Functionalization of gold surfaces with copoly(DMA-NAS-MAPS) by

Sensors and Actuators B 190 (2014) 234–242

Contents lists available at ScienceDirect

Sensors and Actuators B:

Chemical

j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /s n

b

Functionalization of gold surfaces with copoly(DMA-NAS-MAPS)by dip coating:Surface characterization and hybridization tests

D.Petti a ,?,A.Torti a ,F.Damin b ,L.Sola b ,M.Rusnati c ,

E.Albisetti a ,A.Bugatti c ,R.Bertacco a ,M.Chiari b

a

LNESS-Dipartimento di Fisica del Politecnico di Milano,Via Anzani 42,22100Como,Italy

b

Istituto di Chimica del Riconoscimento Molecolare,CNR,Via Mario Bianco 9,20131Milan,Italy c

Department of Molecular and Translational Medicine,University of Brescia,Viale Europa 11,25123Brescia,Italy

a r t i c l e

i n f o

Article history:

Received 5June 2013

Received in revised form 9August 2013Accepted 25August 2013

Available online 4 September 2013

Keywords:

Gold functionalization Copoly(DMA-NAS-MAPS)Chemiluminescence

Surface plasmon resonance Atomic force microscopy

X-ray photoemission spectroscopy

a b s t r a c t

In this work,a new method to functionalize a gold surface by dip coating with a functional copolymer is presented.The coating procedure is simple,robust and can be accomplished in less than one hour.Atomic force microscopy (AFM)scratch tests reveal the presence of a homogeneous polymer coating with a thickness of 2.5nm.X-ray photoemission spectroscopy spectra from C1s,N1s and O1s levels present the typical ?ngerprints of the polymeric overlayer,i.e.the characteristic peaks from CNC O and NC O groups.

Surface plasmon resonance (SPR)binding assays were used to check the coating functional properties.Immobilization of heparin to SPR gold surfaces functionalized with copoly(DMA-NAS-MAPS)-followed by binding analysis with the well known heparin binding protein ?broblast growth factor 2yield binding kinetic parameters comparable to those obtained with commercially available carboxymethyl dextran-functionalized sensorchips,thus con?rming the great potential of the proposed technique.

? 2013 Elsevier B.V. All rights reserved.

1.Introduction

In the last ten years,we assisted to a great development of microarray analysis techniques for proteomics and genomics [1].In this scenario a key-point is the chemical functionaliza-tion of different substrates (glass,silicon,gold)[2]for an effective and selective immobilization of bio-macromolecules such as DNA,oligonucleotides and proteins.

In particular,there is a strong interest in developing methods for gold surface functionalization,as this metal is widely used in biosensing.In surface plasmon resonance (SPR),a thin layer of gold on a high refractive index glass surface absorbs laser light,produc-ing electron waves (surface plasmons)on the gold surface.This occurs only at a speci?c angle and wavelength of incident light which are highly dependent on the surface properties.For this reason,the binding of a target ligand to a receptor immobilized on the gold surface produces a measurable signal related to the concentration of the target [3].

In electrochemical biosensors,an enzymatically catalyzed reac-tion,involving the target analyte,produces or absorbs electrons on the active electrode surface causing either electron transfer across

?Corresponding author.Tel.:+390313327307;fax:+390313327617.E-mail address:daniela.petti@polimi.it (D.Petti).

the double layer (thus producing a current)or contributing to the double layer potential (?nally leading to a voltage)[4].

Several functionalization strategies for gold have been described [2,5]and reference therein]including surface modi?cation with silanes [6]and functional polymers.In SPR the unquestioned coat-ing choice is based on dextran.A stable dextran layer is obtained by covalent binding of the polymer to a functional sublayer formed on the surface by self-assembling of a thiol bearing molecule.The characteristics of the dextran coated surface have been optimized and the quality of this coating has greatly contributed to the success of SPR technology.However,the procedure for binding dextran is cumbersome and requires multistep chemical reactions which may be dif?cult to control.

In the case of the dextran coating as in other bioassays involv-ing gold [7],the coating formation requires spontaneous assembly of chemically reactive alkene thiols leading to formation of so called self-assembled monolayers (SAM)[8–10].By placing a gold substrate into a millimolar solution of an alkanethiol in ethanol a variety of chemically reactive groups can be introduced by a reaction between gold and thiols.This reaction is well-known and widely adopted due to the fact that it is straightforward and requires commercially available chemical reagents.In addition the binding between gold and thiol is very stable owing to its cova-lent character [11,12].However,the formation of a well-assembled monolayer strongly depends on the purity of the alkanethiol being

0925-4005/$–see front matter ? 2013 Elsevier B.V. All rights reserved.https://www.wendangku.net/doc/1e11994544.html,/10.1016/j.snb.2013.08.077

D.Petti et al./Sensors and Actuators B190 (2014) 234–242235

used.The presence of even low levels of contaminants can result in a disordered,non-ideal monolayer.Unfortunately,typical impu-rities in thiol compounds are thiolated precursor molecules,not properly separated during the puri?cation process[13].

In this work we present an alternative approach to surface modi-?cation that is fast,robust and easy to perform even in laboratories that do not have a strong surface chemistry background.In par-ticular,we propose a method based on a polymeric thin coating made of N,N-dimethylacrylamide(DMA)bearing silanating(MAPS) and chemically reactive(NAS)moieties:poly(DMA-NAS-MAPS). The copoly(DMA-NAS-MAPS)was developed for DNA and protein microarray assays on microscope glass slide[14]and on silicon[15]. As shown in previous papers[6],the deposition by“dip and rinse”coating of a thin layer of this polymer represents a fast,inexpensive and robust method to covalently bind proteins and amino-modi?ed DNA probes.In addition,the?lm is stable in aqueous buffers con-taining various additives,even at water boiling temperature and, due to its hydrophilic nature and high homogeneity,it minimizes non-speci?c biomolecular interactions.The last feature is highly desirable for surface recognition bioassays,for which speci?city is dif?cult to achieve.Furthermore,the proposed method,employing non surface-speci?c material modi?cations,can be easily used also to form uniform coatings on many different materials other then gold,such as glass,silicon dioxide,silicon nitride and titanium diox-ide[15],thus being applied to different biosensing problems and techniques.This versatility can be particularly important in appli-cation employing sensors that consist of more than one material. The use of copoly(DMA-NAS-MAPS)allows the derivatization of all the different regions of the device in single step.

The morphological and chemical properties of this polymeric coating on gold surfaces have been studied by means of atomic force microscopy(AFM)and X-ray photoemission spectroscopy(XPS). These techniques reveal that the polymer is immobilized on the sur-face with the proper chemical composition and expected thickness. Furthermore we found a limited impact on the gold underlayer, thus allowing the use of the proposed functionalization method for techniques such as SPR.To demonstrate the compatibility of this derivatization process with standard bioassays,two different experiments have been carried out:a DNA hybridization exper-iment with chemiluminescence and surface plasmon resonance (SPR)to assay the binding of?broblast growth factor(FGF2)in solu-tion to immobilized heparin.Independently from the transducer employed,results comparable to those obtained with commercially available carboxymethyl dextran-functionalized sensorchips were obtained.This indicates the potential of the coating technique to implement robust protocols of biomolecules immobilization onto gold coated surfaces for biosensing applications.

2.Materials and methods

2.1.Chemicals

Tris(hydroxymethyl)aminomethane(TRIS),ethanolamine, saline-sodium citrate(SSC),ammonium sulfate,sodium dodecyl sulfate(SDS),phosphate buffer solution(PBS), N,N dimethylacrylamide(DMA)and[3-(methacryloyl-oxy)propyl] trimethoxysilane(MAPS)were purchased from Sigma(St.Louis, MO).N,N acryloyloxysuccinimide(NAS)was from Polysciences (Warrington,PA).Oligonucleotides were synthesized by MWG-Biotech AG(Ebevsberg,Germany).Streptavidin labeled by HRP (Horse Radish Peroxidase)from Jackson Immunoresearch.Heparin (13.6kDa)was obtained from Laboratori Derivati Organici Spa (Milan,Italy).SuperSignal ELISA Femto maximum Sensitivity kit from ThermoScienti?c.Human recombinant?broblast growth factor2(FGF2)was expressed in Escherichia coli and puri?ed as described in[16].2.2.Gold surface preparation and derivatization

For initial tests,gold?lms on Si were prepared in-house.Silicon wafers were coated with3nm Cr(to promote gold adhesion)and 50nm Au,by means of electron beam evaporation.Copoly(DMA-NAS-MAPS),whose chemical structure is shown in Fig.1,was synthesized and characterized as described in a previous work [17,14].The gold surface was treated for ten minutes in an Oxygen Plasma Generator(Harrick Plasma Cleaner)and then immersed for 30min in a copoly(DMA-NAS-MAPS)solution(1%,w/v in a water solution of ammonium sulfate at a20%saturation level).The silicon slides were then extensively washed with water and dried under vacuum at80?C for15min.

