through the following technical solutions: a kind of electrification based on mobile phone USB-OTG interface
The biochemical detection system that shines is learned, which includes electrochemical luminescence capture analysis system, electrochemical luminescence is adjustable driving source system
With electrochemical luminescence reaction unit.
The electrochemical luminescence capture analysis system includes: mobile phone and electrochemiluminescence analysis unit with camera；
Camera captures electrochemical luminescence image, and electrochemiluminescence analysis unit extracts electrochemical luminescence image, cuts, gray scale
Processing and quantitative analysis, it is final to obtain electrochemiluminescence analysis result.
The adjustable driving source system of electrochemical luminescence includes: that USB-OTG mobile phone end interface, USB-OTG external circuits connect
Mouthful, working electrode interface and to electrode interface；When providing voltage drive using mobile phone, USB-OTG mobile phone end interface is connected to
The USB port of mobile phone connects USB-OTG external circuits interface by connecting line；By the power supply line of USB-OTG external circuits interface
With ground wire respectively as anode and cathode, sequence accesses current foldback circuit, impedance type bleeder circuit and voltage regulator circuit, by steady
Anode connection working electrode interface after volt circuit, cathode are connected to electrode interface.Working electrode interface connects ITO conduction glass
Glass, to electrode interface connection platinum wire ring to electrode.At this time working electrode interface and to can be stable at electrode interface output 1~
4V voltage is for exciting electrochemical luminescence.
The electrochemical luminescence reaction unit includes electrochemical luminescence reactive tank and electrochemical luminescence reaction support sealing bottom
Seat, the electrochemical luminescence reactive tank have in lead to truncated cone-shaped reaction cavity；Electrochemical luminescence reaction support sealed base
Cross section is concave structure, and ITO electro-conductive glass is placed in groove, and sealing ring is placed on the top of ITO electro-conductive glass, is realized and electrification
Learn the sealed connection of luminescence-producing reaction slot；Platinum wire ring is placed in reaction cavity to electrode, and the coreagent of electrochemical luminescence is added
System and corresponding detection substance realize stable electrochemical luminescence detection.
It is a kind of to carry out electrochemical luminescence biochemical detection methods using above system, method includes the following steps:
(1) selection of electrochemical luminescence coreagent system and optimization: selection luminescent substance, coreagent；Determine coreaction
Agent concentration, luminescent substance and coreagent consumption proportion；Electrification is added in the luminescent substance for preparing normal concentration in reaction cavity
Luminous coreagent system is learned, determines optimal luminescent substance concentration.
(2) electrode pre-processes: cutting ITO electro-conductive glass, respectively three times using acetone, ethyl alcohol and water ultrasonic cleaning, finally
With a large amount of ultrapure waters, helium drying is used as working electrode；Platinum wire ring three times, is used as to electrode using ultrapure water.
(3) voltage selects: utilizing electrochemical workstation, the selection of electrochemical luminescence driving voltage is realized, at step (2)
The ITO electro-conductive glass managed is connected with the working electrode port of electrochemical workstation, platinum wire ring and electrochemical workstation to electricity
Extreme mouth is connected, and electrochemical workstation selects cyclic voltammetry mode, applies 1.5V~3.3V voltage, record current signal, electricity
Stream signal proves the generation of electrochemical luminescence reaction from scratch.
(4) the biospecific sex modification of ITO electrode: according to the characteristic of detection target substance, in one layer of spy of ITO surface modification
Anisotropic sensitive membrane.
(5) USB-OTG mobile phone end interface is connected to the USB port of mobile phone, the 5V voltage of mobile phone is connect in working electrode
Mouthful and to output 1~4V voltage stable at electrode interface, to excite electrochemical luminescence.
(6) mobile phone camera captures electrochemical luminescence image.
(7) electrochemiluminescence analysis unit electrochemical luminescence image is extracted, is cut, the pretreatment such as gray proces with
And electrochemical luminescence quantitative analysis, it is final to obtain electrochemiluminescence analysis result；The electrochemical luminescence quantitative analysis specifically:
Image after gray proces is subjected to gray analysis, the average gray of pixel in image is calculated, utilizes least square method meter
Calculate grayscaling curve；Image after gray proces is subjected to RGB analysis, the R average value of pixel in image is calculated, using most
Small square law calculates R calibration curve；Image after gray proces is subjected to RGB analysis, the G for calculating pixel in image is average
Value calculates G calibration curve using least square method；Image after gray proces is subjected to RGB analysis, calculates pixel in image
B average value, utilize least square method calculate B calibration curve.
