ARTICLE IN PRESS

Ultramicroscopy 109 (2009) 916–922

Contents lists available at ScienceDirect

Ultramicroscopy

0304-39

doi:10.1

� Corr

E-m

journal homepage: www.elsevier.com/locate/ultramic

High-sensitivity detection of proteins using gel electrophoresis and atomicforce microscopy

Hiroshi Sekiguchi a,�, Atsushi Hidaka a, Yoshihito Shiga a, Atsushi Ikai b, Toshiya Osada a

a Biodynamics Laboratory, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 B2 Nagatsuta-cho, Midori-ku, Yokohama,

Kanagawa 226-8501, Japanb Innovation Laboratory, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 S2-8 Nagatsuta-cho, Midori-ku, Yokohama,

Kanagawa 226-8501, Japan

a r t i c l e i n f o

PACS:

07.79.Lh

87.15.Kg

87.80.+s

Keywords:

Streptavidin–biotin

Force measurement

Sensitive protein detection

91/$ - see front matter & 2008 Elsevier B.V. A

016/j.ultramic.2009.03.010

esponding author. Tel.: +8145 924 5739; fax:

ail address: hsekiguc@bio.titech.ac.jp (H. Seki

a b s t r a c t

We have developed a method to detect specific proteins with a high sensitivity using a gel

electrophoresis method and force measurement of atomic force microscopy (AFM). Biotinylated

proteins were separated by electrophoresis and fixed with cross-linking chemicals on the gel, followed

by direct force measurement between the biotinylated proteins on the gel and a streptavidin-modified

tip of an AFM cantilever. We were able to achieve a high enough sensitivity to detect the picogram order

of the biotinylated proteins by evaluating the frequency of the interaction force larger than 100 pN in the

force profile, which corresponds to the rupture force of interaction between streptavidin and biotin.

& 2008 Elsevier B.V. All rights reserved.

1. Introduction

Proteome analysis, studying all expressed proteins at a giventime under defined conditions, could be considered the nexttarget for the study of biological systems in the post-genome era.Decoding all genome information has been achieved in severalspecies [1–5] and these achievements could be mostly due to thePCR method which allows nucleic acid molecules, that codegenome information, to be amplified several billion fold. To date,there is no such amplification method for proteins, therefore, atechnique to detect and identify proteins with high sensitivitywould be key for further proteomics research.

In regard to the high sensitive detection of proteins, a newlydeveloped technology, such as atomic force microscopy (AFM),could address proteins at the single molecular level. AFM is auseful device not only to trace topography of biological sampleswith molecular resolution under physiological conditions [6–10]but also to study the interaction force between bio-molecularpairs [11–16] and the mechanical properties of proteins [17–24]at the single molecular level. Moreover, the AFM is used as amanipulator to obtain DNA from chromosomes [25–27], or mRNAfrom local regions of living cells [28,29]. The obtained nucleicacids could be amplified and separated by PCR methodology,therefore it is possible to examine the expression of specific geneswithin individual living cells [28,29].

ll rights reserved.

+8145 924 5828.

guchi).

In this article, we propose a method to detect specific proteinswith a high sensitivity using a gel electrophoresis methodand force measurement of AFM (Fig. 1). We applied the highspecificity of interaction between streptavidin and biotin toidentify biotinylated proteins on an electrophoresis gel withAFM technology. Biotinylated proteins were separated by electro-phoresis and fixed with cross-linking chemicals on the electro-phoresis gel. Then direct force measurement between thebiotinylated proteins on the gel and a streptavidin-modified tipof an AFM cantilever was executed. We compared histogramsof the interaction force detected, respectively, on normal gels, ongels containing normal proteins and on gels containing biotiny-lated proteins. We found that the frequency of detecting aninteraction force larger than 100 pN was much higher on gelscontaining biotinylated proteins than those on normal gels oron gels containing normal proteins. In the end, we were able toachieve a high enough sensitivity to detect the picogram orderof the biotinylated proteins by evaluating the frequency of theinteraction force larger than 100 pN in the force profile.

