CN105378456B

CN105378456B - Label-free method for evaluating chemical cardiac toxic

Info

Publication number: CN105378456B
Application number: CN201380070133.XA
Authority: CN
Inventors: O·D·戴希曼; Y·方; A·M·J·菲里; H·胡; 邬起
Original assignee: Corning Inc
Current assignee: Corning Inc
Priority date: 2012-11-15
Filing date: 2013-11-15
Publication date: 2018-11-06
Anticipated expiration: 2033-11-15
Also published as: CN105378456A; WO2014078646A1

Abstract

A kind of label-free resonance waveguide grating biosensor imaging system is schemed in the presence of and there is no the dynamic mass of the cardiac muscle cell cultivated in the case of drug molecule redistribution (DMR) signal with beating for measuring.The present invention also provides a kind of the method for the DMR signals and beating figure of cardiac muscle cell to evaluate drug-induced cardiac toxic is analyzed using imager system.

Description

Label-free method for evaluating chemical cardiac toxic

The cross reference of pending U. S. application

This application involves the U.S. Provisional Application Serial No. 61/ submitted on November 25th, 2008 for co-owning and transferring the possession of 117838 and in the U.S. Provisional Application Serial No. 12/613,966 that on November 11st, 2008 submits, entitled " hepatotoxicity Experiment " is U.S. Patent Publication No. 20100129854, but does not ask its priority now.It the content of this file and carries herein To publication or all the elements of patent document be all incorporated by reference into herein.This application claims on November 15th, 2012 The priority of the U.S. Patent Application No. 61/726620 of submission, entire contents are totally incorporated herein by reference.

Technical field

The present invention relates to label-free resonance waveguide optical grating (RWG) biosensor apparatus and methods, with assessment, for example, Heart biology or drug-induced cardiac toxic.

Background

In humans and animals, drug-induced side effect be it is known and largely with the major organs such as heart Dirty, liver is related to the dysfunction of kidney.Potential bad drug influence increases safe treatment application complexity level.Class It is similar to hepatotoxicity wind agitation, the assessment to cardiac toxic is the pith of drug discovery and drug discovery process.Drug-induced heart Toxicity be with some drugs (including that some are used to treat chemotherapeutics of blood and solid malignant) relevant adverse events, and And typically result in morbidity and high mortality.In the past ten years, a variety of biochemistries, cell and fabric study have shown that medicine The heart abnormality of object induction is related to the activity change of following factor, for example, ion channel, myocyte's structure, extracellular matrix (ECM) structure and neurohumoral systems.Cardiotoxicity of medicine may also lead to and Ca²⁺Relevant protein exception and signal transduction system System is abnormal.In these factors, the variation of ion channel activity has been considered to the main original of drug-induced cardiac toxic Cause.The ion stream of a variety of overlappings adjusts the vntricular action potential lasting duration and form.Na⁺It is fast by selective sodium channel Speed enters the depolarising for having caused ventricle.Be after this quickly polarized again by what momentary actuation and potassium channel extroversion inactivated, and And it is followed by plateau, mainly entered to determine by L-type calcium channel by calcium ion.During polarizing again, by the mistake of calcium channel It is living and mainly by the net outside potassium of delayed rectifier potassium channel carried with Quick-type component (rapid component) at a slow speed from The increase of subflow makes electronegative membrane potential restore.Inward rectifyimg potassium channel also causes to polarize again.Including Na⁺/K⁺The tune of pump The section factor makes intracellular ion concentration restore to original state.

The evaluation of cardiotoxicity of medicine is important the security development of newtype drug.It is nearly all new in the U.S. Before the approval of medicine, evaluation compound makes the wind of the extended risk in the intervals surface electrocardiogram QT and the arrhythmia cordis of life-threatening Danger is the regulation of a FDA (United States food and drag administration).The intervals QT are in the cardiac electric period, and the starting of Q waves and T waves terminate Between time measured value.In general, the electrical depolarization of QT time interval left and right ventricles and polarizing again.The extended intervals QT are ventricles Tachyarrhythmia such as the Biological indicators of torsades de pointes (TdP), and is the risk factors of sudden death. QT extends most common related to by hERG (the people ether-a-go-go related genes) current loss of potassium-channel, the damage Mistake is the direct blocking due to ion channel caused by the inhibition accidentally that the plasma membrane of drug or channel protein is expressed.Most of In situation, the drug for extending the intervals QT preferentially inhibits to encode the gene hERG or delayed rectification potassium ion of the α-subunit in the channels IKr The Quick-type component of circulation road (I (Kr)).

Generally acknowledged internal cardiotoxicity of medicine evaluation method is electrocardiogram (ECG) test.Surface ECG provides living about electricity Dynamic information, including atrium/ventricular depolarization in heart and ventricular repolarization.In ECG, QT time interval ventricular depolarizations (that is, reduction of membrane potential) and (that is, recovery of resting potential) is polarized again.ECG represents the duration of vntricular action potential And including the intervals QT, which reflects the soak time of two ventricles.Although most drugs induction QT extend with The inhibition of hERG is related, and the reverse correlation at the long intervals QT is caused but without the inhibition in the fatefully channels proof hERG.In addition, Ionic pump activity change and cardiomyocyte cell death may also generate cardiac toxic.The early stage identification of drug-induced adverse risk has Beneficial to evaluating multiple ion channel, cell Proliferation and cell death on a molecular scale.Cardiac toxic test has become drug and opens The core of hair.

In the presence of several other technologies of the in-vitro evaluation for drug-induced cardiac toxic, including use hERG transfections thin The patch clamp technique of born of the same parents or the cardiac muscle cell of separation, Rb⁺Outflow experiment uses Purkinje (Purkinje) fiber or guinea-pig papillary The microelectrode of flesh is tested, and the analysis of the cardiac muscle cell based on bio-impedance.Wherein, patch clamp technique may be that screening is drug-induced Cardiac toxic most widely used tool, allow to monitor single medicine to the direct action of simple target ion channel. Although this technology demonstrates high accuracy, it does not allow to observe multiple ion channel activities simultaneously, and do not allow be Variation in system property displaying cardiotoxicity of medicine, including cell death and cellular signal transduction.Other most of possible techniques with The average measurement value of cell mass is related.Integrate the label-free full cell of unicellular organism and the performance of cardiovascular cell group Test method will be highly advantageous.

Invention content

The present invention provides label-free resonance waveguide optical grating (RWG) the biosensor imaging system of high-frequency and methods, use Divide again in the dynamic mass for measuring the cardiac muscle cell cultivated in the case of existing and chemical substance (such as drug molecule) is not present Cloth (DMR) and beating figure.Label-free device and method use the high-frequency swept wavelength solution of resonance waveguide grating biosensor Transfer detection culture cardiac muscle cell beating figure.In embodiments, present invention provides assess drug-induced Amplatzer duct occluder The data analysing method of the potentiality of property.

Description of the drawings

In embodiments：

Fig. 1 shows the cardiac muscle cell derived from the people iPS- with cultureResonance wave in 384 hole microplates The pseudo-colours high-resolution resonant wavelength image of guide grating biosensor 3x4 arrays.Fig. 2 is shown in the people iPS- with culture The distribution of substrate resonant wavelength at each pixel of the RWG biosensor of derivative cardiac muscle cell.

Fig. 3 A and 3B are respectively shown by analysis buffer (i.e. negative control (Fig. 3 A)) and 160nM isoprels (figure 3B) dynamic mass of cardiac muscle cell derived from the people iPS- of the culture induced redistributes signal.

Fig. 4 is the mean resonance peak for showing the RWG biosensor of cardiac muscle cell derived from the people iPS- with culture.Figure 5 beThe pseudo-colours resonance light intensity map of the 3x4 arrays of RWG biosensor in 384 hole microplates.

Fig. 6 from left to right shows the RWG biologies of cardiac muscle cell derived from the people iPS- with the culture under quiescent condition The pseudo-colours resonance light intensity map of 0 to 27 second time series of sensor.Cardiac muscle is thin derived from the people iPS- of Fig. 7 display cultures The typical beating figure and key parameter of born of the same parents' beating, including：Rise time, slack time, and define the intensity % of beating figure or fight Fatigue resistance.

Fig. 8 provides a series of exemplary plots (A to F), shows that cardiac muscle cell is before contact derived from the people iPS- of culture (baseline；Left column) beating figure and contact or with the stimulation of certain drug molecule later measurement result at any time (referring to beating Intensity % reduced times illustration) in time dependence variation.Fig. 9 provides the Cisapride of display various dose (cisapride) to the series of drawing (a to l) of the effect of the cardiac muscle cell of culture beating：The beating of cardiac muscle cell before stimulation；With The beating of corresponding cardiac muscle cell when 15 minutes after being handled with the Cisapride of various dose.

Figure 10 A and 10B show Yi Shapading (israpadine) induction culture cardiac muscle cell beating rate when Between and dose-dependent variation diagram.Show two independent results (left and right) for repeating experiment.

Figure 11 A and 11B show time and the dose-dependant of the beating intensity of the cardiac muscle cell of the culture of Yi Shapading inductions Variation diagram.Show two independent results (left and right) for repeating experiment.