(8) test substance is placed in reaction cavity, repeat step (5) and (6) obtain electrochemical luminescence image, through with step
Suddenly after (7) identical pretreatment, according to four calibration curves, test substance concentration is obtained.
Further, this method can be used for detecting based on BSA electrochemical luminescence；The coreagent system specifically: three
Bipyridyl ruthenium is luminescent substance, and tripropyl amine (TPA) is coreagent, concentration 1M；The volume ratio of tris (bipyridine) ruthenium and tripropyl amine (TPA) is 1:
9；Optimal tris (bipyridine) ruthenium concentration is 10mM；Optimal excitation voltage is 2.5V.Method includes the following steps:
(a) the anti-BSA modification of ITO electrode: clip area is 1cm2Cellulose nitrate film, be dissolved in the methanol of 1mL
In, stirring, after cellulose nitrate film is completely dissolved；The dissolution cellulose nitrate film of 10uL is taken to drip in ITO electro-conductive glass
Conducting surface, and be uniformly paved into the square modified regions that area is 3cm × 3cm intermediate, stand 1 minute and volatilize to methanol
Afterwards, the sensitizing range of cellulose nitrate film modification is formed in the intermediate region ITO；It is added dropwise the 1mM's of 200uL in the region
Anti-BSA, and the square modified regions that area is 3cm × 3cm are uniformly paved into, 30 minutes are stood, it is thin using nitrocellulose
The suction-operated of film modifies upper anti-BSA in ITO electrode surface, can be special in the reaction of subsequent electrochemical luminescence carries out
Reacting with BSA for property, causes the variation of luminous intensity.
(b) standard solution prepares: compound concentration is respectively 10uM, 20uM, 50uM, 100uM, 200uM, 500uM and
The BSA standard solution of 1000uM, solvent are deionized water.
(c) the BSA electrochemical luminescence detection based on mobile phone: the BSA standard that the 10uM of 200uL is added in reaction cavity is molten
Liquid reacts 10 minutes, adds the tripropyl amine (TPA) of the 10mM tris (bipyridine) ruthenium of the optimal concentration of 200uL and the 1M of 1800uL as electricity
Then chemiluminescent coreagent system provides the excitation electricity of optimal 2.5V using the adjustable driving source system of electrochemical luminescence
Pressure, is finally handled and is analyzed to electrochemical luminescence result according to step (7)；It is right on mobile phone using identical method
The BSA standard solution of 20uM, 50uM, 100uM, 200uM, 500uM and 1000uM carry out electrochemical luminescence result treatment and analysis；
Finally establish the corresponding relationship curve between BSA concentration and gray scale and BSA concentration and RGB.
(d) in the standard curve for establishing the BSA solution of concentration to be measured according to step (c), the dense of BSA solution to be measured is obtained
Degree.
Further, this method can be used for the detection of fibrin ferment electrochemical luminescence, the coreagent system specifically: three
Pyridine ruthenium is luminescent substance, and tripropyl amine (TPA) is coreagent, concentration 1M；The volume ratio of tris (bipyridine) ruthenium and tripropyl amine (TPA) is 1:9；
Optimal tris (bipyridine) ruthenium concentration is 10mM；Optimal excitation voltage is 2.5V.Method includes the following steps:
(A) the Thrombin specificity response of ITO electrode is peptide modified: forming nitrocellulose in the intermediate region ITO first
Film modified sensitizing range；Then the 10 of 200uL are added dropwise in the region-5The Thrombin specificity of M responds polypeptide (Cys-Leu-
Val-Pro-Arg-Gly-Ser-Cys the square modified regions that area is 3cm × 3cm), and are uniformly paved into, stand 30 minutes,
Upper Thrombin specificity, which is modified, in ITO electrode surface responds polypeptide, it can be special in the reaction of subsequent electrochemical luminescence carries out
Property with blood coagulation enzyme reaction, cause the variation of luminous intensity.
(B) standard solution prepares: compound concentration is respectively 10-9M, 10-8M, 10-7M, 10-6M and 10-5The fibrin ferment mark of M
Quasi- solution, solvent are deionized water.
(C) the fibrin ferment electrochemical luminescence detection based on mobile phone: to 10 on mobile phone-9M, 10-8M, 10-7M, 10-6M, and
10-5The thrombin standard solution of M carries out electrochemical luminescence result treatment and analysis；Concentration of thrombin and gray scale are finally established, with
And the corresponding relationship curve between concentration of thrombin and RGB.