2.3.AFM measurements

AFM measurements were performed with a VEECO Innova AFM. The morphological characterization was carried out in tapping mode with512samples/line,frequency of0.8Hz and4–10?m scan range.The thickness of the polymeric layer was evaluated by AFM-tip scratch test[18].This technique consists in scratch-ing the coating with the AFM tip while the normal force applied is set to a value high enough to penetrate the layer but low enough to avoid signi?cant tip or substrate damaging.The scratch was made in contact mode onward and in lift mode backward(start height 0.3?m,lift height0.3?m)with30continuous scan cycles,256sam-ples/line,62.75Hz,1?m scan range.After scratching,the area was imaged in tapping mode with the same tip used for scratching.In this way the area of interest is easily identi?ed within the active scan area of the AFM.

2.4.XPS experiments

XPS experiments were performed in a multichamber ultrahigh-vacuum(UHV)system described in detail elsewhere[19]. Photoelectrons were excited by Al K?(h?=1486.67eV)or Mg K?(h?=1253.6eV)radiation,and analyzed by a150mm hemispher-ical analyzer(SPECS Phoibos150).The angular acceptance set for this work was about±6?,and the spatial resolution was1.4mm. Two modes of operation have been employed:(M1)pass energy of 50eV and Al K?line(total energy resolution of1.643eV)to eval-uate the stoichiometry of the polymer;(M2)pass energy of10eV and Mg K?line(total energy resolution of0.841eV)in order to study the lineshape of the characteristic peaks.The energy scale of the spectra has been calibrated by measuring a Ta foil in good electrical contact with the sample.

2.5.Chemiluminescence test

A23-mer,5 -amine-modi?ed oligonucleotide synthesized by MWG-Biotech AG(Ebevsberg,Germany),100?M stock solution, 5 -NH2-(CH2)6-GCC CAC CTA TAA GGT AAA AGT GA,was dissolved in150mM sodium phosphate buffer at pH8.5.A10?M solution of this oligonucleotide was printed on coated slides to form a15×15 array using a piezoelectric spotter(SciFLEXARRAYER S5;Scienion). Spotting was done at20?C in an atmosphere of50%humidity. The oligonucleotides were coupled to the surfaces by overnight incubation in an uncovered storage box,laid in a sealed cham-ber,saturated with sodium chloride(40g/100mL H2O),at room temperature.After incubation,all residual reactive groups of the coating polymer were blocked by dipping the printed slides in 50mM ethanolamine/0.1M Tris pH9.0at50?C for15min.After discarding the blocking solution,the slides were rinsed two times with water and shaken for15min in4×SSC/0.1%SDS buffer, pre-warmed at50?C,and brie?y rinsed with water.An oligonu-cleotide,complementary to the one spotted on the surface,at a

236 D.Petti et al./Sensors and Actuators B 190 (2014) 234–

242

Fig.1.structure of the copoly(DMA-NAS-MAPS)bound to the gold surface.The copoly presents three functional groups:the polymer backbone interacting with the surface,the pending silane hydrolysable monomers that stabilizing the ?lm and the chemically active monomers allowing the covalent binding of biomolecules.

1.0?M concentration (

2.5?L/cm 2of coverslip)was dissolved in the hybridization buffer (2×SSC,0.1%SDS and 0.2mg/mL BSA)and immediately applied to microarrays.The complementary oligonu-cleotide has the sequence 5 -TCA CTT TTA CCT TAT AGG TGG GC-3 and is labeled with biotin in 5 position.The surfaces,placed in a hybridization chamber,were transferred to a humidi?ed incuba-tor at a temperature of 65?C for 2h.After hybridization,the slides were ?rst washed with 4×SSC at room temperature to remove the coverslip and then with 2×SSC/0.1%SDS at hybridization tem-perature for 5min.This operation was repeated two times and was followed by two washing steps with 0.2×SSC and 0.1×SSC for 1min at room temperature.Streptavidin labeled by HRP (Horse Radish Peroxidase)from Jackson Immunoresearch at the concentration of 1?g/mL in PBS buffer was applied to the gold surface for 60min.After a short rinse in PBS and water,the slides were dried and chemiluminescence was developed using the SuperSignal ELISA Femto Maximum Sensitivity kit from ThermoScienti?c according to the manufacturer instructions.The chemiluminescence signal was registered at 0.2s exposure time using a homemade set up equipped with a Q Imaging CCD Camera.

2.6.Surface plasmon resonance (SPR)

For SPR experiment,a naked gold sensorchip (Xantech Bioanalytics,Dusseldorf,Germany)was functionalized with copoly(DMA-NAS-MAPS)as follows:the gold chip was treated with oxygen plasma in a Plasma Cleaner,Harrick Plasma (Ithaca,NY,USA)to clean the surface.Immediately after the oxygen plasma treatment,the sensorchip was dipped in an aqueous solution of poly(DMA-NAS-MAPS)in ammonium sulphate at 20%of saturation level for 30min,and then it was rinsed with DI water,dried with nitrogen ?ow and cured under vacuum at 80?C for 15min.The polymer was functionalized with streptavidin by spotting 1mg/mL solution of protein dissolved in PBS using a piezoelectric spotter

(SciFLEXARRAYER S5;Scienion).The merged spots,printed at 20?C in an atmosphere of 50%humidity,created a continuous protein ?lm.To promote the protein attachment,the chip was incubated overnight in an uncovered storage box,laid in a sealed chamber,saturated with sodium chloride (40g/100mL H 2O),at room temperature.The functionalized sensorchip was positioned in a BIAcore X instrument (GE-Healthcare,Milwaukee,WI)and the surface was conditioned with 3consecutive 1-min injections of 1M NaCl in 50mM NaOH before heparin immobilization.Biotinylated heparin [100ng/mL in 10mM HEPES buffer pH 7.4containing 150mM NaCl,3mM EDTA,0.005%surfactant P20(HBS-EP)]was injected onto the copoly(DMA-NAS-MAPS)functionalized sensor-chip for 4min at a ?ow rate of 5?L/min.Increasing concentrations of FGF2in HBS-EP were injected over the heparin for 4min at a ?ow rate of 30?l/min (to allow their association with immobilized heparin)and then washed until dissociation was observed.A sensorchip coated with streptavidin was used to evaluate the non-speci?c binding and for blank subtraction.After every run,the sensorchip was regenerated by injection of 2M NaCl.Kinetic parameters were calculated by using the non linear ?tting software package BIAevaluation using a single site model.

3.Results and discussion

3.1.Polymeric coating characterization

A copolymer of dimethylacrylamide,N-hydroxysuccinimide and [3-(methacryloyl-oxy)propyl]trimethoxysilane has been used to form,by dip and rinse coating,a thin ?lm on the gold surface.The copolymer structure is shown in Fig.1,where relative molec-ular weights and molecular weight distributions of the copolymer were determined by gel permeation chromatography (GPC)in pre-vious works [20].It has three functional portions:(i)segments that might interact with the surface by week,non covalent bonds,van

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237

Fig.2.AFM image on a5?m×5?m area of the free gold(panel a)and polymer coated(panel b)surface.The roughness RMS is0.4and0.6nm.

der Waals or hydrophobic forces(polymer backbone-DMA)favored by gold hydrophobicity,(ii)pending silane hydrolysable monomers that stabilizes the?lm by promoting condensation between differ-ent chains(MAPS),and(iii)chemically active monomers that react with amines present in the protein later chains allowing the cova-lent binding of biomolecules to the gold surface(NAS).This polymer forms a?lm on a number of materials of different chemical com-position such as glass[14],silicon oxide[15]and organic polymers [21,22].However,the speci?c mechanism by which the polymer forms a thin layer on the gold surface is not fully understood.

The AFM images taken from the gold and polymer modi?ed sur-face are presented in Fig.2(panels a and b,respectively),for the case of Au/Cr/Si samples.The root mean square(RMS)roughness of the gold surface is about0.36nm on an area of5?m×5?m,while the polymer coated surface presents a RMS roughness of0.60nm. The minor roughness increase suggests a uniform and continu-ous polymeric coating mimicking the gold morphology.In Fig.3a a representative nano-scratch image of the layer and in Fig.3b some depth pro?les(taken along the white lines)are shown.In the scratched area(1?m×1?m)a roughness of0.48nm RMS is recov-ered.The comparison of the AFM phase contrast(data not shown) on the scratched area and on the overlayer,reveals that the two zones are made of different materials.This proves that the scratch has fully removed the polymeric coating.From depth pro?les of Fig.3b,we can then evaluate a layer thickness d=2.5±0.3nm,con-sistent with the thickness evaluated by scratch tests performed on a SiO2surface coated with the same polymer[15].An impor-tant characteristic of this polymeric coating is its ability to swell in aqueous solutions.The swelling,also present in other polymers, depends on the properties of the polymer of interest such as its molecular weight,as well as the environmental conditions such as temperature,or pH[20]

.Fig. 3.Panel a: 4.5?m×4.5?m AFM image of the sample after scratch;the scratched area is1?m2.Panel b:depth pro?les from the lines of panel a.The estimated polymer thickness is2.5±0.3nm,respectively.

