CN105424581A - Microwave flow cytometer and measurement method thereof - Google Patents

Abstract

The invention provides a microwave flow cytometer. The microwave flow cytometer comprises a synchronous signal source, a testing sensor, a comparison sensor, two microwave detectors, a differential amplifier, an A/D converter and a microprocessor. The synchronous signal source is connected with the exciting end of the testing sensor and the exciting end of the comparison sensor, the testing sensor and the comparison sensor are coplanar waveguides of multiple layers of media, wherein conductor bands with two wide ends and the narrow middle are embedded into the coplanar waveguides, U-type liquid tubes are arranged on the waveguides to serve as detection channels, the two microwave detectors are connected with the output ends of the two coplanar waveguides respectively, and the differential amplifier is connected with the microprocessor through the A/D converter. The invention further provides a method of utilizing the microwave flow cytometer for measuring a cell dielectric constant. The method includes the steps of correcting, preparing, testing and calculating. Errors caused by changes of the dielectric constant of a culture solution are reduced, using of a vector network analyzer and other expensive equipment is avoided, a modularized design conception is adopted, functions of all modules are clear, and maintaining is facilitated at the later stage.

Description

Microwave flow cytometer and measuring method thereof

Technical field

The present invention relates to a kind of method utilizing the microwave flow cytometer of cell dielectric constant in Microwave Net scattering parameter inverting suspension and utilize described cell instrument measurement cell dielectric constant, belong to microwave measurement field.

Background technology

Flow cytometer is detecting instrument cell being carried out to automatic analysis and sorting of the advanced person grown up early 1970s.It can a series of important biophysics of the cell dispersion that floats on a liquid of Quick Measurement, storage, display, the characteristic parameter of biological chemistry aspect, and according to the parameter ranges of preliminary election, the cell subsets of specifying therefrom can be sorted out.Flow cytometer mainly contains four part compositions, and they are: flow chamber and liquid fluid system; Lasing light emitter and optical system; Photoelectric tube and detection system; Computing machine and analytic system.The general work principle of flow cytometer: flow cytometer can carry out measuring multiple parameters simultaneously, information spinner will from specific fluorescence signal and non-fluorescence scattered signal.Measurement is carried out in detection zone, and so-called detection zone is exactly that the liquid a fluid stream of illuminating laser beam and squit hole intersects vertically a little.When the individual cells of liquid stream central authorities is by measurement zone, be subject to the whole space scattering light that laser irradiation can be 2 π to solid angle, the wavelength of scattered light is identical with the wavelength of incident light.The intensity of scattered light and space distribution thereof and cell size, form, plasma membrane and cell interior structure closely related because these biological parameters are relevant with the optical characteristics such as reflection, refraction of cell to light again.Flow cytometry is an important technology of analysis of cells, for one of cytology research means, can carry out up to up to ten thousand chromosomal analyses per second to cell and crganelle and biomacromolecule, and multi parameter analysis and the cell sorting of cell can be carried out, this quantitative analysis tech measuring cell with the type of flow, is widely used in the field such as cell biology, medical science at present.Flow cytometry integrates sheath fluid principle, liquid stream drive system, cell dyeing, laser technology and computer technology.Wherein liquid stream drive system is one of core technology of flow cytometry, normal employing sheath fluid wrap dynamic formula.Single cell suspension is held the hole by the certain pore size in flow chamber by sheath fluid stream in cell flow chamber, detection zone is at the center in this hole, cell therewith laser vertical intersects, under the constraint of sheath fluid stream, cell arranges successively by laser detection district in single file, the cell that flow cytometer is surveyed requires to be arranged in single-row, cell suspension is processed under this type of drive, very large to the consumption of sheath fluid stream.The core component flow chamber of flow cytometer has cross seal enclosed, open, the shape such as air injection type, top end opening formula, and the complex structure of these flow chambers, manufacture difficulty is large.

In traditional flow cytometer, often adopt fluoroscopic examination and light scattering principle, cause technical sophistication and expensive, bulky, standing charges are high, especially flow cytometer in the market substantially monopolize by the major company of European and American developed countries, be difficult to common lab promote and use.

Summary of the invention

The object of the invention is to: solve conventional flow cytometer technical sophistication, expensive, bulky problem; Start with from Microwave Measurement Technique field, by directly measuring Microwave Net scattering parameter, inverting cell dielectric constant, and then reach the object of analysis of cells structural property.

