CN101762440B - Electric rotary detecting method for pharmacodynamics research - Google Patents

Abstract

The invention relates to an electric rotary detecting method for pharmacodynamics research. The method comprises the following steps of: (1) filling liquid medium with known conductivity into an electric rotary measuring tank, and keeping perfusion; (2) adding a sample to be measured into the electric rotary measuring tank; (3) enabling the sample to be measured to generate the electric rotary action by applying a uniform rotary electric field, and observing the single sample particle in the center of the electric rotary measuring tank; (4) obtaining the information related to the electric rotation of the sample particles, and making an electric rotary spectrogram. Due to the non-intrusive characteristic, the invention not only can maintain the integrity and the activity of the biological particle in the analyzing process, and use the biological particles to do further integral or destructive analysis, but also can use the same batch of biological particles to complete the whole pharmacodynamics research process, and increase the sensitivity of the pharmacodynamics statistical detection, thereby being more beneficial to obtaining the information of pharmacodynamics research.

Description

The electric rotary detecting method that is used for pharmacodynamic study

Technical field

The present invention relates to a kind of electric rotary detecting method of biologic grain, particularly a kind of method that electric rotary detecting method is applied to pharmacodynamic study.

Background technology

Pharmacodynamics mainly study medicine effect and the mechanism of action, medicine bad reaction and influence pharmaceutically-active factor etc.Its research range comprises the dose-effect relationship (relation of dosage and quantitative response/qualitative response) of the effect of studying medicine and pharmacodynamics effect, pharmaceutically-active duality (therapeutic action and bad reaction), medicine and to the evaluation of drug safety.The method of pharmacodynamic study is a lot, different and different along with disposition of drug object and medicine.Pharmacodynamic analysis with antineoplastic is an example below, and the application of mensuration system in medicine effect research and drug screening that this patent is narrated is described.

Malignant tumour is a kind of disease of serious harm human health, and the mankind are are constantly researching and developing new medicine to suppress the growth of malignant tumour effectively always for many years, make the screening of antineoplastic become an important field of research.It is an important channel of assessment chemotherapeutics drug effect that thereby the detection antineoplastic is made prediction to the susceptibility of medicine to the influence of growth of tumour cell metabolism.

Intervention property dyeing experimental techniques such as antineoplastic in-vitro screening method commonly used at present such as MTT, SRB, CCK-8, crystal violet, placenta indigo plant exist toxicity, complex operation and need the problems such as biological sample of large sample amount.In addition; In case a collection of experiment has been adopted MTT or similarly intervention property method effect with cell sample; Then can when detecting the cell proliferation degree, cause irreversible damaging influence by pair cell, promptly cell thoroughly changes being colored back physiological status and physicochemical property, so that continues the effect of this this investigation of lot sample medicine of use in can't process afterwards; Be not useable for further analyzing and research, and must adopt new sample.Like this, because the intrinsic diversity of biological specimen, when in drug effect, adopting different samples, the variance that in statistical test, is used for comparison has just added the difference variance between sample is criticized, and makes the effect of check difference reduce.

Electricity rotation is a kind of non-destructive, can be used for the new bio physics and the electrobiophysics measuring technique of unicellular measurement; Be a kind of very sensitive method that detects cellular physiological activity, the electrodynamics of using it for pair cell and other biological particle characterizes with optionally controlling and becomes an active research field.Use this technical operation easy; Applying flexible; Not only can overcome mtt assay etc. is the shortcoming that exists in the relevant measurement method on basis with dyeing; Also pharmaceutically-active whole process is monitored with sample with a collection of experiment, be suitable for studying miscellaneous all kinds of biologic grain type, become the important technology that present biological particle is controlled the field owing to thereby the characteristics of its non-invasi are available.Adopted electric rotation technique to observe to comprise the variable grain type of virus, prokaryotic, eukaryotic and part inorganic matter.

At present, extensive day by day for the research of cell dielectric properties under radio frequency, and produced a lot of new practical applications, comprise the on-line determination of biomass and the technology that usefulness is separated, controlled and characterize to the new electrodynamics to individual cells.Although domestic and international research person does not still report electric rotary detecting method is applied to pharmacodynamic study having obtained certain achievement aspect the dielectric property of the electric rotation technique exploration of utilization biologic grain.The mensuration system that adopts this patent to narrate carries out the pharmacodynamic study of medicine; Can be to the variation that detects its electricity rotation spectrum under drug effect with a collection of experimental subjects; Obtain the drug action curve in the whole drug effect process, thereby the efficient of pharmacodynamic study is improved.

Summary of the invention

The objective of the invention is to electric rotary detecting method is applied in the pharmacodynamic study; Can rotate the variation on the spectrogram by detection of biological particle electricity after receiving drug effect; Thereby the susceptibility to medicine is made prediction, and has technical matterss such as toxicity and complex operation to solve the detection that exists in the above-mentioned background technology.

