CN102788831B - Microfluidic chip electrophoretic-electrochemical detecting device with adjustable pH after separation and use thereof - Google Patents
Microfluidic chip electrophoretic-electrochemical detecting device with adjustable pH after separation and use thereof Download PDFInfo
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- CN102788831B CN102788831B CN201210286931.3A CN201210286931A CN102788831B CN 102788831 B CN102788831 B CN 102788831B CN 201210286931 A CN201210286931 A CN 201210286931A CN 102788831 B CN102788831 B CN 102788831B
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Abstract
本发明公开了一种分离后可调pH的微流控芯片电泳-电化学检测装置及其在氨基糖苷类抗生素分析中的应用。该分析装置，主要由双T型进样通道、分离通道、两条辅助通道、W型通道和电极安置通道构成。通过在微流控芯片中分离通道末端增加两条辅助通道和W型通道，将碱性溶液经辅助通道在电泳分离之后加入，再经过W型通道将来自分离通道和辅助通道的溶液混合均匀，提高工作电极测定区域溶液的pH值，使其达到检测糖类物质所需的强碱性条件，同时又不影响在酸性分离缓冲液条件下的电泳分离。本发明还借助金属纳米材料的较大比表面积和其特殊的催化性能，采用电沉积的方法将过渡金属纳米材料修饰在电极表面，获得高性能工作电极，用安培法测定氨基糖苷类抗生素。
The invention discloses a microfluidic chip electrophoresis-electrochemical detection device with adjustable pH after separation and its application in the analysis of aminoglycoside antibiotics. The analysis device is mainly composed of a double T-shaped sampling channel, a separation channel, two auxiliary channels, a W-shaped channel and an electrode placement channel. By adding two auxiliary channels and a W-shaped channel at the end of the separation channel in the microfluidic chip, the alkaline solution is added through the auxiliary channel after electrophoresis separation, and then the solutions from the separation channel and the auxiliary channel are mixed evenly through the W-shaped channel, Increase the pH value of the solution in the measurement area of the working electrode to make it reach the strong alkaline conditions required for the detection of carbohydrates without affecting the electrophoretic separation under the condition of the acidic separation buffer. The present invention also uses the large specific surface area and special catalytic performance of metal nanomaterials to modify the transition metal nanomaterials on the electrode surface by electrodeposition to obtain high-performance working electrodes, and uses the amperometric method to determine aminoglycoside antibiotics.
Description
Technical field
The present invention relates to a kind of separate micro-fluid control chip electrophoretic-electrochemical detection device of rear adjustable pH and the application in aminoglycoside antibiotics is analyzed thereof, belong to the Pharmaceutical Analysis detection technique field of micro-total analysis system.
Background technology
Aminoglycoside antibiotics is the glycoside microbiotic that a class is formed by connecting by oxo bridge by plural amino sugar and aminocyclitol, as miramycin, streptomysin, amikacin, paromomycin, neomycin etc., the infection that is mainly used in resisting Gram-negative and positive bacteria.Aminoglycoside antibiotics is widely used in the various serious bacterial infections for the treatment of clinically, but such medicine shows stronger ototoxicity, renal toxicity, the spinoffs such as N&M blocking-up and allergic reaction, seriously can cause the infringement of hearing and renal function, even threat to life, so, when use, need closely to monitor its concentration in vivo.In addition, in animal farming industry, aminoglycoside antibiotics is also the antibacterials that a class is generally used.Due in animal body residual of these medicines, directly affect the quality and safety of animal derived food.Therefore, clinically with animal-breeding and food production in, exploitation is fast, simple, sensitive and cheaply analytical approach for monitor aminoglycoside antibiotics in vivo with the content of food, have very important significance for guarantee people's health.