Detailed description

Below with reference to the accompanying drawings various embodiments of detailed description of the present invention (if any).With reference to various embodiments It does not limit the scope of the invention, the scope of the invention is limited only by the scope of the appended claims.In addition, arranging in the present specification Any embodiment gone out is all not limiting, and is only listed in many possible embodiments of claimed invention Some embodiments.

In some embodiments, it is special to provide one or more advantages for revealed equipment and method of manufacture and use thereof Sign or aspect, including for example, as described below.The features or aspect listed in any claim are applied generally to this hair Bright all aspects.Any single or multiple features or aspect described in any one claim can combine or with it is any Any other features or aspect displacement described in item or multinomial other claims.

Definition

" central resonance wavelength " refers to the wave for providing the maximum coupling efficiency that irradiation light enters waveguide grating biosensor It is long.

"include", "comprise" or similar terms are meant including but not limited to, that is, are included and nonexclusive.

When describing embodiment of the present invention for modifying the amount, concentration, structure size of ingredient, body in such as composition The numerical value such as product, treatment temperature, processing time, yield, flow velocity, pressure, viscosity and their range " about " refer to quantity Variation, can be happened at for example：Prepare compound, composition, compound, concentrate or the typical of application preparation measure and handle step In rapid；It is not intended to error in these steps；Manufacture, source or for implementing the raw material of the method or the purity of ingredient in terms of In difference；And in similar Consideration.Term " about " further include the aging due to composition or preparation and with it is specific initial Concentration or the different amount of mixture, and due to mixing or processing compositions or preparation and with specific initial concentration or mixture Different amounts.

Unless otherwise stated, otherwise, "one" or "an" of indefinite article used herein and its corresponding definite article "the" indicates an at least (pcs/species) or a (pcs/species) or more (pcs/species).

Abbreviation well known within the skill of those ordinarily skilled can be used (for example, indicating " h " or " hr " of hour, to indicate gram " g " or " gm " indicates " mL " of milliliter, indicates " rt " of room temperature, indicates " nm " of nanometer and similar abbreviation).

Structure, component, ingredient, the disclosed specific and preferred value of additive and similar aspect and its range are only used for Bright, they are not excluded for other numerical value in the numerical value or defined range of other definition.The method of the present invention may include any Any combinations of numerical value or numerical value, concrete numerical value, more specific numerical value and preferred value, including median and range.

The in vitro culture of cell provides the material that can be used for pharmacology, physiology and toxicologic study.Drug screening skill Latest Development in art enables pharmaceutical companies quickly to screen huge compound library for therapeutic targets.

Cardiac toxic is the case where possible leading to myocardial damage.Drug-induced cardiac toxic refers to by the drug influence heart Dirty cardiac toxic, and include direct effect of the drug to heart, but indirectly-acting is may also comprise, for example, since blood is dynamic Force flow variation enhancing or due to Cardioversion.Serious cardiac toxic can lead to cardiomyopathy.Cardiomyopathy is often because for the treatment of (such as chemotherapy Cure) caused by, or can the myocardium disease or illness of damage can be caused to cause by one group.Myocardial damage can cause the heart Dirty pump action is disorderly, and subsequently results in heart failure.

Drug-induced cardiac toxic typically results in left ventricular dysfunction, rhythm disorder and ischemic.These toxic effects State that can be from subclinical abnormality to life-threatening.These effects may be related to medicament contact or medical treatment, it is also possible to length Phase Cardiovascular is related.The medication that usually may result in cardiac toxic or cardiomyopathy includes, for example, anthracene nucleus medicament. Anthracene nucleus medicament including Doxorubicin can be used for treating leukaemia, lymthoma, Huppert's disease, breast cancer, sarcoma With other cancers.The relevant cardiac toxic of Doxorubicin (AAC) can be divided into three kinds of forms by long-recognized：The immediate heart Bao Yan-myocarditis syndrome, early send out chronic progressive form and late send out progressive form chronic.The potential mechanism of AAC it is main Assuming that being the generation of reactive free radical material, the substance and cell membrane interaction and destroying cell membrane.Other are possible Mechanism includes, for example, under the mRNA expression of apoptosis-induced, mitochondrial DNA destroys, ATP is generated variation and sarcoplasmic reticulum calcium atpase It adjusts.

In past many decades, the exploitation of new drug has been discovered that the proarrhythmia of drug, especially anticarcinogen is made It is composed with new, and most important one causes the intervals QT to extend.The extended definition of QTc is different in the literature, but most of thinks Normal QTc is less than or equal to 400 milliseconds (ms), and extended QTc is big in women more than 450ms in male In 470ms.Ventricular arrhythmia, especially torsades de pointes to it is related than or equal to the QTc of 550ms, But the threshold value for extending QT intervals not proarrhythmia risk not being considered under this.About the extended aetologies of QT The hypothesis accepted extensively be interaction with HERG K channels.HERG K channels allow cardiac muscle Quick-type group polarized again Point；When its function of interfering effects of drug, potassium stream, which enters reduction, leads to again polarized extension.Cancer patient may tend to QT extensions, because There is electrolyte disturbance for many in them, while taking drug such as antiemetic, antifungal agent or the antibiosis that can increase the intervals QT Element, or it is abnormal (up to 32% patient) with baseline electrocardiograms.

From early stage nineteen sixties, it has been reported that the accidental sudden death caused by cardiac arrest and the non-cardiac medicine of use Object is related.These cause the drug of sudden death to be focused in torsades de pointes (TdP), and one kind can develop into The polymorphic ventricular arrhythmia cordis of ventricular fibrillation and sudden death.The extension at the intervals QTc is the alternative finger for the ability that drug leads to TdP Mark.If drug-induced QTc extensions coexist with other risk factors, the risk factors such as private medical service, congenital length The presence of QT syndromes, heart failure, bradycardia, electrolyte imbalance, excess QTc- extend drug, female gender, restraint (restraint), old, liver or kidney injury and slow metabolism state are then more likely to that arrhythmia cordis occurs.Pharmacodynamics and Pharmacokinetic interaction can also increase the risk of arrhythmia cordis.

The main reason for drug reduces during pharmacology safety issue is clinical development, and account for whole drug reductions About 35% to 40%.In this, cardiovascular relevant toxicity accounts for the 19% of the drug of exclusion.The major part of these toxicity is Due to function and dose-dependent effect.The Arrhythmia that is about typically due in 19% exclusion causes, including latent Life-threatening state, such as drug-induced torsades de pointes (TdP).In addition, extend about QTc, It is suitable for large-scale drug between TdP and sudden death the problem of relationship.Due to the Cardiovascular Toxicity effect in people, from city Several cardiac drugs and non-cardiac drug are recalled on.Although toxic effect is not observed in animal, Drug Administration Mechanism has extended the requirement of cardiac toxic test.

In embodiments, the present invention provides detecting drug-induced heart effect in a kind of method of no label, Including：

The cultured myocardial on the surface of at least one resonance waveguide optical grating (RWG) biosensor；

Use the swept wavelength demodulation resonance waveguide grating biosensor imager of the first light source with first wave length Irradiate at least one RWG biosensor；

Resonance spectrum is acquired at all pixels position of at least one biosensor and with swept wavelength solution Resonance waveguide grating biosensor imager is adjusted to determine the center of all pixels position of at least one RWG biosensor The average value of resonant wavelength；

At least one RWG biosensor is irradiated using the second light source with second wave length；

It is anti-to monitor the resonance generated by second light source irradiation from least one RWG biosensor in real time with imager It penetrates the intensity of light and is composed with the beating for obtaining cardiac muscle cell under quiescent condition；

The cardiac muscle cell of quiescent condition is contacted with drug molecule；

The cardiac muscle cell through medicament contact on the surface of at least one RWG biosensor is irradiated using second light source；

It is anti-to monitor the resonance generated by second light source irradiation from least one RWG biosensor in real time with imager The intensity for penetrating light, the beating to obtain the cardiac muscle cell through medicament contact are composed；And

What the beating of the beating spectrum and the cardiac muscle cell through medicament contact of extracting and compare the cardiac muscle cell of quiescent condition was composed At least one beating parameter, the drug-induced variation of wherein at least one beating parameter are the indexs of the heart effect of drug.

In embodiments, cardiac muscle cell is that people's primary cardiomyocytes, animal primary cardiomyocytes, human embryo stem cell spread out The pluripotency of cardiac muscle cell derived from raw cardiac muscle cell, people's inductivity pluripotent stem cell (iPS cells) and animal induction is dry Cardiac muscle cell and combinations thereof derived from cell (iPS cells).

In embodiments, the resonate surface of waveguide grating biosensor is contained with extracellular matrix protein, or cardiac muscle The coating of cell compatibility synthetic material.Extracellular matrix protein can be, for example, fibronectin, matrigel, collagen I, collagen iv With gelatin and combinations thereof.Cardiac muscle cell's compatibility synthetic material can be that (the co- MAA-PEO4- glass of HEMA- connects egg for example, poly- Peptide conjugate in vain).

In embodiments, the waveguide grating biosensor imager that resonates can be, for example, swept wavelength Demodulation Imaging Analysis System.In embodiments, biosensor imager can have, for example, 3 microns to 500 microns, for example, 3 microns, it is 6 micro- Rice, 12 microns, 25 microns, 50 microns, the spatial resolutions of 100 microns or 500 microns, including median and range.Preferred Embodiment in, biosensor imager can be with 12 microns of spatial resolution.In embodiments, resonate Waveguide Grid biosensor imager can be, for example, the resonance waveguide grating biosensor of the Wavelength demodulation based on optical fiber system System, wherein the system can be used for scanning biosensor.