A kind of electric rotary detecting method that is used for pharmacodynamic study comprises the steps:

(1) in electric wheel measuring pond, is full of the liquid medium of known conductivity, and keeps perfusion;

(2) the biological sample particle to be measured before and after the drug effect is joined in the electric wheel measuring pond;

(3) make sample particle that electric circling behavior take place through applying uniform rotating electric field, observe the single sample particle that is positioned at central area, electric wheel measuring pond;

(4) obtain the information of relevant sample particle electricity rotation, and make electricity rotation spectrogram.

Wherein, the perfusion rate in the step (1) is set at 0.002mL/min to 4mL/min.

Wherein, said perfusion rate is 0.16mL/min.

Wherein, apply uniform rotating electric field in the step (3) for applying uniform sine voltage signal on equally distributed a plurality of microelectrodes around the electric wheel measuring pond.

Wherein, this microelectrode is four, and this sine voltage signal is followed successively by u ₁=U ₀Cos (ω t), u ₂=U ₀Cos (ω t+90 °), u ₃=U ₀Cos (ω t+180 °) and u ₄=U ₀The sine voltage signal of cos (ω t+270 °).

Wherein, said microelectrode is a platinum electrode.

Wherein, this central area is that electric wheel measuring pond central diameter is the border circular areas of 0.3x, and wherein x is the distance between each microelectrode tip.

Wherein, only the sample particle that separates at least three diameter distances each other being done the electricity rotation in the step (3) observes.

Wherein, this biological sample particle is virus, prokaryotic or eukaryotic.

The present invention utilizes electric pivot analysis method to carry out pharmacodynamic study, and it is easy and simple to handle, and step such as need not dye is not applied to the toxicity organic reagent yet.Can be used for the different physiological statuss of dynamic monitoring cell, be used for numerous research fields such as drug screening, cell cycle and Apoptosis and cell physiological state monitoring.

A kind of electric rotary detecting method that is used for pharmacodynamic study provided by the invention; Can provide information about single individuality in the biologic grain colony; And the required sample volume of this miniaturized devices is than much less required in the routine monitoring method; Promptly do not need the large number of biological particle as research object; Only use very small amount of sample and reagent and detection time seldom just can accomplish whole testing process, make it become the ideal tools of a small amount of biosome of research, can be used to carry out fast and the biological diagnosis of low consumption.Itself can be used as breadboard sensing and analytic unit on a kind of chip, also can combine, be used for biologic grain is carried out express-analysis with other electrodynamics technology as dielectrophoresis and row ripple dielectrophoresis that kind.

Another characteristics that are superior to other routine techniquess of electricity rotation technique are the characteristics from its non-invasi; Make its not only can in analytic process, keep biologic grain complete, with the experiment before the same situation and the activity of state, make this biologic grain sample can be used to carry out further integral body or destructive analysis; And adopt the enough same batch of biologic grains of this method ability to accomplish the process of whole pharmacodynamic study, increase the sensitivity of drug effect statistical test, thereby more help obtaining the information of drug efficacy study.In addition, it still is a kind of general analytical technology, can be used for observing many dissimilar organisms.And owing to used perfusion device, also can detect and study the situations of change of biologic grain under drug effect such as cell in real time, promptly in the pharmacodynamics check, realize real-time administration, have very big advantage and potential use as a kind of new drug screening method.

Description of drawings

Fig. 1 a is the used detection system structure vertical view of electric rotary detecting method that is used for pharmacodynamic study provided by the invention;

Fig. 1 b is used for the detection system perspective view of the electric rotary detecting method of pharmacodynamic study for the present invention;

Fig. 2 is the electricity rotation spectrogram of the HeLa cell in the glycocoll/PBS medium that is in the different electric conductance;

Fig. 3 is the electricity rotation spectrogram (in conductivity is in glycocoll/PBS medium of 600 μ S/cm, and cis-platinum concentration is 15 μ g/mL) of answering the HeLa cell behind the cis-platinum effect different time that the electricity consumption rotary detecting method obtains;

Fig. 4 is the electricity rotation spectrogram of answering the HeLa cell after the variable concentrations cis-platinum effect that the electricity consumption rotary detecting method obtains (is in glycocoll/PBS medium of 600 μ S/cm in conductivity, cis-platinum action time be 48h);

Fig. 5 for the variable concentrations cis-platinum that records with mtt assay at the growth inhibited curve of different time to the HeLa cell.

Description of reference numerals

1-electricity wheel measuring pond; The 2-microelectrode; The 3-substrate; The 4-cofferdam; The 5-water inlet pipe; The 6-rising pipe.

Embodiment

For characteristics of the present invention can be understood better, below will enumerate preferred embodiment and combine accompanying drawing to be elaborated.

