L'Ancien d'Algérie, Oct 2011

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Enzyme-Assisted Nanolithography

Leif Riemenschneider, Sven Blank, and Manfred Radmacher*

Institute of Biophysics, UniVersity of Bremen, 28359 Bremen, Germany

Received September 21, 2004; Revised Manuscript Received July 20, 2005

ABSTRACT

We have chemically immobilized alkaline phosphatase molecules onto the apex of a tip of an atomic force microscope. When the substrate

BCIP is dephosphorylated by alkaline phosphatase, it will precipitate in the presence of NBT. By bringing the tip in the vicinity of a suitable

sample, we could locally deposit this complex on the sample. Thus we combined the activity of an enzyme with the accuracy in positioning

a tip in scanning probe microscopy to demonstrate a novel technique referred to as enzyme-assisted nanolithography. By use of other

enzymes, this method will open the possibility to chemically modify surfaces on a nanometer scale.

Future applications in nano-biotechnology will ask for

techniques that allow surface structure modification on a

nanometer scale. Developing a versatile and flexible tech-

nique for this type of modifications will be essential for

potential functional materials. This includes the design of

nanoreactors as the ultimate stage of miniaturized labs-on-

a-chip, of miniaturized assays,1 or of intelligent materials

which could be used as smart drug-delivery systems. 2 The

key issue for all these applications will be a molecular-

defined surface which exhibits addressability and molecular

function. These requirements raise the need to locally modify

molecular groups on a surface. The most versatile molecular

tool kit for directed and controlled chemical modifications

can be found in nature: enzymes. Thus we implemented andtested a technology which allows combining the chemical

versatility of enzymes found in nature and the nanometer

precision in positioning objects in scanning probe techniques

such as AFM (atomic force microscopy).

The technique introduced here may become an important

contribution in the field of nano-biotechnology but also be

the basis for alternative processes in semiconductor fabrica-

tion. In both fields, there is a constant need for decreasing

structure sizes beyond the current state of the art eventually

modifying surfaces at a nanometer, i.e., molecular, scale. At

this length scale, structuring is equivalent with adding or

removing single molecules or modifying single chemical

groups. So, a bottom-up approach, where surfaces aremodified in a chemical sense, e.g. by emplyoing enzymes,

may be most appropriate.

One possible route to implement a method for structuring

surfaces at a nanometer scale is employing scanning probe

techniques. For example, soft polymeric films can be

modified by mechanical interaction of an atomic force

microscope tip.3,4 Also the dip pen technology,5 where a

suitable ink is adsorbed on an AFM tip and then locally

released on a sample, has attracted much interest.

Enzymes have been used to chemically modify surfaces

locally, e.g., by applying them with a micropipet to a sample

coated with a substrate of this particular enzyme.6 By

hydrolyzing the substrate, it became soluble and thus the

surface has been modified on a length scale of several tens

of micrometers. Recently enzyme molecules immobilized to

an AFM tip were used to modify a sample with adsorbed

enzyme molecules. Here the specific binding of the substrate

molecule to the enzyme molecule has been used to physically

remove the substrate from the sample.7 However, in this

example the enzymatic activity itself has not been used to

modify a surface. In another application phospholipasepresent in the bulk medium was used to modify a lipid film.

Here local disturbances of the packing density of the lipid

film caused by the mechanical interaction with an AFM tip

were used to modify the sample locally.8 However, in all

these cases surfaces coated with the enzyme’s substrate were

needed. It is not clear how these schemes can be generalized

to modify arbitrary surfaces.

As has been introduced with the enzyme lysozyme9 and

demonstrated for many other systems, an AFM tip positioned

on top of an enzyme molecule can pick up conformational

changes and thus monitor the activity of enzymes.10 The

activity of alkaline phosphatase molecules adsorbed on a

mica support (Figure 1a) can also be observed directly by

atomic force microscopy (AFM). Using the substrate BCIP,

which after dephosphorylation forms a precipitate in the

presence of the cofactor NBT, we observed directly the

activity of individual enzyme molecules adsorbed on the mica

support. Little piles of precipitate were formed near active

enzymes.