(D) in the standard curve for establishing the thrombin solution of concentration to be measured according to step (C), it is molten to obtain fibrin ferment to be measured
The concentration of liquid.
The beneficial effects of the present invention are:
1, realize excitation and analyze integrated mobile phone electrochemiluminescence analysis, and by the technology be applied to BSA and
In the electrochemical luminescence detection process of fibrin ferment, have device portable general, stability is good, operates the advantages that convenient, Neng Goushi
Answer the requirement of instant biochemistry detection.
2, the present invention realizes the design of electrochemical luminescence driving source, design benefit using mobile phone USB-OTG interface for the first time
The 5V voltage exported with mobile phone USB-OTG interface, can be with after current foldback circuit, impedance type bleeder circuit and voltage regulator circuit
Stable output 1~4V voltage, for exciting electrochemical luminescence, while the electrochemical luminescence intensity can be caught by mobile phone camera
It obtains.
3, electrochemiluminescence analysis unit is developed, the quantitative analysis for electrochemical luminescence；In the electrochemistry of practical BSA
During luminesceence analysis, the high matching degree of luminous intensity and gray value is established；In the electrochemiluminescence analysis of practical fibrin ferment
In the process, the high matching degree of luminous intensity and gray value and R parameter is established；Demonstrate tris (bipyridine) ruthenium, tripropyl amine (TPA) electrification
It learns in the coreagent system that shines, the R parameter in the sum of the grayscale values RGB parameter of luminescent image has better concentration dependent,
G, B parameter is weaker with electrochemical luminescence intensity corresponding relationship.
4, the electrochemiluminescence analysis based on ITO electro-conductive glass is constructed, due to its good translucency, is reduced luminous
Loss of the signal in transmittance process；Its good electric conductivity provides necessary condition for the generation of electrochemical luminescence simultaneously；This
Its outer bright and clean smooth surface is more advantageous to the modification on surface, reduces the environmental error in test process；Last ITO conduction glass
Glass, can be disposable, and environmental pollution is small, more conducively the exploitation of portable electrochemical luminescence biosensor.
5, by cellulose nitrate film, the good functional membrane of stability is formed in electrode surface, there is repeatability height, robustness
The advantages that good, simplifies modification, realizes nontoxic, green and efficient modification.
6, requirement of this method to use condition is small, easy to operate, stability is good, versatile, easy to spread makes for detection
With.
Detailed description of the invention
Fig. 1 is the electrochemical luminescence detection system frame diagram based on mobile phone USB-OTG interface；
Fig. 2 is USB-OTG interface internal line map；
Fig. 3 (a) is one angled arrangement figure of electrochemical luminescence reaction unit；
Fig. 3 (b) is another angled arrangement figure of electrochemical luminescence reaction unit；
Fig. 4 is based on mobile phone electrochemiluminescence analysis software design flow chart；
Fig. 5 is electrochemical luminescence corresponding current result in the case where applying different voltages；
Fig. 6 is that mobile phone captures and handles the luminous result figure of tris (bipyridine) ruthenium under resulting various concentration；
Fig. 7 is the luminous gray scale result figure of tris (bipyridine) ruthenium under the various concentration that cell phone software is analyzed；
Fig. 8 is the luminous R result figure of tris (bipyridine) ruthenium under the various concentration that cell phone software is analyzed；
Fig. 9 is the luminous G result figure of tris (bipyridine) ruthenium under the various concentration that cell phone software is analyzed；
Figure 10 is the luminous B result figure of tris (bipyridine) ruthenium under the various concentration that cell phone software is analyzed；
Figure 11 is that mobile phone captures and handles the luminous result figure of BSA under resulting various concentration；
Figure 12 is the luminous gray scale result figure of BSA under the various concentration that cell phone software is analyzed；
Figure 13 is the luminous R result figure of BSA under the various concentration that cell phone software is analyzed；
Figure 14 is the luminous G result figure of BSA under the various concentration that cell phone software is analyzed；
Figure 15 is the luminous B result figure of BSA under the various concentration that cell phone software is analyzed；
Figure 16 is that mobile phone captures and handles the luminous result figure of fibrin ferment under resulting various concentration；
Figure 17 is the luminous gray scale result figure of fibrin ferment under the various concentration that cell phone software is analyzed；
Figure 18 is the luminous R result figure of fibrin ferment under the various concentration that cell phone software is analyzed；
Figure 19 is the luminous G result figure of fibrin ferment under the various concentration that cell phone software is analyzed；
Figure 20 is the luminous B result figure of fibrin ferment under the various concentration that cell phone software is analyzed.