In embodiments, resonance waveguide grating biosensor may include biosensor array.Preferably implementing In mode, biosensor array can be, for example, the titer plate that biomolecular screening association-is compatible.

In embodiments, the wavelength of second light source can be, for example, all pictures of at least one RWG biosensor More than the average value of the central resonance wavelength of elementization position 100 micromicrons to 1500 micromicrons (pm), for example, 100pm, 200pm, 300pm, 400pm, 500pm, 600pm, 800pm, 1000pm or 1500pm, including median and range.

In embodiments, the wavelength of second light source can be all pixels position of at least one RWG biosensor 100 micromicrons below the average value for the central resonance wavelength set to 1500 micromicrons (pm), for example, 100pm, 200pm, 300pm, 400pm, 500pm, 600pm, 800pm, 1000pm or 1500pm, including median and range.

In embodiments, the wavelength of irradiation light can be all pixels position of at least one RWG biosensor Central resonance wavelength mean wavelength.Central resonance wavelength is to provide the maximum that irradiation light enters waveguide grating biosensor The wavelength of coupling efficiency.When average value of the wavelength of irradiation light higher than the central resonance wavelength of biosensor, the heart of gained The beating spectrum of myocyte is made of a series of lowest troughs generated from cellular beating.On the contrary, when the wavelength of irradiation light is less than biology When the average value of the central resonance wavelength of sensor, the beating spectrum of the cardiac muscle cell of gained is a series of by what is generated from cellular beating Maximum peak forms.When the wavelength of irradiation light is identical as the average value of central resonance wavelength of biosensor, the cardiac muscle of gained The system that the beating spectrum of cell is generated by the slave cellular beating of the substrate resonant wavelength pixelation position depending on biosensor Row peak and valley forms, this is attributed to wider distribution of the substrate resonant wavelength in the different pixels position of biosensor.

In embodiments, the long-term cultivation of cardiac muscle cell can be carried out, until it is thin to reach most of cardiac muscles in sensor Born of the same parents synchronize beating.Incubation time depends on the type of cardiac muscle cell, but ranging from 5 days to 2 weeks usual.In embodiments, The Short-term Culture of cardiac muscle cell, typically less than 1 week can be carried out, to spontaneous beat only occurred.

In embodiments, drug molecule can be, for example, the drug molecule of the marketization, the preclinical drug used are waited Select molecule, testing drug molecule or combinations thereof.

In embodiments, the temporal resolution of cardiac muscle cell's beating spectrum is 0.01 second to 1 second, for example, 0.01 second, 0.05 Second, 0.1 second, 0.2 second, 0.5 second, 1 second, including median and range.

In embodiments, can after being exposed to drug molecule about 1 minute to 5 days, such as 1 after being handled with drug molecule Minute, 15 minutes, 30 minutes, 60 minutes, 240 minutes, 8 hours, 12 hours, 16 hours, 1 day, 2 days, 4 days and 5 days are (in including Between value and range) obtain cardiac muscle cell beat spectrum.The cardiac muscle cell's of the culture obtained when different time points after drug-treated Beating spectrum can be used for classifying to the toxicity of drug molecule.

In embodiments, at least one beating parameter of the beating spectrum of cardiac muscle cell includes at least one below：It fights The integral area at beating interval, beating intensity, rise time, slack time, each beating peak between dynamic frequency, adjacent beats or A combination thereof.

It in embodiments, can be thin in the standard of physiological condition, i.e. cardiac muscle cell at 35 to 40 DEG C and at preferably 37 DEG C The beating spectrum of cardiac muscle cell is obtained under born of the same parents' condition of culture.

In embodiments, label-free method may additionally include cardiac muscle cell and acquire dynamic before and after medicament contact Quality redistribution (DMR) signal, wherein RWG biosensor imager are during experiment in about 30 minutes to 5 days when multiple Between point on acquire certain time, for example, 10 seconds to 1 minute cardiac muscle cells beating spectrum.

In embodiments, can be in short term, for example, 5 minutes, 1 minute, 30 seconds and 10 seconds, including median and range. In embodiments, " redistribution of long-term dynamics quality " experiment may include by track at any time the central resonance wavelength of resonance light come The DMR signals of the cell of response stimulation are monitored in real time." long-term " refers to being more than, for example, about 5 minutes, 15 minutes, 30 points Clock, 1 hour, 4 hours, 6 hours, 12 hours, 1 day, 2 days, 5 days, 10 days constant duration (including median and range) appoint What period.

It in embodiments, can be by the beating parameter phase with cardiac muscle cell's (that is, before drug-treated) under quiescent condition The exception of the beating parameter of ratio defines the heart effect of drug molecule.If drug-treated, which generates, to be had than under quiescent condition At least one beating interval of equispaced big 25%, then drug molecule can be considered leading to the irregular beating of cardiac muscle cell. It can be considered as the effector molecule for interfering cardiac muscle cell's function to lead to any drug molecule of beating Parameters variation.Lead to cardiac muscle The drug molecule of cell exception can be considered as cardiotoxic drugs molecule.

The present invention also provides a kind of label-free method of replacement, the Amplatzer duct occluder for evaluating drug molecule or compound Property, including：

At least one RWG biosensor is irradiated using the first swept wavelength demodulation scheme, wherein the first swept wavelength model It is at least 5nm to enclose and frequency sweep cycle continues at least 1 second；

The swept wavelength demodulation resonance waveguide optical grating imager of use space parsing measures at least one RWG biosensor All pixels position at central resonance wavelength and make acquisition average value；

At least one RWG biosensor is irradiated using the second swept wavelength demodulation scheme, wherein the second swept wavelength model Be trapped among the 3nm ranges of the average value of the central resonance wavelength at all pixels position of at least one RWG biosensor with It is interior, and frequency sweep cycle continued less than 0.3 second；

The central resonance wavelength for tracking RWG biosensor by using the second swept wavelength demodulation scheme has to establish The baseline dynamic mass of at least one RWG biosensor of cardiac muscle cell redistributes (DMR) signal；

Cell is set to be contacted with drug molecule；

Monitor the DMR signals of drug molecule induction in real time with imager；With

Oscillation mode in DMR that oscillation mode in DMR baseline periods is induced with drug molecule is compared, with determination The cardiac toxic of drug molecule.

In embodiments, the oscillation mode in the interim DMR signals of comparison base and the DMR signals of drug molecule induction can With for example, being completed at 1 minute after contacting cell with drug molecule to 5 days, for example, 1 point after cell is contacted with compound Clock, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 4 hours, 8 hours, 1 day, 2 days, 5 days when etc., including median and Range.

It is thin with low frequency DMR and high-frequency beating for evaluate the present invention also provides difunctional label-free method Born of the same parents' response, this method include：

There is provided at least one resonance waveguide optical grating (RWG) biosensor and on the surface of at least one biosensor Upper culture cell；

RWG biosensor is irradiated with adjustable light source, wherein adjustable light sources are covering at least one RWG biologies biography Illumination wavelength is scanned in the particular range of the resonant wavelength of sensor；

The spectrogram of the resonant reflection light from least one RWG biosensor is acquired using high speed optical camera Picture synchronizes to obtain a series of spectrum pictures wherein when the wavelength of the inswept particular range of adjustable light sources；

To spectrum picture handled with：

Obtain the central resonance wavelength at all pixels position of at least one RWG biosensor；And

Extract the two-dimensional space parsing resonant wavelength image of at least one RWG biosensor；

Monitor the resonant wavelength image of at least one biosensor in real time using low frequency swept wavelength demodulation modes, and And the low frequency is 0.001 to 1 hertz；

Data are acquired from low frequency swept wavelength demodulation modes and are switched to high-frequency intensity monitoring pattern, wherein using tool There is the second light source of specific wavelength to irradiate at least one RWG biosensor, and high-frequency is 1 to 100 hertz；

The intensity of 5 second to 1 minute reflected light of the monitoring from least one RWG biosensor is to obtain high frequency in real time Rate luminous intensity adjusts image；

Swept wavelength demodulation modes are switched back into monitor the resonant wavelength image of at least one RWG biosensor in real time； And

Resonant wavelength image data is handled and adjusts spectrum and low-frequency resonance wavelength to obtain high-frequency luminous intensity Dynamic mass redistributes (DMR) signal, wherein adjusting spectrum using high-frequency luminous intensity to monitor the high-frequency of the cell of culture Beating spectrum, and monitor the DMR that cell response induces using low-frequency resonance wavelength DMR signals.

In embodiments, this method may also include：

Make cell and molecule contacts；

The high-frequency luminous intensity of acquisition molecule treated specific time adjusts spectrum, and it is remaining after being handled with molecule when Between low-frequency resonance wavelength DMR signals；

Compare and adjust spectrum before being handled with molecule with high-frequency luminous intensity later, and before being handled with molecule with later Low-frequency resonance wavelength DMR signals, the variation of high frequency luminous intensity shaping modes and low-frequency resonance wavelength DMR signals Variation be index that the molecule acts the cell of culture.In embodiments, adjustable light sources can be from covering biology Scanning wavelength in the particular range of the resonant wavelength of sensor.In embodiments, which can be, for example, 825nm To 840nm.In embodiments, completing the time of single sweep operation can be, for example, 3 seconds, for example, during length scanning When obtaining 150 frame image in total.