We have modified the experimental setup such that we

immobilize the enzyme molecule on the apex of an AFM

tip allowing us to create deposits at arbitrary locations. So,* Correspondence and requests for materials should be addressed to

M.R. ([email protected]).

NANO

LETTERS

2005Vol. 5, No. 91643-1646

10.1021/nl0484550 CCC: $30.25 © 2005 American Chemical SocietyPublished on Web 07/28/2005



we have combined the chemical activity of enzyme molecules

and the high accuracy in positioning a tip by scanning probe

microscopy to demonstrate the first implementation of

enzyme-assisted nanolithography.

The AFM tip was first silanized and then biotinylated using

standard procedures. Alkaline phosphatase conjugated withstreptavidin could be immobilized onto the tip via specific

biotin-streptavidin interaction. First experiments where tip

and cantilever were coated with phosphatase by immersing

the tip in a phosphatase solution were not satisfactory. In

this case many phosphatase molecules were active including

those on the cantilever legs and even on the supporting chip.

This resulted in an excessive production of precipitate which

then deposited everywhere on the entire sample. Therefore

we needed to design an experimental procedure which allows

us to coat only the very apex of an AFM tip. We found a

simple and reliable way for achieving this goal. A silicon

wafer was biotinylated using the same procedure as used

for coating AFM tips. Then the biotinylated wafer wasincubated in a solution containing a phosphatase-streptavidin

conjugate at low concentrations. This resulted in a sample

sparsely populated with phosphatase molecules (about 10 per

µm2) as proven by AFM imaging (Figure 2a). This sample

was mounted in an AFM and imaged for about 1000 s with

a biotinylated tip covering an area of 4 µm2. On average we

found 40 alkaline phosphatase molecules in this field of view.

Streptavidin exhibits four binding sites for biotin. For

geometric reasons it is conceivable that it will bind with up

to two sites to the support, exposing the other two binding

sites to the AFM tip. Thus, streptavidin molecules on the

sample could also bind to biotin molecules on the tip. While

scanning across the surface streptavidin will bind to the tip,

and one bond will be separated, either the bond between the

tip and streptavidin or the bond between streptavidin and

support. On average 50% of the streptavidin molecules will

bind to the tip (Figure 2b-d). Since the spacer molecules

used here are rather short, streptavidin molecules will only

be able to bind to the very apex of the AFM tip, presumably

only to the very last 5-10 nm.

Thus we were able to coat an AFM tip specifically at its

very apex with streptavidin phosphatase molecules. Since

our experimental procedure involves the formation and forced

rupture of biotin streptavidin bonds, there is a high prob-

ability for denaturing streptavidin. However, since phophatase

molecules are not mechanically loaded by this process, the

activity of the enzyme is maintained, as can be seen in AFM.

The activity of the immobilized enzyme phosphatase was

tested in a control measurement employing a standard

photometric assay. We coated a silicon oxide wafer with the

same procedure as used for coating tips and silicon wafers.

When hydrolyzing the substrate p-nitrophenyl-phosphate

(pNPP) absorption of yellow light will increase proportional

to the concentration of the product p-nitrophenolate. As-

suming a complete coverage of the silicon nitride wafer, we

observed a maximum activity of 3200 reactions per second.

The manufacturer claims an activity of 4200 reactions per

second in solution, indicating that the activity of immoblized

enzyme molecules is only marginally reduced. The func-

tionalized silicon nitride wafers exhibited significant enzy-

Figure 1. Alkaline phosphatase molecules have been adsorbed onto mica and imaged in buffer solution by AFM (a). In this image, thehighest features ( z-range 5 nm) correspond to intact enzyme molecules, the smaller features correspond to fragments which are alwayspresent in protein samples. When the substrate NBT is added to the buffer solution, active enzyme molecules will dephosphorylate BCIP.In the presence of NBT a precipitate is formed in the vicinity of the enzyme molecules (b). Growth of these precipitates will stop when allsubstrate is hydrolyzed but will start over when new substrate is added to the buffer solution (c). (In (b) and (c) the z-range has beenchanged to 20 nm due to larger height of aggregates compared to enzymes.)