The chemical characterization of the polymeric coating has been carried out by XPS.Wide scans taken on coated samples show the characteristic peaks of four chemical elements:gold from the substrate and carbon,oxygen,nitrogen coming from the polymer. In Table1,the intensity ratios O1s/C1s,and N1s/C1s are shown, after normalization to the cross section,analyzer transmission and probing depth.To this scope,we normalized the peak intensity to the function f( )= *(1?exp(?d/ )),where is the electron escape depth[23].The surface was analyzed at two collection angles from the sample normal:0?and60?,the last one providing higher surface sensitivity due to the?nite escape depth of the emitted photoelectrons.

Table1

Relative intensities of N1s,O1s and C1s peaks taken at0?,60?collection angle and relative nominal atomic ratios(from the polymer composition).

Collection

angle(?)

N1s/C1s

intensity ratio

O1s/C1s

intensity ratio XPS00.17±0.020.16±0.02

600.14±0.020.15±0.02 copoly(DMA-NAS-MAPS)

composition

–0.190.20

238 D.Petti et al./Sensors and Actuators B190 (2014) 234–

242

Fig.4.XPS spectrum of C1s taken at0?(panel a)and60?(panel b)collection angle; the deconvolution of the peaks reveals the presence of the speci?c group of the polymer:NC O and CNC O.

The nitrogen and oxygen content,evaluated from the intensity of the characteristic peaks,are,respectively,17%and16%of that of the carbon.In the surface sensitive con?guration(60?collection angle)the signal coming from N1s decreases to14%of that of C1s. Note that this decrease is close to the limit of the uncertainty of the XPS measurement(about10%of the ratios between concentrations of different elements),but could be compatible with the presence of some additional adventitious carbon(e.g.physisorbed CO2)at the surface.These results reveal a slightly higher carbon concen-tration with respect to oxygen and nitrogen,when compared to the expected atomic ratio for the copoly reported in Table1(19% and20%,respectively),which may be due again to surface carbon contamination.This contamination arises mainly to the extended exposure of the sample to atmospheric environment before the measurements.A rough estimation of the amount of adventitious carbon can be obtained by comparing the measured and nominal N/C and O/C ratios of Table1:a value of contaminant of about6%of the total elemental composition is estimated,consistent with data obtained on similar functionalization[24].

The deconvolution of the C1s peak shown in Fig.4allows dis-entangling the contributions from the different coordinations of C atoms in the polymer.The?tting has been performed with a Voigt line-shape for each component,with a?xed FWHM of1.56eV.This is consistent with the FWHM of the C1s peak from adventitious car-bon,previously measured in the same experimental conditions on other samples[25]

.Fig.5.XPS spectrum of N1s(panel a),O1s(panel b)and Au4f(panel c)at60?collection angle;while the?rst two spectra show the features coming from NC O groups,the Au4f peak lineshape presents the typical metallic character of a free gold.

Panel(a)(0?collection angle)and panel(b)(60?collection angle) of Fig.4show common features.The main peak(A1)is located at 285.6eV and represents about63%of the entire C1s signal.As it can be ascribed both to adventitious carbon(carbon partially bounded to O and N[25])and C C bonds,it cannot be unambiguously

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239

Fig.6.Scheme of the bioassay surface.

connected to the polymer coating.Two other contributions are visible in both panels of Fig.4:A2(286.7eV),which is due to the presence of C O and C N C O chemical bonds[26]and A3 (288.4eV)coming from CO2,NC O and methoxy(O CH3)groups. [26–28]The experimental weight of A2and A3are22%and15%of the whole C1s peak,respectively,but also in this case they can be only partially attributed to C atoms within the polymer,due to some possible CO and CO2contaminations contributing to A2and A3. These values must be compared with the polymer chemical struc-ture,from which the relative weight of the different carbon bonds can be evaluated.In a nominal polymer coating C C bonds(con-tributing to A1)represent50.5%of the entire carbon bonds,C N and C O bonds(A2)31.5%,while NC O,O C=O,O CH3groups (indicated as OMe in the polymer chemical structure of Fig.1)18.3%.

The discrepancy between the expected ratios form the chemical formula and the experimental ones is consistent with the oxy-gen and nitrogen de?ciency found in the composition analysis of Table1.Adventitious carbon contamination can clearly account for these results and prevents from a precise estimation of the coating composition via quantitative XPS analysis.

Finally note that,at0?collection angle(bottom panel of Fig.4), an additional feature(A4)at290eV is needed to?t the C1s peak. Due to the increased probing depth at normal emission,this feature can be attributed to some contaminants far away from the surface, most probably carbonates on the bare gold surface which can form during the coating in wet environment.

Spectra at60?collection angle of N1s and O1s,are presented in Fig.5.Also in this case the peaks have been decomposed using Voigt functions with?xed FWHM of1.62eV and1.53eV,respectively, [25,26].

The N1s spectrum presents two characteristic contributions:the main one at400.7eV(B1),corresponding to C N C and N C O bonds from the polymer[26]and a minor one at399.1eV(B2),cor-responding to C N bounds from adventitious carbon[29].B2is only 5%of the entire peak and can be attributed to contamination from adventitious carbon,as already seen from C1s spectra deconvo-lution.Regarding oxygen,three features give rise to the O1s peak,but here the attribution is not straightforward.While the feature at 531.7eV(C1)is due only to NC O groups[26],the prominent con-tribution at533.3eV(C2)can arise from the overlap of the signals from H2O(533.3eV),O CH3(533.2eV)[28]and C O(533.6eV) groups[30].The minor feature at534.9eV(C3)might instead be due to physisorbed CO2.All these features in the O1s peak are consistent with the presence of the polymeric groups found from the deconvolution of N1s and C1s peaks,but a quantitative analy-sis here is prevented from the superposition of the characteristic signals.

In order to evaluate the impact of the polymer coating on the gold surface,we present in Fig.5c the Au4f peaks collected at60?in order to maximize the surface sensitivity.The continuous line rep-resents a?t performed using the lineshape measured on the clean gold surface,before dip coating,in the same experimental condi-tions.The results seem to suggest that the gold surface is unchanged upon dip coating,without detectable traces of oxidization.How-ever,previous studies have demonstrated that the Au surface can be oxidized by oxygen plasma treatments[31],therefore we can not exclude that,at least a small portion of the methoxysilane on the polymer might condensate with oxidized gold atoms strengthening the binding between the polymer and the surface which primar-ily is due to non covalent interactions such as van der Waals or hydrophobic forces.

In conclusion,both the AFM scratch test and the XPS analysis reveal that a continuous polymer is immobilized on the surface. In particular XPS shows the presence of the characteristic chemi-cal groups from copoly(DMA-NAS-MAPS)on top of a gold surface preserving its chemical integrity upon coating.

3.2.Hybridization experiments

A hybridization test was carried out to prove that the func-tional polymer bind amino modi?ed oligonucleotides with great ef?ciency and low background noise.A simple DNA hybridiza-tion experiment was performed to prove that the coating has introduced chemical reactive groups on the gold surface.A scheme

240 D.Petti et al./Sensors and Actuators B 190 (2014) 234–242

Table 2

The association rate (k on )and dissociation rate (k off )are reported and the dissociation constant (K d )was derived from the k off /k on ratio.The results on Carboxymethyl dextran are representative of two independent experiments with similar results.

Functionalization

FGF2/heparin interaction kinetic parameters:

References

k on (s ?1M ?1)

k off (s ?1)K d (nM)Copoly(DMA-NAS-MAPS)8.22×104 1.54×10?318.0Present work Carboxymethyl dextran

9.0×103 3.8×10?442.5[37]8.14×103

3.2×10?4

38.0

[38]

of a typical array experiment is shown in Fig.6.Replicates of an oligonucleotide ligand are spotted on the polymer coated gold using a piezoelectric spotter.After washing and blocking the sur-face unreacted sites,the array is probed with a sample containing a complementary target oligonucleotide labeled with biotin.A detection step with streptavidin labeled with horse radish per-oxidase (HRP)is then performed.The hybridization reaction was revealed by chemiluminescence using an enzyme,HRP,that catal-yses the conversion of the enhanced chemiluminescent substrate into a sensitized reagent in the vicinity of the molecule of inter-est,which on further oxidation by hydrogen peroxide,produces a triplet (excited)carbonyl,which emits light when it decays to the singlet carbonyl.Enhanced chemiluminescence allows detec-tion of minute quantities of a biomolecule.The strong signal to noise ratio of the oligonucleotide spots proves that the surface bind cova-lently amino-modi?ed DNA molecules (Fig.7).Chemiluminescence was chosen to prevent ?uorescence quenching that would have occurred labeling the DNA with a ?uorophore [32].In this approach,luminol,one of the most widely used chemiluminescent reagents,is oxidized by peroxide and it results in creation of an excited state product called 3-aminophthalate.This product decays to a lower energy state by releasing photons of light.The distance from the surface is enough to prevent quenching.

