CN109682902A - Micro-fluidic chip extracts fluidic chip-mass spectrometry analytical equipment and the method for declining - Google Patents
Micro-fluidic chip extracts fluidic chip-mass spectrometry analytical equipment and the method for declining Download PDFInfo
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- CN109682902A CN109682902A CN201811556995.4A CN201811556995A CN109682902A CN 109682902 A CN109682902 A CN 109682902A CN 201811556995 A CN201811556995 A CN 201811556995A CN 109682902 A CN109682902 A CN 109682902A
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Classifications
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
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- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
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- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The present invention provides a kind of micro-fluidic chip, fluidic chip-mass spectrometry analytical equipment and the method for declining are extracted, belongs to field of biological detection.The micro-fluidic chip includes: substrate, at least one sample microchannel for placing the sample extraction slot of solid phase extraction material and being connected to each sample extraction slot is provided on substrate；Substrate is additionally provided with spraying microchannel；Wherein, each sample microchannel is set to the circumferential direction for the fluid spray beam that spraying microchannel sprays far from the end of sample extraction slot, so that the axis of sample microchannel and the axis of spraying microchannel intersect.The extraction declines fluidic chip-mass spectrometry analytical equipment, including electrospray mass spectrometry and above-mentioned micro-fluidic chip.This analytical equipment and method separate test substance without using any chromatography equipment, can be realized and are directly analyzed by mass spectrometry the detection field by bed to the other biological sample of micro updating and have broad application prospects.
Description
Technical field
The present invention relates to field of biological detection, join in particular to a kind of micro-fluidic chip, micro-fluidic chip-mass spectrum
With analytical equipment and method.
Background technique
Micro-fluidic chip, also referred to as chip lab, be using chip as operating platform, while based on analytical chemistry,
With the micro- support of micro electro mechanical processing technology, using microchannel network as structure feature.Micro-fluidic chip have ultra-high-speed mixing with separate
Efficiency, sample consumption are few, the first characteristics such as integrated of multifunctional single.In recent years, biology, chemistry, a fluid are had been developed as
With the new hot spot of the interdisciplinary researchs such as material.
Electrospray ionization mass spectrum (ESI-MS) have benefited from its outstanding sensitivity and specificity and its to the wide of numerous compounds
Detection performance is composed, one of the common technology for being most widely used in bioanalysis has been become.However due to biological sample matrix meeting
There is inhibiting effect to mass signal, significantly limits application of the ESI-MS in clinical detection.In general, potential in order to eliminate
Matrix effect, processing before needing to carry out biological sample before being analyzed by mass spectrometry, including extracted from biological sample matrix
Determinand and/or to sample substrate carry out chromatography.This kind of pre-treatment step is complicated for operation, time-consuming and to work people
The Specialized Quality of member requires high, it is difficult to promote the use of in clinical detection (especially detection by bed).
Micro-fluidic chip and highly selective, sensitivity mass spectrometric hyphenated technique, there is high application in bioanalysis
Prospect.In most of micro-fluidic chips-mass-spectrometric technique research and development, ESI ionization source is succinct with its interface and becomes the most frequently used ionization
Source.Micro-fluidic chip-ESI-MS, which also becomes, studies the macromolecular and small molecule in various models with biomedical meaning
Hot technology.However, seriously restrict micro-fluidic chip-due to interference of the matrix effect to mass signal of biological sample
Development of the ESI-MS in field of bioanalysis.
Summary of the invention
The first object of the present invention is to provide a kind of micro-fluidic chip, can be directly to biology by this micro-fluidic chip
Sample extracts, to eliminate interference of the matrix effect to mass signal of biological sample.
The second object of the present invention is to provide a kind of extraction and declines fluidic chip-mass spectrometry analytical equipment, the equipment
It is cheap, speed is fast for analysis, on-line checking result accuracy is high, can be widely used in by bed in detection (POCT).