In embodiments, the time series of the resonant wavelength image of biosensor can be used for generating dynamic mass and divide again Cloth (DMR) signal.In embodiments, the time series of the intensity image of the light reflected from biosensor can be used for generating use The not oscillation mode of the cell of treated with medicaments.In embodiments, the specific wavelength of the second radiation source can be at least More than the average value of the central resonance wavelength of all pixels position of one RWG biosensor 100 micromicrons to 1500 micromicrons, For example, 100pm, 200pm, 300pm, 400pm, 500pm, 600pm, 800pm, 1000pm or 1500pm, including median and model It encloses.In embodiments, the wavelength of radiation source can be the central resonance wavelength of all pixels position of biosensor The above 500pm of mean wavelength.In embodiments, the specific wavelength of the second radiation source can be at least one RWG biologies About 100 micromicrons below the mean wavelength of the central resonance wavelength of all pixels position of sensor to about 1500 micromicrons, for example, 100pm, 200pm, 300pm, 400pm, 500pm, 600pm, 800pm, 1000pm or 1500pm, including median and range.

The present invention also provides a kind of label-free method, the cardiac toxic for evaluating drug molecule, including：

In the first mode, at least one RWG biosensor is irradiated using the first swept wavelength demodulation scheme, wherein the One frequency sweep wave-length coverage is at least 5nm and frequency sweep cycle continues at least 1 second；And the swept wavelength demodulation of use space parsing Resonance waveguide optical grating imager monitors the central resonance wave at all pixels position of at least one RWG biosensor in real time It is long；

In a second mode, at least one RWG biosensor is irradiated using the second swept wavelength demodulation scheme, wherein the Central resonance wavelength average value at all pixels position of ranging from least one RWG biosensor of two swept wavelengths Within 3nm ranges, and frequency sweep cycle continued less than 0.3 second；And imager is used, using the swept wavelength solution of space analysis Resonance waveguide optical grating imager is adjusted to monitor the central resonance at all pixels position of at least one RWG biosensor in real time Wavelength；

In the third mode, at least one RWG biosensor is irradiated using the third light source with specific wavelength, wherein The specific wavelength of third light source is above, less than or equal to all pixels position of at least one RWG biosensor at The specific wavelength of central resonance wavelength average value；And it is monitored using imager and is come from least by what third light source irradiated in real time The intensity of the resonant reflection light of one RWG biosensor；

The cell of culture is monitored in the imager for using first mode with the slow dynamic quality for the cell cultivated Redistribute (DMR) response；

Imager is switched to second mode from first mode, makes the quick DMR responses of the cell of imager monitoring culture； And

By imager from the first or second pattern switching to the third mode, make the high-frequency of the cell of imager monitoring culture Luminous intensity Regulate signal, wherein slowly can be used for studying drug point with quick DMR signals and high-frequency luminous intensity Regulate signal Effect of the son to the cell of culture.

The present invention also provides label-free resonance waveguide grating biosensor imaging systems, for measuring in presence and not Dynamic mass redistribution (DMR) signal of the cardiac muscle cell of culture in the case of there are drug molecule and beating are schemed, the system Including：

Adjustable light sources, the scanning irradiation wave in the specific range of resonance for covering at least one RWG biosensor It is long；

Charge coupled device (CCD) camera, such as record is passed from least one RWG biologies in the form of pixelation The reflected light of sensor；

Computer program calculates the centre wavelength of all pixels position of at least one RWG biosensor；

Operating system allows to irradiate at least one RWG biosensor with three kinds of different detection patterns, including：

The first mode of at least one RWG biosensor is irradiated using the first swept wavelength demodulation scheme, wherein the One frequency sweep wave-length coverage is at least 5nm and frequency sweep cycle continues at least 1 second；

The second mode of at least one RWG biosensor is irradiated using the second swept wavelength demodulation scheme, wherein the The average value of central resonance wavelength of the two swept wavelength ranges at all pixels position of at least one RWG biosensor 3nm ranges within, and frequency sweep cycle continue less than 0.3 second；With

The third mode of at least one RWG biosensor is irradiated using the third light source with specific wavelength, wherein the Three light sources have be higher than, less than or equal at least one RWG biosensor all pixels position central resonance wavelength Average value specific wavelength；And

Computer and display are used to extract and show the data obtained using three kinds of different detection patterns.

1. cardiac muscle cell

Cardiac muscle cell, also referred to as myocardium muscle cell, is the cell for including cardiac muscle.Myocardium (heart flesh) is that heart is (special Myocardium) histological basis, and be a kind of nonvoluntary striated muscle found in the wall of heart.Cardiac muscle is three kinds main One of muscle types, remaining two kinds are skeletal muscle and smooth muscle.Cardiac muscle cell can contain there are one, two or rarely contain very much Three or four nucleus.The collaboration of Heart center myocyte is shunk is extrapolated to left side/body/entirely by blood from atrium and ventricle In the blood vessel of the body circulatory system and right side/lung/pulmonary circulation system.As in body institute in a organized way, cardiac muscle cell rely on In enough blood supplies to deliver oxygen and nutriment and remove waste, such as carbon dioxide.Cardiac muscle is shown by thick and thin egg The crossbanding that the alternating segment of white silk is formed.As skeletal muscle, myocardium major structural protein is actin and flesh ball Albumen.

Primary cardiomyocytes are commonly used in the testing in vitro of drug-induced cardiac toxic.These primary cardiomyocytes have Assemble the advantage of all cardiac ion channels.Although cardiac muscle cell provide it is a kind of for study whole cell physiology be System, there are significant interspecific differences in leading to again polarized electric current and pharmacology sensitivity.The missing of predictive ability hinders Data derived from animal model are directly transformed into people.This critical shortage because of in vitro Human Cardiomyocytes for test purposes And it is further complicated.In addition, Human adult cardiomyocytes have extremely low proliferative capacity through final differentiation, limits it and needing Purposes in the Large-scale Screening application of a large amount of volume cells.Separation process is to take, is difficult and high-cost, and once locating In culture, in vitro Human adult cardiomyocytes quickly dedifferente, and the phenotype hindered in long-term cultivation maintains.

In recent years, Human embryo or the stem cell-derived cardiac muscle cells of iPS have the potentiality for providing Human Cardiomyocytes source, this It potentially contributes to overcome the problems, such as species difference.These derivative cardiac muscle cells can be formed with some heart tissue features Cluster, this also allows for evaluation organizational parameter.In addition, human stem cell has unrestricted proliferation and can be used as taking in theory Cell origin not to the utmost.When being proliferated under suitable conditions, hESC is stablized on caryogram and epigenetic in many years , stable and dependable source is provided, is especially important in the vital drug screening of repeatability.Derived from hESC Or the cardiac muscle cell of iPS is very lasting under standard condition of in vitro culture, allows to inhibit the acute effect such as channels hERG, And it exposes relevant longer-term effect such as ion channel transport with extended drug and is evaluated.As single layer prepared product or In the aggregation or cluster of bigger, these derivative cardiac muscle cells show the functional of contraction and synchronize, and this facilitate to cell With cell coupling, signal transduction and the research of polarization characteristic again.In addition, these cardiac muscle cells are with cluster or aggregation mode combinations It is very steady when together and allows to redistribute the detection device that disengaging is not directed to long term cell culture design.

2. resonate waveguide grating biosensor

RWG biosensor is by such as base material (such as glass), the waveguide film and biological cell that have embedded optical grating construction Layer composition.Photoresonance is coupled into waveguide by RWG biosensor by way of diffraction grating, is caused on solution-surface circle Total internal reflection at face, and then generate electromagnetic field at interface.This electromagnetic field essence fades, and indicates it from sensor surface Exponential decay；The distance that it decays to the 1/e of initial value is known as the depth of penetration, it sets with specific RWG biosensor It counts and changes, but usually in about 200 nanoscales.This kind of biosensor is identified using this evanescent wave, such as sensor sheet The variation that cellular layer on face or close to the surface is induced by ligand.

The waveguide optical grating imaging system 3. swept wavelength resonates

Be developed recently swept wavelength resonance waveguide optical grating imaging system (referring to, Ferrie, A.M. etc., Resonant Waveguide grating imager for live cell sensing (for living cells induction resonance waveguide optical grating at As instrument) .Appl.Phys.Lett.2010,97：223704；And Ferrie, A.M. etc., High resolution resonant waveguide grating imager for cell cluster analysis under physiological Condition (the high-resolution resonance waveguide optical grating imager for cell kmeans cluster in physiological conditions) .Appl.Phys.Lett.2012,100：223701).These RWG imagers are with such as 12 microns or 80 microns of space point Resolution.For the high resolution R WG imagers of the spatial resolution with 12 microns, the light from frequency sweep adjustable light sources is guided Beam focuses on the 3x4 biosensor arrays in such as 384 hole microplates；And use high-speed cmos (supplement metal oxide Semiconductor) digital camera records the resonance light of escape and reflection.The camera has 1400x1024 pixels, Pixel Dimensions It is 7.4 microns, and image forming optics have 1.6 times of amplification factors, generates 12 microns of effective spatial resolution.From During the single cycle of 825 to 840nm length scanning, 150 spectrum pictures in total are obtained within every 3 seconds, then by spectrum picture Heap is processed into real time sensor resonant wavelength or DMR images, leads to 3 seconds temporal resolutions.It is known as base in all pixels The starting resonant wavelength of bottom resonant wavelength after zero standardization to obtaining DMR images.With every 20 millisecond of 100 micromicron gradually into traveling wave Long scan.Therefore, which can carry out the resonance of time resolution, and wherein resonance light is coupled in specific time in specific position To waveguide.This generates improved spatial resolution, although resonance light can be propagated before its leakage in waveguide.The imager Overlay area is small, it is made to can be placed in desk-top cell culture couveuse.However, about 3 seconds of swept wavelength RWG imagers when Between resolution ratio hinder its detection quick response such as cardiac muscle cell beating in application.