The electric rotary detecting method that is used for pharmacodynamic study of the present invention, its principle is:

A biologic grain is understood generating electrodesization in the time spent of doing that receives external electric field, and polarizability is used for weighing the complexity of a molecule CHARGE DISTRIBUTION of external electrical field distortion.The polarizability of living cells and their composition, form and phenotype (observable physics or biochemical character on the organism are by genetic constitution or environmental impact decision) have much relations, with the frequency that applies electric field very strong relation are arranged also.The electricity rotation technique is the part in the AC electrical mechanical technology; According to electric spin theory; When a biologic grain is suspended in a certain amount of fluid, receives uniform rotating electric field by one group of microelectrode that has applied alternating electric field and do the time spent, the electric dipole moment that can obtain to induce.This dipole moment and electric field interact, and can produce moment, and this biologic grain is rotated.The sensitivity function of the dielectric properties (conductivity and specific inductive capacity) that the speed of rotation and direction are biologic grain and its residing suspending medium.Speed through measuring electricity rotation is rotated spectrogram with the electricity that the relation of apply between the rotating electric field frequency can obtain biologic grain; So as to knowing its dielectric property; Realization is to the sign of biologic grain, thereby sees clearly the drug action to it of vigor, physiological status and the medicine of this biologic grain.

After cancer therapy drug is applied to tumour cell, make the dielectric properties of each aspect of cell change, and on electricity rotation spectrum, cause the change that can observe.Specifically, the dielectric properties of biologic grain is relevant with its physicochemical property, and the film of an active particle can let ion and other kinds matter selective ground pass through; And when cell was deathward, the active transport mechanism of cell was lost gradually, and transmembrane gradient dissipates thereupon, and change has all taken place for inner CHARGE DISTRIBUTION and dielectric parameter; Cell can be more and more freely exchanges material with the suspending medium of outside, causes the conductivity of nonactive cell interior to reduce, thereby causes the significant difference of electricity rotation spectrogram.In addition, also there is difference between active and active its specific inductive capacity of cell that descends.Above mechanism description electric rotation spectrogram shape variation be because due to the physiological status difference really, thereby a kind of simple and external pharmacodynamic study method fast are provided.

For a spheric grain that is in the electric field that intensity is E, induce dipole moment

can be expressed from the next:

$\stackrel{\→}{m}\left(\mathrm{\ω}\right)=4\mathrm{\π}{\mathrm{\ϵ}}_{m}f({\mathrm{\ϵ}}_p^{*},{\mathrm{\ϵ}}_m^{*})R^3{\stackrel{\→}{E}}_{\mathrm{RMS}}---\left(1\right)$

Wherein R is the radius of particle, ε _mBe the specific inductive capacity of medium, f (ε _p ^*, ε _m ^*) be the Clausius-Mossotti factor:

$f({\mathrm{\ϵ}}_p^{*},{\mathrm{\ϵ}}_m^{*})=\frac{{\mathrm{\ϵ}}_p^{*}-{\mathrm{\ϵ}}_m^{*}}{{\mathrm{\ϵ}}_p^{*}+{2\mathrm{\ϵ}}_m^{*}}---\left(2\right)$

Parameter ε ^* _pAnd ε ^* _mRepresent the complex permittivity of particle and medium respectively, shape is:

${\mathrm{\ϵ}}_i^{*}={\mathrm{\ϵ}}_i-j{\mathrm{\σ}}_i/\mathrm{\ω}---\left(3\right)$

Wherein subscript i represents particle (p), medium (m) respectively, and σ is a conductivity, j=(1) ^1/2

The electricity rotation of viewed particle is the result of induce dipole moment and rotating electric field interphase interaction.Depend on following relation arranged between moment of torsion (Γ (ω)) and the induce dipole moment (seeing formula 1) of electric field frequency:

$\mathrm{\Γ}\left(\mathrm{\ω}\right)=\stackrel{\→}{m}\left(\mathrm{\ω}\right)\stackrel{\→}{E}---\left(4\right)$

Observed electricity rotation is by the imaginary part decision of Clausius-Mossotti factor, and it is all relevant with the relative conductivity and the specific inductive capacity of suspending medium and particle, also relevant with electric field intensity that applies and frequency thereof.As Im (f (ε ^* _p, ε ^* _m))＞0 o'clock (seeing formula 2 and 5), the electric field that the induced dipole hysteresis applies is less than half period, so just there is anti-driving torque to affact on the particle.On the contrary, as Im (f (ε ^* _p, ε ^* _m))＜0 o'clock, the induced dipole electric field apply that lags behind surpasses half period, so just have along torsional interaction to particle.