2. Materials and methods

2.1. AFM tip modification and sample preparation

Gold-coated AFM tips (OMCL-TR400PB, Olympus, Japan)were functionalized with streptavidin through processes utilizingself-assembled mono-layers and PEG (polyethylene glycol)

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H. Sekiguchi et al. / Ultramicroscopy 109 (2009) 916–922 917

cross-linkers [30], as schematized in Fig. 2. We arranged thetip modification procedure shown in Ref. [30] for our experi-ment. First, gold-coated AFM tips were cleaned in a UV ozonecleaner (NL-UV253, Nippon Laser & Electronics Lab., Japan),and immersed in an ethanolic thiol solution containing 1 mM1,8-octanedithiol (Sigma-Aldrich, St. Louis, MO) and 10 mM6-mercapto hexane-1-ol (Sigma-Aldrich) for 18 h. Second, theAFM tips were removed from the thiol solution, rinsed withethanol, dried with nitrogen gas, and placed into a solutioncontaining 1 mg/ml of N-hydroxysuccinimidyl polyethylene-gly-col maleimide (NHS-PEG-MAL, m:w: ¼ 5000, Nektar Therapeutics,Huntsville, AL) in dimethyl sulfoxide and ethanol for 30 min.After washing the tips with MilliQ water, they were immersedin PBS (phosphate buffered saline) with 1 mg/ml streptavidin(MP Biomedicals, Solon, OH) for 1 h. Streptavidin-modified tipswere washed with TBS (tris buffered saline) and stored in TBSbefore use.

Gel electrophoresis in a PhastSystem (GE Healthcare) wasconducted with a specified amount of biotinylated BSA (bovineserum albumin) (A6043, Sigma) and BSA (A7638, Sigma) usingPhastGel Gradient gel 8–25% (17-0542-01, GE Healthcare). Afterthe electrophoresis, the gel was first immersed in 1% glutaralde-hyde solution for 5 min to cross-link proteins each other in the gel,and then it was washed with PBS. After PBS washing, the gel wasstained with CBB (Coomassie brilliant blue), and was de-stainedaccording to the standard protocol. After that, the gel was treatedwith PBS containing glycine (1.5 mg/ml) for 30 min at room

Fig. 1. Experimental overview of the AFM SDS-PAGE method. The streptavidin-

modified tip was used to detect biotinylated proteins on an electrophoresis gel.

Fig. 2. Functionalization of the gold-coated AFM tip with streptav

temperature, and was washed with PBS. After 1 h of incubationof the gel in PBS with blocking reagent (2% w/v, Roche) and Tween20 (0.1% v/v, P9416, Sigma), force measurement of AFM wasexecuted.

The sample protein solutions were stamped onto the electro-phoresis gel surface in the PhastSystem, not loaded into thewell of the electrophoresis gel, therefore a certain proportionof the protein ran near the gel surface. To confirm the existence ofprotein near the surface of electrophoresis gel, the cross-sectionof CBB stained electrophoresis gel was imaged by optical micro-scopy (Fig. 3). The cross-section of CBB stained protein’s band wasobtained by scissors’ cut at the position of protein’s band (Fig. 3left). The color of the obtained image was converted to gray scale.

For the analysis of detection limit of BSA with CBB and silverstaining method, the gel was stained by a standard protocol of CBBand silver staining method (EzStain, ATTO, Tokyo, Japan) after theelectrophoresis of BSA.

2.2. Atomic force microscopy

Force measurements between the biotinylated proteins on thegel and a streptavidin-modified tip of cantilever were carried outwith an AFM NVB-100 AFM (Olympus, Inc., Tokyo, Japan) whichwas set on an inverted optical microscope (IX70, Olympus, Inc.,Tokyo, Japan). The electrophoresis gel was immobilized on aplastic dish with a magnet on the gel to the steel-stage belowthe dish. The modified AFM tips were placed, respectively, ona normal gel, on a gel containing BSA and on a gel containingbiotinylated-BSA, using an optical microscope, and force curvemeasurements were executed on different positions with a scanspeed of around 2300 nm/s (1000 nm in scan size and 1.16 Hzin frequency) and with a final applied force of 1.5 nN underPBS containing Tween 20 (0.1% v/v). The force curves from about120 positions within 1mm squares were recorded in eachexperimental condition to make a histogram of the rupture forcein force curves.