In figure, driving source system 1 that electrochemical luminescence is adjustable, electrochemical luminescence is adjustable driving source system 2, camera 3, electrification
Learn luminesceence analysis unit 4, USB-OTG mobile phone end interface 5, connecting line 6, USB-OTG external circuits interface 7, current foldback circuit
8, impedance type bleeder circuit 9, voltage regulator circuit 10, working electrode interface 11, in electrode interface 12, USB-OTG mobile phone end interface
Portion port 13, USB-OTG external circuits interface internal port 14, electrochemical luminescence reactive tank 15, electrochemical luminescence reaction support
Sealed base 16, reaction cavity 17, groove 18.
Specific embodiment
Below in conjunction with drawings and the specific embodiments, the present invention is described in detail, but is not the limitation present invention.
A kind of electrochemical luminescence biochemical detection system based on mobile phone USB-OTG interface provided by the invention, the system packet
Include electrochemical luminescence capture analysis system (1), electrochemical luminescence is adjustable driving source system (2) and electrochemical luminescence reaction unit.
Electrochemical luminescence capture analysis system (1) includes: as shown in Figure 1, having the mobile phone and electrification of camera (3)
It learns luminesceence analysis unit (4)；Camera (3) captures electrochemical luminescence image, and electrochemiluminescence analysis unit (4) sends out electrochemistry
Light image extracts, cuts, gray proces and quantitative analysis, final to obtain electrochemiluminescence analysis result.
The adjustable driving source system (2) of electrochemical luminescence, as shown in Figure 1, include USB-OTG mobile phone end interface (5),
USB-OTG external circuits interface (7), working electrode interface (11) and to electrode interface (12)；Swash providing voltage using mobile phone
When encouraging, USB-OTG mobile phone end interface (5) is connected to the USB port of mobile phone, connects USB-OTG external circuits by connecting line (6)
Interface (7)；It is suitable using the power and ground of USB-OTG external circuits interface (7) as anode and cathode (shown in Fig. 2)
Sequence accesses current foldback circuit (8), impedance type bleeder circuit (9) and voltage regulator circuit (10), by after voltage regulator circuit (10) just
Pole connects working electrode interface (11), and cathode is connected to electrode interface (12).Working electrode interface (11) connects ITO conduction glass
Glass, to electrode interface (12) connection platinum wire ring to electrode.Working electrode interface (11) and to can be at electrode interface (12) at this time
Stable output 1~4V voltage is for exciting electrochemical luminescence.
The electrochemical luminescence reaction unit includes that electrochemical luminescence reactive tank (15) and electrochemical luminescence reaction support are close
Back cover seat (16) (shown in Fig. 3), the electrochemical luminescence reactive tank (15) have in lead to truncated cone-shaped reaction cavity (17)；Electrification
The cross section for learning luminescence-producing reaction support sealed base (16) is concave structure, places ITO electro-conductive glass in groove (18), ITO is led
Sealing ring is placed on the top of electric glass, realizes the sealed connection with electrochemical luminescence reactive tank (15)；It is put in reaction cavity (17)
Platinum wire ring is set to electrode, and be added electrochemical luminescence coreagent system and corresponding detection substance, realize stable electricity
Chemiluminescence detection.
Preferably, platinum wire ring is 2.5cm to the diameter of electrode, and reaction cavity (17) bottom diameter of a circle is 2cm, platinum wire ring
Vertical range to electrode and ITO electro-conductive glass is 4mm~8mm.
A kind of electrochemical luminescence biochemical detection system based on mobile phone USB-OTG interface, method includes the following steps:
(1) selection of electrochemical luminescence coreagent system and optimization: selection luminescent substance, coreagent；Determine coreaction
Agent concentration, luminescent substance and coreagent consumption proportion；The luminescent substance for preparing normal concentration is added in reaction cavity (17)
The coreagent system of electrochemical luminescence determines optimal luminescent substance concentration.
(2) electrode pre-processes: the ITO electro-conductive glass of 10cm × 10cm is cut into 3cm × 5cm using glass cutter, point
Not Shi Yong acetone, ethyl alcohol and water ultrasonic cleaning three times, finally use a large amount of ultrapure waters, helium drying, as working electrode；Directly
The platinum wire ring that diameter is 2.5cm three times, is used as to electrode using ultrapure water.