Figure 2. AFM image (tapping mode) of single alkaline phos-phatase molecules bound to a silicon wafer (a). The wafer isbiotinylated and phosphatase is specifically bound to the wafer viastreptavidin-biotin interactions. When the sample is imaged slowlyin contact mode with a biotinylated tip (b), streptavidin will alsobind to the tip (c), and consequently the very apex of the AFM tipis coated with phosphatase molecules (d).

1644 Nano Lett., Vol. 5, No. 9, 2005



matic activity over a course of several days. Inferring from

this observation a sufficient stability of the enzyme im-

mobilized to AFM tips can be assumed. This test of the

activity of immobilized phosphatese on silicon nitride

employing silane chemistry serves also as a control verifying

the activity of phosphatase on the silicon nitride AFM tips.Since silicon nitride exhibits a native oxide layer, it was to

be expected that binding via silane will also be possible on

this support.

The sample was now replaced by a piece of freshly cleaved

mica to be modified and the buffer solution was replaced

by a solution with the substrate BCIP and the cofactor NBT.

Prior to the writing process, the surface is imaged in tapping

mode to verify that it is clean. To start the actual deposition

of the precipitate, the AFM tip is brought into contact with

the surface by engaging it in contact mode (Figure 3a). We

could form single spots by keeping the tip for a short time

(20 s) in one location, then moving it to another location to

form a new spot (Figure 3b). Alternatively the tip could be

moved slowly across the sample (velocity 10 nm/s). This

resulted in a line of deposit, which can have any arbitrary

shape by steering the tip accordingly (Figure 3c). The deposit

on the surface had a typical lateral dimension of 150-170

nm and a height of 10 nm. The deposits were then imaged

with the same tip in tapping mode in liquids.

Summary and Outlook. We have demonstrated that

enzyme molecules linked to an AFM tip can be used to

locally modify a sample. With the enzyme alkaline phos-

phatase we were able to create features of about 150 nm in

diameter. However, by minimizing the contact time, con-

centration of substrate, or number of enzyme molecules

immobilized to the tip, it is conceivable to create smaller

features, ultimately of molecular size. This opens the

possibility for an enzyme-assisted nanolithography. Here we

have demonstrated the topographic modification of the

sample. It is conceivable, by employing different enzymes,

to also achieve a chemical modification of the sample

surface. Due to the modular setup of our enzyme im-

mobilization scheme, it is possible to move to other enzymesystems, by using the corresponding conjugate of an enzyme

with streptavidin. It is conceivable that the method described

here can be used to modify samples possibly on a scale of

single molecular reactions to form the basis for miniaturized

devices and chemical nanoenvironments. This technique may

become very important in the emerging field of nano-

biotechnology.

Methods. Precipitation near Adsorbed Alkaline Phos-

phatase. Alkaline phosphatase in buffer solution (Tris 40

mmol plus 1 mM MgCl2, pH 9.8) at a concentration of 0.02

mg/mL was incubated for 10 min on a piece of freshly

cleaved mica. The sample was thoroughly flushed with purebuffer and mounted in an AFM (Nanoscope III, Digital

Instruments). Imaging was performed in tapping mode in

liquids using soft silicon nitride cantilevers (Oriented Twin

Tips, Digital instruments, force constant 60 mN/m) at a

frequency of 29 kHz. The buffer has been replaced by a

buffer with BCIP and NBT at a concentration of 0.5 mmol.

Functionalization of AFM Tip. Soft silicon nitride AFM

cantilevers (force constant 60 mN/m) (NP-STT, Veeco

Instruments, Santa Barbara, CA) were cleaned by UV

irradiation and functionalized with biotin following a pro-

cedure adopted from Baselt et al. (Proc. IEEE 1997, 85 (No.

