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NANO LETTERS

Magnetomechanical Detection of the Specific Activities of Endonucleases by Cantilevers


Yossi Weizmann, Roey Elnathan, Oleg Lioubashevski, and Itamar Willner*
Institute of Chemistry, The Hebrew UniVersity of Jerusalem, Jerusalem 91904, Israel
Received February 1, 2005

2005 Vol. 5, No. 4 741-744

ABSTRACT
Thiolated nucleic acids 1 or 2 are immobilized on Au-coated cantilevers and hybridized with the complementary nucleic acids 1a or 2a associated with magnetic particles. The duplexes 1/1a or 2/2a include specific sequences for the scission by Apa I or Mse I, respectively. The cantilevers positioned in a flow cell are subjected to an external magnetic field, leading to the deflection of the cantilevers. Upon the specific scission of the DNA duplexes by Apa I or Mse I, the magnetic particles are disconnected from the cantilevers leading to their retraction to the rest position. The deflection/retraction of the cantilevers are followed by a conventional atomic force microscope optical detection system.

The controlled deflection of cantilevers by physical or chemical stimuli attracts recent interest directed to the development of miniaturized devices such as valves, robots, machines, or sensors.1 For example, the surface stress exerted on cantilevers modified with redox-active polymer films, such as polyaniline,2 or electroactive monolayers such as a ferrocene-tethered monolayer3 was used for the cyclic electrochemically induced deflection and retraction of cantilevers. The electrochemical charging of these electroactive films yielded electrostatic repulsions between surfaceconfined components, resulting in surface stress and, therefore, deflection of the cantilevers. Electrochemical removal of the charges reduced the surface stress and led to the retraction of the cantilevers to their original positions. The electrostatic repulsive interactions between surface-confined biomolecular components were also employed for nanomechanical biosensing.4 For example, the specific hybridization of DNA with its complementary nucleic acid on an array of cantilevers led to electrostatic repulsions and the specific deflection of the cantilever where hybridization occurred. Other biorecognition processes were followed by the mechanical deflection of cantilevers.5 A major goal in the development of biosensors involves the tailoring of sensitive and specific analytical processes. The coupling of amplification paths to the biorecognition events is a general method to enhance the sensitivity of biosensors. The use of enzyme conjugates that generate redox-active products or stimulate the formation of insulating films on electrode surfaces was reported as a general means to follow antigen-antibody6 or DNA7 recognition processes
* To whom correspondence should be addressed. Tel. 972-2-6585272, Fax 972-2-6527715, e-mail willnea@vms.huji.ac.il. 10.1021/nl050204j CCC: $30.25 Published on Web 03/04/2005 2005 American Chemical Society

by electrochemical means. Nanoparticles provide versatile labels for the amplification of biosensing events.8 Antibody or nucleic acid-functionalized metal or semiconductor nanoparticles were employed as labels for the amplified electrochemical9,10 or microgravimetric11 detection of biorecognition events. Recently, the amplified magnetomechanical detection of DNA and single base mismatches was demonstrated on cantilevers.12 For example, M13 phage DNA was analyzed by its hybridization with a nucleic acid primer linked to a magnetic particle. The replication of the analyte while incorporating biotin labels into the replica was followed by the attachment of the labeled magnetic particles to an avidinmodified cantilever. The application of an external magnetic field on the cantilever resulted in its deflection, controlled by the magnetic field strength. Also, magnetic beads associated with a biotin/avidin complex linked to cantilevers were used to amplify the biorecognition process by following the oscillations of the piezocantilever in an alternating magnetic field.13 Here we report on the magnetomechanical detection of the specific activities of endonucleases. Related studies have followed the scission of dsDNA with endonucleases by imaging the height of the dsDNA on the surface before and after scission,14a by the labeling of the two strands with magnetic particles and following the magnetic relaxation upon scission,14b and by labeling of the strands with Au nanoparticles and following their de-aggregation.14c Scheme 1 depicts the concept for the magnetomechanical detection of the endonuclease activity. The setup to follow the activities of the endonucleases is depicted in Scheme 1A and described in the experimental section. The relocation of the magnet on a translator permits to switch ON and OFF the magnetic field acting on the cantilever, modified with magnetic particles, and thus to deflect or retract the canti-

Scheme 1.