At present, the analytical approach of aminoglycoside antibiotics mainly contains microbiological method, enzyme immunoassay, thin-layered chromatography, high performance liquid chromatography and Capillary Electrophoresis etc.Although they are widely used and develop in aminoglycoside antibiotics analysis, all there is limitation in various degree.Wherein, microorganism detection method time and effort consuming, can only provide the rough result of total aminoglycoside antibiotics, and accuracy and reappearance are poor; Enzyme immunoassay is subject to the restriction of enzyme and the condition of reagent own, generally cannot measure multiple aminoglycoside antibiotics simultaneously; High effective liquid chromatography for measuring aminoglycoside antibiotics is comparatively general, but, aminoglycoside antibiotics lacks ultraviolet chromophore and fluorophor, cannot directly adopt ultraviolet method or fluorescence method to detect it, need to take before post or post column derivatization reaction, introduce chromophore or fluorophor, again it is detected, and derivative reaction generally need to be longer time (0.5-1.5h), and experimental procedure is loaded down with trivial details, analysis cost is higher; Mass spectroscopy and chromatograph joint usedly can carry out good qualitative and quantitative analysis to aminoglycoside antibiotics, but instrument costliness, operating cost are high; Capillary Electrophoresis has that separation efficiency is high, analysis speed is fast, sample and the advantage such as reagent consumption is few, is also widely used in the analysis of aminoglycoside antibiotics, wherein majority be adopt separate before derivatization, then pass through laser-Induced Fluorescence Detection.But, because kapillary and galvanochemistry and mass detector are subject to the restriction of interface problem, therefore, about the application report of this respect less at present.
Micro flow control chip capillary electrophoresis is as a kind of capillary electrophoresis technique of microminiaturization; not only include the basic function of Capillary Electrophoresis; and its size is less, reagent consumption still less, analysis speed faster and easily realize hyperchannel parallel analysis and can and other operating unit integration etc.; be particularly suitable for microanalysis and the functional study of biological sample; simultaneously also in life science; new drug development; medical diagnosis on disease; environmental protection; Food Inspection; criminal investigation qualification, the fields such as space probation show huge application prospect.In the detection method of many and micro-fluid control chip electrophoretic coupling, electrochemical detection method because of its have highly sensitive, selectivity good, equipment is simple, be easy to the features such as integrated and microminiaturized, in micro-fluid control chip electrophoretic research, play an important role, become a kind of detection method extensively adopting.People utilize multiple transition metal nanometer material modified electrode, by the larger specific surface area of nano material and special physico-chemical character, promote oxidation and the electronics transmission of sugar at electrode surface, improve sensitivity and the stability of electrode, for measuring the materials such as sugar and amino acid.
For the neutral glucide of majority, only in the strong basicity solution of (pH value is greater than 12), sugar alcohol base (hydroxyl) just can be dissociated into negative ion, and it is carried out to electrophoretic separation.But, use larger separation voltage under highly basic condition time, in microchannel, produce larger electric current and Joule heat, easily cause the generation of bubble, thereby destroy electrophoretic separation.Because containing multiple amino, aminoglycoside antibiotics there is certain alkalescence, easily positively charged under acid and neutrallty condition, therefore, be highly suitable for and in acidic buffer, carry out electrophoretic separation, and by add cationic surfactant in dissociating buffer, as cetyl trimethyl ammonia bromide (CTAB), can further improve separating effect.Because Electrochemical Detection is sugared and aminoglycoside antibiotics all needs highly basic condition, its reaction mechanism is that under highly basic condition, high valence state copper (trivalent) oxide is participated in sugared electrochemical oxidation reactions process, promotes electronics transmission, reduces reaction activity.In order to adopt better microchip electrophoretic separation and Electrochemical Detection aminoglycoside antibiotics and glucide, we design a kind of new microfluidic chip device, can add strong base solution at the end of split tunnel, improve the pH value in determination of electrode region, both met the needed alkali condition of aminoglycoside antibiotics Electrochemical Detection, do not affect again its electrophoretic separation under acid condition, this method contribute to aminoglycoside antibiotics clinically rationally use and monitor its residual in food.
Summary of the invention
The object of this invention is to provide a kind of separate micro-fluid control chip electrophoretic-electrochemical detection device of rear adjustable pH and the application in aminoglycoside antibiotics is analyzed thereof, micro-fluid control chip electrophoretic-electrochemical detection device of adjustable pH after separation provided by the invention, can make working electrode measure region and reach the required strong alkaline condition of detection glucide, and do not affect electrophoretic separation, realize under non-strong basicity background buffer solution condition, micro-fluid control chip electrophoretic separates and electrochemical gaging aminoglycoside antibiotics, and by the chemical modification to electrode, obtain the good working electrode of performance, set up fast with this, sensitive, reliably, the method of aminoglycoside antibiotics in micro-fluid control chip electrophoretic separation cheaply and amperometric detection biological sample.