Finally,to prove the practical exploitability of copoly(DMA-NAS-MAPS),a SPR assay was set up.Streptavidin was immobilized to a copoly(DMA-NAS-MAPS)-functionalized gold sensorchip and used to capture biotinylated heparin.Then,heparin immobilized to the gold surface was evaluated for its capacity to bind FGF2,one of the most important angiogenic growth factors largely studied for its heparin-binding capacity [33,34].Injection of 100ng/mL of hep-arin onto a copoly(DMA-NAS-MAPS)/streptavidin-functionalized sensorchip allows the immobilization of 233resonance units (RU)equal to 17.1fmol/mm 2of heparin.Relevant to note,in previous experiments,the injection of heparin onto

streptavidin

Fig.7.Chemiluminescence signals of oligonucleotide spots after hybridization with complementary oligonucleotide labeled with biotin followed by incubation with streptavidin labeled with Horse Radish

Peroxidase.

Fig.8.SPR analysis of FGF2/heparin interaction.(A)Sensorgrams showing the bind-ing of FGF2(300nM)to a copoly(DMA-NAS-MAPS)-functionalized gold layer coated with streptavidin alone (hatched line)or with streptavidin and biotinylated-heparin (straight line).(B)Blank-subtracted sensorgram overlay showing the binding of increasing concentrations of FGF2(300,150,75,36,18e 9nM from top to bottom)to a copoly(DMA-NAS-MAPS)-functionalized gold layer coated with streptavidin and biotinylated-heparin.In both the panels the response (in RU)was recorded as a function of biographies time.

immobilized to a carboxymethyl dextran-functionalized sensor-chip lead to the immobilization of comparable amounts of heparin (from 5.8to 9.5fmol/mm 2)[35,36].We then studied the capac-ity of copoly(DMA-NAS-MAPS)/streptavidin-immobilized heparin to bind FGF2.As shown in Fig.8A,when immobilized to a surface via copoly(DMA-NAS-MAPS)/streptavidin,heparin retains its capacity to bind FGF2in a speci?c way,as demonstrated by the absence of interaction with immobilized streptavidin.Increasing concentra-tions of FGF2were injected over the heparin surface to evaluate the kinetic parameters of FGF2/heparin interaction.As reported in Table 2,very similar values of dissociation (k off )and associa-tion (k on )rates and of dissociation constant (K d )can be obtained from the analyses performed onto copoly(DMA-NAS-MAPS)-or carboxymethyl dextran-functionalized gold layers.

4.Conclusions

In this paper,a new method for functionalizing a gold surface with copoly(DMA-NAS-MAPS)is presented.The polymer is simply

D.Petti et al./Sensors and Actuators B190 (2014) 234–242241

deposited by dip coating after an oxygen plasma treatment, followed by washing in water and drying under vacuum.

AFM scratch tests reveal the presence of a uniform polymeric coating with a thickness of2.5nm.The XPS spectra from C1s N1s and O1s levels present the typical?ngerprints of the poly-meric overlayer,i.e.the features arising from NC O and OMe groups.The chemical composition found by XPS reveals a slight de?ciency of nitrogen and oxygen with respect to carbon.This can be due to a limited carbon contamination of the polymeric coming in wet and atmospheric environment.The analysis of the surface chemical properties of the gold substrate by XPS indicates no modi?cations induced by the coating process.The lim-ited impact on the Au properties seen by XPS is con?rmed also by functional tests performed on the polymer coated gold sub-strates.

Indeed,we showed the capability of the coating in the immo-bilization of biomolecules in different experiments.In particular, oligonucleotides were used as probes for the capture of comple-mentary oligonucleotide strands detected by chemiluminescence. To assess the possibility to use copoly(DMA-NAS-MAPS)in SPR analysis,heparin was immobilized onto the polymer-coated gold surfaces of an SPR sensor and evaluated for its capacity to bind to FGF2,an important angiogenic factor.Three considerations can be drawn from the results of these experiments:(i)copoly(DMA-NAS-MAPS)allows the immobilization of heparin in amount that are comparable(if not higher)to those routinely immobilized onto different commercially available carboxymethyl dextran sur-faces(i.e.CM5and F1sensorchips[37,38]);(ii)in our analyses, copoly(DMA-NAS-MAPS)turned out to be devoid of signi?cant binding capacity,as shown by the very low level of aspeci?c bind-ing of FGF2to a copoly(DMA-NAS-MAPS)surface devoid of heparin (see Fig.8A);(iii)the speci?c binding of FGF2to copoly(DMA-NAS-MAPS)-immobilized heparin occurs with kinetic parameters that are comparable to those measured onto commercially available car-boxymethyl dextran surfaces(Table2).This is at variance with the higher simplicity and robustness of the process we introduced in this paper,which could?nd applications in many different bio-chemical procedures.

Acknowledgements

This work was partially supported by Fondazione Cariplo via the project SpinBioMed(Project No.2008.2330).M.R and A.

B.acknowledge Ministero dell’Istruzione,dell’Universita’e della Ricerca,Istituto Superiore di Sanita’(AIDS Project),and Fondazione CARIPLO(project No.2008.2198)for?nancial support. References

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Biographies

Daniela Petti received B.S.and M.S.degrees in Physics Engineering from Politecnico di Milano in2004and2006.In2010she obtained her PhD in Physics from Politecnico di Milano,where she is actually Assistant Professor in the Physics Department.She is co-author of more than25research papers and2patents.She started her activity in the?eld of spintronics.Currently,the main research activity is related to the applications of nanomagnetism to biology and medicine.Her interests include the development of magnetic platforms for biosensing and for on chip?ne manipulation of functionalized magnetic nanoparticles for in-vitro applications.

Andrea Torti received B.S.,M.S.degrees in Physics Engineering and PhD in Physics from Politecnico di Milano in2006,2009and2013,respectively.At the beginning of2013he joined the Electronic Department of Politecnico di Milano to work on the integration of biosensors and CMOS technology.Actually,he is Technical Device Engineer at ST Microelectronics,Agrate Brianza(Italy).He started his activity in the ?eld of Nanomagnetism and Nanomedicine.Currently his research interests are in the smart power integrated devices?eld.

Francesco Damin was born in1971in Busto Arsizio(VA,Italy),graduated in Bio-logical Science,specialty Molecular Biology,at Universitàdegli Studi di Milano in 1998.He was recipient of several fellowships and research contracts from ICRM, C.N.R.where he was appointed Research Scientist in2011.His research interests, documented by more than35publications on peer-reviewed journals and several communications at congresses,deal with the development of surfaces to the anal-yses of DNA and proteins with the microarray technology.

Laura Sola earned her M.S.Degree in Chemistry and Pharmaceutical Technolo-gies and her Ph.D.in Drug Chemistry at University of Milan in2008and2012, respectively.She currently holds a post-doctoral appointment at the Institute of Chemistry for Molecular Recognition,CNR where she develops novel polymeric coatings for immobilizing biomolecules and techniques for molecular recognition on supports made of various materials.These are used in biomedical applications of biosensors and Lab on Chip https://www.wendangku.net/doc/1e11994544.html,ura specializes in chemical synthesis of monomers,polymers and linear matrices for DNA,DNA sequencing by cap-illary electrophoresis and novel capillary polymer coatings enabling improved analysis.

Marco Rusnati gained his degree at the Institute of Biochemistry,University of Milan,Italy,in1985.He is now associated professor,Section of Experimen-tal Oncology and Immunology,University of Brescia.His researches focused on: 1983–85:puri?cation and biochemical characterization of d-amino acid oxidase. 1986–1992:puri?cation of angiogenic factors,identi?cation of their functional domains.1992–present:interaction of angiogenic factors with receptors and extracellular binders.1996–present:biological activity of HIV-1Tat,study of its interaction with receptors and extracellular binders.2001–present:study of inter-actions of viral proteins by surface plasmon resonance.He is author of110scienti?c publications in peer-reviewed journals.

Edoardo Albisetti obtained B.S.and M.S.degrees in Physics Engineering from Politecnico di Milano in2008and2010.Currently,he is a PhD student in the Physics Department of Politecnico di Milano.His research activity mainly concerns the development of spintronic devices and their application in biology.In this framework,he developed a sensing platform based on magnetoresistive sensors for detection of molecular recognition events and a system based on magnetic domain wall conduits for providing local mixing of solutions in micro?uidic systems.Magnetic domain wall conduits are also used for ultra-?ne spatial manipulation of magnetic nanoparticles for in-vitro applications.

Antonella Bugatti obtained her B.S.degree in Biomedical Laboratory Technician from University of Verona.Since2006she is Research fellow at the Unit of Exper-imental Oncology and Immunology,University of Brescia.She is co-author of25 scienti?c publications in peer-reviewed journals.Her expertise is related to cell and molecular biology assays,protein-protein interaction(SPR technology),in-vitro assays(endothelial cell proliferation,migration,invasion and morphogenesis).Her research interests are associated to the study of protein-protein interaction by sur-face plasmon resonance.