(3) voltage selects: electrochemical workstation is utilized, realizes the selection of electrochemical luminescence driving voltage, it will be in step (2)
It handles ITO electro-conductive glass well to be connected by conducting wire with the working electrode port of electrochemical workstation, platinum wire ring passes through conducting wire and electricity
Chem workstation is connected to electrode ports.As shown in figure 5, electrochemical workstation selects cyclic voltammetry mode, apply 1.5V
~3.3V voltage, record current signal, current signal prove the generation of electrochemical luminescence reaction from scratch.
(4) the biospecific sex modification of ITO electrode: according to the characteristic of detection target substance, in one layer of spy of ITO surface modification
Anisotropic sensitive membrane.
(5) USB-OTG mobile phone end interface (5) is connected to the USB port of mobile phone, by the 5V voltage of mobile phone in working electrode
Interface (11) and to output 1~4V voltage stable at electrode interface (12), to excite electrochemical luminescence.
(6) mobile phone camera (3) captures electrochemical luminescence image.
(7) electrochemiluminescence analysis unit (4) electrochemical luminescence image is extracted, is cut, the pre- place such as gray proces
Reason and electrochemical luminescence quantitative analysis, it is final to obtain electrochemiluminescence analysis result (shown in Fig. 4)；The electrochemical luminescence is fixed
Amount analysis specifically: the image after gray proces is subjected to gray analysis, calculates the average gray of pixel in image, is utilized
Least square method calculates grayscaling curve；Image after gray proces is subjected to RGB analysis, calculates the R of pixel in image
Average value calculates R calibration curve using least square method；Image after gray proces is subjected to RGB analysis, calculates picture in image
The G average value of vegetarian refreshments calculates G calibration curve using least square method；Image after gray proces is subjected to RGB analysis, is calculated
The B average value of pixel in image calculates B calibration curve using least square method.
(8) test substance is placed in reaction cavity (17), repeats step (5) and (6) and obtain electrochemical luminescence image, passes through
With step (7) after identical pretreatment, according to four calibration curves, test substance concentration is obtained.
It is a kind of to carry out electrochemical luminescence biochemical detection methods, in the step (1), coreagent system using above system
Specifically: tris (bipyridine) ruthenium is luminescent substance, and tripropyl amine (TPA) is coreagent, concentration 1M；Tris (bipyridine) ruthenium and tripropyl amine (TPA)
Volume ratio is 1:9；Optimal excitation voltage is 2.5V in the step (3).Method includes the following steps:
(a) standard solution prepares: compound concentration is respectively 0.1mM, 1mM, 2mM, 3mM, 5mM, 7mM, 10mM and 20mM
Tris (bipyridine) ruthenium standard solution, solvent are deionized water.
(b) various concentration tris (bipyridine) ruthenium electrochemical luminescence detects: the 0.1mM of 200uL being added in reaction cavity (17)
Coreagent system of the tripropyl amine (TPA) of the 1M of tris (bipyridine) ruthenium and 1800uL as electrochemical luminescence, then utilizes based on mobile phone
Driving source system that electrochemical luminescence is adjustable (2) provides the driving voltage of the optimal 2.5V selected by step (3), finally according to step
(7) electrochemical luminescence result is handled and is analyzed；Using identical method, to 1mM, 2mM, 3mM, 5mM on mobile phone,
The tris (bipyridine) ruthenium standard solution of 7mM, 10mM and 20mM carry out electrochemical luminescence result treatment and analysis；Finally establish three pyrroles
Corresponding relationship curve (Fig. 8,9,10 between pyridine ruthenium concentration and gray scale (Fig. 6,7 shown in) and tris (bipyridine) ruthenium concentration and RGB
It is shown).
(c) according to step (2), by the analysis to luminescent image, corresponding concentration when luminous intensity maximum is found, with true
Fixed optimal luminescent substance concentration.
Embodiment 1: the BSA electrochemical luminescence detection based on mobile phone, specifically includes the following steps:
(A) the anti-BSA modification of ITO electrode: clip area is 1cm2Cellulose nitrate film, be dissolved in the methanol of 1mL
In, stirring, after cellulose nitrate film is completely dissolved；The dissolution cellulose nitrate film of 10uL is taken to drip in ITO electro-conductive glass
Conducting surface, and be uniformly paved into the square modified regions that area is 3cm × 3cm intermediate, stand 1 minute and volatilize to methanol
Afterwards, the sensitizing range of cellulose nitrate film modification is formed in the intermediate region ITO；It is added dropwise the 1mM's of 200uL in the region
Anti-BSA, and the square modified regions that area is 3cm × 3cm are uniformly paved into, 30 minutes are stood, it is thin using nitrocellulose
The suction-operated of film modifies upper anti-BSA in ITO electrode surface, can be special in the reaction of subsequent electrochemical luminescence carries out
Reacting with BSA for property, causes the variation of luminous intensity.