4. dynamic mass redistribution experiment

The cellular response of stimulation can be encoded by the room and time dynamics of downstream signaling network by cell target. For this purpose, the integration of real time monitoring cell signalling can be provided for cell biology letter related to the physiology that physiology understands Breath.Optical biosensor includes resonance waveguide optical grating (RWG) biosensor, and it is related to detect the redistribution of dynamic cellular substance Integrator cell response, to provide research cell signalling Noninvasive means.All optical biosensors are total to It is the variation of their energy measurement sensor surfaces or the local indexes of refraction very close to the surface with point.In principle, nearly all Optical biosensor can be used in cell sensing, because they can use the variation of ligand induction in evanescent wave identification of cell.

Recently, the parameter that the description of theoretical and mathematical model measures optical signalling in living cells responds ligand stimulation is developed And characteristic.Combination of these models based on 3 layers of Wave guide system Yu known cell biological physics by the optical signal of ligand induction and is led to Receptor-mediated specific cells process is crossed to connect.Because biosensor measurement is flat positioned at the cell of incident light irradiation area It responds, cell height fused layer may be used to obtain best test result.Due to the short depth of penetration with biosensor Larger compared to cell size, sensor configuration is regarded as unconventional three-tier system：Base material, the guide membrane with optical grating construction, and Cellular layer.Therefore, ligand induction effective refractive index (that is, the signal measured) variation can be, in the form of single order, directly with carefully Born of the same parents' layer bottom variations in refractive index is proportional：

Δ N=S (C) Δs n_c

Wherein S (C) is the sensitivity to cellular layer, Δ n_cFor the cellular layer part of the ligand induction of biosensor sensing Variations in refractive index.Because the refractive index of intracellular given volume is mainly determined by the concentration of biomolecule such as protein, Δ n_cIt can It is directly proportional with the ligand induction variation of molecular assemblies in local cells target concentration or sensing volume to be assumed to.It considers The optical signal of the exponential decay property that evanescent wave extends from sensor surface, ligand induction is dominated by following formula：

Wherein, Δ Z_cTo enter the depth of penetration of cellular layer, α is specific refraction increment (protein is about 0.18/mL/g), z_iThe distance and d that occur for quality redistribution are the imaginary thickness of lamella in cellular layer.Here, cellular layer is divided into vertical direction On slice at equal intervals.Above-mentioned equation show the optical signal of ligand induction be the quality that occurs from sensor surface different distance again The sum of distribution, respectively differs the contribution of overall response.In addition, using the measured signal of wavelength or angle change form mainly to hair The raw quality redistribution in sensor surface vertical direction is sensitive.Because of its dynamic nature, it is also referred to as dynamic mass and divides again Cloth (DMR) signal.

The non-invasive manner of detection cellular response in real time is provided using the DMR tests of optical biosensor.Therefore, DMR can be used for characterize be related to cell adherence, cell Proliferation and the wide scope of cell death cell processes.In recent years, label-free Optical biosensor is gradually popular in research bio-molecular interaction and cell biology.It has shown that and is based on microplate Resonance waveguide optical grating (RWG) biosensor receptor-ligand can be interacted and translate into not tape label and/or cell and grasp Behavioral characteristics (that is, dynamic mass redistribution) in the living cells of work.DMR signals are real-time and the response of intact cell phenotype, Therefore mode not seen before is provided for research cell biology.However, as almost all of microplate reads instrument technology, Existing label-free system is mostly at room temperature without CO₂It operates in the case of control, and is surveyed from the cell mass of height fusion Measure average response.

The present invention provides a variety of advantages, including provide following ability：1) there is the beating to the cardiac muscle cell of culture The high-frequency detection method of figure；2) while detection and the drug-induced cellular response of the cardiac muscle cell of culture are relevant label-free Dynamic mass redistributes signal；3) cellular response is detected in unicellular and individual cells level；4) under the conditions of studying physiological Heart biology；5) cardiac toxic of drug, including beating figure, receptor signal conduction and final cell are evaluated in single experiment Dead variation；And 6) cardiotoxicity of medicine is detected in the case of synchronizing beating in the cardiac muscle cell with and without culture.

Material and method：

Chemicals.Obtained from the bright of sigma chemical company (Sigma Chemical Co.) (St. Louis) Glue, isradipine (isoradipine), a hydration Cisapride, E-4031 and astemizole.Isoprel is obtained from Tocris Biological Science Co., Ltd (Tocris Biosciences Co) (St. Louis).

The coating of resonance waveguide optical grating (RWG) biosensor.384 hole cell culture compatibilities Plate is obtained from Corning Incorporated's (NY, USA).By by the freshly prepared phosphate-buffered of plate and 10 microlitres of 20 micrograms/mL Gelatin solution in brine is incubated and is dried in vacuum overnight to obtain gelatin coatingPlate.Or or simultaneously, for example, In experiment using cardiac muscle cell derived from IPS, by will be in not coating plate and 40 microlitres of 0.1% gelatin (A types) solution It is coating that overnight incubation obtains gelatinPlate, which is made of distilled water and high pressure sterilization 30 minutes is with degerming.Then Will before motive myocyte inoculation with 40 microlitres of plate of the distillation water washing through incubation three times.It is prepared using scheme below Poly- (the co- MAA-PEO4-VN of HEMA-)-is coating384 aperture biosensors, the program are co-owning and are turning before It is disclosed in the U.S. Patent Application No. 13/420,735 that the 15 days Mays in 2012 allowed submit.VN is vitronectin peptide (KGGPQVTRGDVFTMP) abbreviation.First, cellular adhesion peptide-copolymer of functionalization is prepared.By 60mg's (4.6mmol) The amber equipped with magnetic stirring bar is added in the MAA-PEO4-VN of hydroxyethyl methacrylate (HEMA) and 1000mg (0.05mmol) In 75mL ethyl alcohol in amber color flask.Then, the 2 of 90mg, 2 '-two-(2- methylbutyronitriles) of azo are added and stir to completely molten Solution.Deoxidation is carried out 10 minutes to solution with argon gas purging.Then the flask of sealing is heated 20 hours under stiring at 68 DEG C, And it protects and is protected from light.After cooling to room temperature, it dilutes poly- (the co- MAA-PEO4-VN of HEMA-) polymer with 200mL DMSO and passes through Crude reaction medium is poured into 1000mL ethyl acetate to detach.The white solid of gained is washed three times with acetone and vacuum is dry It is dry.It is arranged by the size coupled with refractive index detector, light scattering detector, photodiode array detector and viscosity detector Hinder the molecular weight that chromatography (SEC) measures poly- (the co- MAA-PEO4-VN of HEMA-) copolymer.Movement is mutually trifluoroethane and trifluoro second Sour potassium.It is 138,284 and PDI is 2.38 that average Mn, which is 58,170, Mw,.Secondly, by by poly- (the co- MAA- of HEMA- of 0.5mg PEO4-VN) copolymer is dissolved in prepares coating composition in 10mL deionizations (DI) water.Finally, it prepares coating 384 hole cell based assays plates.For this purpose, disperseing 25 microlitres of coating composition in each hole.Then plate is placed in 50 DEG C of couveuses In 30 minutes to allow copolymer to adsorb.After cooling to room temperature, mildly to elute plate by originally flow primary.Then, to each 30 microlitres of 1%SDS solution is added in hole and is incubated about 30 minutes.Then plate is eluted 4 times with deionized water.Finally, by (plate being placed upside down) is rotated under 800rpm 1 minute plate to be dried.Use the BCA protein quantification reagents from Uptima Box measures fixed peptide density by dihomocinchonine sour (BCA) experiment.Standard items are prepared using the VN peptides in PBS.?Absorbance is read on Synergy 4.BCA shows that the amount of fixed peptide is about 15pmol/mm²。

Resonate waveguide grating biosensor imaging system.Optical read device is based on swept wavelength imaging technique.

First mode irradiates resonance waveguide optical grating biology with normal incidence by LED (light emitting diode) beams collimated and passes Sensor passes through the tunable optical filter for passing through bandwidth with 1nm.By the sensor region through irradiation in high-speed cmos (supplement Metal-oxide semiconductor (MOS)) it is imaged in camera.When the wavelength model of the resonance of the inswept covering biosensor of tunable optical filter When enclosing, synchronization gain spectrum picture.Image stack spectrum containing sensor in all pixels, wherein with 2D image zooming-out resonance waves It is long.In general, in each acquisition, length scanning is from 825nm to 840nm or 820nm to 835nm is for 3 seconds, and is swept in wavelength 150 frame images are obtained during retouching.Typical long-term DMR is tested, renewal rate puts down Multiple-Scan from 15 seconds to a few minutes At individual data point.This is the first operator scheme for obtaining DMR responses, and response is slower than 3 seconds；This pattern is known as slow Slow resonant wavelength imaging DMR patterns or slow DMR patterns (sDMR).Three kinds of operation modes are summarized in table 1.