The essence of observed rotational speed (R (ω)) is moment of torsion and the result of the viscous force balance in a particular medium who affacts on the particle, can be defined by following formula:

$R\left(\mathrm{\&omega;}\right)=-\frac{{\mathrm{\&epsiv;}}_{\mathrm{<figure-callout\; id="2"\; label="m\; E"\; filenames="H2009102444595E00011.png"\; state="\{\{state\}\}">m</figure-callout>}}{E}^{}{}^2}{2\mathrm{\&eta;}}\mathrm{Im}\left(f({\mathrm{\&epsiv;}}_p^{*},{\mathrm{\&epsiv;}}_m^{*})\right)---\left(5\right)$

Wherein η is the dynamic viscous coefficient of medium.Because being the form with phase shift voltage, electric field applies, so R (ω) also can be expressed as:

$R\left(\mathrm{\&omega;}\right)=-\frac{{\mathrm{\&epsiv;}}_m{k}^2{\mathrm{<figure-callout\; id="2"\; label="V"\; filenames="H2009102444595E00011.png"\; state="\{\{state\}\}">V</figure-callout>}}^2}{2\mathrm{\&eta;}}\mathrm{Im}\left(f({\mathrm{\&epsiv;}}_p^{*},{\mathrm{\&epsiv;}}_m^{*})\right)---\left(6\right)$

Wherein k be one with relevant proportionality constants such as the geometric configuration of applying RMS voltage (V), electrode and particle position.

Concrete; The structure of detection system that realizes the electric rotary detecting method that is used for pharmacodynamic study of the present invention is shown in Fig. 1 a and Fig. 1 b; The substrate 3 that comprises the microelectrode circuit board, the material of this substrate 3 can be for tygon, polypropylene, PVC, polystyrene, acrylonitrile-butadiene-styrene (ABS) co-polymer, polyamide, polysulfones, polyoxymethylene, polycarbonate, Noryl, methylpentene polymer, ethenol copolymers, gather that amino span comes acid amides, gathers triazine, crosslinked polyimide, polyethersulfone, polyphenylene sulfide, polyimide, polyetheretherketone, fluoroplastic and organosilicon material one of them.

The central authorities of this substrate 3 are provided with cofferdam 4 in case the solution stopping body flows out.Cofferdam 4 central authorities are electric wheel measuring pond 1, and the diameter range in electric wheel measuring pond 1 is 500 μ m to 2mm, and particulate to be measured is positioned at this electricity wheel measuring pond 1.Be evenly distributed with a plurality of microelectrodes 2 around the electricity wheel measuring pond 1, microelectrode 2 is four in the present embodiment, diameter 100 μ m, and this microelectrode 2 is platinum electrode preferably, and the spacing between each microelectrode 2 is 100 μ m to 1mm.

On cofferdam 4, be provided with perfusion device, this perfusion device comprises that perfusion with kapillary water inlet pipe 5 and rising pipe 6, reaches the peristaltic pump that is connected with rising pipe 6 with water inlet pipe 5, and the internal diameter of kapillary water inlet pipe and water outlet pipe is 250 microns.Make medium solution circulate in electric wheel measuring pond 1 through this perfusion device, reach the purpose of cooling off electric wheel measuring pond 1 interior medium and keeping its ingredient stability, avoid simultaneously because the increase of the dielectric conductance rate that water evaporates causes.

Known that electrolyte solution heating under electric field action not only can pair cell exerts an influence even makes cell because of overheated and deadly, concentration and the viscosity that also can change medium makes and has non-pharmaceutically-active effect among the result who records.Make medium solution circulate in electric wheel measuring pond 1 through the kapillary inlet tube and outlet tube among the present invention, reach the purpose of liquid in the electric wheel measuring of the cooling pond 1, avoid simultaneously because the increase of the dielectric conductance rate that water evaporates causes.Perfusion can also rotate change at any time in the mensuration process at electricity with the solution compolision in the kapillary in addition, in the pharmacodynamics check, realizes real-time administration.Regulating suitable flow rate pump is that biologic grain provides the fresh liquid environment with the assurance, does not influence the electric circling behavior of biologic grain simultaneously.

Have two leading-out terminals on the substrate 3 of microelectrode circuit board, the sine voltage signal generator is wired on described two leading-out terminals.The sine voltage signal generator produces the uniform sine voltage signal of multichannel phase differential; Sine voltage signal (1 μ Hz to 20MHz; 4mVpp to 20Vpp) frequency and amplitude are controlled; Through substrate 3 the multichannel sine voltage signal is evenly distributed on a plurality of microelectrodes 2, makes and produce uniform rotating electric field in the electric wheel measuring pond.Two-channel digital storage oscilloscope (RIGOL DS-5022M) connects the sine voltage signal generator, with monitoring waveform situation.Because biologic grain not only stands uniform rotating electric field in electric field; Also possibly stand dielectrophoretic force; Making them move to electrode or away from the direction of electrode, is that rotating electric field should be more evenly good more so measure a requirement that proposes for reliable electricity rotation.

Fig. 1 a and Fig. 1 b are depicted as the present invention and are used for pharmacodynamic study when test, and it is in the middle of the rotating electric field E that forms of 90 ° sinusoidal voltage that particle p is suspended in four electrode phase differential.The dielectric properties that depends on particle and suspending medium adds the frequency of electric field, and biologic grain will be with identical with the direction of electric field rotation (altogether) or opposite (anti-'s) direction rotation.Be anti-rotation shown in Fig. 1 a.