For achieving the above object, the invention provides a kind of microwave flow cytometer, comprise: source of synchronising signal, testing sensor, control sensor, two microwave detectors, differential amplifier, A/D converter, microprocessor, the excitation end of described source of synchronising signal connecting test sensor and control sensor, described testing sensor and control sensor are the co-planar waveguide of the multilayered medium being embedded with the narrow conductor belt in wide centre, two ends, described waveguide is settled U-shaped liquid line as sense channel, described two microwave detectors are connected with the output terminal of described two co-planar waveguides respectively, signal from two sensors is delivered to differential amplifier, described differential amplifier is connected with microprocessor by an A/D converter.

The narrow design in wide centre, conductor belt two ends had both taken into account the requirement of port Impedance 50 ohm, reached again the object improving measuring accuracy.Because centre is test section, centre is narrow is more concentrated in order to fetter middle electromagnetic field;

As optimal way, the co-planar waveguide of described multilayered medium comprises: the conductor belt comprising wide portion, two ends and middle narrow portion hit exactly above the medium substrate above the ground plate of bottom, ground plate, medium substrate, the copper coin of described conductor belt both sides, be close to the bonding film above described conductor belt, the glass plate above bonding film, described conductor belt and copper coin are positioned at same plane and both are separated with gap, U-shaped through hole is provided with bottom glass plate, settle U-shaped liquid line in U-shaped through hole, the sweep in the middle of U-shaped through hole is positioned at the top of narrow portion in the middle of center conductor band.

As optimal way, the gap-fill phosphate buffer PBS in the middle of conductor belt between narrow portion and copper coin, the gap-fill air between wide portion, conductor belt two ends and copper coin.Central filler high dielectric material PBS be because coplanar waveguide structure to measure the material precision of high dielectric higher, and nutrient solution is low dielectric, so can increase total specific inductive capacity of test zone like this, increases accuracy of measurement.

As optimal way, the long 25.4mm of co-planar waveguide of described multilayered medium, wide 12.7mm, wide portion long 15mm, the wide 1.92mm respectively at described conductor belt two ends, the long 5mm of middle narrow portion, wide 0.4mm, wide portion is connected by straight line with narrow portion.

As optimal way, the gap between wide portion, conductor belt two ends and copper coin is 0.15mm, and the gap in the middle of conductor belt between narrow portion and copper coin is 0.06mm.

Above-mentioned size is through that optimization Simulation repeatedly finally determines, measuring accuracy is the highest.

The present invention also provides a kind of method utilizing above-mentioned microwave measured by flow cytometry cell dielectric constant, comprises the steps:

(1) calibrate: when using this microwave flow cytometer first, start described source of synchronising signal, sine wave output signal, feedback signal is sent to microwave detector by described two sensors, rectified signal is sent to differential amplifier by described microwave detector, the amplitude of described differential amplifier to two rectified signal differences is modulated and amplifies, and send to A/D converter, described A/D converter sends to microprocessor after the signal received is changed into digital signal, the signal received is calculated calibration data by corresponding program and is stored in internal register by described microprocessor, close source of synchronising signal,

(2) prepare: start in the U-shaped liquid line of described testing sensor, inject the suspension containing cell to be detected, in the U-shaped liquid line of control sensor, inject pure nutrient solution, be adjusted to flow rate of liquid consistent, ensure synchronously to contrast measurement;

(3) test: open source of synchronising signal, sine wave signal is sent to the excitation port of two sensors, described two microwave detectors detect the feedback signal of respective sensor output terminal, and rectified signal is sent to differential amplifier, the amplitude of described differential amplifier to two rectified signal differences is modulated and amplifies, and sending to A/D converter, described A/D converter sends to microprocessor after the signal received is changed into digital signal;

(4) calculate: embedded corresponding program in described microprocessor, the digital signal received is carried out Inversion Calculation, obtains cell dielectric constant, finally by corresponding software analysis of cells structural property.

In co-planar waveguide device, the relational expression of scattering parameter and propagation constant and specific inductive capacity is as follows:

$A=\frac{(1+S_{11})(1-S_{22})+S_{12}{S}_{21}}{2S_{21}}---\left(1\right)$

$D=\frac{(1-S_{11})(1+S_{22})+S_{12}{S}_{21}}{2S_{21}}---\left(2\right)$

$\mathrm{\γ}=\frac{1}{L}\·\ln \[\frac{(A+D)\±\sqrt{{(A+D)}^2-4}}{2}\]---\left(3\right)$

$\mathrm{\ϵ}={\left(\frac{c\·\mathrm{\γ}}{2\mathrm{\π}f}\right)}^2---\left(4\right)$

Wherein: S ₁₁for excitation end reflection coefficient, S ₂₂for output terminal reflection coefficient, S ₁₂for output terminal is to the transmission coefficient of excitation end, S ₂₁for excitation end is to the transmission coefficient of output terminal, γ is propagation constant, and L is the length of co-planar waveguide device, and ε is specific inductive capacity, and c is the light velocity, and f is frequency of operation.