Second mode, if cardiac muscle cell's beating those of causes, needs corresponding to detect the redistribution of quick dynamic mass Ground increases data acquisition rate.In order to accurately be sampled to beat signals, detection renewal rate needs the Nai Kui than about 4Hz Si Te (Nyquist) frequency is fast, which is four times of the Beating Rate of about 1Hz.Therefore, resonance waveguide optical grating imaging is established Instrument can support second operator scheme.The operation of this second operator scheme is with close or be faster than nyquist frequency Swept wavelength is imaged renewal rate.Second mode is known as quick resonant wavelength imaging DMR patterns or quick DMR patterns (fDMR). This can be realized by using high-speed camera and fast tunable section light source.Second mode can also be used with about 60 frame per second The conventional cameras of speed.If such as 12 frames are only absorbed in single pass, renewal rate can increase to about 4Hz, and The central resonance wave-length coverage tight fit of adjustable range and sensor resonance.Although sensor spectrum is sparse sampling, altogether Shaking detection noise may not be ideal like that into first mode, but increases detection speed and keep quantifying for sensor simultaneously Resonant wavelength.When detection renewal rate is slightly below or is slower than nyquist frequency, detection is no longer smart due to crenellated phenomena Really.However, the frequency and amplitude of light beam still can provide qualitative information.

1. operation mode of table

The third mode, can be by setting optical source wavelength to the waist point (waist of resonance spectrum in third operation mode Point) renewal rate is made to be further increased to far more than nyquist frequency, the variation of wherein resonant wavelength is converted into instead The intensity adjustment of irradiating light beam.The third mode is known as intensity (or energy) imaging pattern.For example, if the sensor light spectrum width of detection Degree is 3nm, linearly maintains about ± 0.5nm, then can obtain the adjusting on wider.It can be by widening tunable optical filter Width or sensor resonance further increase linear dynamic range, cost is that the slope of Strength Changes drops in wavelength shift It is low.Monitoring optical energy reference may also be made to the offset of (reference out) intensity of light source.Renewal rate in the detection scheme It is only limited by the frame of camera speed, can be, such as 30Hz to 1000Hz, depend on the speed of the digital camera of selection Degree.Camera can preferably have the deep full-well capacity for reducing shot noise (shot noise).It can be real in this operation mode Existing multiple variations.For example, can push-and-pull operate on the both sides of central resonance between two wavelength toggle lights, wherein The common mode noise from light source can be deleted.If the wavelength change of sensor is more than opereating specification, which also can be Switch between multiple wavelength.

Rapid detection method in above-mentioned second mode and the third mode can be combined with first operator scheme, more to provide DMR information in a time scale.Specifically, for Cardiomyocyte Assays, compared with prior art, while capture with it is single thin The slow DMR dynamics of born of the same parents or the relevant quick DMR dynamics of beating and routine in single cell cluster resolution ratio provides solely Special advantage.The Beating Rate of cardiac muscle cell is typically 1Hz, and nyquist frequency is 4Hz.In this embodiment, at 12 seconds Renewal rate under obtain routine DMR images.Measurement fDMR images, each data point are averagely under 10Hz renewal rates, for example, 6 frames.If desired, renewal rate can faster, cost is to increase the size of data file.

The culture of cardiac muscle cell derived from iPs.Cardiac muscle cell is obtained from cell dynamic international corporation (Cellular Dynamics International).Cardiac muscle cell derived from these iPS is the highly purified popular feeling derived from iPS cells Myocyte.These cells express monomer red fluorescent protein (mRFP) and blasticidin S resistance, both in α-myosin Under the control of heavy chain (Myh6) promoter, to allow cardiac muscle cell to purify simultaneously and identification.Cardiac muscle cell is that have Typical electrophysiological characteristics and the spontaneous electrical activity heart that expected electro physiology and biochemical response are shown after being exposed to exogenous agent The myocyte in room, section and ventricular-like.Because heart tissue shows the protein expression pattern of species specificity, for drug discovery For toxotest, it is contemplated that using the Human Cardiomyocytes finally broken up, rather than other alternative models are (for example, corpse or dynamic Object cell, and conversion immortalized cell line) generate the result for more accurately predicting to respond in relevant human body. The use of cardiac muscle cell can save time, resource and compound.Cardiomyocyte Differentiation from people's pluripotent stem cell, It keeps in vitro, therefore the model system for being easy to obtain and have physiological relevance is provided, for evaluating compound to people The electrophysiological effect of heart cell.Cardiac muscle cell shows the standard biochemical and electro physiology of Normal Human Heart's cell Feature is learned, forms the syncytial layer of the synchronous beating of electrical connection, and verified in a variety of biochemical tests and arrhythmia cordis There is practicability in test.According to the scheme that supplier recommends, useCardiac muscle cell's bed board culture medium, will obtain Point cellifugal refrigeration suspension be directly plated onIn plate, and it is maintained atCardiac muscle cell maintains training It supports in base.The beating of 4 days detection cardiac muscle cells after incubation, and cardiac muscle cell is becoming synchronous later.Cardiac muscle Cell is survived in culture to be up to 2 weeks, therefore enabled assessment is short-term and long term toxicity is tested.In functional cell culture couveuse Inherent 37 DEG C obtain all detected values.

As a result it and discusses

Use RWG imagers with 384 coating holes of gelatin first3x4 biologies are obtained in biosensor plate The substrate resonant wavelength image of sensor array.Image is obtained when 4 days after cultivating cardiac muscle cell derived from iPS.Cardiac muscle cell Single layer is formed on the surface of biosensor.Fig. 1 shows the pseudo-colours substrate resonant wavelength image of biosensor array. Due to its high spatial resolution (12 microns), individual cells can be differentiated.High resolution R WG imagers are recently developed based on us With 80 microns of spatial resolution whole plate imager (A.M.Ferrie etc., Appl Phys Lett, 97,223704 (2010)).Different from irradiation entire plate, the light beam from frequency sweep adjustable light sources focuses on the biography of the 3x4 biologies in 384 hole microplates On sensor array.The resonance light of reflection is recorded using high-speed cmos (supplement metal-oxide semiconductor (MOS)) digital camera.Instead The resonance light penetrated is escaped or is leaked out out of waveguide film due to resonance light.The camera has 1400x1024 pixels, as Plain size is 7.4 microns, and image forming optics have 1.6 times of amplification factors, generates 12 microns of effective spatial resolution. During the single cycle of the length scanning from 825 to 840nm, 150 spectrum pictures in total are obtained within every 3 seconds, and by spectrum Image stack is processed into real time sensor resonant wavelength or DMR images, leads to 3 seconds temporal resolutions.Title on all positions DMR images are obtained later to zero standardization for the starting resonant wavelength of substrate resonant wavelength.Length scanning is with every 20 microsecond, 100 skin Rice (pm) gradually carries out.For resonant wavelength in discovery sensor in particular range, this depends on Multiple factors, including, for example, Difference in sensor construction, cell fusion, cell adherence degree, cellular response or combinations thereof.The imager provides introducing The mode of the resonance of time resolution, wherein resonance light are coupled into waveguide in specific time in specific position.This generates improvement Spatial resolution, although resonance light can be propagated before its leakage in waveguide.In addition, the overlay area of the imager is small, make It can be placed in desk-top cell culture couveuse.

Then, test that two different face coats, gelatin are coating and vitronectin is coatingBio-sensing Influence of the device microplate to the cell growth, cellular beating and synchronous beating of the cardiac muscle cell of culture.The results show that two kinds of surface productions The cellular morphology of cardiac muscle cell, cell viability derived from raw iPS, with start beating time and reach synchronous beating when Between quite.

Then, the basic resonance at all pixels position of single biosensor in array shown in FIG. 1 is analyzed The distribution of wavelength.As shown in Fig. 2, the distribution shows that the single peak centered on about 830.7nm has the peak width of about 1.5nm, table Basic resonance wavelength after clear-cells single layer is formed is very uniform.Fig. 2 is shown in the people iPS- with culture and derives Cardiac muscle cell RWG biosensor each pixel at basic resonance wavelength distribution.Basic resonance wavelength is in particular range The quantity of pixel in (that is, bin size (bin size), usually 100pm) determines total counting.

Then, the cardiac muscle cell for having detected culture responds beta 2-adrenergic receptor agonist compound isoproterenol The DMR signals of the stimulation of element.The results show that compared with experiment supporting agent solution leads to the negative control of small DMR signals, in physiology item Under part, the isoprel of 160nM leads to DMR letters more longlasting in cardiac muscle cell at 35 to 40 DEG C, and at preferably 37 DEG C Number (Fig. 3).It is interesting that compared with baseline and the DMR of experiment buffer solution induction, in the thorn by being contacted with isoprel After swashing, the oscillation mode of DMR signals becomes smaller, but has higher frequency, this shows that isoprel causes to train Foster cardiac muscle cell, which is beaten, to be increased, but has smaller amplitude.