The detection method of electricity rotation detection system that is used for pharmacodynamic study more than the application is specific as follows said, for describe convenient for the purpose of, the pharmacodynamic study that acts on the HeLa cell with cis-platinum is that example is explained.Method when detection method of the present invention is applied to pharmacodynamics and detects other drug reagent and act on other biological sample particle object like virus, prokaryotic and eukaryotic etc.; The pharmacodynamics detection method that acts on the HeLa cell with the detection cis-platinum is identical, so repeat no more.

The model biologic grain of using in the present embodiment is HeLa cell (a human cervical cancer cell).Control group is employed in the hyclone that contains 10% heat fire extinguishing, 100U/mL penicillin, and the 100U/mL streptomysin, conventional condition of culture in the DMEM nutrient culture media of PH7.3 (37 ℃, 5%CO ₂, saturated humidity) and the following HeLa cell of cultivating.What experimental group adopted is to be respectively 2.5,5 in cis-platinum concentration, 10,15,20 and 25 μ g/mL, the HeLa cell of each self-applying after 12,24,48 hours.Treat that promptly cell is inoculated in 96 well culture plates (100 μ L/ hole), every hole 3 * 10 when getting into exponential phase ³0.9%NaCl (the 25 μ L/ hole) solution of individual cell, the constant temperature culture antineoplastic cis-platinum that the experimental port adding prepares in advance after 4 hours makes the final concentration of every hole cis-platinum be respectively 2.5; 5,10,15; 20 and 25 μ g/mL put into incubator and are cultured to required action time; The DMEM nutrient solution that control wells then adds equivalent continues to cultivate, 6 multiple holes of every settings.

When the electricity consumption rotary detecting method detects, specifically comprise the steps:

Step 1: make the liquid medium that is full of a certain amount of known conductivity in the electric wheel measuring pond 1 through perfusion device, and in experiment, keep perfusion.

Regulate the flow velocity of water inlet pipe and water outlet pipe through the peristaltic pump that links to each other with water inlet pipe and water outlet pipe.It is 250 microns that perfusion uses the internal diameter of kapillary water inlet pipe and water outlet pipe, and the solution compolision in the electric wheel measuring pond 1 can be rotated change at any time in the mensuration process at electricity, in the pharmacodynamics check, realizes real-time administration.The flow range of peristaltic pump is 0.002mL/min to 4mL/min, and regulating suitable flow rate pump is that biologic grain provides the fresh liquid environment with the assurance, does not influence the electric circling behavior of biologic grain simultaneously.

Through experiment, when being set at 1.0rpm (0.16mL/min), perfusion rate is optimum.Can guarantee that under this flow velocity the liquid in the measuring cell is that biologic grain provides the fresh liquid environment, not influence the electric circling behavior of biologic grain simultaneously.

Choosing the liquid medium of known conductivity in this experiment: be suspending medium with the glycocoll, regulate conductivity, measure with DDSJ-308A type electric conductivity appearance and thunder magnetic DJS-1C type conductance electrode through adding phosphate buffer (PBS).Select 10kHz, 50kHz, 100kHz, 200kHz when measuring frequency; 400kHz, 800kHz, 1MHz, 2MHz; 4MHz, 8MHz, 10MHz, 15MHz; A series of frequencies such as 20MHz obtain being in the information of the HeLa cell in the glycocoll/PBS medium of different electric conductance, and use software to generate electricity rotation spectrogram (Fig. 2) in view of the above.

Such as expection, find that medium conductivity is an important parameters for electric rotation test.By the characteristic frequency that the increase that can be clear that the external agency conductivity among the figure can cause electricity to rotate, promptly anti-rotation crest frequency f _cIncrease.Can see that characteristic frequency is 100kHz when the dielectric conductance rate is 200 μ S/cm; And characteristic frequency is 1MHz when the dielectric conductance rate is 1000 μ S/cm.In addition, significant change has also taken place in rotational speed.In fact the increase of external agency conductivity can reduce the time that makes the biologic grain film charged, increases with the characteristic frequency of sending a telegraph rotation.

Glycocoll/PBS medium that selected in the present embodiment conductivity is 600 μ S/cm is as the perfect medium of measuring administration front and back cell, so that biologic grain has the speed of rotation of being convenient to measure in our selected frequency range.In the experiment that detects the other biological particle, select liquid mediums and conductivity thereof according to the character of the biologic grain that detects, the selection of conductivity is so that the biologic grain that is detected has the speed of rotation of being convenient to measure in selected frequency range be as the criterion.

Step 2: the testing sample that will be suspended in the same liquid medium joins in the circular electric wheel measuring pond on the microelectrode circuit board with liquid-transfering gun.