In the negative control experiments, the force measurementswere performed in an experimental buffer with free biotin(80 nM).

To calibrate the response of the cantilever deflection signal as afunction of piezo movement, standard force curve measurements

idin through self-assembled mono-layer and PEG cross-linker.

ARTICLE IN PRESS

gelplastic plate

0.5 mm

gel

plastic plate

Fig. 3. Cross-sectional image of the electrophoresis gel at the CBB stained band. Black color in the gel indicates high intensity of CBB staining. The surface and near surface

of the electrophoresis gel was stained with CBB. Therefore a certain amount of BSA protein ran near the gel surface which could be accessible from the streptavidin on the

AFM tip. 500 ng of BSA protein was applied for the gel electrophoresis.

Can

tilev

er D

efle

ctio

n

Z piezo movement

200 nm

500 pN

approach release

(0)

(ii)

(iii)

(i)

(iv)

Fig. 4. Obtained force curves. Typical force curves obtained on the bottom of a dish

(0) and on the gel surfaces ((i)–(iv)). Retraction curves obtained on gel surfaces

could be classified into four types by the shape of profile: (i) with no attractive

interaction force, (ii) with an adhesive interaction near the sample surface, (iii)

with a single sharp attractive interaction and finally (iv) those with multiple

attractive interactions.

H. Sekiguchi et al. / Ultramicroscopy 109 (2009) 916–922918

were carried out on a bottom of the bottom of the dish, and thespring constant of the functionalized cantilevers was individuallycalibrated by the thermal vibration method [31–33].

All rupture events of the interaction between the tip of thecantilever and gel surface were analyzed off-line considering theirmorphological characteristics in time series of releasing forcecurve [34,35]. The time series of force data were convoluted withthree different filters, which were vertical segment, v-shaped, andright angle detecting filters that were described in the article [34].The convolution of the force data with three different filters gave

three new series of data, and the threshold was set in each seriesof the convoluted data considering the background noise. Thoseinteraction forces that came out above the threshold values of allthe three filtering processes were selected to be included in theconstruction of the final histograms. Most of the curves showingadhesive interactions, such as the curves given in Fig. 4(ii), wereeliminated by these procedures. When multiple peaks weredetected in a force curve, as the curve given in Fig. 4(iv), peaksthat met the above criteria were also included in the histogram.The selection procedure described above were precessed auto-matically by custom software written for IGOR Pro (Wavemetrics,Lake Oswego, OR).

2.3. The dot-blot and SDS-PAGE analysis

In the dot-blot experiment, a specified amount of biotinylatedBSA was dropped on a nitrocellulose membrane (Hybond ECLRPN68D, GE Healthcare UK Ltd.) and dried. And the spottednitrocellulose membrane was stained in blue color according tothe standard protocol of NBT/BCIP (11681451001, Roche) andstreptavidin-AP (alkaline phosphatase) conjugate (11089 161001,Roche).

A usual image scanner (HP Officejet 7210) without correctionsettings was used to capture images of the stained electrophoresisgel and the nitrocellulose membrane. The captured images wereconverted to gray scale, and the density of the band in the gel andspots in the membrane was analyzed by IGOR Pro (Wavemetrics,Lake. Oswego, OR).

3. Results and discussion

A streptavidin-modified tip was used to detect biotinylatedproteins on an electrophoresis gel (Fig. 1). As a first trial,biotinylated BSA was used as target proteins, which was separatedby electrophoresis and fixed with glutaraldehyde on the gel. Thendirect force measurement with a streptavidin-modified tip wasexecuted on the site of the band corresponding to the positionof biotinylated BSA.