Utilize the relational expression of interfacial polarization theory calculate cell dielectric constant as follows:

${\mathrm{\ϵ}}_{sample}={\mathrm{\ϵ}}_{fluid}(1+n\frac{\mathrm{\φ}\frac{(1/n)({\mathrm{\ϵ}}_{cell}-{\mathrm{\ϵ}}_{fluid})}{{\mathrm{\ϵ}}_{fluid}+(1/n)({\mathrm{\ϵ}}_{cell}-{\mathrm{\ϵ}}_{fluid})}}{1-\mathrm{\φ}\frac{(1/n)({\mathrm{\ϵ}}_{cell}-{\mathrm{\ϵ}}_{fluid})}{{\mathrm{\ϵ}}_{fluid}+(1/n)({\mathrm{\ϵ}}_{cell}-{\mathrm{\ϵ}}_{fluid})}})$

Wherein: φ is cell volume integration, n is shape factor, ε _samplefor the specific inductive capacity of cell suspension to be measured, ε _cellfor cell dielectric constant, ε _fluidfor the specific inductive capacity of nutrient solution.

Beneficial effect of the present invention:

(1) sense channel of testing sensor and control sensor injects cell suspension and pure nutrient solution respectively, and synchro measure real time calibration specific inductive capacity, reduces the error that nutrient solution change in dielectric constant is brought.

(2) multiport circuit and differential amplification principle is adopted, scattering parameter is the ratio of incident, reflection and transmitting energy in essence, even if because these ratios also have very high precision in microwave frequency, and because differential amplifier has high cmrr, can be decayed undesired signal effectively, and therefore the present invention has very high resolution.

(3) avoid the use of the expensive device such as vector network analyzer, sensor bulk is small and exquisite, and circuit chip size belongs to grade, and all there is sale in market, with low cost.

(4) adopt modular design concept, each functions of modules is clear, is easy to later maintenance service work.

Accompanying drawing explanation

Fig. 1 is described microwave flow cytometer structural representation

Fig. 2 is the co-planar waveguide stereographic map of described multilayered medium

Fig. 3 is the main pseudosection of co-planar waveguide of described multilayered medium

Fig. 4 is the co-planar waveguide top plan view of described multilayered medium

Fig. 5 be described microwave flow cytometer for detecting time schematic diagram

Wherein, 1-source of synchronising signal, 2-testing sensor, 3-control sensor, 4-microwave detector, 5-differential amplifier, 6-A/D converter, 7-microprocessor, the co-planar waveguide of 8-multilayered medium, 9-glass plate, 10-bonding film, 11-U type liquid line, 12-conductor belt, 13-medium substrate, 14-ground plate, 15-phosphate buffer PBS, 16-gap, 17-copper coin.

Embodiment

Below by way of specific instantiation, embodiments of the present invention are described, those skilled in the art the content disclosed by this instructions can understand other advantages of the present invention and effect easily.The present invention can also be implemented or be applied by embodiments different in addition, and the every details in this instructions also can based on different viewpoints and application, carries out various modification or change not deviating under spirit of the present invention.

Refer to Fig. 1 and Fig. 2, the invention provides a kind of microwave flow cytometer, comprise: source of synchronising signal 1, testing sensor 2, control sensor 3, two microwave detectors 4, differential amplifier 5, A/D converter 6, microprocessor 7, the excitation end of described source of synchronising signal 1 connecting test sensor 2 and control sensor 3, described testing sensor and control sensor are the co-planar waveguide 8 of the multilayered medium being embedded with the narrow conductor belt in wide centre, two ends, described waveguide is settled U-shaped liquid line 11 as sense channel, described two microwave detectors are connected with the output terminal of described two co-planar waveguides respectively, signal from two sensors is delivered to differential amplifier, described differential amplifier 5 is connected with microprocessor 7 by an A/D converter 6.

Conductor belt two ends are wide is requirement in order to arrive port Impedance 50 ohm, centre is test section, middle narrow be more concentrate to fetter middle electromagnetic field (electromagnetic field be in the vertical be mainly distributed in conductor belt both sides gap in the middle of), to improve the precision of measurement;

The co-planar waveguide of described multilayered medium comprises: the ground plate 14 of the brass material making of bottom, medium substrate 13 above ground plate, the conductor belt 12 comprising wide portion, two ends and middle narrow portion hit exactly above medium substrate, the copper coin 17 of described conductor belt both sides, be close to the bonding film 10 above described conductor belt, glass plate 9 above bonding film 10, described conductor belt and copper coin are positioned at same plane and both are separated with gap 16, U-shaped through hole is provided with bottom glass plate, U-shaped liquid line 11 is settled in U-shaped through hole, sweep in the middle of U-shaped through hole is positioned at the top of narrow portion in the middle of center conductor band.