Be that the HeLa cell sample to be measured that about 2 μ L are suspended in the same media is joined in the circular electric wheel measuring pond on the microelectrode circuit board with liquid-transfering gun in the present embodiment.

Step 3: make it to take place electric circling behavior through applying uniform rotating electric field, observe the single sample particle that is positioned at central area, electric wheel measuring pond.

Need to prove; When carrying out electric wheel measuring; The shape of resulting electricity rotation spectrogram and amplitude receive the influence that has fragment on position and the particle surface of biologic grain in measuring cell; So only the sample particle of position in the central area of electrode observed, and measured cell electricity rotation spectrum.The central area is meant that electric wheel measuring pond central diameter is the interior zone of circle scope of 0.3x (wherein x is the distance between each eletrode tip) defined.If therefore the position of cell is beyond the zone at measuring cell center 1/3rd, perhaps in the process of record, moved more than three times of they diameters, perhaps on its surface fragment is arranged, will they be got rid of outside analytic target.In addition, the heterogeneity of the field that the dipole that near the rotation particle having been found that is induced causes can cause the dielectrophoretic force between the particle, influences the electricity rotation response of particle to be measured.For this influence is reduced to minimum, only the sample particle that separates at least three diameter distances is each other done the electricity rotation and observe.These careful choice criteria have all guaranteed the reliability and stability of data.

Step 4: use the camera recorded video that is connected with microscope, through cell analysis software the video of being recorded is analyzed then, thereby obtain the information that relevant sample particle electricity rotates, and make electricity rotation spectrogram.

In glycocoll/PBS medium (600 μ S/cm), gather electricity rotation spectrogram.Through observing and record HeLa cell different with the change curve of frequency of electric angular velocity of rotation before and after administration, detect the drug action of cancer therapy drug cis-platinum.The observation of cell electricity rotation before and after the administration and the measurement of the cell anglec of rotation are made with CFM-500 type inverted fluorescence microscope and CF-2000XB biological cell analysis software.

Because the composition of cell, form are with structural different, the dielectric properties of cell has intrinsic heterogencity, makes the different cells of same type have different speed.Through speed and the direction that the observed and recorded biologic grain at least 20 points of each frequency rotates, obtain electricity rotation spectrogram, each point on the spectrogram is represented the similar cell of size is done the mean value of measuring for 20 times.See also Fig. 3, shown in Figure 4, it detects the electricity rotation spectrogram that obtains for the present invention for the HeLa cell before and after the administration, and wherein, horizontal ordinate is the logarithm value of electric field frequency among the figure, and ordinate is a HeLa cell speed of rotation.Horizontal ordinate is above for being total to a rotation; Below the horizontal ordinate anti-rotation.

Before the experiment in beginning step 1, can also comprise step: choose suitable experiment parameter in addition based on research object.

Choose distance between the suitable microelectrode 2 according to research object.For selecting the microelectrode design of doing this research usefulness for use, can come biologic grain is brought out the electricity rotation with distance between the very big microelectrode of scope.Spacing between each microelectrode 2 is 100 μ m to 1mm; Preferred each microelectrode spacing of employing is the electrode of 400 μ m; This distance can produce a suitable monitored area and rotational speed down, and use the influence that less microelectrode spacing can avoid checkout equipment local pyrexia effect that the biologic grain film is caused.The size of microelectrode spacing can change be observed dissimilar biologic grains, and needn't change circuit or analytic system.

The ac signal that the offset of sinusoidal voltage signal generator is applied is chosen suitable amplitude, and on a plurality of microelectrodes, applies uniform sine voltage signal successively.Driving voltage on four microelectrodes among Fig. 1 (diameter 100 μ m) is followed successively by u ₁=U ₀Cos (ω t); u ₂=U ₀Cos (ω t+90 °); u ₃=U ₀Cos (ω t+180 °); u ₄=U ₀Cos (ω t+270 °).The sine voltage signal generator produces frequency 1 μ Hz to 20MHz, the controlled sine voltage signal of voltage 4mVpp to 20Vpp.In order to reduce electrocaloric effect, the field intensity of rotating electric field should be low as far as possible, and preferably the peak-to-peak voltage value with each frequency maintains 12V.

After the experiment in step 4, the electricity rotation spectrogram that obtains in the step 4 is analyzed:

As shown in Figure 3; It is to answer the electricity rotation spectrogram of the HeLa cell behind the cis-platinum effect different time that the electricity consumption rotary detecting method obtains (in conductivity is in glycocoll/PBS medium of 600 μ S/cm; Cis-platinum concentration is 15 μ g/mL), be respectively the electricity rotation spectrogram of cis-platinum effect after 12 hours, 24 hours and 48 hours.Observation to administration after the cell record of the different time electricity rotation spectrum of getting off, can see wherein has along the part of a rotation with anti-rotation.Act in the process of tumour cell at cancer therapy drug, the variation of cell dielectric character has been reflected in the variation of electric rotation spectrum.The variation of most critical is characteristic frequency f in the spectrogram _cMove and the difference of peak height.Visible by figure, along with the increase of drug treating time, f _cMove to high frequency direction, and there is notable difference in the rotational speed under the same frequency.This can distinguish the activity of biologic grain through the marked difference of characteristic frequency and peak height fully with regard to pointing out us.