The third object of the present invention is to provide a kind of extraction and declines fluidic chip-mass spectrometry analysis method, this method
Using the micro-fluidic mass spectrometric hyphenated technique of paper chromatography-, efficiently separates protein and eliminate matrix effect, it can be achieved that right in a short time
Biological sample is quickly analyzed.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
In a first aspect, the present invention provides a kind of micro-fluidic chip comprising:
Substrate, the sample for being provided at least one sample extraction slot on substrate and being connected to each sample extraction slot are micro- logical
Road；Substrate is additionally provided with spraying microchannel；Wherein, each sample microchannel is set to micro- by spraying far from the end of sample extraction slot
The circumferential direction for the fluid spray beam that channel sprays, so that the axis of sample microchannel and the axis of spraying microchannel intersect.
Further, in preferred embodiments of the present invention, the substrate for surrounding spraying microchannel is sprayed along spraying microchannel
The direction of fluid spray beam be arranged in spininess, surround the substrate of each sample microchannel along the circumferential direction towards spraying microchannel
Direction is arranged in spininess, so that the liquid outlet of each sample microchannel is located at the week for the fluid spray beam that spraying microchannel sprays
To.
Further, in preferred embodiments of the present invention, the axis of the axis of above-mentioned sample microchannel and spraying microchannel
Angle between line is 80~110 degree.
Further, in preferred embodiments of the present invention, the liquid outlet of above-mentioned sample microchannel and by spraying microchannel
Vertical range between atomizing nozzle is 0.2-0.8mm；
Preferably, the vertical range between liquid outlet and substrate edge is 0.5-1.5mm；
Preferably, the vertical range between fluid spray beam that liquid outlet and spraying microchannel spray is 0.2-0.8mm；
Further, in preferred embodiments of the present invention, above-mentioned sample extraction slot is arranged in parallel with spraying microchannel；
Preferably, the vertical range between the middle line of sample extraction slot and spraying microchannel is 6-14mm.
Further, in preferred embodiments of the present invention, there are two above-mentioned sample extraction slots, two sample extraction slots pair
Claim the two sides for being set to spraying microchannel.
Further, in preferred embodiments of the present invention, each arc-shaped setting in sample microchannel, and the sample of the arc
Side of the center of circle of product microchannel close to spraying microchannel.
Second aspect extracts the fluidic chip-mass spectrometry analytical equipment that declines the present invention also provides a kind of comprising EFI
The atomizing nozzle of mist mass spectrum and above-mentioned micro-fluidic chip, spraying microchannel is aligned with the injection port of electrospray ionization mass spectrum and atomizing nozzle
Vertical range between injection port is 1-4mm.
The third aspect is extracted the fluidic chip-mass spectrometry analysis method that declines the present invention also provides a kind of, is utilized above-mentioned
Extraction declines fluidic chip-mass spectrometry analytical equipment comprising:
Biological sample is added dropwise in the sample extraction slot equipped with solid phase extraction material, adds sample in sample extraction slot
After extracting solution, voltage is applied to spraying microchannel and sample microchannel respectively, makes the spraying solvent in spraying microchannel with fluid
The form of fuel spray, which sprays and carries the sample extracting solution sprayed from sample microchannel, enters mass spectrographic injection port, carries out matter
Spectrum detection.
It further, is 3-5KV to the voltage that spraying microchannel applies in preferred embodiments of the present invention；To sample
The voltage that extraction tank applies is 60-120V.
Preferably, the solid phase extraction material includes chromatographic paper, reverse phase silica gel and ion exchange resin.
Compared with prior art, the invention has the benefit that
Micro-fluidic chip provided by the invention and micro-fluidic chip-mass spectrometry analytical equipment, can be in sample extraction slot
After extracting processing to biological sample using chromatography paper slip, make the sample extraction containing determinand in such a way that voltage drives
Liquid stream intersects through sample microchannel, and with the spray liquids sprayed from spraying microchannel, by spray liquids by sample extracting solution
Delivery is tested and analyzed to mass spectrographic injection port.