4)). The cantilevers are immersed for 30 min in a solution

containing 9 mL of methanol, 370 µL of deionized water,

80 µL of concentrated acidic acid, and 230 µL of N -(2-

aminoethyl)(3-aminopropyl)trimethoxysilane (Sigma/Merck

catalog # 8.19172.0100). Then the cantilevers are rinsed well

in methanol and dried in a stream of liquid nitrogen. In the

next step they are cured in an oven for 3 min at 120 °C and

then immersed for 2 h in a solution of 4 mL of DMSO

(Fluka/41640) containing 1 µg of NHS-Biotin (Biotin- N -

hydroxysuccinimide, Sigma/H-1759). Then the cantilevers

are rinsed well in ethanol and dried in a stream of nitrogen.

Preparation of Alkaline Phosphatase Populated Sur-

face. Oxidized silicon wafers (Crystec, Berlin/S 3012) were

cut in square pieces (12 mm × 12 mm), cleaned for 10 minin a mixture of concentrated sulfuric acid and hydrogen

peroxide (ratio 3:1) for 10 min, and then rinsed well in

deionized water. They were biotinylated following the same

procedure as used for modifying AFM tips described above.

To populate the wafer sparsely with enzymes, a 200 µL

droplet of a 0.2 nM streptavidin-phosphatase conjugate in

a buffer solution is applied to the surface for 10 min. The

buffer contains 40 mM TRIS and 1 mM magnesium chloride

at a pH of 9.8. It proved necessary to remove enzymes that

were not thoroughly immobilized by immersing the wafer

Figure 3. The enzyme alkaline phosphatase was immobilized ontoan AFM tip. The substrate BCIP was present in the surrounding

medium. The product of the enzymatic reaction will form togetherwith NBT a water-insoluble complex which will precipitate on thesample (a). Surface modification was done by two procedures: thetip was resting for 20 s in one spot, then moved rapidly to the nextspot to deposit the next dot (b). Alternatively the tip was slowlymoved with a velocity of 10 nm/s across the sample to form acontinuous line (c). Imaging was done after the enzymatic reactionwith the same tip in tapping mode.

Nano Lett., Vol. 5, No. 9, 2005 1645



in a 1 mM solution of p-nitrophenyl phosphate (PNPP, Sigma

N-4665) in buffer and keeping it on a stirrer for 10 min.

Verification of Immobilization of Alkaline Phosphatase.

Silicon oxide wafers were cleaned and biotinylated as

described above. Biotinylated wafer were incubated in a

solution of 100 mmol streptavidin-phosphatase conjugate

for 10 min. The sample was flushed with buffer, and then

the absorbency at 405 nm was recorded using a photometer

(Shimadzu UV2102-PC, Shimadzu Scientific Instruments

Columbia, MD) for 25 min in the presence of the substratepNNP at a concentration of 2.7 mM. As a control, the

absorbency of a solution of alkaline phosphatese conjugated

with streptavidin in the presence of pNPP was recorded.

Enzyme-Assisted Nanolithography. Mica was purchased

from Plano Wetzlar, Germany. The substrate solution is a

1:1 mixture of fractions A and B. Fraction A contains 40

mM TRIS and 1 mM MgCl2 at pH 9.8. The B fraction is

the stock solution of the substrate BCIP/NBT as provided

by the supplier (Sigma B-6404). It contains the BCIP at a

concentration of 0.56 mM and NBT at a concentration of

0.48 mM. The concentration of substrate was not sufficient

to produce distinct features and had to be increased by

thermal evaporation of water by a factor of 5-10.Imaging and deposition were done with a commercial

AFM (MFP-3D, Asylum research, Santa Barbara, CA).

Images of deposits were taken in tapping mode in liquids at

a drive frequency of around 30 kHz and a scan rate of 1

line per second.

Acknowledgment. This work was supported by the

Volkswagen Stiftung under code I/78790. We thank Monika

Fritz for helpful discussions.

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NL0484550

1646 Nano Lett., Vol. 5, No. 9, 2005

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L'Ancien d'Algérie, Oct 2011