Riccardo Bertacco got his degree in Electronic Engineering at Politecnico di Milano in1994.He obtained a permanent position at the Physics Department of Politecnico di Milano in1999and the PhD in Physics in2000.Actually,he is Associate Professor at the Physics Department of Politecnico di Milano,where he leads the NanoBiotech-nology and Spintronics group of the LNESS center.He is the technical responsible for the realization of a new clean-room for micro and nanofabrication at Politecnico di Milano.He is author of about100papers in international journals,a chapter of a book and6patent applications.

Marcella Chiari graduated in Chemistry and Pharmaceutical Techniques at the Isti-tuto di Chimica Organica,Facoltàdi Medicina,Universitàdi Milano in1982.She received the Diploma in Clinical Biochemistry,Universitàdi Milano in1990.Since 1992she has been Senior Researcher at the ICRM,CNR,where she leads the labora-tory“Development of Analytical Microsystems¨.Her research activity is documented by more than100publications and several patents.She has been a contractor of the EC in the framework of different projects and responsible for several national research programs.

酵母双杂交技术

酵母双杂交系统 1．原理 酵母双杂交系统的建立得力于对真核细胞调控转录起始过程的认识。研究发现，许多真核生物的转录激活因子都是由两个可以分开的、功能上相互独立的 结构域(domain)组成的。例如，酵母的转录激活因子GAL4，在N端有一个由147个氨基酸组成的DNA结合域(DNA binding domain,BD)，C端有一个由113 个氨基酸组成的转录激活域(transcription activation domain,AD)。GAL4分子的DNA结合域可以和上游激活序列(upstream activating sequence，UAS)结合，而 转录激活域则能激活UAS下游的基因进行转录。但是，单独的DNA结合域不 能激活基因转录，单独的转录激活域也不能激活UAS的下游基因，它们之间只 有通过某种方式结合在一起才具有完整的转录激活因子的功能。 2．试验流程 酵母双杂交系统正是利用了GAL4的功能特点，通过两个杂交蛋白在酵母细 胞中的相互结合及对报告基因的转录激活来捕获新的蛋白质，其大致步骤为: 2．1、视已知蛋白的cDNA序列为诱饵(bait)，将其与DNA结合域融合，构建 成诱饵质粒。 2．2、将待筛选蛋白的cDNA序列与转录激活域融合，构建成文库质粒。2．3、将这两个质粒共转化于酵母细胞中。 2．4、酵母细胞中，已分离的DNA结合域和转录激活域不会相互作用，但诱 饵蛋白若能与待筛选的未知蛋白特异性地相互作用，则可激活报告基因的转录；反之，则不能。利用4种报告基因的表达，便可捕捉到新的蛋白质。 3．特点 优点 蛋白--蛋白相互作用是细胞进行一切代谢活动的基础。酵母双杂交系统的建立 为研究这一问题提供了有利的手段和方法。 缺点 尽管该系统己被证实为一种非常有效的方法，但它也有自身的缺点和问题。1、它并非对所有蛋白质都适用，这是由其原理所决定的。双杂交系统要求两种杂 交体蛋白都是融合蛋白，都必须能进入细胞核内。因为融合蛋白相互作用激活 报告基因转录是在细胞核内发生的。2、假阳性的发生较为频繁。所谓假阳性，即指未能与诱饵蛋白发生作用而被误认为是阳性反应的蛋白。而且部分假阳性 原因不清，可能与酵母中其他蛋白质的作用有关。3、在酵母菌株中大量表达外源蛋白将产生毒性作用，从而影响菌株生长和报告基因的表达。 使用酵母双杂交技术应注意的问题 真正明了酵母双杂交技术的主要原理及筛选方法是进行酵母双杂交实验的前提，构建成功的诱饵质粒及大量的材料准备是进行酵母双杂交实验的保证。只 有明了双杂交的原理，才有可能设计实验进程、才能有目的的进行材料准备， 并能对实验结果作出预测与分析，尤其要对具体实验中各种选择性压力培养基 的使用目的要十分清楚。大量的材料准备、较长的实验流程是酵母双杂交有别

一种大屏幕拼接拼接缝的消除方法

一种大屏幕拼接拼接缝的消除方法 1 引言 图像镶嵌技术（mosai ）是图像融合技术的一种，一般指的是同种类型图像的融合。他把多幅具有重叠信息部分的图像衔接在一起，得到一幅完整的、范围更大的图像，并且去除其中的冗余信息。图像镶嵌技术的应用非常广泛。例如，虚拟现实中的全景图显示及遥感图像的处理等领域，都有广泛的应用。图像镶嵌的评价标准是镶嵌后得到的图像，不但具有良好的视觉效果，而且还要尽可能地保持图像光谱特征。通俗地说，就是镶嵌的图像越“无缝”，效果就越好。当然，这里的“无缝”，不是绝对意义上的，而是人眼分辨力以内的“无缝”。 一般情况下，进行图像拼接时，在拼接的边界上，不可避免地会产生拼接缝。这是因为两幅待拼接图像在灰度上的细微差别都会导致明显的拼接缝。而在实际的成像过程中，这种细微差别很难避免。因此图像镶嵌技术的难点就在于准确寻找图像之间的位置关系，并把两幅以上的图像平滑地衔接在一起，获取一幅全局的图像。本文的基本思想就是突破以往在寻找拼接线时，只要找到一个最佳拼接点，以此点做一条直线作为拼接线的不合理性，而是取一个闭值，在闭值范围内寻找出每个拼接点，把这些点连成的折线作为拼接线，进行拼接。 2 拼接缝消除的方法 传统的拼接缝消除的方法有很多，其中用得较多的方法有；中值滤波法、利用小波变换的方法、加权平均法等 2 . 1 中值滤波法消除拼接缝 中值滤波法是对接缝附近的区域进行中值滤波。对与周围灰度值差比较大的象素取与周围象素接近的值，从而消除光强的不连续性。中值滤波器处理接缝附近的狭长地带。该方法速度快，但质量一般。平滑的结果会使图像的分辨率下降，使图像细节分辨不出，产生图像模糊。 2 . 2 利用小波变换的方法消除拼接缝 小波变换方法也是目前比较常用的一种方法，他充分利用小波变换的多分辨率特性，很好地解决了拼接图像的接缝问题。其原理为：由于小波变换具有带通滤波器的性质，在不同尺度下的小波变换分量，实际上占有一定的频宽，尺度j 越大，该分量的频率越高，因此每一个小波分量所具有的频宽不大，把要拼接的两幅图像先按小波分解的方法将他们分解成不同频率的小波分量，只要分解得足够细，小波分量的频宽就能足够小。然后在不同尺度下，选取不同的拼接宽度，把2 个图像按不同尺度下的小波分量先拼接下来，然后再用恢复程序，恢复到整个图像。这样得到的图像可以很好地兼顾清晰度和光滑度2 个方面的要求。但是，小波变换也存在缺点，如小波变换的算法比较复杂，需要在小波变换域内先进行拼接处理，在计算过程中涉及到大量的浮点运算和边界处理问题，对实际生产中的大容量图像进行处理时计算机内存开销很大，且处理时间较长，拼接速度慢。 2 . 3 利用加权平滑的方法消除拼接缝