Fluidic chip-mass spectrometry analytical equipment and the method for declining are extracted using this paper chromatography, in micro-fluidic chip
On the basis of, sample separating step is simplified come the small molecule in enriched biological sample to the suction-operated of substance using chromatography paper slip,
Influence of the matrix effect to Mass Spectrometer Method is effectively eliminated, and further increases the sensitivity and accuracy of detection.This analysis is set
Standby and method, operation is simple, without carrying out complicated pre-treatment step to biological sample, also without using including high-efficient liquid phase color
Any chromatography equipment including spectrometer separates test substance, can be realized direct to the other biological sample of micro updating
It is analyzed by mass spectrometry；In addition its testing cost is cheap, is easy to minimize, therefore has wide application by the bed in detection field
Potentiality.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 is the structural schematic diagram of the micro-fluidic chip and mass spectrometry that provide in the embodiment of the present invention 1；
Fig. 2 is to analyze blood sample using decline fluidic chip-mass spectrometry analytical equipment of the extraction in the embodiment of the present invention 2
The step schematic diagram of product；
Fig. 3 is that the flow control nanoESI-MS analytical concentration that declines is extracted in experimental example of the present invention is 6.0x 10-6M ATP、 ADP
And the mass spectrogram of AMP standard items；
Fig. 4 is the mass spectrogram of ATP, ADP, AMP and 8- bromine adenosine in experimental example of the present invention；
Fig. 5 is the time-optimized curve for extracting nucleotide in experimental example of the present invention from blood sample；
Fig. 6 is the equation of linear regression of ATP, ADP and AMP in experimental example of the present invention；
Fig. 7 is experimental example of the present invention MS scanning figure complete to blood sample.
Label: 100- micro-fluidic chip；110- substrate；120- sample extraction slot；121- sample microchannel；122- goes out liquid
Mouthful；130- is sprayed microchannel；131- atomizing nozzle；132- fluid spray beam；200- mass spectrum；210- injection port.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Feature and performance of the invention are described in further detail with reference to embodiments:
Embodiment 1
The present embodiment provides a kind of micro-fluidic chips 100, and (i.e. paper chromatography extracts the flow control core that declines with the combination of mass spectrum 200
Piece-mass spectrometry analytical equipment) as shown in Figure 1.The micro-fluidic chip 100, comprising:
Substrate 110 uses PMMA material.PMMA, full name polymethyl methacrylate, is commonly called as acrylic glass, for dress
Most economical one of the material with most simple processing and fabricating in pool circle.The processing of PMMA material is simple, can carry out laser ablation, drilling with
And a series of processing methods including cutting, and can be made into arbitrary shape after heating.In the present embodiment, using PMMA as miniflow
The building material of chip 100 is controlled, which is made of 2 pieces of 24*20mm acrylic glass chips, to reduce ESI spray
The size of mouth, this work use with a thickness of 1.5mm PMMA chip.
Substrate 110 is provided with spraying microchannel 130, and depth is 20 μm, width is 80 μm.In actual application,
It is placed with spraying solvent in spraying microchannel 130, under conditions of plus high-pressure, spraying solvent is spouting from atomizing nozzle 131,
Form fluid spray beam 132.
Preferably, the substrate 110 for surrounding spraying microchannel 130 is arranged along the direction of fluid spray beam 132 in spininess, i.e.,
The atomizing nozzle 131 of spraying microchannel 130 is located at the spiny tip.This spiny design helps to make from atomizing nozzle
The 131 fluid spray beams 132 sprayed keep column, it is made to be easier to deliver the sample introduction that the extracting solution containing sample enters mass spectrum 200
Mouth 210, to eliminate spraying osmosis.
At least one sample extraction slot 120 and sample microchannel 121 are additionally provided on substrate 110.Sample extraction slot
120 be strip, length 0.8-15mm, width 0.9-1.3mm.In the present embodiment, the length of sample extraction slot 120 is
11mm, width 1.2mm.The axis of sample extraction slot 120 and the axis of spraying microchannel 130 are arranged in parallel.It is more in order to realize
It is detected while a difference biological sample, sample extraction slot 120 is settable multiple.In the present embodiment, sample extraction slot 120 has
Two, two sample extraction slots 120 are symmetrically disposed on the two sides of spraying microchannel 130.