Described testing sensor 2 is identical with the structure of control sensor 3, and purposes is different, and the U-shaped liquid line 11 of testing sensor 2 is used as the passage of cell suspension, and the U-shaped liquid line 11 of control sensor 3 is used as the passage of pure nutrient solution.

Gap-fill phosphate buffer (PBS) 15 in the middle of conductor belt between narrow portion and copper coin, the gap-fill air between wide portion, conductor belt two ends and copper coin.Central filler high dielectric material PBS be because coplanar waveguide structure to measure the material precision of high dielectric higher, and nutrient solution is low dielectric, so can increase total specific inductive capacity of test zone like this, increases accuracy of measurement.

The long 25.4mm of co-planar waveguide of described multilayered medium, wide 12.7mm, wide portion long 15mm, the wide 1.92mm respectively at described conductor belt two ends, the long 5mm of middle narrow portion, wide 0.4mm, wide portion is connected by straight line with narrow portion.

Gap between wide portion, conductor belt two ends and copper coin is 0.15mm, and the gap in the middle of conductor belt between narrow portion and copper coin is 0.06mm.

The present invention also provides a kind of method utilizing above-mentioned microwave measured by flow cytometry cell dielectric constant, comprises the steps:

(1) calibrate: when using this microwave flow cytometer first, start described source of synchronising signal, sine wave output signal, feedback signal is sent to microwave detector by described two sensors, rectified signal is sent to differential amplifier by described microwave detector, the amplitude of described differential amplifier to two rectified signal differences is modulated and amplifies, and send to A/D converter, described A/D converter sends to microprocessor after the signal received is changed into digital signal, the signal received is calculated calibration data by corresponding program and is stored in internal register by described microprocessor, close source of synchronising signal,

(2) prepare: start in the U-shaped liquid line of described testing sensor, inject the suspension containing cell to be detected, in the U-shaped liquid line of control sensor, inject pure nutrient solution, be adjusted to flow rate of liquid consistent, ensure synchronously to contrast measurement;

(3) test: open source of synchronising signal, sine wave signal is sent to the excitation port of two sensors, described two microwave detectors detect the feedback signal of respective sensor output terminal, and rectified signal is sent to differential amplifier, the amplitude of described differential amplifier to two rectified signal differences is modulated and amplifies, and sending to A/D converter, described A/D converter sends to microprocessor after the signal received is changed into digital signal;

(4) calculate: embedded corresponding program in described microprocessor, the digital signal received is carried out Inversion Calculation, obtains cell dielectric constant, finally by corresponding software analysis of cells structural property.

In co-planar waveguide device, the relational expression of scattering parameter and propagation constant and specific inductive capacity is as follows:

$A=\frac{(1+S_{11})(1-S_{22})+S_{12}{S}_{21}}{2S_{21}}---\left(1\right)$

$D=\frac{(1-S_{11})(1+S_{22})+S_{12}{S}_{21}}{2S_{21}}---\left(2\right)$

$\mathrm{\γ}=\frac{1}{L}\·\ln \[\frac{(A+D)\±\sqrt{{(A+D)}^2-4}}{2}\]---\left(3\right)$

$\mathrm{\ϵ}={\left(\frac{c\·\mathrm{\γ}}{2\mathrm{\π}f}\right)}^2---\left(4\right)$

Wherein: S ₁₁for excitation end reflection coefficient, S ₂₂for output terminal reflection coefficient, S ₁₂for output terminal is to the transmission coefficient of excitation end, S ₂₁for excitation end is to the transmission coefficient of output terminal, γ is propagation constant, and L is the length of co-planar waveguide device, and ε is specific inductive capacity, and c is the light velocity, and f is frequency of operation.