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酵母双杂交系统的发展和应用 随着对多种重要生物的大规模基因组测序工作的完成，基因工程领域又迎来了一个新的时代---功能基因组时代。它的任务就是对基因组中包含的全部基因的功能加以认识。生物体系的运作与蛋白质之间的互相作用密不可分，例如：DNA合成、基因转录激活、蛋白质翻译、修饰和定位以及信息传导等重要的生物过程均涉及到蛋白质复合体的作用。能够发现和验证在生物体中相互作用的蛋白质与核酸、蛋白质与蛋白质是认识它们生物学功能的第一步。 酵母双杂交技术作为发现和研究在活细胞体内的蛋白质与蛋白质之间的相互作用的技术平台，在近几年来得到了广泛运用。酵母双杂交系统是在真核模式生物酵母中进行的，研究活细胞内蛋白质相互作用，对蛋白质之间微弱的、瞬间的作用也能够通过报告基因的表达产物敏感地检测得到，它是一种具有很高灵敏度的研究蛋白质之间关系的技术。大量的研究文献表明，酵母双杂交技术既可以用来研究哺乳动物基因组编码的蛋白质之间的互作，也可以用来研究高等植物基因组编码的蛋白质之间的互作。因此，它在许多的研究领域中有着广泛的应用。本文就酵母双杂交的技术平台和应用加以介绍。 酵母双杂交系统的建立是基于对真核生物调控转录起始过程的认识。细胞起始基因转录需要有反式转录激活因子的参与。反式转录激活因子，例如酵母转录因子GAL4在结构上是组件式的（modular），往往由两个或两个以上结构上可以分开，功能上相互独立的结构域（domain）构成，其中有DNA结合功能域(DNA binding domain,DNA-BD)和转录激活结构域（activation domain，DNA-AD）。这两个结合域将它们分开时仍分别具有功能，但不能激活转录，只有当被分开的两者通过适当的途径在空间上较为接近时，才能重新呈现完整的GAL4转录因子活性，并可激活上游激活序列（upstream activating sequence, UAS）的下游启动子，使启动子下游基因得到转录。 根据这个特性，将编码DNA-BD的基因与已知蛋白质Bait protein的基因构建在同一个表达载体上，在酵母中表达两者的融合蛋白BD-Bait protein。将编码AD的基因和cDNA文库的基因构建在AD-LIBRARY表达载体上。同时将上述两种载体转化改造后的酵母，这种改造后的酵母细胞的基因组中既不能产生GAL4，又不能合成LEU、TRP、HIS、ADE，因此，酵母在缺乏这些营养的培养基上无法正常生长。当上述两种载体所表达的融合蛋白能够相互作用时，功能重建的反式作用因子能够激活酵母基因组中的报告基因HIS、ADE、LACZ、MEL1，从而通过功能互补和显色反应筛选到阳性菌落。将阳性反应的酵母菌株中的AD-LIBRARY 载体提取分离出来，从而对载体中插入的文库基因进行测序和分析工作。在酵母双杂交的基础上，又发展出了 酵母单杂交、酵母三杂交和酵母的反向杂交技术。它们被分别用于核酸和文库蛋白之间的研究、三种不同蛋白之间的互作研究和两种蛋白相互作用的结构和位点。 基于酵母双杂交技术平台的特点，它已经被应用在许多研究工作当中。

酵母双杂交系统

酵母双杂交技术的研究与应用 摘要：酵母双杂交系统是在20世纪90年代初发展起来的利用遗传学方法在酵母真核细胞体内研究蛋白质之间相互作用的一种高度灵敏的分子生物学技术，它可以有效分离新基因或新的能与一种已知蛋白相互作用的蛋白质及其编码基因，被广泛应用于蛋白质组学、细胞信号转导和功能基因组学等领域，已成为分子生物学研究领域的重要实验手段之一，获得了许多有价值的重要发现。 关键词：酵母双杂交系统；蛋白质组学；功能基因组学Abstract:Yeast two-hybrid system is a highly sensitive molecular biology technique,which uses genetic methods to study protein-protein interaction in eukaryotic yeast cells,developed in the early 1990s.It can effectively separate new genes or new protein which has interaction with a known protein and protein-coding genes, is widely used in the field of proteomics, cellular signal transduction and functional genomics, has become one of the important experimental methods in the molecular biology areas, gained a lot of valuable important discovery. Key words:Yeast two-hybrid system;Proteomics;Functional Genomics.

(完整版)酵母双杂交原理

酵母双杂交系统原理 酵母双杂交系统(Yeast Two-hybrid System)由Fields和Song等首先在研究真核基因转录调控中建立。典型的真核生长转录因子，如GAL4、GCN4、等都含有二个不同的结构域: DNA 结合结构域(DNA-binding domain)和转录激活结构域(transcription-activating domain)。前者可识别DNA上的特异序列，并使转录激活结构域定位于所调节的基因的上游，转录激活结构域可同转录复合体的其他成分作用，启动它所调节的基因的转录。二个结构域不但可在其连接区适当部位打开，仍具有各自的功能。而且不同两结构域可重建发挥转录激活作用。酵母双杂交系统利用杂交基因通过激活报道基因的表达探测蛋白－蛋白的相互作用。主要有二类载体: a 含DNA -binding domain的载体; b 含DNA-activating domain的载体。上述二类载体在构建融合基因时，测试蛋白基因与结构域基因必须在阅读框内融合。融合基因在报告株中表达，其表达产物只有定位于核内才能驱动报告基因的转录。例如GAL4-bd具有核定位序列(nuclear-localization sequence)，而GAL4-ad没有。因此，在GAL4-ad氨基端或羧基端应克隆来自SV40的T-抗原的一段序列作为核定位的序列。 双杂交系统的另一个重要的元件是报道株。报道株指经改造的、含报道基因(reporter gene)的重组质粒的宿主细胞。最常用的是酵母细胞，酵母细胞作为报道株的酵母双杂交系统具有许多优点: 〈1〉易于转化、便于回收扩增质粒。〈2〉具有可直接进行选择的标记基因和特征性报道基因。〈3〉酵母的内源性蛋白不易同来源于哺乳动物的蛋白结合。一般编码一个蛋白的基因融合到明确的转录调控因子的DNA－结合结构域(如GAL4-bd，LexA-bd)；另一个基因融合到转录激活结构域(如GAL4-ad，VP16)。激活结构域融合基因转入表达结合结构域融合基因的酵母细胞系中，蛋白间的作用使得转录因子重建导致相邻的报道基因表达(如lacZ)，从而可分析蛋白间的结合作用。 酵母双杂交系统能在体内测定蛋白质的结合作用，具有高度敏感性。主要是由于：①采用高拷贝和强启动子的表达载体使杂合蛋白过量表达。②信号测定是在自然平衡浓度条件下进行，而如免疫共沉淀等物理方法为达到此条件需进行多次洗涤，降低了信号强度。③杂交蛋白间稳定度可被激活结构域和结合结构域结合形成转录起始复合物而增强，后者又与启动子DNA结合，此三元复合体使其中各组分的结合趋于稳定。④通过mRNA产生多种稳定的酶使信号放大。同时，酵母表型，X-Gal及HIS3蛋白表达等检测方法均很敏感。 酵母双杂交筛选原理 双杂交系统的建立得力于对真核生物调控转录起始过程的认识。细胞起始基因转录需要有反式转录激活因子的参与。80年代的工作表明, 转录激活因子在结构上是组件式的(modular), 即这些因子往往由两个或两个以上相互独立的结构域构成, 其中有DNA结合结构域(DNA binding domain, 简称为DB，？BD)和转录激活结构域(activation domain, 简称为AD), 它们是转录激活因子发挥功能所必需的。单独的DB虽然能和启动子结合, 但是不能激活转录。而不同转录激活因子的DB和AD形成的杂合蛋白仍然具有正常的激活转录的功能。如酵母细胞的Gal4蛋白的DB与大肠杆菌的一个酸性激活结构域B42融合得到的杂合蛋白仍然可结合到Gal4结合位点并激活转录。 Fields等人的工作标志双杂交系统的正式建立。他们以与调控SUC2基因有关的两个蛋白质Snf1和Snf2为模型, 将前者与Gal4的DB结构域融合, 另外一个与Gal4的AD结构域的酸性区域融合。由DB和AD形成的融合蛋白现在一般分别称之为“诱饵”(bait)和“猎物”或靶蛋白(prey or target protein)。如果在Snf1和Snf2之间存在相互作用, 那么分别位于这两个融合蛋白上的DB和AD就能重新形成有活性的转录激活因子, 从而激活相应基因的转

酵母双杂交系统及其应用

酵母双杂交系统及其应用 Yeast Two-hybrid System and Its Application 1. 酿酒酵母（Saccharomyces cerevisiae）的生物学特性 （1）单细胞真核生物 尽管酵母细胞比较简单，但它们具有所有真核生物细胞的主要特征，如含有一个独立的细胞核、多条线性染色质包装成染色体、细胞质包含了全部的细胞器和细胞骨架结果（如肌动蛋白纤维）。 （2）与其它真核生物相比，它们的基因组较小，基因数目也较少； 1996 年已完成酵母全基因组测序（1.5 x 10 7 bp ），是第一个被测序的真核生物。大约有6000个基因。目前已经建立了一个6000 个菌株的文库，每一个菌株中只删除了一个基因。其中5000 多株在单倍体状态时能够存活，表明大多数酵母基因时非必需的。 （3）易于培养和操作,可以在实验室快速繁殖 在指数生长期每90 分钟繁殖一代，从单个细胞可以繁殖称克隆群体。 （4）单倍体和双倍体的存在使酿酒酵母便于进行遗传分析 酿酒酵母可以以单倍体状态和双倍体状态生长。单倍体和双倍体之间的转换是通过交配和孢子形成来实现的。 有两种单倍体细胞类型，分别为a 型和α型。在一起生长时，这些细胞因交配而形成a/ α双倍体细胞。在营养匮乏时，a/ α双倍体发生减数分裂，产生一个子囊的结构，每个子囊含有4 个单倍体孢子（两个a－孢子和两个α－孢子）。但当生长条件改善时，这些孢子可以出芽并以单倍体细胞的形式生长或交配而重新形成双倍体。一个酵母细胞可同时兼容几种不同质粒bud，芽, 蓓蕾starvation ，饥饿, 饿死ascus，n.［微生物］子囊meiosis，n.减数分裂, 成熟分裂 haploid，n.［生物］单倍体, 仅有一组染色体的细胞adj.单一的diploid ，adj.双重的, 倍数的, 双倍的n.倍数染色体ascospore，n.［植］囊孢子rupture，v.破裂, 裂开, 断绝（关系等）, 割裂。n.破裂, 决裂, 敌对, 割裂 germinate，v.发芽, 发育, 使生长 spore，n.孢子vi. 长孢子