Specifically, if make comparisons with 48h experimental group and control group, the characteristic frequency that can see cellular control unit is greatly about 400kHz; And significantly moving to high frequency direction taken place greatly about 1MHz in the characteristic frequency of cell behind the cis-platinum effect 48h.In addition from rotating speed, can find will be starkly lower than the control group rotating speed at the rotating speed of the frequency window experimental group cell of 10kHz-400kHz, can make rapid judgement to the susceptibility of medicine according to the rotating speed difference of cell under this electric field frequency.

In addition, the HeLa cell shown in the figure is in the middle of near the rotation of the electricity 10MHz spectrum, and the cell of 48h experimental group and control group is with opposite direction rotation, and this just provides a unifrequency activity test method easily.

As shown in Figure 4, it is the electricity rotation spectrogram of answering the HeLa cell after the variable concentrations cis-platinum effect that the electricity consumption rotary detecting method obtains (is in glycocoll/PBS medium of 600 μ S/cm in conductivity, cis-platinum action time for being 48h).We have chosen the trend that four concentration are explained variation here, are specially the electricity rotation spectrogram under control group, cis-platinum concentration 5 μ g/ml, 15 μ g/ml and the 25 μ g/ml.Can see the increase along with drug concentration, fc moves to high frequency direction, and there is notable difference in the rotational speed under the same frequency.Relatively cis-platinum concentration is the electricity rotation spectrogram of the experimental group cell of 5 μ g/mL and 25 μ g/mL, and visible the former characteristic frequency is greatly about 800kHz, and the latter's characteristic frequency is then greatly about 2MHz, and the rotating speed under the characteristic frequency the former be approximately 1.5 times of the latter.The speed that this just points out us can measure rotation at the place, top that rotates near anti-field is in order to the cell of differentiation different activities.The rotational speed when if comparison frequency is 400kHz, the rotating speed that can find cellular control unit is 3.5 times of 25 μ g/ml experimental group cell rotating speeds.

Mtt assay is the common method in the antineoplastic in-vitro screening, as with the comparison of electric spinning solution, done the MTT experiment in the present embodiment simultaneously.Use mtt assay and detect the toxic action of cis-platinum, observe variable concentrations cis-platinum (2.5,5 the HeLa cell line proliferation; 10,15,20 and 25 μ g/mL) act on HeLa clone 12; 24; The inhibiting effect of on cell proliferation after 48 hours is confirmed HeLa cell activity before and after the administration, in order to validity and the versatility that shows electric rotation technique.Even medicine acts on the MTT solution 25 μ L that in each hole of 96 orifice plates, add 5mg/mL after 12,24,48 hours respectively; Continue to cultivate after 4 hours, discard liquid in the hole, add DMSO (100 μ L/ hole); Put low-speed oscillation 10min on the shaking table; Bluish violet Jia Za crystal is fully dissolved, and the OD value in that enzyme-linked immunosorbent assay instrument 570nm (630nm calibration) locates to measure each hole calculates the inhibiting rate (Fig. 5) under each concentration of medicine.

As shown in Figure 5, its for the variable concentrations cis-platinum that records with mtt assay at the growth inhibited curve of different time to the HeLa cell.The result shows that cis-platinum truly has inhibiting effect to the HeLa cell line proliferation, and is time and dose dependent.Under the same drug concentration of this experiment condition, drug effect 48 hours is the strongest to the growth inhibited effect of HeLa cell.

Hence one can see that, and the conclusion of electric rotary detecting method has obtained support with the check of reactive dye method mtt assay.When answering the electricity consumption rotary detecting method, we have two kinds of simple approach to come promptly to confirm cell activity, and one is the sense of rotation of only passing through particle under the CF; Another is the difference through rotational speed in the certain frequency window.Some drug effect does not cause its modal variation behind biologic grain, but can make it produce similar electricity rotation spectrogram with known non-activity or the active cell that descends, and we just can detect this variation through electric rotation technique.

When the process of a cell from activity to nonactive variation the variation of dielectric properties very clearly the electricity consumption spinning solution detect.Can find out different with administration time and dosage; The response difference of electricity rotation is very big; Difference between the spectrogram has been clearly illustrated that with this technology and in individual cells, has identified careful biochemical variation by the physical change owing to cell membrane and tenuigenin characteristic very much.