Later, the mean resonance peak of biosensor is analyzed.All formants at each pixel to biosensor The formant equalized after being averaged.As shown in figure 4, formant is relatively wide.Dotted line in Fig. 4 is shown for building Vertical intensity monitoring operation scheme, that is, the wavelength of the third operation mode of biosensor imager system, and selection is for monitoring The operative wavelength of the beating figure of the cardiac muscle cell of culture.It is produced using the light of the wavelength with the central resonance wavelength higher than equalization (Fig. 7) is composed in the beating of the cardiac muscle cell of the raw culture being made of small peaks a series of.On the contrary, using the center having less than equalization The light of the wavelength of resonant wavelength generates a series of beating figure being made of posivtive spikes.This by gained and in fig. 5 and fig. shown in it is strong Degree image proves.

Fig. 5 is shown in coating 384 holes of VN peptides of the cardiac muscle cell with monolayer cultivation3x4 lifes in microplate The pseudo-colours intensity map of object sensor array.It is (i.e. biological in the light settings that will be used for the irradiation of biosensor to centre wavelength The resonant wavelength of sensor) more than value after, which is obtained by the monitoring luminous intensity that resonates, as shown in Figure 4.

Fig. 6 is shown with a kind of (excitement of endogenous adrenergic receptor in cardiac muscle cell of 1 micromolar adrenaline Agent) stimulation after, have culture cardiac muscle cell single biosensor pseudo-colours intensity map.Cardiac muscle cell passes in biology It is cultivated 4 days in sensor surfaces and not yet establishes synchronous beating.The results show that under the conditions of this asynchronous beating, imager The beating of the single cardiac muscle cell of individual can be detected, such as by increasing at particular point in time, i.e., 3 seconds, 21 seconds, 24 seconds and 27 seconds Intensity (that is, blush or deeper shadow spots) confirmed.Fig. 6 is from left to right shown with the culture under quiescent condition The pseudo-colours resonance light intensity map of 0 to 27 second time series of the RWG biosensor of cardiac muscle cell derived from people iPS-.? The Strength Changes of resonance light at each pixel are the instructions of single cardiac muscle cell's beating.Luminous intensity at given pixel is (red Color) increase the cellular beating indicated in corresponding position.Observe by non-uniform variation institute table in resonance light intensity image The uneven beating shown shows that cardiac muscle cell derived from the iPS of culture does not reach synchronous pulsatile status.Synchronous pulsatile status is anti- The state of cardiac muscle cell while beating has been reflected, the characteristic beating figure of resonance luminous intensity is generated.

Then, the beating figure for the cardiac muscle cell that monitoring is cultivated after cell has built up synchronous beating.The results show that Under the conditions of the synchronous beating obtained after culture in 7 days, cardiac muscle cell shows about 55 Beating Rates per minute and -0.6 and fights The rule of fatigue resistance and repeatable beating.It is obtained using the wavelength irradiation light higher 400pm than the central resonance wavelength of equalization Beating figure.Fig. 7 be show cardiac muscle cell derived from the people iPS- of culture beat (b1 to b2 and b2 to b3) typical beating figure and Include the figure of following key parameter：Rise time (710), slack time (720), and definition beating figure intensity % (730) or Beating intensity.As shown in fig. 7, dynamic parameter can be extracted from the beating performance of analysis cardiac muscle cell, it is thus possible to determine by changing Object is closed, the potential cardiac toxic induced such as drug molecule or drug candidate.Dynamic parameter includes：1) it beats the time；2) phase twice Interval between neighbour's beating；3) rise time respectively beaten；4) slack time respectively beaten；With the beating intensity knead dough at 5) each peak Product.Then statistical analysis can be carried out determine the cardiac muscle cell of culture any beating spectrum scrambling.

Later, a series of influence of drug molecules to the beating spectrum of cardiac muscle cell is examined.For each drug, the heart is obtained first The baseline beating of myocyte composes and determines the influence of each compound or drug molecule as reference.Later, after medicine irritation Each specific time at (for example, 1 minute, 5 minutes, 15 minutes), observe multiple beating and compose, so as to examine drug molecule Short-term and long term toxicity.Fig. 8 provides a series of figures (A to F), shows that cardiac muscle cell derived from the people iPS- of culture is contacting Before (baseline；Left column) beating figure time dependence variation and contact or with the stimulation of certain drug molecule later at any time Testing result (referring to beating intensity % reduced times illustration)：(1 minute after 2 row=stimulation, 5 minutes after 3 row=stimulation, 15 minutes after 4 row=stimulation)：10 micromole's isradipines (A rows)；10 micromoles (-)-isoproterenol (B rows)；10 Micromole one is hydrated Cisapride (C rows)；10 micromole E-4031 (D rows)；10 micromole's astemizoles (E rows)；With 0.025% DMSO (F rows).As shown in figure 8, result shows that 10 micromole's isradipines substantially inhibit the beating of the cardiac muscle cell of culture, and Isradipine inhibition be quick and in cell just with medicament contact when occur.On the contrary, isoproterenol increases beating Frequency but inhibit its pulsatile range or intensity；And the variation in isoproterenol induction beating figure continues for some time. Cisapride changes the beating figure of the cardiac muscle cell of culture in a manner of time dependence, and finally beating is made to stop.E-4031 Also lead to the beating figure of variation with astemizole, while negative control (the supporting agent solution containing 0.025%DMSO) is several to beating figure Do not influence.

Finally, it is determined that the dose-dependent effects of Cisapride and Yi Shapading.Fig. 9 provides display various dose The series of drawing (a to 1) of influence of the Cisapride to cardiac muscle cell's beating of culture：Cardiac muscle cell before stimulation is beaten (that is, not having Any processing or control) (left column：A is to f)；With when 15 minutes after being handled with the Cisapride of various dose corresponding cardiac muscle it is thin Beating (the right row of born of the same parents：G is to 1)；Scheming (g) has 0 micromolar Cisapride (that is, only testing buffer solution)；Scheming (h) has 0.16 Micromolar Cisapride；Scheming (i) has 0.62 micromolar Cisapride；Scheming (j) has 2.5 micromolar Cisaprides； Scheming (k) has 10 micromolar Cisaprides；Scheming (l) has 40 micromolar Cisaprides.It is being proved in Fig. 9 the results show that Cisapride reduces beat rates and amplitude with dosage-dependent manner, the IC with about 160nM₅₀.Similarly, such as Figure 10 and 11 Described, Yi Shapading also blocks the beating of the cardiac muscle cell of culture, the IC with about 100nM with dosage-dependent manner₅₀.Specifically Ground, Figure 10 A and 10B show the time of the beat rates of the cardiac muscle cell of the culture of Yi Shapading inductions and dose-dependent change Change figure.Show two independent results (left and right) for repeating experiment.Figure 11 A and 11B show the culture of Yi Shapading inductions Cardiac muscle cell beating intensity time and dose-dependent variation diagram.Show that two independent results for repeating experiment are (left And the right side).The result shows that high-frequency imager can detect the cardiac toxic of cardiac muscle cell's beating and drug molecule induction.

The present invention is described by reference to various specific implementation modes and technology.However, it is possible within the scope of the invention Make many changes and improvements.

Claims

1. a kind of label-free method detecting drug-induced heart effect, the method includes：

It is irradiated using the swept wavelength demodulation resonance waveguide grating biosensor imager of the first light source with first wave length At least one RWG biosensor；

The resonance spectrum at all pixels position of at least one biosensor is acquired, and uses swept wavelength solution Resonance waveguide grating biosensor imager is adjusted to determine in whole pixelation positions of at least one RWG biosensor The average value of heart resonant wavelength；

At least one RWG biosensor is irradiated using the second light source with second wave length, and with the imager The intensity of monitoring resonant reflection light in real time, the beating to obtain the cardiac muscle cell under quiescent condition are composed；

The cardiac muscle cell of the contact drug at least one RWG biosensor surface is irradiated with the second light source, and And the intensity of resonant reflection light is monitored in real time with the imager and is composed with the beating for obtaining the cardiac muscle cell of the contact drug；

With

Extract and compare at least the one of the beating spectrum of the cardiac muscle cell of quiescent condition and the beating spectrum of the cardiac muscle cell of contact drug A beating parameter, the drug-induced variation of at least one beating parameter are the indexs of the heart effect of drug.

2. the method as described in claim 1, which is characterized in that the cardiac muscle cell includes at least one of following cell： People's primary cardiomyocytes, animal primary cardiomyocytes, the cardiac muscle cell of derived from human embryonic stem, people's inductivity pluripotency are dry thin Cardiac muscle cell derived from born of the same parents, cardiac muscle cell derived from animal inductivity pluripotent stem cell or combinations thereof.

3. the method as described in any one of claim 1-2, which is characterized in that the resonance waveguide grating biosensor Coating is contained on surface, and the coating has extracellular matrix protein, cardiac muscle cell's compatibility synthetic material or combinations thereof.

4. method as claimed in claim 3, which is characterized in that the coating includes at least one of following substance：Fibre is even Albumen, matrigel, collagen I, collagen iv, gelatin, poly- (the co- MAA-PEO4- vitronectins peptides of HEMA-) conjugate or combinations thereof.

5. the method as described in claim 1, which is characterized in that the space of the resonance waveguide grating biosensor imager Resolution ratio is 3 microns to 500 microns.