At the beginning of the experiments, we obtained cross-sectional images of CBB stained electrophoresis gel with BSA(Fig. 3) to confirm whether proteins separated by electrophoresisexisted on the gel surface or not. In our method being proposed(Fig. 1), the biotinylated protein should exist on or near thegel surface so that they are accessible from the AFM tip modifiedwith streptavidin. Fig. 3 shows the cross-sectional image of theelectrophoresis gel at CBB stained band. The CBB stained part ofthe gel was in black color in Fig. 3 right (the CBB’s purple color in

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original image was converted to gray scale). We found that the gelsurface together with the inside of the BSA band was stained byCBB (Fig. 3 right). Therefore a certain amount of BSA was presentnear the surface of the gel and biotinylated proteins run byelectrophoresis were accessible from the tip of cantilever on thegel surface.

Then, force curves were recorded with a streptavidin-modifiedtip of an AFM cantilever on the electrophoresis gel surface. InFig. 4, typical force curves obtained on the bottom of the dish(Fig. 4(0)) and on the gel surface (Figs. 4(i)–(iv)) are presented.The relationships between the force applied on the cantilever(ordinate) and the sample position along the z-axis (abscissa)were plotted on these force curves as described below.

When the AFM tip approaches the sample, the force curve isinitially flat until it reaches the sample surface, where it starts tosense repulsive force. If the tip pushes a solid substrate likebottom of the dish, the force signal increases proportionally to thetip movement Fig. 4(0). Alternatively, if the tip pushes an elasticmaterial like electrophoresis gel, the force signal increased with adistinct curvature (Figs. 4(i)–(iv)). After the tip starts to retract atthe left end of the diagram, the curve closely follows the previousapproach curve until the deflection returns to the initial level.When the tip moves away further from the gel surface, thecantilever deflection varies depending on the interaction betweenthe AFM tip and the sample surface. Retraction curves obtained ongel surfaces could be classified into four types by the shapeof profile: (1) with no attractive interaction force (Fig. 4(i)), (2)with an adhesive interaction near the sample surface (Fig. 4(ii)),(3) with a single sharp attractive interaction (Fig. 4(iii)) and finally(4) those with multiple attractive interactions (Fig. 4(iv)).

The adhesive curves shown in Fig. 4(ii) were sometimesobtained on all kinds of the gel surfaces, and were regarded asnon-specific curve. Non-specific adhesive interaction is consid-

100 nm

200 pN

biotin-BSA

normal BSA

200 pN

100 nm

Fig. 5. Force curves analyzed on the various conditions of gel surfaces; on gels containin

on gels containing normal BSA (C), and on normal gels. Approach and release curves ar

4(ii)) were obtained at all conditions, and such curves were excluded in analysis proce

ered to be a significant problem in force measurement of AFM.Significant efforts have been reported to reduce such interaction,for example, various surfactants were added to an experimentalbuffer [36,37] and flexible long cross-linkers were used toimmobilize biomolecules of interest [13,38–40]. In our experi-ment, PEG linkers were used to immobilize streptavidin on thetip of the cantilever, and surfactants were added in the experi-mental buffer during force measurement. However, we could notcompletely reduce the non-specific interaction between the tip ofthe cantilever and the gel surface. Such adhesive curves wereexcluded out analytically by the selection procedures described at‘‘Materials and methods’’ section. The roughness of the gel surfacecould affect the results of our experiments; however, the gelsurface was too soft to be imaged by AFM. The AFM images we gotwere too vague for discussion of our results.

In Fig. 5, typical force curves obtained on gel surfaces underdifferent conditions are given. The probability of getting suchcurves was 30–50% of all the attempts of force curve measure-ment. And one streptavidin-modified tip endured 500 times of theforce measurement, at least, since we did not observe clearreduction of the hit probability on gels contained biotinylated BSA(data not shown). The shapes of the release curves undereach condition were not so different; however, the magnitudeof rupture force required to break the interaction between the tipand the sample was different. Fig. 6 shows the histograms of thedetected interaction force on normal gels (gray dashed lines),on gels containing BSA (gray lines) and on gels containingbiotinylated-BSA (black lines), respectively. A specified amountof biotinylated BSA and normal BSA was applied for gelelectrophoresis, namely, 500 ng for (A) and (B), 500 pg for (C)and 5 pg for (D). As a negative control experiment, free biotin wasadded in the experimental buffer for force measurements toconstruct the histogram in (B).

normal gel

200 pN

100 nm

biotin-BSA-inhibit

200 pN

100 nm

g biotinylated BSA (A), on gels containing biotinylated BSA in inhibition condition,

e shown in gray and black color, respectively. The curves with adhesive force (Fig.

ss by three filters described at ‘‘Materials and methods’’.