Sample microchannel 121 is connected with sample extraction slot 120, and depth is 20 μm, and width is 80 μm.Sample microchannel
121 one end far from sample extraction slot 120 are liquid outlet 122, liquid in sample extraction slot 120 under voltage driving with
The form of fluid beam is flowed out from liquid outlet 122.Each sample microchannel 121 is set to stream far from the end of sample extraction slot 120
The circumferential direction of spray body beam 132, i.e., the liquid outlet 122 of each sample microchannel 121 are located at the stream sprayed from spraying microchannel 130
The circumferential direction of spray body beam 132 is ensured so that the axis of sample microchannel 121 and the axis of spraying microchannel 130 intersect
It can intersect from the fluid beam flowed out in sample microchannel 121 with the fluid spray beam 132 sprayed in spraying microchannel 130,
Extraction formula electrospray ionisation to the fluid beam containing sample is completed by fluid spray beam 132, and sample ions are sent into mass spectrum
200 injection port 210 carries out Mass Spectrometer Method.
When sample extraction slot 120 and spraying microchannel 130 are arranged in parallel, make to flow out from sample microchannel 121
Sample extracting solution can intersect with fluid spray beam 132, and camber is arranged in each sample microchannel 121 by inventor, and is allowed
Side of the center of circle of the sample microchannel 121 of the arc close to spraying microchannel 130.
Preferably, surrounding circumferential direction of the substrate 110 of each sample microchannel 121 along the spraying microchannel 130 of direction is in
Spininess setting, i.e., the liquid outlet 122 of each sample microchannel 121 are located at opening for the spiny tip and liquid outlet 122
Mouth direction is located at the circumferential direction for the fluid spray beam 132 that spraying microchannel 130 sprays.This spiny design helps to make to flow
Spray body beam 132 contacts extraction and electricity more complete, and then that promote sample entirety with the liquid outlet 122 of sample microchannel 121
From efficiency, so that more samples enter mass spectrum 200.
For the fluid spray beam 132 and sample extracting solution that spray spraying microchannel 130 mixed effect more preferably, to be formed
It is good spraying, obtain sensitiveer, stable mass signal.In the present embodiment, the axis of sample microchannel 121 and spraying
The axis of microchannel 130 is mutually perpendicular to, more preferably, close to the axis of the sample microchannel 121 at 122 end of liquid outlet and spraying
The axis of microchannel 130 is mutually perpendicular to, so that the fluid spray beam that the air-flow flowed out in liquid outlet 122 and atomizing nozzle 131 spray
132 are mutually perpendicular to.
To be further formed good spray effect, better 200 sample introduction effect of mass spectrum is realized, inventor is micro-fluidic to this
Design is optimized in the size of chip 100, specifically:
A. the vertical range between the liquid outlet 122 of sample microchannel 121 and the atomizing nozzle 131 of spraying microchannel 130 is
0.2-0.8mm is perhaps 0.3-0.7mm or is 0.4-0.6mm, this vertical range between the two is in the present embodiment
0.5mm。
B. the vertical range between the liquid outlet 122 of sample microchannel 121 and the edge of substrate 110 is 0.5-1.5 mm,
It perhaps be 0.6-1.4mm is perhaps that 0.7-1.3mm is perhaps 0.8-1.2mm or is 0.9-1.1mm, in the present embodiment
This vertical range between the two is 1.0mm.
Hanging down between the fluid spray beam 132 that C. liquid outlet 122 of sample microchannel 121 and spraying microchannel 130 spray
Straight distance is 0.2-0.8mm, perhaps be 0.3-0.7mm or be 0.4-0.6mm, in the present embodiment this therebetween vertical
Straight distance is 0.5mm.
D. the vertical range between the middle line of sample extraction slot 120 and spraying microchannel 130 is 6-14mm, or is 7-
13mm is perhaps 8.0-12mm or is 9.0-11mm, this vertical range between the two is 10.0mm in the present embodiment.
E. be sprayed microchannel 130 atomizing nozzle 131 be aligned with the injection port 210 of electrospray ionization mass spectrum 200 and therebetween
Vertical range is 1-4mm, is perhaps 1.5-3mm or is 2.0-2.5mm, in the present embodiment this it is between the two it is vertical away from
From for 2.0mm.