文库构建及酵母双杂交技术

第四章基因文库构建及酵母双杂交技术 1 基因组文库的构建 基因组文库（genomic library）将某种生物细胞的整个基因组DNA切割成大小合适的片断，并将所有这些片断都与适当的载体连接，引入相应的宿主细胞中保存和扩增。 理论上讲，这些重组载体上带有了该生物体的全部基因，称为基因文库。 1. 1构建基因文库的载体选用 载体能够容载的DNA片断大小直接影响到构建完整的基因文库所需要的重组子的数目。

第四章基因文库构建及酵母双杂交技术1.1.1对载体的要求：载体容量越大，所要求的DNA片断数目越少，所需的重组子越少。 1.1.2目前常用的载体： 载体系列（容量为24 kp ）、cosmid载体（容量为50 kb ）、YAC（容量为1 Mb ）、BAC （容量为300 kb） 1.2 基因文库构建的一般步骤 1.2.1染色体DNA大片段的制备：断点完全随机，片断长度合适于载体连接。不能用一般的限制性内切酶消化法，使用物理切割法或不完全酶切法。 1.2.2载体与基因组DNA大片段的连接：直接连接、人工接头或同聚物加尾。

噬菌体载体构建基因组文库

第四章基因文库构建及酵母双杂交技术2 cDNA文库的构建 cDNA克隆的基本过程是通过一系列，酶酶催作用，使poly(A) mRNA转变成双链cDNA群体并插入到适当的载体分子上，转化大肠杆菌寄主细胞，构建包含所有基因编码序列的cDNA基因文库。 2.1高质量mRNA的制备 应用Promega PolyAT tract mRNA Isolation System 分离Poly(A)RNA。将Biotinylated Oligo(dT)引物与细胞总RNA共温育，加入与微磁球相连的Streptavidin，用磁场吸附与PMP相连的SA-Biotinylated Oligo(dT)-mRNA。

蛋白的酵母双杂交操作手册

蛋白的酵母双杂交实验 ——以钓饵蛋白筛选cDNA 文库研究蛋白相互作用 第一部分 系统简介 1. 实验原理 蛋白的酵母双杂交实验是以酵母的遗传分析为基础，研究反式作用因子之间的相互作用 对真核基因转录调控影响的实验。很早就已知道，转录活化蛋白可以和DNA 上特异的序列结合而启动相应基因的转录反应。这种DNA 个相互独立的结构域即DNA 结合结构域(Binding Domain, BD)和转录活化结构域(Activation Domain, AD)分别来完成的，并且这两个结构域对于基因的转录活化都是必须的。目前酵母双杂交实验采用的系统有LexA 系统和Gal4 LexA DNA 结合结构域由一个完整的原核蛋白LexA 构成，转录活化结构域则由一个88个氨基酸的酸性的大肠杆菌多肽B42构成，它在酵母中可以活化基因的转录; 在Gal4系统中，BD 和AD 分别由Gal4蛋白上不同的两个结构域(1-147aa 与768-881aa)构成。cDNA 文库或DNA 与文库蛋白或要验证的蛋白相结合。一般情况下，单独的BD 可以与GAL4上游活化序列（）结合但不能引起转录，单独的AD 则不能与GAL UAS 结合，只有当BD 与AD BD 与AD 才能与GAL UAS 结合并且引起报道基因的转录。在BD 与AD 要导入的酵母菌AH109通过基因工程的方法在GAL4 UASs 和启动子的下游构建了3个报道基因——ADE2，HIS3，MEL1（或LacZ ），否存在相互作用。GAL4系统的原理如图所示： 图一：酵母双杂交系统工作原理 Kan r Amp r pGBKT7-bait pACT2-cDNA

酵母双杂交实验流程

模块七蛋白质之间的相互作用 1. 实验目的 本实验以重组质粒和酵母细胞为材料，学习检测蛋白质相互作用的基本原理和技术方法。主要介绍酵母双杂交的基本原理与操作技术；让学生了解和掌握酵母双杂交系统的应用；掌握酵母感受态的制备的基本原理和主要的操作步骤。 2. 实验原理 1989年Fields和Song等人根据当时人们对真核生物转录起始过程调控的认识（即细胞内基因转录的起始需要转录激活因子的参与），提出并建立了酵母双杂交系统。该系统作为发现和研究活细胞体内的蛋白质与蛋白质之间的相互作用的技术平台，近几年得到了广泛的运用和发展。 相比于其它蛋白质筛选系统，酵母双杂交系统具有以下优点：（1）检测在真核活细胞内进行，在一定程度上代表细胞内的真实情况。（2）作用信号是在融合基因表达后，在细胞内重建转录因子的作用而给出的，省去了纯化蛋白质的繁琐步骤。（3）检测结果是基因表达产物的积累效应，因而可检测存在于蛋白质之间的微弱或暂时的相互作用。（4）酵母双杂交系统可采用不同组织、器官、细胞类型和分化时期材料构建cDNA文库，能分析细胞质、细胞核及膜结合蛋白等多种不同亚细胞部位及功能蛋白。（5）通过mRNA产生多种稳定的酶使信号放大。同时，酵母表型、X-Gal 及HIS3 蛋白表达等检测方法均很敏感。 酵母双杂交系统也具有一定的局限性。首先，经典的双杂交系统分析蛋白间的相互作用定位于细胞核内，因而限制了该系统对某些细胞外蛋白和细胞膜受体蛋白的研究。酵母双杂交系统的另一个局限性是“假阳性”。在酵母双杂交系统建立的初期阶段，由于仅仅采用β-半乳糖苷酶这一单一的报告基因体系，这种报告基因的表达往往不能十分严谨地被控制，因此容易产生假阳性。由于某些蛋白本身具有激活转录的功能或在酵母中表达时发挥转录激活作用，使DNA结合结构域融合蛋白在无特异激活结构域的情况下也可被激活转录。另外某些蛋白表面含有对多种蛋白质的低亲和力区域，能与其他蛋白形成稳定的复合物，从而引起报告基因的表达，产生“假阳性”结果。产生“假阴性”结果的原因可能有许多蛋白质间的相互作用依赖于翻译后加工如糖基化、磷酸化和二硫键形成，还有些蛋白的正确折叠和功能有赖于某些非酵母蛋白的辅助等。 现在的酵母双杂交系统大都采用多种报告基因，如AH109酵母株含有三类报告基因—ADE2、HIS3、MEL1/lacZ，这三类报告基因受控于三种完全不同、异源性的GAL4-反应元件和三类启动子元件-GAL1、GAL2以及MEL1（如图6-1-1）。通过这种方法就消除了两类最主要的假阳性，一类是融合蛋白可以直接与GAL4结合位点结合或者是在结合位点附近结合所带来的假阳性；另一类是融合蛋白和某种转录因子结合后再结合到特定的TA TA盒上所带来的假阳性。ADE2一种报告基因就已经能够提供较强的营养选择压力，这时选择性地使用HIS3报告基因，一来可以降低假阳性率；二来可以控制筛选的严格性（如果需要筛选与诱饵蛋白具有较强结合的蛋白，就可以同时使用ADE2、HIS3两种报告基因；如果只需要筛选与诱饵蛋白具有中等强度或较弱结合的蛋白，就可以使用ADE2或HIS3两者中的一种）。MEL1和lacZ分别编码α-半乳糖苷酶和β-半乳糖苷酶，可以作用于相应的底物

酵母双杂交实验步骤

LexA酵母双杂交系统简介 一、LexA酵母双杂交系统的设计原理 报告质粒p8op-LacZ的GAL4 UAS编码序列被完全去除，因此在缺乏LexA融合激活剂的情况下，报告基因LacZ的转录活性为零，该基因的筛选标志为URA3，可以作为有自主复制能力的质粒存在于酵母EGY48菌株中，也可以被整合到EGY48基因组DNA上。 质粒pLexA的筛选标志为HIS3，在双杂交系统中用于表达DNA-BD(202个氨基酸残基组成的LexA蛋白)与目标蛋白(钓饵，Bait)的融合蛋白，该融合体的表达受酵母强启动子ADH1的调控，选择与报告基因的操纵子LexA×8结合。 质粒pB42AD的筛选标志为TRP1，在其供外源基因插入的多克隆位点(EcoR I与Xho I)上游，含有SV40核定位(SV40 nuclear localization)、HA(血凝素)及AD(来自于的88个氨基酸残基组成的B42蛋白)等几种编码序列，共同组成可以启动报告基因转录表达的激活成份。在酵母EGY48的基因组中还整合有另一个报告基因Leu，它与LacZ报告基因具有相同的操纵子-LexA，但两者启动子不同。 根据双杂交系统的原理，如果某一复合物同时具有DNA-BD和AD的活性，即可激活报告基因的转录和表达。分别将待测蛋白X、Y的编码序列插入pLexA质粒载体和pB42AD质粒载体的多克隆位点中，然后共同转入含有报告基因的酵母菌株，如果蛋白X与Y能相互作用，则启动报告基因的转录和表达，通过检测报告基因的表达情况，就可以间接反映蛋白X、Y是否具有相互作用以及作用的强弱。 如果将蛋白Y换为取自组织或血液的cDNA文库，则可用X从该文库中筛选出能与其相互作用的蛋白，并且可以获得编码这些蛋白的cDNA。 二、商品化酵母双杂交系统的组成 1. 载体质粒：pLexA、pB42AD、p8op-LacZ、pB42AD-DNA文库 2. 酵母菌株：EGY48、EGY48（p8op-LacZ）、YM4271（EGY48的伴侣菌株） 3. 大肠杆菌菌株： KC8株 4. 对照质粒：