(B) standard solution prepares: compound concentration is respectively 10uM, 20uM, 50uM, 100uM, 200uM, 500uM and
The BSA standard solution of 1000uM, solvent are deionized water.
(C) the BSA mark of the 10uM of 200uL the BSA electrochemical luminescence detection based on mobile phone: is added in reaction cavity (17)
Quasi- solution reacts 10 minutes, add the 10mM tris (bipyridine) ruthenium of the optimal concentration of 200uL selected by step (3) with
Coreagent system of the tripropyl amine (TPA) of the 1M of 1800uL as electrochemical luminescence, then utilizes the electrochemical luminescence based on mobile phone can
Driving source system (2) are adjusted to provide the driving voltage of the optimal 2.5V selected by step (3), finally according to step (7) to electrochemistry
Luminous result is handled and is analyzed；Using identical method, to 20uM, 50uM, 100uM on mobile phone, 200uM, 500uM and
The BSA standard solution of 1000uM carries out electrochemical luminescence result treatment and analysis；Finally establish BSA concentration and gray scale (Figure 11,12
It is shown) and BSA concentration and RGB between corresponding relationship curve (shown in Figure 13,14,15).
(D) in the standard curve for establishing the BSA solution of concentration to be measured according to step (C), the dense of BSA solution to be measured is obtained
Degree.
Embodiment 2: the fibrin ferment electrochemical luminescence detection based on mobile phone, specifically includes the following steps:
(A) the Thrombin specificity response of ITO electrode is peptide modified: clip area is 1cm2Cellulose nitrate film,
It is dissolved in the methanol of 1mL, stirs, after cellulose nitrate film is completely dissolved；The dissolution cellulose nitrate film of 10uL is taken to drip
The square modified regions that area is 3cm × 3cm are uniformly paved into the conducting surface of ITO electro-conductive glass, and intermediate, stand 1 point
Clock forms the sensitizing range of cellulose nitrate film modification after methanol volatilization, in the intermediate region ITO；It is added dropwise in the region
The 10 of 200uL-5The Thrombin specificity of M responds polypeptide (Cys-Leu-Val-Pro-Arg-Gly-Ser-Cys), and is uniformly paved into
Area is the square modified regions of 3cm × 3cm, 30 minutes is stood, using the suction-operated of cellulose nitrate film, in ITO
On electrode face finish Thrombin specificity respond polypeptide, subsequent electrochemical luminescence reaction carry out in can specificity with
Blood coagulation enzyme reaction causes the variation of luminous intensity.
(B) standard solution prepares: compound concentration is respectively 10-9M, 10-8M, 10-7M, 10-6M and 10-5The fibrin ferment mark of M
Quasi- solution, solvent are deionized water.
(C) the 10 of 200uL the fibrin ferment electrochemical luminescence detection based on mobile phone: is added in reaction cavity (17)-9M's is solidifying
Hemase standard solution reacts 10 minutes, adds the 10mM tris (bipyridine) ruthenium for the optimal concentration of 200uL selected by step (3)
Coreagent system with the tripropyl amine (TPA) of the 1M of 1800uL as electrochemical luminescence, then utilizes the electrochemical luminescence based on mobile phone
Adjustable driving source system (2) provides the driving voltage of the optimal 2.5V selected by step (3), finally according to step (7) to electrification
The result that shines is learned to be handled and analyzed；Using identical method, to 10 on mobile phone-8M, 10-7M, 10-6M and 10-5M's is solidifying
Hemase standard solution carries out electrochemical luminescence result treatment and analysis；Finally establish concentration of thrombin and gray scale (Figure 16,17 institutes
Show) and concentration of thrombin and RGB between corresponding relationship curve (shown in Figure 18,19,20).
(D) in the standard curve for establishing the thrombin solution of concentration to be measured according to step (C), it is molten to obtain fibrin ferment to be measured
The concentration of liquid.