Embodiment 2
Fluidic chip-mass spectrometry the analysis method that declines is extracted the present embodiment provides a kind of, is declined using said extracted
Fluidic chip-mass spectrometry analytical equipment is come as shown in Figure 2 the step of analyzing biological sample.In the present embodiment with blood sample
For be illustrated.
The analysis method specifically includes the following steps:
A. acquiring 3 μ L sample of blood is test sample, and the 5 μ L PBS that contains 50mmol/L EDTA is added dropwise (EDTA is anticoagulation
Agent, and inhibit ATP metabolic enzyme).
B. by above-mentioned mixed blood sample point in one end of chromatography paper slip (long 1cm, width 1mm), and it is placed on micro-fluidic core
In the sample extraction slot of piece, one end with blood sample is placed far from sample microchannel.Chromatography paper slip is sample absorption carrier, can
Adsorbed proteins, lipid and other matrix.
C. sample extracting solution is added in sample extraction slot, it is made to flood chromatography paper slip.
D. spraying solvent is placed in spraying microchannel, and applies 3-5KV far from one end of atomizing nozzle in spraying microchannel
High pressure, spray spraying solvent in the form of fluid spray beam；Meanwhile adding that 60-120 V's is low in one end of sample extraction slot
Pressure, sample flows through chromatography paper slip by capillarity, and passes through the absorbed layer on chromatography paper slip.In the process, extra
Matrix can be adsorbed on chromatography paper slip, and determinand will constantly move ahead, and confuse Extraction solvent flow through sample microchannel with
The form of fluid beam is projected from liquid outlet, and enters mass spectrographic injection port under the carrier band of fluid spray beam, carries out Mass Spectrometer Method.
Experimental example
Atriphos (ATP) is all life nucleotide constituent element and biological respinse energy source.This experimental example
Fluidic chip-mass spectrometry (hereinafter referred to as " extraction decline flow control nanoESI-MS ") is declined using the extraction of above-mentioned foundation
Analytical equipment and method analyze blood sample nucleotide content.
This experimental example detects blood sample using the analysis method of above-described embodiment 2, wherein sample extracting solution is to contain perchloric acid
Methanol aqueous solution.Perchloric acid is often to quote polar extractive solvent, is strong acid, is widely used in from intact cell and extracts ATP
Related compound, and inhibit the activity of ATP enzyme, while precipitating proteins.25-75% methanol aqueous solution has similar extraction effect
Fruit, and it is more preferable with electron spray solvent compatibility.Formalin is a kind of broad spectrum activity electrolyte.This experimental example is high using 0.4%
Chloric acid, 50% methanol and 0.1% formalin are as sample extracting solution, to extract ATP, ADP and AMP in whole blood.
One, mass spectrum operating parameter and data processing:
This experimental example application paper chromatography extraction decline fluidic chip-mass spectrometry analytical equipment by micro-fluidic chip,
ESI- ion trap mass spectrometer (LCQDeca, ThermoFinnigan, San Jose, CA) and Hamilton sample introduction needle
(Hamilton, Las Vegas, NV) is constituted, and the latter is used to fluid spray introducing nano-electrospray mouth.Xcalibur software
It (ThermoFinnigan) is system controlling software.
Optimum of operation is as follows: MS/MS is with respect to impact energy 20-30%；Separate width 1.0u；Firing time 30ms；Set
Tube voltage 55V；Electron spray channel pressurization+4.0kV is used for ionization spray.Tandem mass spectrum result is analyzed using collision induced dissociation.
It chooses the highest fragment of abundance in each compound and carries out quantitative analysis.Three kinds of compound parent ions and fragment ion are successively are as follows:
AMP (m/z 348 → 136), ADP (m/z 428 → 348), ATP (m/z 508 → 428) and internal standard 8- bromine adenosine (m/z
348→216)。
MS/MS mass spectral results are collected, while quantitative analysis is carried out to four kinds of substances above-mentioned in blood sample.Internal standard is dissolved in methanol,
And diluted with pure water, 100 times are diluted to final concentration of 2x10 with Extraction solvent again later-5Mol/L, and be added dropwise in chromatography paper slip
On.The average absolute signal intensity rate of internal standard and sample is taken to do quantitative analysis.Detection is limited to three times blank sample standard deviation and removes
With the slope of standard curve.