A kind of micro-fluid control chip electrophoretic-electrochemical detection device that separates rear adjustable pH provided by the present invention, it comprises micro-fluidic chip body; Described micro-fluidic chip body comprises split tunnel, and one end of described split tunnel is connected with sample cell, buffer solution pond and sample waste liquid pool respectively, and the other end is connected with accessory channel, W type passage, electrode arrangement passage and damping fluid waste liquid pool successively; In described electrode arrangement passage, be provided with working electrode; The other end of described accessory channel is connected with alkaline solution liquid storage tank; Described accessory channel is located near described W type passage place.
After above-mentioned separation in micro-fluid control chip electrophoretic-electrochemical detection device of adjustable pH, the solution that can utilize the static pressure of the poor generation of liquid level in described alkaline solution liquid storage tank and other liquid storage tank to promote in described alkaline solution liquid storage tank enters the region crossing with described split tunnel along accessory channel, after realizing separation, add alkalescence or other derivative reagent at split tunnel end, reach the needed condition of Electrochemical Detection (or other detection modes).
After above-mentioned separation in micro-fluid control chip electrophoretic-electrochemical detection device of adjustable pH, the described W type passage being connected between the connectivity part of described accessory channel and described split tunnel and described electrode arrangement passage, can make can mix from the solution in split tunnel and accessory channel, overcome simple laminar flow diffusion couple solution in straight type passage and mixed inhomogeneous problem, thereby effectively realized the electrophoretic separation of micro-fluidic chip under non-strong basicity background buffer solution condition and the Electrochemical Detection aminoglycoside antibiotics under strong alkaline condition.
After above-mentioned separation in micro-fluid control chip electrophoretic-electrochemical detection device of adjustable pH, described 2 accessory channels are specifically arranged symmetrically centered by described split tunnel, when use, utilize the fluid pressure promotion strong base solution of the poor generation of liquid level in the alkaline solution liquid storage tank being connected with two accessory channels to enter the region crossing with split tunnel, realize after separation (split tunnel end) and add alkaline solution, make working electrode measure region and reach the required strong alkaline condition of detection glucide.
After above-mentioned separation, in micro-fluid control chip electrophoretic-electrochemical detection device of adjustable pH, between described sample cell, buffer solution pond and sample waste liquid pool and described split tunnel, form double-T shaped passage.
The height of described accessory channel and width all can be 45～55 μ m, and length can be 0.2～1.0cm; The width of described W type passage and highly all can be 45～55 μ m, length can be 1.5～2.5mm.
After above-mentioned separation, in micro-fluid control chip electrophoretic-electrochemical detection device of adjustable pH, along in described split tunnel direction, the angle between described accessory channel and described split tunnel can be 0 °～180 °, as 45 °, but is not 0 ° and 180 °.
After above-mentioned separation, in micro-fluid control chip electrophoretic-electrochemical detection device of adjustable pH, described working electrode is the Pt silk that surface is coated with Cu-Cr-Sn Nanoalloy layer; The diameter of described Pt silk can be 20～30 μ m, and the average thickness of described Cu-Cr-Sn Nanoalloy layer can be 1.5～3.0 μ m.
After above-mentioned separation, in micro-fluid control chip electrophoretic-electrochemical detection device of adjustable pH, described working electrode is according to comprising following condition preparation: the electroplate liquid that the Pt silk cleaning is placed in to polycomponent slaine is electroplated; Electroplate liquid used is by CuSO
4, Cr
2(SO
4)
3, SnCl
2, H
2sO
4respectively according to 50mM, 0.2mM, 0.2mM and 10mM ratio composition; The time of described plating can be 120～150s; Described plating current potential can be-0.5～-0.3V.