Magnetomechanical Detection of Endonucleases Activities on a Functionalized Cantilever: (A) Instrument Setup and (B) Magnetomechanical Detection of Specific Endonucleases Action

lever, respectively. Two endonucleases were examined, Apa I and Mse I. For analyzing the Apa I activity, the primer 1 was immobilized on a Au-coated cantilever, while primer 2 was assembled on the cantilever for the analysis of Mse I activity. The complementary nucleic acids 1a and 2a were linked to magnetic particles (5 m) and hybridized with the respective 1- or 2-modified surfaces. The sequence-specific scission modes of dsDNA of the respective endonucleases are shown in Scheme 1. The application of the external magnetic field on the cantilevers that includes the hybridized nucleic acid-functionalized magnetic particles 1a or 2a results in the deflection of the cantilevers. Addition of the specific endonuclease is anticipated to cleave off the dsDNA and to release the magnetic particles, a process that results in the retraction of the cantilever to its original state. This enables the magnetomechanical detection of the endonuclease activity. Figure 1 shows the magnetomechanical detection of the Apa I activity. It should be noted that this concept to follow
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the endonuclease activities has advantages over the previously described method to follow DNA hybridization events on cantilevers using surface stress interactions.4 While in the reported method the induction of the surface stress resulting in the deflection of the cantilever requires a high surface coverage of the double-stranded DNA, the present method includes an intrinsic amplification principle. That is, already a low coverage of the magnetic particle-labeled doublestranded DNA stimulates the cantilever deflection under an action of the external magnetic field. Thus, the scission of the labeled DNA would be detected already at a low surface coverage. The 1a-functionalized magnetic particles were hybridized with the 1-modified cantilever, and free magnetic particles were washed off. At point a, the external magnet is positioned close to the cantilever (state ON) and the cantilever is subjected to a force that results in its deflection. At point b the external magnet is removed (state OFF), resulting in
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Figure 1. Analysis of the magnetic-field-induced forces exerted on a cantilever functionalized with 1/1a/magnetic particles upon the specific scission with Apa I endonuclease. Steps a-s are detailed in the text.

Figure 2. Analysis of the magnetic-field-induced forces exerted on a cantilever functionalized with 2/2a/magnetic particles upon the specific scission with Mse I endonuclease. Steps a-p are outlined in the text.

the elimination of the magnetic force and the retraction of the cantilever to its original state. At points c and d the effect of the external magnet is re-examined to demonstrate the cyclic ON and OFF deflection and retraction of the cantilever by means of the external magnetic field, respectively.15 At point e the external magnetic field is reapplied on the cantilever, and at point f the enzyme Mse I, that does not recognize the sequence, is added to the system. The cantilever remains in its deflected position, implying that the enzyme does not remove the magnetic particles. The system remains structurally intact, and the cantilever responds to the absence or presence of external magnetic field by its retraction, deflection, and re-retraction, points g-i. At point j the buffer required for Apa I activity is exchanged in the system. The cantilever continues to be deflected and retracted upon the application of the external magnetic field, points k-o. At point p the enzyme Apa I is added to the system while the external magnetic field is applied on the cantilever. The cantilever is retracted to its initial position, implying the elimination of the external magnetic force. These results indicate that the magnetic particles are removed from the cantilever by the biocatalytic scission of the double-stranded assembly. To confirm that the retraction of the cantilever originates from the removal of the hybridized magnetic
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particles, the external magnet was removed and reapplied on the system, points r and s. No magnetically induced motion of the cantilever is observed, consistent with the fact that the magnetic particles were removed. To confirm the specific magnetomechanical detection of the endonuclease activity, the process was applied to analyze Mse I (Figure 2). The nucleic acid 2 assembled on the cantilever was hybridized with the 2a-modified magnetic particles. At points a-c the cantilever is deflected, retracted, and re-deflected by the external magnet, indicating the repeated deflection/ retraction of the modified cantilever. At point d the enzyme Apa I is added. The position of the cantilever is unaffected, and it is further affected by the external magnet, points e-h. These results indicate that Apa I has no catalytic effect on the scission of the 2/2a system that is labeled with the magnetic particles. At point i, the buffer in the system is exchanged with the composition required for Mse I. The system reveals magnetoswitchable retraction and deflection features, points j-m. At point n, the Mse I endonuclease is added to the system when the cantilever is in its deflected state. Addition of the enzyme results in the retraction of the cantilever to its rest position. The enzyme-induced retraction of the cantilever is attributed to the scission of the double743