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Fig. 6. Comparison of unbinding force distribution. Histogram of unbinding force on normal gel (gray dashed lines), on the gel containing BSA (gray lines) and on the gel

containing biotinylated-BSA (black lines). Specified amounts of proteins were applied for gel electrophoresis, such as 500 ng for (A) and (B), 500 pg for (C) and 5 pg for (D).

As a negative control experiment, free biotin was added in the experimental buffer for force measurement to make the histogram of (B).

H. Sekiguchi et al. / Ultramicroscopy 109 (2009) 916–922920

The force from 20 to 80 pN was detected in all conditions(Fig. 6); however, the frequency of obtaining a strong force, suchas one larger than 100 pN, was higher on gels that containedbiotinylated BSA than on normal gels or on a gel that containednormal BSA ((A), (C) and (D)). Such strong forces were inhibitedwhen free biotin was added in the experimental buffer (B). Weconcluded, therefore, that most of the strong forces, larger than100 pN, originated from unbinding events between streptavidinon the tip and biotin on the electrophoresis gel.

The rupture force for the interaction between streptavidin andbiotin using AFM or other single molecular method was reportedpreviously, with different values for the unbinding force[15,41–44]. The difference in the reported values revealedthat the force value depended on the force loading rate andtemperature condition. Our experimental conditions consistedof about 5 nN/s for the force loading rate and around 290 K for thetemperature. Under these conditions, the rupture force for singlestreptavidin–biotin pair was reported around 100 pN [15,43,44].Those reported values were consistent with our findings that mostof the detected interaction forces larger than 100 pN came fromunbinding of single or multiple streptavidin–biotin pairs.

When it come to the unbinding length, it sometimes exceeded100 nm as shown in Fig. 5A. Streptavidin was modified on the tipof AFM cantilever through a self-assembled mono-layer and PEG

cross-linker. The length of the PEG linker we used was estimatedas around 40 nm at most considering the molecular weight andbackbone length of PEG [45]. The force curve with longerextension than 100 nm could not be explained by the length ofPEG linker, the electrophoresis gel or unfolded BSA protein in thegel matrix might be stretched during the measurement.

Next, we compared the detection limit of proteins usingdifferent techniques in Fig. 7. The detection limit of BSA in theCBB and silver stained method in SDS-PAGE (A) were around 50and 10 ng, respectively, based on the optical density profile. Andthe detection limit of dot-blot (B) methods were around 10 pg.These values corresponded to the value in the literature [46].

In our proposed AFM method, the ratio of the frequency toobtain the interaction force larger than 100 pN was evaluated.The frequency to obtain an interaction force larger than 100 pNwas divided by the total frequency to obtain an interaction forceto get f 100ratio values, which were shown in Fig. 7(C). The f 100ratio

value on the gel which contains biotinylated BSA was clearlyhigher than that on a gel that contains normal BSA or on a normalgel. The detection limit of our proposed method was at least 5 pgfrom graph (C), and this value was lower than one by CBB or silverstained SDS-PAGE and dot-blot method.

We estimated the critical detection limit of our proposedmethod by focusing on the relationship between the concentration

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Fig. 7. Comparison of the detection limit of proteins with different methods. Scanned images and optical density profiles of CBB and silver stained electrophoresis gel (A),

and stained dot-blot (B). The f 100ratio values in our proposed method (C). The frequency to obtain an interaction force larger than 100 pN was divided by the total frequency

to obtain an interaction force to get f 100ratio values.