Two, signal density and detection limit
Analyte detection limit to be measured is by its molecular characterization and sample substrate joint effect.Inventor is to molecular weight between 135-600
Small molecule analysis show detection limit fluctuation range may span across 3 orders of magnitude.Sample such as urine sample, waste water, blood plasma and blood sample etc.
In matrix components it is more complicated, the signal density of determinand also accordingly reduces.In mass spectral analysis, it is limited to chromatographic paper size
Size and extraction/spraying solvent volume, if sample volume, more than several milliliters, detection limit will be reduced no longer.If expanding chromatography paper slip
Size, then analysis time and sample substrate content can all increase on year-on-year basis.With paper chromatography then can efficient selective enrichment it is to be measured
Object, while sample segment matrix being adsorbed on paper.
After being handled using chromatographic paper, the absolute signal strengths of the characteristic ion fragment peak of three kinds of adenosine phosphate are than directly detecting
When signal strength be higher by about 5 times.The result shows that can be eliminated from bulk sample selective enrichment testing molecule using chromatographic paper
Sample ions inhibiting effect further decreases detection limit.
The flow control nanoESI-MS that declines is extracted in this experimental example to limit three kinds of sample detections, as shown in table 1:
Linear regression parameters, the LOD, the rate of recovery of 1 three kinds of nucleotide of table.
aNucleotide peak resolution ratio is by software statistics；
bAll samples measure three times；
cRegression equation formula Y=(mean ± SD) X+ (mean ± SD)；
dThe rate of recovery (%)=100* (measured value-initial value)/additive amount, as a result takes and measures average value three times.
As shown in Table 1, flow control nanoESI method is declined to three kinds of nucleotide using this extraction that designs of the present invention
Detection, which is restricted water supply, to be put down in 3.6-6.8x10-7Mol/L range can meet three kinds of nucleotide in clinical sample such as serum, urine completely
The demand of quantitative analysis.
The present embodiment using it is established extraction decline flow control nanoESI-MS analysis method have detected ATP in blood sample,
ADP and AMP concentration.
Fig. 3 is that 6.0x 10 is analyzed in mixed solution-6The MS/MS mass spectrogram of M ATP, ADP and AMP: (a) [AMP]+m/
z348；(b) m/z136 is verified as AMP, [ADP]+m/z428；(c) m/z136,348, [ATP]+m/z508； (d)m/z136,
428, internal standard [8- bromine adenosine]+m/z348；(e)m/z216.Experiment condition: nanoESI voltage+4kV；(a) it is mentioned with (b) sample
Take tank voltage ,+75V；Electron spray solvent flow rate, 250nL/min.
Fig. 4 is the mass spectrogram of ATP, ADP, AMP and 8- bromine adenosine.(a)[AMP]+M/z348, m/z136； (b)
[ADP]+M/z428, m/z136,348, (c) [ATP]+M/z508, m/z136,428；(d) internal standard [8- bromine adenosine]+m/z348。
Each sample test three times, regression curve, linearly dependent coefficient r2, RSD etc. be shown in Table 1.
This experimental example is also further optimized the experiment parameter for extracting the flow control nanoESI-MS analysis method that declines,
Test the effect of 20-80% methanol concentration in spraying solvent, the results showed that optimal methanol concentration is between 60-80%.It is spraying molten
Formic acid is added in agent and makees electrolyte, and concentration is affected to detection sensitivity and quantitative effect.Comprehensively consider, this experimental example is adopted
Make spraying solvent with the aqueous solution of 80% methanol, 0.25% formic acid.Meanwhile this experimental example has been investigated between 0.1-2.0 μ L/min
When influence of the flow velocity to result, the results showed that optimal flow rate is 0.2-0.5 μ L/min.
To sum up, the final experiment condition of this experimental example are as follows: the spraying solvent of aqueous solution work of 80% methanol, 0.25% formic acid,
Flow velocity 250nL/min, electron spray voltage+4kV, sample extraction tank voltage+75V.