6. the method as described in claim 1, which is characterized in that the wavelength of the second light source is following at least one：

More than whole pixelation place-centric resonant wavelength average values of at least one RWG biosensor 100 to 1500 Micromicron；

100 to 1500 below whole pixelation place-centric resonant wavelength average values of at least one RWG biosensor Micromicron；Or

Whole pixelation place-centric resonant wavelength average values of at least one RWG biosensor.

7. the method as described in claim 1, which is characterized in that the cardiac muscle cell is multiple cells, and to the multiple The culture of cell continues 5 days to 2 weeks time until reaching synchronous beating at 35 to 40 DEG C.

8. the method as described in claim 1, which is characterized in that the drug is at least one of following substance：It is commercially available Drug molecule；Preclinical molecule drug candidate；Exploratory molecule drug candidate；Or combinations thereof.

9. the method as described in claim 1, which is characterized in that the temporal resolution of the beating spectrum of the cardiac muscle cell is 0.01 Second was to 1 second.

10. the method as described in claim 1, which is characterized in that obtained in 1 minute to 5 days after contacting the drug molecule Contact the beating spectrum of the cardiac muscle cell of drug.

11. the method as described in claim 1, which is characterized in that at least one beating ginseng of the beating spectrum of the cardiac muscle cell Number includes following at least one：When beating interval between Beating Rate, adjacent beats, beating intensity, rise time, relaxation Between, the integral area or combinations thereof at each beating peak.

12. the method as described in claim 1, which is characterized in that the method further include the acquisition cardiac muscle cell with it is described Dynamic mass before and after medicament contact redistributes signal, wherein the RWG biosensor imager was at 30 minutes to 5 The beating spectrum of 10 seconds to 1 minute cardiac muscle cells is acquired on multiple time points during it experiment, and the RWG biologies pass The dynamic mass of sensor imager acquisition experiment remaining time redistributes signal.

13. a kind of label-free method of the cardiac toxic of evaluation drug molecule, the method includes：

At least one RWG biosensor is irradiated using the first swept wavelength demodulation scheme, wherein the first frequency sweep wave Long range is at least 5nm, and frequency sweep cycle continues at least 1 second；

The swept wavelength demodulation resonance waveguide optical grating imager of use space parsing measures at least one RWG biosensor All pixels position at central resonance wavelength and obtain average value；

At least one RWG biosensor is irradiated using the second swept wavelength demodulation scheme, wherein the second frequency sweep wave The 3nm models of long range central resonance wavelength average value at whole pixelation positions of at least one RWG biosensor Within enclosing, and frequency sweep cycle continued less than 0.3 second；

Track the central resonance wavelength of RWG biosensor by using the second swept wavelength demodulation scheme has cardiac muscle to establish The baseline dynamic mass of at least one RWG biosensor of cell redistributes signal；

Cell is set to be contacted with drug molecule；

Monitor the dynamic mass redistribution signal of the drug molecule induction in real time with imager；And

Dynamic mass is redistributed into the oscillation in the oscillation mode in baseline period and the dynamic mass redistribution of drug molecule induction Pattern is compared to determine the cardiac toxic of the drug molecule.

14. method as claimed in claim 13, which is characterized in that the cell contact drug molecule after 1 minute to 5 days it is complete The oscillation mode of dynamic mass redistribution signal and the dynamic mass redistribution signal of drug molecule induction in pairs of baseline period Compare.

15. a kind of exempt to mark for evaluating the difunctional of cellular response with the redistribution of low frequency dynamic mass and high-frequency beating Note method, the method includes：

It is radiated at least one resonance waveguide optical grating (RWG) biosensor with adjustable light sources, the RWG biosensor The cell on surface with culture, wherein the adjustable light sources scan illumination wavelength, the particular range in particular range Include the resonant wavelength of at least one RWG biosensor；

The spectrum picture is handled, to：

Obtain the central resonance wavelength at least one RWG biosensor all pixels position；And

Monitor the resonant wavelength image of at least one biosensor, institute in real time using low frequency swept wavelength demodulation modes It is 0.001 to 1 hertz to state low frequency；

Data are acquired from low frequency swept wavelength demodulation modes and are switched to high-frequency intensity monitoring pattern, wherein using having spy The second light source of standing wave length irradiates at least one RWG biosensor, and the high-frequency is 1 to 100 hertz；

5 second to 1 minute intensity of reflected light of the monitoring from least one RWG biosensor is to obtain high-frequency in real time Luminous intensity adjusts image；

Swept wavelength demodulation modes are switched back into monitor the resonant wavelength image of at least one RWG biosensor in real time； With

Resonant wavelength image data is handled and adjusts spectrum and low-frequency resonance wavelength dynamic to obtain high-frequency luminous intensity Quality redistributes signal, wherein spectrum is adjusted using high-frequency luminous intensity to monitor the high-frequency beating spectrum of the cell of culture, and And it redistributes signal using low-frequency resonance wavelength dynamic mass and is redistributed to monitor the dynamic mass of cell response induction.

16. method as claimed in claim 15, the method further include：

Make the cell and molecule contacts；

Acquisition and the high-frequency luminous intensity of specific time after molecule processing adjust spectrum, and acquire and the molecule contacts The low-frequency resonance wavelength dynamic mass of remaining time redistributes signal afterwards；

Compare and adjust spectrum with the high-frequency luminous intensity before and after the molecule contacts, and compares and the molecule contacts Before and after low-frequency resonance wavelength dynamic mass redistribute signal, the variation of high frequency luminous intensity shaping modes and low The variation of frequency resonance wavelength dynamic mass redistribution signal is the index of effect of the molecule to the cell of culture.

17. the method as described in any one of claim 15-16, which is characterized in that the wavelength of second radiation source is It is following at least one：

More than whole pixelation place-centric resonant wavelength average values of at least one RWG biosensor 100 to 1500 The specific wavelength of micromicron；

Below whole pixelation place-centric resonant wavelength mean wavelengths of at least one RWG biosensor 100 to The specific wavelength of 1500 micromicrons；And

The specific wavelength of whole pixelation place-centric resonant wavelength average values of at least one RWG biosensor.

18. a kind of label-free method of the cardiac toxic of evaluation drug molecule, the method includes：

In the first mode, at least one RWG biosensor, wherein institute are irradiated using the first swept wavelength demodulation scheme It states the first swept wavelength ranging from least 5nm and frequency sweep cycle continues at least 1 second；And the swept wavelength of use space parsing During demodulation resonance waveguide optical grating imager is monitored in real time at all pixels position of at least one RWG biosensor Heart resonant wavelength；

In a second mode, at least one RWG biosensor is irradiated using the second swept wavelength demodulation scheme, wherein described the The 3nm of central resonance wavelength average value at ranging from least one RWG biosensor whole pixelation position of two swept wavelengths Within range, and frequency sweep cycle continued less than 0.3 second；And the swept wavelength demodulation resonance waveguide optical grating of use space parsing Imager monitors the central resonance wavelength at all pixels position of at least one RWG biosensor in real time；

In the third mode, at least one RWG biosensor is irradiated using the light source with specific wavelength, wherein described specific Wavelength is higher than, being averaged less than or equal at least one RWG biosensor whole pixelation place-centric resonant wavelength Value；And the resonant reflection light intensity from least one RWG biosensor is monitored in real time using imager, the light source Degree；

Using the cell of the imager monitoring culture using first mode, to obtain the slow dynamic quality of the cell of the culture Redistribution response；

The imager is switched to second mode from first mode, and monitored by the imager culture cell it is fast Fast dynamic mass redistribution response；

The imager is switched to the third mode from first mode or second mode, the culture is monitored by the imager The high-frequency luminous intensity Regulate signal of cell；And

Slow dynamic quality redistribution response signal, quick dynamic mass redistribution response signal and high-frequency luminous intensity are adjusted Signal is handled to determine effect of the drug molecule to the cell of the culture.

19. it is a kind of for detect in the presence of and there is no drug molecule in the case of the dynamic mass of cardiac muscle cell cultivated redistribute The label-free resonance waveguide grating biosensor imaging system of signal and beating figure, the system include：

Adjustable light sources, scan illumination wavelength in particular range, and particular range covering at least one RWG biologies pass The resonant wavelength of sensor；

Charge coupled device (CCD) camera, record comes from least one RWG biosensor in the form of pixelation Reflected light；

Processor calculates the centre wavelength of all pixels position of at least one RWG biosensor；

Operating system is used to irradiate at least one RWG biosensor and provides three kinds of different detection patterns, including：

The first slow pattern of at least one RWG biosensor is irradiated using the first swept wavelength demodulation scheme, wherein described First swept wavelength range is at least 5nm and frequency sweep cycle continues at least 1 second；

The second quick mode of at least one RWG biosensor is irradiated using the second swept wavelength demodulation scheme, wherein described Second swept wavelength range central resonance wavelength average value at least one RWG biosensor whole pixelation position Within 3nm ranges, and frequency sweep cycle continued less than 0.3 second；And

The third intensity mode of at least one RWG biosensor is irradiated using the third light source with specific wavelength, Described in third light source have be higher than, be less than or equal at least one RWG biosensor whole pixelation place-centric The specific wavelength of resonant wavelength average value；And

Computer and display are used to analyze and show the data from three kinds of detection patterns.