The concrete application of micro-fluid control chip electrophoretic-electrochemical detection device that the present invention also provides adjustable pH after above-mentioned separation in aminoglycoside antibiotics is analyzed, as the mensuration in milk sample such as miramycin, streptomysin, amikacin, paromomycin and neomycin.Analyze and measure in the method for aminoglycoside antibiotics in the present invention, specifically can select pH is that 5.0 sodium acetate solution is as buffer solution; The concentration of sodium acetate buffer solution specifically can be 6.0mM; In sodium acetate buffer solution, the concentration of CTAB specifically can be 0.6mM; Specifically can be-1200V of separation voltage; Detect specifically can be+0.65V(vs of current potential Ag/AgCl) as detecting current potential.Under above-mentioned preferably testing conditions, above-mentioned five kinds of antibiotic minimum detectable levels within the scope of 2.1～4.6 μ M, the relative standard deviation of peak current and transit time all respectively lower than 7.9% and 2.0%(n=30).
Tool of the present invention has the following advantages:
(1) the present invention can add alkaline solution after split tunnel end is realized separation, in the electrophoretic separation not affecting under acid condition, change the potential of hydrogen of determination of electrode region solution, the desired alkali condition of electrochemical reaction that makes it reach glucide.
(2) working electrode in the present invention is to utilize the method for electro-deposition that metal nano alloy (Cu-Cr-Sn) is deposited on to the good metal of electric conductivity (platinum) silk surface, stability and the sensitivity of working electrode are improved, obtain the modified electrode of better performance, realized the target of micro-fluid control chip electrophoretic separation-electrochemical detection aminoglycoside antibiotics.
(3) the present invention, for rationally using clinically aminoglycoside antibiotics and effectively monitoring this type of medicament residue in animal derived food, provides a kind of quick, sensitive, pharmaceutical analysis apparatus and method reliably.
Brief description of the drawings
Fig. 1 is polymerization dimethylsilane (PDMS) micro-fluid control chip electrophoretic-electrochemical detection device of adjustable pH after separation provided by the invention; In figure, each mark is as follows: 1 sample cell, 2 buffer solution ponds, 3 sample waste liquid pools, 4,5 alkaline solution liquid storage tanks, 6 damping fluid waste liquid pools, 7 double-T shaped passages, 8 split tunnels, 9 accessory channels, 10W type passage, 11 working electrodes, 12 electrode arrangement passages, 13 detection zones.
Fig. 2 is the scanning electron microscope diagram sheet of electrode, and wherein Fig. 2 (A), Fig. 2 (B), Fig. 2 (C) and Fig. 2 (D) are respectively the scanning electron microscope diagram sheet (SEM) of Cu/Pt, Cu-Cr/Pt, Cu-Sn/Pt and Cu-Cr-Sn/Pt modified electrode.
Fig. 3 is that electrode sweep speed in the 50mM NaOH solution that contains 50 μ M amikacins is 100mV s
-1cyclic voltammetry curve, wherein curve a, curve b, curve c and curve d are respectively the cyclic voltammetry curve of Pt, Cu/Pt, Cu-Cr/Pt and Cu-Cr-Sn/Pt electrode; Interior illustration is Cu-Cr-Sn/Pt modified electrode cyclic voltammogram in 50mM NaOH solution blank and that contain 50 μ M amikacins.
Fig. 4 is the impact on five kinds of aminoglycoside antibiotics separation determinations of the buffer solution of different pH.
Fig. 5 is the impact on five kinds of aminoglycoside antibiotics separation determinations of the sodium acetate buffer solution of variable concentrations.
Fig. 6 is the impact on five kinds of aminoglycoside antibiotics separation determinations of the buffer solution of different CTAB concentration.
Fig. 7 is the impacts of different separation voltages on five kinds of aminoglycoside antibiotics separation determinations.
Fig. 8 is the different impacts that current potential is measured five kinds of aminoglycoside antibioticss that detect.