Claims (4)
1. a kind of electrochemical luminescence biochemical detection system based on mobile phone USB-OTG interface, which is characterized in that the system includes electricity
Chemiluminescence captures analysis system (1), electrochemical luminescence is adjustable driving source system (2) and electrochemical luminescence reaction unit；
Electrochemical luminescence capture analysis system (1) includes: mobile phone and electrochemiluminescence analysis unit with camera (3)
(4)；Camera (3) captures electrochemical luminescence image, and electrochemiluminescence analysis unit (4) mentions electrochemical luminescence image
It takes, cut, gray proces and quantitative analysis, it is final to obtain electrochemiluminescence analysis result；
The adjustable driving source system (2) of electrochemical luminescence includes: USB-OTG mobile phone end interface (5), USB-OTG external circuits
Interface (7), working electrode interface (11) and to electrode interface (12)；When providing voltage drive using mobile phone, USB-OTG mobile phone
End interface (5) is connected to the USB port of mobile phone, connects USB-OTG external circuits interface (7) by connecting line (6)；By USB-
For the power and ground of OTG external circuits interface (7) respectively as anode and cathode, sequence accesses current foldback circuit (8), resistance
Anti- formula bleeder circuit (9) and voltage regulator circuit (10) are born by anode connection working electrode interface (11) after voltage regulator circuit (10)
Pole is connected to electrode interface (12)；Working electrode interface (11) connects ITO electro-conductive glass, connects platinum wire ring to electrode interface (12)
To electrode；At this time working electrode interface (11) and to can be stable at electrode interface (12) output 1~4V voltage for exciting
Electrochemical luminescence；
The electrochemical luminescence reaction unit includes electrochemical luminescence reactive tank (15) and electrochemical luminescence reaction support sealing bottom
Seat (16), the electrochemical luminescence reactive tank (15) have in lead to truncated cone-shaped reaction cavity (17)；Electrochemical luminescence reaction branch
The cross section for supportting sealed base (16) is concave structure, places ITO electro-conductive glass, the top of ITO electro-conductive glass in groove (18)
Sealing ring is placed, realizes the sealed connection with electrochemical luminescence reactive tank (15)；Platinum wire ring is placed in reaction cavity (17) to electricity
Pole, and be added electrochemical luminescence coreagent system and corresponding detection substance, realize stable electrochemical luminescence detection.
2. a kind of carry out electrochemical luminescence biochemical detection methods using system described in claim 1, which is characterized in that this method packet
Include following steps:
(1) selection of electrochemical luminescence coreagent system and optimization: selection luminescent substance, coreagent；Determine that coreagent is dense
Degree, luminescent substance and coreagent consumption proportion；Electrification is added in the luminescent substance for preparing normal concentration in reaction cavity (17)
Luminous coreagent system is learned, determines optimal luminescent substance concentration；
(2) electrode pre-processes: ITO electro-conductive glass is cut, respectively three times using acetone, ethyl alcohol and water ultrasonic cleaning, finally with big
Ultrapure water is measured, helium drying is used as working electrode；Platinum wire ring three times, is used as to electrode using ultrapure water；
(3) voltage selects: utilizing electrochemical workstation, realizes the selection of electrochemical luminescence driving voltage, step (2) is handled well
ITO electro-conductive glass be connected with the working electrode port of electrochemical workstation, platinum wire ring and electrochemical workstation to electrode tip
Mouth is connected, and electrochemical workstation selects cyclic voltammetry mode, applies 1.5V~3.3V voltage, record current signal, electric current letter
The generation of electrochemical luminescence reaction is proved number from scratch；
(4) the biospecific sex modification of ITO electrode: according to the characteristic of detection target substance, in one layer of specificity of ITO surface modification
Sensitive membrane；
(5) USB-OTG mobile phone end interface (5) is connected to the USB port of mobile phone, by the 5V voltage of mobile phone in working electrode interface
(11) and to output 1~4V voltage stable at electrode interface (12), to excite electrochemical luminescence；
(6) mobile phone camera (3) captures electrochemical luminescence image；
(7) electrochemiluminescence analysis unit (4) electrochemical luminescence image is extracted, is cut, the pretreatment such as gray proces with
And electrochemical luminescence quantitative analysis, it is final to obtain electrochemiluminescence analysis result；The electrochemical luminescence quantitative analysis specifically:
Image after gray proces is subjected to gray analysis, the average gray of pixel in image is calculated, utilizes least square method meter
Calculate grayscaling curve；Image after gray proces is subjected to RGB analysis, the R average value of pixel in image is calculated, using most
Small square law calculates R calibration curve；Image after gray proces is subjected to RGB analysis, the G for calculating pixel in image is average
Value calculates G calibration curve using least square method；Image after gray proces is subjected to RGB analysis, calculates pixel in image
B average value, utilize least square method calculate B calibration curve；
(8) test substance is placed in reaction cavity (17), repeats step (5) and (6) and obtain electrochemical luminescence image, through with step
Suddenly after (7) identical pretreatment, according to four calibration curves, test substance concentration is obtained.