Though the pharmacodynamic study that acts on the HeLa cell with cis-platinum among the present invention is that example is explained; But not as limit; Method when detection method of the present invention is applied to pharmacodynamics and detects other drug reagent and act on other biological sample particle object like virus, prokaryotic and eukaryotic etc.; The pharmacodynamics detection method that acts on the HeLa cell with the detection cis-platinum is identical, only need glycocoll/PBS liquid medium be replaced with the liquid medium that is complementary with detected object and get final product according to the different detection object; And select for use which kind of liquid medium with the matching detection object be those skilled in the art should know, so repeat no more.

In sum; The present invention has shown that electric rotation technique can be used for measuring the activity change of biologic grain in the administration process; Detected biologic grain owing to use the variation of the electricity rotation response that antineoplastic caused, this conclusion has obtained reactive dye method mtt assay result's confirmation.Promptly through observing and the different electric circling behavior of record cell before and after administration; We have detected can be obviously observed in the electricity rotation spectrum of cell before and after medication, can be owing to the variation of medicine pair cell effect, be used for the dielectric properties of characterising biological particulate; Thereby the susceptibility to medicine is made prediction; Reach the purpose that detects the antineoplastic drug action, had in drug screening and the potential use in analyzing, can be used for developing into new pharmacodynamic analysis method.

The present invention utilizes electric pivot analysis method to carry out pharmacodynamic study, and it is easy and simple to handle, and step such as need not dye is not applied to the toxicity organic reagent yet.Can be used for the different physiological statuss of dynamic monitoring cell, be used for numerous research fields such as drug screening, cell cycle and Apoptosis and cell physiological state monitoring.

A kind of electric rotary detecting method that is used for pharmacodynamic study provided by the invention; Can provide information about single individuality in the biologic grain colony; And the required sample volume of this miniaturized devices is than much less required in the routine monitoring method; Promptly do not need the large number of biological particle as research object; Only use very small amount of sample and reagent and detection time seldom just can accomplish whole testing process, make it become the ideal tools of a small amount of biosome of research, can be used to carry out fast and the biological diagnosis of low consumption.Itself can be used as breadboard sensing and analytic unit on a kind of chip, also can combine, be used for biologic grain is carried out express-analysis with other electrodynamics technology as dielectrophoresis and row ripple dielectrophoresis that kind.

Electricity rotation technique another characteristics that are superior to other routine techniquess be it not only can in analytic process, keep biologic grain complete, with test preceding the same situation and the activity of state, make this biologic grain sample can be used to carry out further integral body or destructive analysis; And adopt the enough same batch of biologic grains of this method ability to accomplish the process of whole pharmacodynamic study, increase the sensitivity of drug effect statistical test, thereby more help obtaining the information of drug efficacy study.In addition, it still is a kind of general analytical technology, can be used for observing many dissimilar organisms.And owing to used perfusion device, also can detect and study the situations of change of biologic grain under drug effect such as cell in real time, promptly in the pharmacodynamics check, realize real-time administration, have very big advantage and potential use as a kind of new drug screening method.

The above description of this invention is illustrative, and nonrestrictive, and those skilled in the art is understood, and within spirit that claim limits and scope, can carry out many modifications, variation or equivalence to it, but they will fall in protection scope of the present invention all.

Claims (4)

1. an electric rotary detecting method that is used for pharmacodynamic study is characterized in that, comprises the steps:

(1) in electric wheel measuring pond, be full of the liquid medium that known conductivity is 600 μ S/cm, and keep perfusion, perfusion rate is set at 0.002mL/min to 4mL/min;

(2) the biological sample particle to be measured before and after the drug effect is joined in the electric wheel measuring pond, this biological sample particle is virus, prokaryotic or eukaryotic;

(3) make sample particle that electric circling behavior take place through applying uniform rotating electric field; Observation is positioned at the single sample particle of central area, electric wheel measuring pond; Apply uniform rotating electric field for applying uniform sine voltage signal on equally distributed a plurality of microelectrodes around the electric wheel measuring pond; This microelectrode is four, and this sine voltage signal is followed successively by u ₁=U ₀Cos (ω t), u ₂=U ₀Cos (ω t+90 °), u ₃=U ₀Cos (ω t+180 °) and u ₄=U ₀Cos (ω t+270 °), sine voltage signal frequency 1 μ Hz to 20MHz, voltage 4mVpp to 20Vpp, this central area is that electric wheel measuring pond central diameter is the border circular areas of 0.3x, wherein x is the distance between each microelectrode tip;

(4) obtain the information of relevant sample particle electricity rotation, and make electricity rotation spectrogram.

2. the electric rotary detecting method that is used for pharmacodynamic study as claimed in claim 1 is characterized in that said perfusion rate is 0.16mL/min.

3. the electric rotary detecting method that is used for pharmacodynamic study as claimed in claim 1 is characterized in that said microelectrode is a platinum electrode.

4. the electric rotary detecting method that is used for pharmacodynamic study as claimed in claim 1 is characterized in that, only the sample particle that separates at least three diameter distances is each other done the electricity rotation in the step (3) and observes.

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