Embodiment
The experimental technique using in following embodiment if no special instructions, is conventional method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
Micro-fluid control chip electrophoretic-electrochemical detection device of adjustable pH after embodiment 1, separation
As shown in Figure 1, after separation provided by the invention, micro-fluid control chip electrophoretic-electrochemical detection device of adjustable pH comprises a micro-fluidic chip body, this micro-fluidic chip body comprises a split tunnel 8, one end of this split tunnel 8 is connected with sample cell 1, buffer solution pond 2 and sample waste liquid pool 3 respectively, and forms double-T shaped passage 7; The other end of this split tunnel 8 is connected with W type path 10, electrode arrangement passage 12 and damping fluid waste liquid pool 6 successively; The width of this W type path 10 and be highly 50 μ m, length is 2mm; In electrode arrangement passage 12, be provided with working electrode 11, this working electrode 11 is the surperficial Pt silk that is coated with Cu-Cr-Sn Nanoalloy layer, and the diameter of this Pt silk is 25 μ m, and the average thickness of Cu-Cr-Sn Nanoalloy layer is 2.5 μ m; Also be connected with 2 accessory channels 9 with the connectivity part of W type path 10 at split tunnel 8, the height of 2 accessory channels 9 and width are 50 μ m, length is 0.5cm, and be arranged symmetrically centered by split tunnel 8, in buffer solution pond 2, to the direction of electrode arrangement passage 12, the angle between 2 accessory channels 9 and split tunnel 8 is 45 °; The other end of 2 accessory channels 9 is connected with alkaline solution liquid storage tank 4 and 5 respectively.
Device provided by the invention can be made by following method:
(1) structure (shown in Fig. 1) of utilizing computer aided design software (CAD) design and drawing micro-fluidic chip passage, with high precision printing device making photomask.
(2) prepare silicon chip masterplate
Get the silicon chip of a diameter 100mm left and right, use the concentrated sulphuric acid (98%): the Piranha solution of hydrogen peroxide (30%)=3:1 cleans, dry.On clean silicon chip, be coated with photoresist (photoresist) SU-82035, form the thick rete of approximately 50 μ m.Then by being close on the silicon chip that scribbles photoresist with the photomask (black and white film) that designs chip pattern (passage is light transmission part), through high-intensity ultraviolet light exposure-processed; 1-Methoxy-2-propyl acetate for masterplate (propylene glycol methyl ether acetate) after exposure is soaked to 15min, remove the photoresist of unexposed portion, leave protruding microchannel, rush Xian with isopropyl alcohol again, dry up with nitrogen stream, be placed in heat under 150 DEG C of conditions and dry 30min, naturally cooling rear for subsequent use as the formpiston of making chip.
(3) preparation of high performance operation electrode (surface is coated with the Pt silk of Cu-Cr-Sn Nanoalloy layer)
The electroplate liquid that preparation contains required metallic ion: electroplate liquid consist of 50mM CuSO
4, 0.2mM Cr
2(SO
4)
3, 0.2mM SnCl
2and the H of 10mM
2sO
4; Tinsel pre-service: the diameter that intercepts suitable length is the Pt silk of 25 μ m, uses respectively 0.1mM HNO
3, the each 10min of second alcohol and water ultrasonic cleaning, be then placed under 105 DEG C of conditions of baking oven and toast 30min.Electrochemical Modification electrode: the Pt silk of above-mentioned processing is inserted in above-mentioned electroplate liquid, in three-electrode system (taking Ag/AgCl as contrast electrode, Pt be working electrode as auxiliary electrode and 25 μ m Pt silks), taking-0.3V as constant potential, electroplating time is 120～150s, and Cu-Cr-Sn Nanoalloy is plated on to Pt silk surface.Electroplated is complete, takes out Pt silk and puts under 105 DEG C of conditions of baking oven and toast 1h left and right, make the modification of Cu-Cr-Sn Nanoalloy Pt silk working electrode (Cu-Cr-Sn/Pt, diameter 30 μ m), as shown in Fig. 2 (D).
(4) making of PDMS micro-fluidic chip
Getting a certain amount of PDMS monomer and hardening agent mixes by the mass ratio of 10:1, degasification, be poured on the silicon chip masterplate that contains passage of above-mentioned preparation, 65 DEG C of heating 2h solidify above, after cooling, peel from masterplate the PDMS film that contains passage, with card punch at buffer pool, sample cell, sample cell waste liquid pool, damping fluid waste liquid pool, and punch respectively in Liu Ge position, two alkali lye ponds, the hole that formation diameter is 5mm, then by the above-mentioned Pt silk (Cu-Cr-Sn/Pt that is coated with Cu-Cr-Sn Nanoalloy, diameter 30 μ m) are placed in electrode arrangement passage, together be placed in plasma cleaning device with the smooth PDMS film of another sheet and process 30s, then, take out rapidly two PDMS pressing, form the PDMS microfluidic chip analysis device of sealing.