Select 0.8,1.6,2.4,3.2 and 4.0min as paper chromatography ATP extraction time.As a result as shown in Fig. 5, when
When extraction time is less than 4min, have benefited from the suction-operated of paper chromatography, extracts reagent and transport determinand to mass spectrum spout, ATP
Signal strength increases with extraction time and is promoted, until signal strength is to peak when 4min.It is greater than 4min, protein between upon extracting
And part inhibits the stroma ground substance of signal also to be migrated together to mass spectrum spout, and inhibits signal strength, therefore chooses 4min and be
Best sample extraction time.
Three, regression curve equation, detection limit and quantitative limit
Regression curve equation measures concentration selection based on three kinds of nucleotide real standards in blood sample.The result shows that dense in selection
Three kinds of nucleotide have preferable linear relationship in degree range.It is storage liquid with nucleosides aqueous acid, and is diluted to regression curve
Working concentration needed for equation, each measurement of concetration are denoted as concentration three times, with peak area.
As a result Fig. 6 is seen, each regression curve equation has preferable related coefficient (r2>=0.99), show each determinand
Good relationship between concentration and peak area.The standard items of measurement serial dilution are repeated 3 times, are LOD in terms of 3 times of signal-to-noise ratio.Such as
Shown in table 1, the detection limit of institute's sample is below 3.6-6.8*10-7mol/L。
Four, bloodstain detects
Using the experiment condition after optimization, each nucleotide concentration in bloodstain is tested, as a result sees Fig. 7.The result shows that our
Method can be used for the detection of ATP, ADP and AMP content in single-point bloodstain.
Further to verify this experimental result, result in three kinds of nucleotide mass spectral results and standard mass spectrometry database is carried out
Comparison.As shown in figure 4, result height meets cleavage of mass spectrum rule.Wherein, m/z 409 shows that ATP has lost one after rearrangement
A phosphate group (H3PO4, 99Da).
It can be seen that this method successfully eliminates the matrix effect in blood sample, and have detected wherein ATP, ADP and AMP
Content, as a result as shown in Figure 7.Each nucleotide content is shown in Table 2 in blood sample.
ATP, ADP and AMP concentration (μm ol/L in 2 blood sample of tablea)
aAs a result measurement average value ± SD three times is taken；
bBlood sample derives from two healthy volunteers of this seminar.
Five, conclusion
As known to those skilled in the art, chemiluminescence is ATP traditional quantitative methods, is limited to anion in sample and interferes,
Luciferase reagent easy in inactivation, and also there is the defects of sample preparation time is long, troublesome in poeration, make it that can not detect three seed nucleus simultaneously
Thuja acid content.And this experimental example use voltage assisted extraction decline flow control nanoESI-MS can detect simultaneously ATP, ADP and
Tri- kinds of nucleotide of AMP, and measuring stability is high, sensitivity is good, high specificity, separating step is the determinand based on paper chromatography
Extraction and enrichment is horizontal high, easy to operate.Thus illustrate, the extraction flow control nanoESI-MS that declines can become a kind of analysis people
The powerful of ATP level in source blood sample and energetic supersession access.
Meanwhile this extraction flow control nanoESI-MS method that declines constructed by the present invention is reproducible, accuracy is high, tool
There are the potentiality for high throughput analysis of samples.It is simple using microfluidic chip structure in the equipment, it can be used such as PMMA and paper
Equal low costs material is made, therefore potential is applied to by disposable bed in detection (POCT) micro-fluidic chip.In addition,
Binary channels sample detection in the micro-fluidic chip can also be used for the dynamic analysis in biosystem, and binary channels is used to analyze
The variation of chemical component can obtain more structurally sound experimental result in control group and experimental group.
Flow control nanoESI-MS analytical equipment and method in conclusion this novel extraction provided by the invention declines, tool
Have following advantage significant:
One, chromatographic paper used is cheap, used having a size of millimeter rank, can make a one-time use only；The spray of PMMA chip
Mist mouth structure is hard, and for paper spout, long service life, durable, stability is good；
Two, this analytical equipment and method have practice potentiality, using protein in the adsorbable whole blood of paper chromatography, and
It can extract in conjunction with Extraction solvent and convey determinand to electron spray spout.