3. electrochemical luminescence biochemical detection methods according to claim 2, which is characterized in that for being based on BSA electrochemistry
Shine detection；The coreagent system specifically: tris (bipyridine) ruthenium is luminescent substance, and tripropyl amine (TPA) is coreagent, concentration
For 1M；The volume ratio of tris (bipyridine) ruthenium and tripropyl amine (TPA) is 1:9；Optimal tris (bipyridine) ruthenium concentration is 10mM；Optimal excitation voltage
For 2.5V；Method includes the following steps:
(a) the anti-BSA modification of ITO electrode: clip area is 1cm2Cellulose nitrate film, be dissolved in the methanol of 1mL, stir
It mixes, after cellulose nitrate film is completely dissolved；The dissolution cellulose nitrate film of 10uL is taken to drip in the conduction of ITO electro-conductive glass
Face, and the square modified regions that area is 3cm × 3cm are uniformly paved into intermediate, 1 minute is stood after methanol volatilization, in ITO
Intermediate region forms the sensitizing range of cellulose nitrate film modification；The anti-BSA of the 1mM of 200uL is added dropwise in the region, and
The square modified regions that area is 3cm × 3cm are uniformly paved into, stand 30 minutes, are made using the absorption of cellulose nitrate film
With, modify upper anti-BSA in ITO electrode surface, in the reaction of subsequent electrochemical luminescence carries out can specificity and BSA
Reaction, causes the variation of luminous intensity；
(b) standard solution prepares: compound concentration is respectively 10uM, 20uM, 50uM, 100uM, 200uM, 500uM and 1000uM
BSA standard solution, solvent are deionized water；
(c) the BSA electrochemical luminescence detection based on mobile phone: the BSA standard that the 10uM of 200uL is added in reaction cavity (17) is molten
Liquid reacts 10 minutes, adds the tripropyl amine (TPA) of the 10mM tris (bipyridine) ruthenium of the optimal concentration of 200uL and the 1M of 1800uL as electricity
Then chemiluminescent coreagent system provides the excitation of optimal 2.5V using the adjustable driving source system (2) of electrochemical luminescence
Voltage is finally handled and is analyzed to electrochemical luminescence result according to step (7)；It is right on mobile phone using identical method
The BSA standard solution of 20uM, 50uM, 100uM, 200uM, 500uM and 1000uM carry out electrochemical luminescence result treatment and analysis；
Finally establish the corresponding relationship curve between BSA concentration and gray scale and BSA concentration and RGB；
(d) in the standard curve for establishing the BSA solution of concentration to be measured according to step (c), the concentration of BSA solution to be measured is obtained.
4. electrochemical luminescence biochemical detection methods according to claim 2, which is characterized in that sent out for fibrin ferment electrochemistry
Light detection, the coreagent system specifically: tris (bipyridine) ruthenium is luminescent substance, and tripropyl amine (TPA) is coreagent, and concentration is
1M；The volume ratio of tris (bipyridine) ruthenium and tripropyl amine (TPA) is 1:9；Optimal tris (bipyridine) ruthenium concentration is 10mM；Optimal excitation voltage is
2.5V；Method includes the following steps:
(A) the Thrombin specificity response of ITO electrode is peptide modified: forming cellulose nitrate film in the intermediate region ITO first
The sensitizing range of modification；Then the 10 of 200uL are added dropwise in the region-5The Thrombin specificity of M responds peptide C ys-Leu-Val-
Pro-Arg-Gly-Ser-Cys, and the square modified regions that area is 3cm × 3cm are uniformly paved into, 30 minutes are stood, in ITO
On electrode face finish Thrombin specificity respond polypeptide, subsequent electrochemical luminescence reaction carry out in can specificity with
Blood coagulation enzyme reaction causes the variation of luminous intensity；
(B) standard solution prepares: compound concentration is respectively 10-9M, 10-8M, 10-7M, 10-6M and 10-5The thrombin standard of M is molten
Liquid, solvent are deionized water；
(C) the fibrin ferment electrochemical luminescence detection based on mobile phone: to 10 on mobile phone-9M, 10-8M, 10-7M, 10-6M and 10-5M's
Thrombin standard solution carries out electrochemical luminescence result treatment and analysis；Finally establish concentration of thrombin and gray scale and blood coagulation
Corresponding relationship curve between enzyme concentration and RGB；
(D) in the standard curve for establishing the thrombin solution of concentration to be measured according to step (C), thrombin solution to be measured is obtained
Concentration.
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