Embodiment 2, the application of PDMS micro-fluid control chip electrophoretic-electrochemical detection device provided by the invention in aminoglycoside antibiotics is analyzed
As shown in Figure 1, use time of the present invention, alkaline solution liquid storage tank 4 and 5 places that are being connected with two accessory channels 9, add high concentration basic solution (0.1M NaOH), and the height that makes its liquid level is greater than other liquid storage district (sample cell 1, buffer pool 2, sample waste liquid pool 3 and damping fluid waste liquid pool 6) approximately 1 times of the height of liquid level, utilize the differential static pressure of solution to order about alkaline solution liquid storage tank 4 and 5 place's strong alkali solutions and import split tunnel 8 ends from the acidic buffer of split tunnel, mix through W type path 10, make the pH in detection zone 13 be greater than 12, thereby can effectively realize under non-strong basicity separation buffer solution condition, micro-fluid control chip electrophoretic separates the target with Amperometric Determination aminoglycoside antibiotics.
Below with five kinds of aminoglycoside antibioticss (miramycin, streptomysin, amikacin, paromomycin, neomycin) for example, illustrate micro-fluid control chip electrophoretic-electrochemical detection device provided by the invention aminoglycoside antibiotics analyze in application.Experimental result is as follows:
(1) platinum filament surface characteristics comparison under different plating conditions
SEM result in Fig. 2 shows, under difference plating condition, marked change has occurred the pattern of working electrode electrodeposited coating.Shown in Fig. 2 (A), when only containing Cu
2+when the electroplate liquid of metallic ion, in the surface attachment of Pt silk one deck relatively evenly but comparatively loose Cu nano-structured particles, stable (A) when this modified electrode is measured is poor; Shown in Fig. 2 (B), when at Cu
2+electroplate liquid in add Cr
2+time, compared with Fig. 2 (A), can significantly observe electrodeposited coating and become compact and firm, this modified electrode shows good stability, but sensitivity declines to some extent; Shown in Fig. 2 (C), when by Sn
2+join Cu
2+electroplate liquid in, with the result comparison of Fig. 2 (A) and Fig. 2 (B), the surface of this modified electrode has obvious columnar protrusions structure, possesses relatively large specific surface area, show higher sensitivity, but stability is general, and coating is easy to come off.Based on above result, in order to obtain good stability and sensitivity high working electrode again, the present invention adopts and contains Cu
2+, Cr
3+and Sn
2+hybrid metal ion-conductance plating solution, as shown in Fig. 2 (D), the compactness of layer electrodes and specific surface area have all obtained good improvement.Therefore, it is that working electrode is measured aminoglycoside antibiotics that the present invention adopts the modified electrode of plating Cu-Cr-Sn Nanoalloy, improves stability and sensitivity to analyte determination.
(2) galvanochemistry of Cu-Cr-Sn/Pt modified electrode and electro-catalysis behavior
The electrochemical properties of modified electrode and to the electrochemical behavior of amikacin as shown in Figure 3.
Naked Pt silk electrode (curve a) at-0.2V when scanning between+0.8V current potential, almost without any redox peak, illustrate that amikacin does not have electrochemical activity at this potential region on naked Pt silk electrode; Cu/Pt electrode can have been observed an obvious oxidation peak in+0.65V left and right, and (curve b), is the oxidation peak of amikacin; On Cu-Cr/Pt electrode, although the stability of electrode has obtained larger raising, (curve c) in the oxidation peak reduction of amikacin; On Cu-Cr-Sn/Pt electrode, the oxidation peak that can observe amikacin obviously increases that (curve d), illustrates Sn
2+increase the specific surface area of Cu Nanoparticle Modified Electrode, the sensitivity that has improved modified electrode.