Utilize the relational expression of interfacial polarization theory calculate cell dielectric constant as follows:

${\mathrm{\ϵ}}_{sample}={\mathrm{\ϵ}}_{fluid}(1+n\frac{\mathrm{\φ}\frac{(1/n)({\mathrm{\ϵ}}_{cell}-{\mathrm{\ϵ}}_{fluid})}{{\mathrm{\ϵ}}_{fluid}+(1/n)({\mathrm{\ϵ}}_{cell}-{\mathrm{\ϵ}}_{fluid})}}{1-\mathrm{\φ}\frac{(1/n)({\mathrm{\ϵ}}_{cell}-{\mathrm{\ϵ}}_{fluid})}{{\mathrm{\ϵ}}_{fluid}+(1/n)({\mathrm{\ϵ}}_{cell}-{\mathrm{\ϵ}}_{fluid})}})$

Wherein: φ is cell volume integration, n is shape factor, ε _samplefor the specific inductive capacity of cell suspension to be measured, ε _cellfor cell dielectric constant, ε _fluidfor the specific inductive capacity of nutrient solution.

Above-described embodiment is illustrative principle of the present invention and effect thereof only, but not for limiting the present invention.Any person skilled in the art scholar all without prejudice under spirit of the present invention and category, can modify above-described embodiment or changes.Therefore, such as have in art usually know the knowledgeable do not depart from complete under disclosed spirit and technological thought all equivalence modify or change, must be contained by claim of the present invention.

Claims (6)

1. a microwave flow cytometer, it is characterized in that, comprise: source of synchronising signal, testing sensor, control sensor, two microwave detectors, differential amplifier, A/D converter, microprocessor, the excitation end of described source of synchronising signal connecting test sensor and control sensor, described testing sensor and control sensor are the co-planar waveguide of the multilayered medium being embedded with the narrow conductor belt in wide centre, two ends, described waveguide is settled U-shaped liquid line as sense channel, described two microwave detectors are connected with the output terminal of described two co-planar waveguides respectively, signal from two sensors is delivered to differential amplifier, described differential amplifier is connected with microprocessor by an A/D converter.

2. microwave flow cytometer according to claim 1, it is characterized in that: the co-planar waveguide of described multilayered medium comprises: the ground plate of bottom, medium substrate above ground plate, the conductor belt comprising wide portion, two ends and middle narrow portion hit exactly above medium substrate, the copper coin of described conductor belt both sides, be close to the bonding film above described conductor belt, glass plate above bonding film, described conductor belt and copper coin are positioned at same plane and both are separated with gap, U-shaped through hole is provided with bottom glass plate, U-shaped liquid line is settled in U-shaped through hole, sweep in the middle of U-shaped through hole is positioned at the top of narrow portion in the middle of center conductor band.

3. microwave flow cytometer according to claim 2, is characterized in that: the gap-fill phosphate buffer PBS in the middle of conductor belt between narrow portion and copper coin, the gap-fill air between wide portion, conductor belt two ends and copper coin.

4. microwave flow cytometer according to claim 2, it is characterized in that: the long 25.4mm of co-planar waveguide of described multilayered medium, wide 12.7mm, wide portion long 15mm, the wide 1.92mm respectively at described conductor belt two ends, the long 5mm of middle narrow portion, wide 0.4mm, wide portion is connected by straight line with narrow portion.

5. microwave flow cytometer according to claim 2, is characterized in that: the gap between wide portion, conductor belt two ends and copper coin is 0.15mm, and the gap in the middle of conductor belt between narrow portion and copper coin is 0.06mm.

6. utilize the method for the microwave measured by flow cytometry cell dielectric constant described in claim 1 to 5 any one, it is characterized in that comprising the steps:

(1) calibrate: when using this microwave flow cytometer first, start described source of synchronising signal, sine wave output signal, feedback signal is sent to microwave detector by described two sensors, rectified signal is sent to differential amplifier by described microwave detector, the amplitude of described differential amplifier to two rectified signal differences is modulated and amplifies, and send to A/D converter, described A/D converter sends to microprocessor after the signal received is changed into digital signal, the signal received is calculated calibration data by corresponding program and is stored in internal register by described microprocessor, close source of synchronising signal,

(2) prepare: start in the U-shaped liquid line of described testing sensor, inject the suspension containing cell to be detected, in the U-shaped liquid line of control sensor, inject pure nutrient solution, be adjusted to flow rate of liquid consistent, ensure synchronously to contrast measurement;

(3) test: open source of synchronising signal, sine wave signal is sent to the excitation port of two sensors, described two microwave detectors detect the feedback signal of respective sensor output terminal, and rectified signal is sent to differential amplifier, the amplitude of described differential amplifier to two rectified signal differences is modulated and amplifies, and sending to A/D converter, described A/D converter sends to microprocessor after the signal received is changed into digital signal;

(4) calculate: embedded corresponding program in described microprocessor, the digital signal received is carried out Inversion Calculation, obtains cell dielectric constant, finally by corresponding software analysis of cells structural property.