Three, this method analysis speed is fast, easy to operate, examines in conjunction with the selective ion of electrospray techniques and mass spectrum software
Survey mode can eliminate matrix interference effect automatically and detect numerous chemical constituents.
Four, this analytical equipment can also be combined with chromatographic paper, by taking different solvents, selective enumeration method variety classes
Sample.And sample substrate effect can be eliminated, improve detection sensitivity, at the same provide stablize and reliable testing result (RSD <
4.8%).
Five, this analytical equipment is cheap, analysis speed is fast, on-line checking result accuracy is high, can be widely used in
In the other detection (POCT) of bed.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (10)
1. a kind of micro-fluidic chip characterized by comprising
Substrate, the sample for being provided at least one sample extraction slot and being connected to each sample extraction slot on the substrate
Microchannel；The substrate is additionally provided with spraying microchannel；Wherein, each sample microchannel is far from the sample extraction slot
End is set to the circumferential direction for the fluid spray beam that the spraying microchannel sprays so that the axis of the sample microchannel with it is described
The axis of spraying microchannel intersects.
2. micro-fluidic chip according to claim 1, which is characterized in that surround the substrate of the spraying microchannel described in
The direction for the fluid spray beam that spraying microchannel sprays is arranged in spininess, surrounds the substrate of each sample microchannel along court
It is arranged to the circumferential direction of the spraying microchannel in spininess, so that the liquid outlet of each sample microchannel is positioned at described
The circumferential direction for the fluid spray beam that spraying microchannel sprays.
3. micro-fluidic chip according to claim 2, which is characterized in that the axis of the sample microchannel with it is described spraying
Angle between the axis of microchannel is 80~110 degree.
4. micro-fluidic chip according to claim 2, which is characterized in that the liquid outlet of the sample microchannel and the spray
Vertical range between the atomizing nozzle of mist microchannel is 0.2-0.8mm；
Preferably, the vertical range between the liquid outlet and the substrate edge is 0.5-1.5mm；
Preferably, the vertical range between fluid spray beam that the liquid outlet and the spraying microchannel spray is 0.2-
0.8mm。
5. micro-fluidic chip according to claim 2, which is characterized in that the sample extraction slot and the spraying microchannel
It is arranged in parallel；
Preferably, the vertical range between the middle line of the sample extraction slot and the spraying microchannel is 6-14mm.
6. micro-fluidic chip according to claim 5, which is characterized in that there are two the sample extraction slots, described in two
Sample extraction slot is symmetrically disposed on the two sides of the spraying microchannel.
7. micro-fluidic chip according to claim 2, which is characterized in that each arc-shaped setting in sample microchannel,
And the center of circle of the sample microchannel of the arc is close to the side of the spraying microchannel.
8. a kind of extraction declines fluidic chip-mass spectrometry analytical equipment, which is characterized in that it includes electrospray ionization mass spectrum and such as
The described in any item micro-fluidic chips of claim 1-7, the atomizing nozzle of the spraying microchannel and the electrospray ionization mass spectrum into
Sample mouth is aligned and the vertical range between the atomizing nozzle and the injection port is 1-4mm.
Fluidic chip-mass spectrometry analysis method 9. a kind of extraction declines, which is characterized in that it is using as claimed in claim 8
Extraction decline fluidic chip-mass spectrometry analytical equipment comprising:
Biological sample is added dropwise in the sample extraction slot equipped with solid phase extraction material, is added in the sample extraction slot
After sample extracting solution, voltage is applied to the spraying microchannel and the sample extraction slot respectively, is made in the spraying microchannel
Spraying solvent sprayed in the form of fluid spray beam and carry the sample extracting solution sprayed from the sample microchannel
Into the mass spectrographic injection port, Mass Spectrometer Method is carried out.
Fluidic chip-mass spectrometry analysis method 10. extraction according to claim 9 declines, which is characterized in that described
The voltage that spraying microchannel applies is 3-5KV；It is 60-120V to the voltage that the sample extraction slot applies；
Preferably, the solid phase extraction material includes chromatographic paper, reverse phase silica gel and ion exchange resin.
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