In addition, interior illustration is Cu-Cr-Sn/Pt modified electrode cyclic voltammetry curve in the 50mM NaOH background electrolytic solution that does not contain and contain amikacin, as can be seen from the figure, in the time not containing amikacin in solution, there is obvious oxidation current at+positive potential scanning direction that 0.4V starts, corresponding Cu (II) is oxidized to the electric current of Cu (III), when to negative potential scanning direction, + obviously sagging reduction peak of 0.65V current potential place appearance, corresponding Cu (III) is reduced to the electric current of Cu (II); In the middle of amikacin being joined to solution time, when positive potential scanning, there is characteristic peak at+0.65V place, illustrate that this peak is the oxidation peak of amikacin, and in the time that negative potential scans, the reduction peak of sinking at+0.65V current potential place obviously diminishes, illustrate that Cu (III) has participated in the oxidation of amikacin, self be reduced to Cu (II), this phenomenon has also further been verified the catalytic oxidation effect of Cu (III)/Cu (II) to aminoglycoside antibiotics simultaneously.
(3) impact of different buffer solution pH on five kinds of aminoglycoside antibiotics separation determinations
In Capillary Electrophoresis, because the pH value of damping fluid affects the dissociation degree of carrying capacity and the Acidity of Aikalinity medicine of channel surface.Electroosmotic flow (EOF) drives the power of solution as electrophoresis process, the carried charge of its size and channel surface is proportional.Owing to having multiple amino in aminoglycoside antibiotics structure, therefore the pH value of buffer solution is one of important parameter affecting electrophoretic separation detection.
Fig. 4 has investigated in pH 4.0～6.0 scopes, and separation buffer pH value of solution separates the impact detecting on five kinds of aminoglycoside antibioticss.Its test condition is, runtime buffer solution: sodium acetate 6mM+CTAB 0.6mM; Separation voltage :-1200V; Sample introduction voltage :-400V/+100V; Sample injection time: 30s; Detect current potential :+0.65V(vs.Ag/AgCl).Wherein, the sample introduction concentration of aminoglycoside antibiotics is: miramycin (Spe) 134.6 μ M, streptomysin (Str) 91.5 μ M, amikacin (Ami) 85.3 μ M, paromomycin (Par) 93.4 μ M, neomycin (Neo) 73.3 μ M.
As shown in Figure 4, along with the continuous reduction of pH, five kinds of antibiotic transit times extend gradually, and degree of separation increases gradually.In the sodium-acetate buffer of pH 5.0, five kinds of microbiotic have reached better baseline separation.In an application of the invention, through considering the factors such as analysis time, separation efficiency and signal to noise ratio (S/N ratio), the sodium acetate solution that selection pH is 5.0 is as dissociating buffer.
(4) impact of different buffer concentrations on five kinds of aminoglycoside antibiotics separation determinations
Peak shape on the selectivity separating, measured object and separation efficiency have important impact to the composition of buffer solution with concentration.Generally, the concentration that increases buffer solution will reduce electrostatic double layer, reduces zeta electromotive force and can make EOF reduce thereupon, causes the prolongation of transit time, and the degree of separation between separated object increases.Under identical pH condition, investigate the impact that it is measured five kinds of Antibiotics separations, the same above-mentioned steps of other test condition (3) by changing sodium-acetate buffer concentration (2～10mM).
As shown in Figure 5, five kinds of microbiotic can reach baseline separation under the condition of different buffer concentrations, and still, along with the increase of sodium acetate concentration, five kinds of antibiotic degree of separation increase gradually, especially the degree of separation between amikacin and paromomycin.In the time that concentration is increased to 6.0mM, measured object can reach better baseline separation.In the time that buffer concentration exceedes 6.0mM, although degree of separation has increased slightly, baseline noise increases and peak current also starts to reduce.This phenomenon is mainly because the ionic strength along with damping fluid strengthens, and EOF is reduced, and Joule heat increases.Therefore, cause the reduction of separation efficiency, sensitivity and stability.Based on above situation, in an application of the invention, can select sodium acetate concentration is the buffer solution of 6.0mM.
(5) impact of different CTAB concentration on five kinds of aminoglycoside antibiotics separation determinations in buffer solution
Aminoglycoside antibiotics belongs to alkalescence sugar, and pKa is greatly between 6～9, and it can be positively charged in neutral and acid solution.In cathode direction electrophoretic separation pattern, positively charged material should go out peak early than EOF.If but add cationic surfactant in solution, as CTAB, EOF changes direction, now in anode direction electrophoretic separation pattern, EOF flows to detection zone, now, and the electrophoresis direction of positively charged material and EOF opposite direction, when EOF speed while being greater than the electrophoretic velocity of positively charged material, lotus electropositive substance is the final detection zone that arrives under the driving of EOF.In buffer solution, adding cationic surfactant to be actually has increased the residence time of separated object in passage, has been equivalent to extend the effective length of split tunnel, thereby improves separating effect.