stranded assembly and to the biocatalytic removal of the magnetic particles from the cantilever. Indeed, points o and p correspond to the responses of the cantilever to the removal and application of the external magnet, respectively. The cantilever is not influenced by the external magnet, indicating that no magnetic particles are associated with the cantilever. It should be noted that the forces exerted on the cantilever in the Apa I system (Figure 1) are about twofold higher than those observed in the Mse I system (Figure 2). This might originate from different spring constants of the cantilevers used in this system, and eventually a different content and position of magnetic particles on the cantilevers. Thus, the present study has employed the magnetomechanical detection of the specific scission of sequencespecific dsDNA using cantilevers as the sensing element. Experimental Section. Materials and Methods. Aminefunctionalized borosilicate-based magnetic particles (5 m, MPG long chain alkylamine, CPG, Inc.) were employed. The heterobifunctional cross-linker, 3-maleimidepropionic acid N-hydroxysuccinimide ester, and the functionalized oligonucleotides were purchased from Sigma. Preparation of DNA-Functionalized Magnetic Particles. A total of 30 mg of the amino-functionalized magnetic particles were activated by the reaction with the heterobifunctional cross-linker 3-maleimidepropionic acid N-hydroxysuccinimide ester (5 mg, Sigma) in 1 mL of dimethylsulfoxide (DMSO). After 4 h of incubation at room temperature, the particles were collected with an external magnet and washed with DMSO and 10 mM phosphate buffer, pH 7.4. The maleimido-activated particles were then reacted with 20-30 O.D. (optical density) of the thiolated oligonucleotide in 10 mM phosphate buffer, pH 7.4, for a period of 8 h. (The thiolated nucleotide was freshly reduced with dithiothreitol and separated on a Sephadex G-25 column prior to the reaction with the functionalized particles). Finally, the magnetic particles were washed with 10 mM phosphate buffer, pH 7.4. The DNA-modified particles could be stored for periods longer than 6 months, in phosphate buffer that included 1% (w/v) sodium azide, at 4 C. Detection System. The experimental setup shown in Scheme 1A includes a home-built instrument consisting of a cantilever mounted in a flow cell and coupled to an optical detection system.12 The flow cell enables the rinsing of the cantilever and the exchange of the cell buffer and its content. We used commercially produced Si microcantilevers of a length 350 m and thickness 1.0 m (CSC12, cantilever E, MicroMasch, Estonia). The spring constant of the levers was nominally specified as 0.03 N/m. The back of the cantilevers was Al-coated to allow optical reflection from a laser diode, and the front side was gold-coated to allow immobilization of thiolated DNA strands. The reflected laser beam then passed onto a linear photodiode, which is used to measure the lever deflection. An external permanent magnet (neodymium iron boron, NdFeB, 50 50 10 mm) is positioned on a translator. The applied magnetic field is directed perpendicular to the cantilever surface. The magnet could be repositioned along the instrument in parallel to the cell wall, on the translator stage.
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CantileVer Modification. Cantilevers were incubated with the oligonucleotides (freshly reduced), solubilized in 0.2 M phosphate buffer, pH 7.4, for 12 h to obtain the ssDNA monolayer. The resulting cantilevers were washed with phosphate buffer, pH 7.4, prior to their use. Hybridization Conditions. A total of 1.5 mg of magnetic particles modified with the ssDNAs were hybridized with the complementary ssDNAs on the cantilever Au surface. Hybridization was performed in 10 mM phosphate buffer, pH 7.4, and 0.3 M NaCl at room temperature for 4 h. Compositions of the Buffers for Different Endonucleases. The compositions of the buffers for the different endonucleases are, for Apa I buffer, 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, and 100 g/ mL bovine serum albumin (BSA), pH 7.9 and, for Mse I buffer, 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 100 g/mL BSA, pH 7.9. A total of 10 units of each of the endonucleases was injected into a flow cell to affect the respective scission processes. Acknowledgment. This research was supported by the German-Israel Program (DIP). References
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