酵母双杂交及其衍生系统_黄欣媛

?技术与方法? 生物技术通报 BIOTECHNOLOGY BULLETIN 2014年第1期 生物大分子间的相互作用是有机体细胞活动的基础，在蛋白质层面上揭示生命活动本质是现代生物学的一个主要目标。1989年诞生的酵母双杂交（Yeast two -hybrid，Y2H）系统以及随后出现的各种衍生系统是研究蛋白质之间以及蛋白质和RNA、小分子化合物之间相互作用的最重要的工具，已成功揭示了大量的蛋白相互作用，在细胞信号转导、蛋白质组学、病理学、药物研发等方面有着广泛应用［1］。本文对Y2H 系统的基本原理、衍生系统和应用进展等方面进行介绍。 1 酵母双杂交系统的原理 酵母GAL4转录因子由两个可以分开的结构域组成：DNA 结合域（DNA binding domain，BD）负责结合基因的上游激活序列，将转录激活域（Transcriptional activation domain，AD）募集到启动 收稿日期：2013-08-03基金项目：特色果蔬质量安全控制湖北省重点实验室开放基金项目（2013K04）作者简介：黄欣媛，女，博士，研究方向：生物化学与分子生物学；E -mail ：huangxycn@https://www.wendangku.net/doc/1e11994544.html, 酵母双杂交及其衍生系统 黄欣媛1 范红波2 （1.湖北工程学院特色果蔬质量安全控制湖北省重点实验室，孝感 432000；2.湖北职业技术学院，孝感 432000） 摘 要： 酵母双杂交系统是一种研究蛋白质相互作用的分子生物学方法，过去20多年里，大量衍生系统的出现使得这套双杂交技术体系更加完善和高效，成为研究蛋白质-配体相互作用的重要技术手段，广泛应用于功能基因组学、蛋白质组学、病理学等研究领域。对酵母双杂交及其衍生系统的基本原理和应用进展进行综述。 关键词： 酵母双杂交系统 蛋白质相互作用 双杂交技术体系 The Yeast Two -Hybrid System and Its Several Derived Systems Huang Xinyuan 1 Fan Hongbo 2 （1. Hubei Key Laboratory of Quality Control of Characteristic Fruits and Vegetables ，Hubei Engineering University ，Xiaogan 432000；2.Hubei Polytechnic Institute ，Xiaogan 432000） Abstract: The Yeast two -hybrid（Y2H）system is a molecular biology approach to detect protein -protein interactions. A diverse series of technologies derived from it have emerged in the past 20 years, which makes this two -hybrid methodology system more complete and efficient. They have been among the most important and powerful tools to study ptotein -ligand interactions that widely used in functional genomics, proteomics and pathology study. This article provides an overview on the basic principles and application progress of Y2H system and some derived techniques. Key words: Yeast two -hybrid（Y2H）system Protein -protein interaction Two -hybrid methodology system 子区域从而激活基因的转录。两者分开时不能激活基因转录，只有BD 和AD 通过一定方式在空间上足够靠近，才能发挥完整的GAL4转录因子活性。基于此特点，Fields 和Song ［2］设想以一对能相互作用的蛋白质为桥梁，将空间上分开的BD 和AD 彼此拉近，从而重建出具有活性的转录因子，以此创立了Y2H 系统。在该系统中，将BD 和AD 基因分别和诱饵蛋白（Bait）和猎物蛋白（Prey）基因构建成融合蛋白表达载体，使其在酵母中共表达出BD -Bait 和AD -Prey，借助Bait 和Prey 的物理性相互作用使BD 和AD 相互靠近而产生具有功能的转录因子，进而激活1个或多个报告基因（如lacZ 、HIS3和URA3等）的转录。 2 酵母双杂交系统的衍生系统 Y2H 系统是在酵母菌这种真核细胞环境中研究

酵母双杂交

酵母双杂交 酵母双杂交系统由Fields和Song等首先在研究真核基因转录调控中建立。典型的真核生物转录因子，如GAL4、GCN4、等都含有二个不同的结构域：DNA结合结构域(DNA-binding domain)和转录激活结构域(transcription-activating domain)。前者可识别DNA上的特异序列，并使转录激活结构域定位于所调节的基因的上游，转录激活结构域可同转录复合体的其他成分作用，启动它所调节的基因的转录。 1、基本思想 酵母双杂交由Fields在1989年提出.他的产生是基于对真核细胞转录因子特别是酵母转录因子GAL4性质的研究.GAL4包括两个彼此分离的但功能必需的结构域.位于N端1-147位氨基酸残基区段的DNA结合域（DNA binding domain，DNA-BD）和位于C端768-881位氨基酸残基区段的转录激活域（Activation domain,AD).DNA-BD能够识别位于GAL4效应基因（GAL4-responsivegene）的上游激活序列(Upstream activating sequence,UAS)，并与之结合.而AD则是通过与转录机构（transcriptionmachinery）中的其他成分之间的结合作用，以启动UAS下游的基因进行转录.DNA-BD和AD单独分别作用并不能激活转录反应，但是当二者在空间上充分接近时，则呈现完整的GAL4转录因子活性并可激活UAS下游启动子，使启动子下游基因得到转录. Fields建立了一个双杂交系统，BD与X蛋白融合，AD与Y蛋白融合，如果X、Y之间形成蛋白-蛋白复合物，使GAL4两个结构域重新构成，则会启动特异基因序列的转录.他们利用Snf1与Snf4的相互作用，将Snf1与BD融合，Snf4与AD融合，构建在穿梭质粒上.其中Snf1是一种依赖于丝氨酸、苏氨酸的蛋白激酶，Snf4是他的一个结合蛋白.研究者将二种穿梭质粒转化酵母GGY：171菌株，该菌株含有LacZ报告基因，并已去除相应转录因子基因.该实验的结果表明由Snf1和Snf4相互作用使得AD和BD在空间上接近，激活了报告基因LacZ的转录.一般地，将BD-X的融合蛋白称作诱饵（bait),X往往是已知蛋白，AD-Y称作猎物（prey），能显示诱饵和猎物相互作用的基因称报告基因，通过对报告基因的检测，反过来可判断诱饵和猎物之间是否存在相互作用. 2、基本原理 双杂交系统的建立得力于对真核生物调控转录起始过程的认识。细胞起始基因转录需要有反式转录激活因子的参与。80年代的工作表明，转录激活因子在结构上是组件式的（modular）,即这些因子往往由两个或两个以上相互独立的结构域构成,其中有DNA结合结构域（DNA binding domain，简称为BD）和转录激活结构域（activation domain，简称为AD），它们是转录激活因子发挥功能所必需的。前者可识别DNA上的特异序列，并使转录激活结构域定位于所调节的基因的上游，转录激活结构域可同转录复合体的其他成分作用，启动它所调节的基因的转录。两个结构域不但可在其连接区适当部位打开，仍具有各自的功能。而且不同两结构域可重建发挥转录激活作用。酵母双杂交系统利用杂交基因通过激活报道基因的表达探测蛋白－蛋白的相互作用。单独的BD虽然能和启动子结合，但是不能激活转录。而不同转录激活因子的BD和AD形成的杂合蛋白仍然具有正常的激活转录的功能。如酵母细胞的Gal4蛋白的BD与大肠杆菌的一个酸性激活结构域B42融合得到的杂合蛋白仍然可结合到Gal4结合位点并激活转录。主要有二类载体：a含DNA-binding domain的载体；b含DNA-activating domain的载体。一般编码一个蛋白的基因融合到明确的转录调控因子的DNA－结合结构域(如GAL4-bd，LexA-bd）；另一个蛋白的基因融合到转录激活结构域（如GAL4-ad，VP16）。上述二类载体在构建融合基因时，测试蛋白基因与结构域基因必须在阅读框内融合。融合基因在报告株中表达，其表达产物只有定位于核内才能驱动