Fig. 6 has investigated the CTAB(0.2～1.0mM of variable concentrations) impact on five kinds of aminoglycoside antibiotics separation determinations, the same above-mentioned steps of other test condition (3).
From Fig. 6, can observe, five kinds of microbiotic can reach better baseline separation under the condition of variable concentrations CTAB, but along with the continuous increase of CTAB concentration, five kinds of antibiotic transit times shorten gradually, this is mainly the increase due to CTAB concentration, the positively charged amount of channel surface is increased, and EOF increases, and the migration velocity of measured object in passage increases.But in the time that CTAB concentration is greater than 0.6mM, CTAB reaches balance in the absorption of channel surface, and the transit time of separated medicine is relatively stable, but baseline noise starts to increase, and peak current reduces.Consider the factors such as transit time, detection sensitivity and baseline noise, in an application of the invention, can select concentration is the CTAB of 0.6mM.
(6) impact of different separation voltages on five kinds of aminoglycoside antibiotics separation determinations
In capillary electrophoresis separation, separation voltage is also to improve important parameter of detection sensitivity.
Fig. 7 is the electrophoretic separation figures of five kinds of microbiotic under different separation voltages (1400～-800V) condition, the same above-mentioned steps of other test condition (3).As shown in Figure 7, along with the reduction of voltage, five kinds of antibiotic transit times shorten gradually; Simultaneously the peak shape of measured object also become gradually sharply and more symmetrical.But when separation voltage is during lower than-1200V, degree of separation also reduces gradually and noise obviously increases.Consider analysis time, separation efficiency, the factors such as the stability of signal to noise ratio (S/N ratio) and baseline, in an application of the invention, can select-1200V is separation voltage.
(7) impact that different detection current potentials are measured five kinds of aminoglycoside antibioticss
Because ampere detection method has the highly sensitive advantages such as integrated and microminiaturized that are easy to, the present invention adopts an ampere detection method to measure measured object.
Fig. 8 has investigated under different detection current potential (+0.4～+ 0.9V) condition, five kinds of antibiotic peak current change curves, the same above-mentioned steps of other test condition (3).As shown in Figure 8, along with the beginning that detects current potential increases gradually, five kinds of antibiotic peak currents increase gradually.When detecting reach+0.7V(of current potential for miramycin, reach+0.6V) time, it is maximum that antibiotic peak current reaches.But in the time continuing to increase detection current potential, noise also starts to increase.Because too high detection current potential will cause higher background current, thereby the peak current of measured object is reduced, as shown in Figure 8.Therefore select in test process+0.65V of the present invention (vs Ag/AgCl), as detecting current potential, so not only can reach maximum signal to noise ratio (S/N ratio), and the working electrode of modifying also can have good stability and reappearance under this detection current potential.
Based on above measurement result, optimize testing conditions under, five kinds of antibiotic detectabilities can reach within the scope of 2.1～4.6 μ M, the relative standard deviation of peak current and transit time all respectively lower than 7.9% and 2.0%(n=30).In addition the present invention also analyzes the aminoglycoside antibiotics in actual sample (milk), and after mark-on, average recovery rate can reach more than 70%.These results suggest that, by micro-fluid control chip electrophoretic-electrochemical detection device of the present invention, after separation, add alkaline solution, make working electrode measure region and reached the required strong alkaline condition of detection glucide, and do not affect its electrophoretic separation, effectively realized under non-strong basicity separation buffer solution condition, micro-fluid control chip electrophoretic separates the target with Amperometric Determination aminoglycoside antibiotics; And the working electrode method of modifying of the present invention's exploitation, has also significantly improved the stability and the sensitivity that detect glucide.This invention, for rationally using clinically aminoglycoside antibiotics and effectively monitoring residual aminoglycoside antibiotics in animal derived food, provides a kind of quick, sensitive, Pharmaceutical Analysis method reliably.
Claims (8)
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| CN112871229B (en) * | 2021-01-21 | 2022-06-28 | 中国科学技术大学 | A chip for dielectrophoretic bacterial sorting in water |
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