H. Sekiguchi et al. / Ultramicroscopy 109 (2009) 916–922 921

of the biotin molecules in the electrophoresis gel and the volume(V tip) in which the tip of the cantilever was involved during theforce measurement of AFM, assuming that the streptavidin wasmodified on the tip of the cantilever in a dense homogeneouscondition.

The square pyramidal shape of the tip was used in theexperiments, and the volume (V tip) in which the tip was involvedin the measurement was estimated from the following equation:

V tip ¼4

3I3 tan2 y

2(1)

where I is the indentation of the tip to the electrophoresis gel andy is the face-to-face angle of the tip.

The indentation depth of the tip to the electrophoresis gel wasestimated by subtracting the deflection of the cantilever from theZ travel distance of the piezo to push from the AFM tipfs contactpoint. The Z travel distance was estimated as 300–350 nm fromFig. 4. The deflection of the AFM cantilever was around 18 nmsince the spring constant of the cantilever we used was around

0.08 N/m and maximum applied force during force measurementwas 1.5 nN. Therefore usual indentation depth in our experimentwas around 300 nm. The face-to-face angle of the tip which weused was around 40� (catalog value), therefore the tip whichinvolved volume V tip was estimated to be around 5� 10�21 m3.

If a biotin molecule exists within the volume of the tip V tip,an avidin-modified tip can access the biotin during the indenta-tion process during force measurement of AFM, which is thecritical limit of detecting the biotin molecule in our proposedmethod.

Assuming that most of the biotinylated BSA molecules aredistributed within the volume of Vprotein homogeneously in thecritical situation described above and the label efficiency of biotinto the protein is b mol/mol, the critical detection limit of thebiotinylated BSA in our proposed method was estimated usingfollowing equation:

Critical detection limit ¼Vprotein

V tip

Mw

NA

1

b(2)

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H. Sekiguchi et al. / Ultramicroscopy 109 (2009) 916–922922

where Mw is the molecular weight of the protein and NA isAvogadro’s constant.

The width of the protein’s band was 2 mm which is determinedby the width of lanes in the electrophoresis system, and assumingthe length and depth were 50mm, the distribution volume ofproteins Vprotein was roughly estimated as 5� 10�12 m3. Themolecular weight of BSA and the biotin labeling efficiencyof biotinylated BSA were 66 000 and 8 mol/mol (from the catalogvalue), respectively. The critical detection limit is estimated asabout 14 pg for one time force measurement. Though thereare many assumptions in the calculation above, we think thatthe critical detection limit in the current system of our proposedmethod is around picogram order, which was achieved by actualmeasurement (Fig. 7).

The ratio of the tip involved the volume, and proteins’distributing volume Vprotein=V tip had an effect on the detectionlimit.

Our proposed method should be improved when a large anddull tip, for example, a colloidal tip, is used for the detectionand deeper indentation is executed during force measurementof AFM. However, a larger tip and the deep indentation mightenhance the unwanted physical adhesive force between the tipand the substrate surface, which could affect the signal-to-noiseratio for the detection. The tip dimension for our proposedmethod needs to be optimized.

With regard to the proteins’ distributed volume (Vprotein), lesstime for electrophoresis and a more narrow width of lanes couldimprove the sensitivity of our method. In that sense, the currentsetting of our proposed method does not utilize the nano-meterspatial resolution of AFM properly, and it also should be optimizedfurther.

4. Conclusion

We have achieved a method with sufficiently high sensitivityto detect the picogram order of the biotinylated proteins byevaluating the frequency of the interaction force larger than100 pN using the force mode of AFM. Our proposed method basedon AFM can be applied for any biotinylated proteins, such asbiotinylated antibodies or secondary antibodies. We hope that acombination of the immunoblot method and our AFM methodwill be applied for detection of rare but important proteins in anadvanced proteomics research.

Acknowledgments

This work was supported by a Grant-in-Aid for CreativeScientific Research (no. 19GS0418) from the Japan Society for thePromotion of Science and by the Foundation Advanced TechnologyInstitute.

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