A highly sensitive microsystem based on nanomechanical biosensors for genomics applications

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Montevideo, 27-29 de setiembre de 2006 IBERSENSOR 2006

ISBN: 9974-0-0337-7 1/4

A highly sensitive microsystem based on nanomechanical biosensors for genomics applications

M. Moreno, M. Álvarez, L.G. Carrascosa, J.A. Plaza, K. Zinoviev, C Domínguez, and L.M. Lechuga*, Microelectronics National Center (CNM). CSIC

Isaac Newton 8, Tres Cantos, 28760 (Spain)

*Corresponding autor: [email protected] Tel: +34 918060700, Fax: +34 918060701 Abstract

A new DNA biosensor microsystem based on nanomechanical transducers is presented. The microcantilever- based biosensors are a promising tool that is actually being used to detect biomolecular interactions in a direct way, without labelling, and with a very high accuracy. We show the development of a portable biosensor microsystem which will be able to detect nucleic acid hybridization with high sensitivity. The microsystem comprises an array of 20 micromechanical cantilevers with a microfluidic system for delivery of the samples, an array of 20 lasers of vertical emission and the photodetectors with the CMOS circuitry integrated for signal acquisition and conditioning. Robust immobilization and regeneration procedures have been also implemented for the oligonucleotide receptor sequences.

Keywords: Microsystem, Si technology, biosensor, genomics.

Systems based on micro/nanotechnologies are a promising tool to develop ultrasensitive and miniaturised biosensors and biochips. The application of this new micro- and nanotechnology techniques will result in both miniaturization of biochip format and an increase of the sensitivity of the assays performed. Biosensors based on nanomechanics have been demonstrated to be able to specifically detect single-base mismatches with high resolution, performing label-free molecular recognition measurements [1, 2]. In these assays, nucleic acids are immobilized on a side of a micromachined lever (active side). Exposure of the cantilever to a sample containing complementary nucleic acid gives rise to a cantilever bending (deflection) of a few nanometres (see Figure 1). The nanomechanical response is due to the surface stress change of the active side with respect to the other side, in which DNA is not immobilized [3]. Surface stress arises from the electrostatic, van der Waals and steric intermolecular interactions on the surface [4]. The deflection is measured with sub-nanometer resolution by an optical system (similar to that of an AFM), in which a laser beam reflects off the back of the cantilever to a position sensitive photodetector.

In the framework of an European project, (OPTONANOGEN) [4] we are developing a portable multibiosensor microsystem able to detect hybridization of nucleic acids partly complementary to human genes with femtomolar sensitivity and with the ability to discern single base variations. The biosensor microsystem consists of: i) an array of twenty microcantilevers as biosensing tranducers, ii) an optical detection system (lasers and photodetectors) capable of measuring the cantilever movement with sub-nanometer resolution and iii) a

polymer microfluidic system for reagents delivery to the biosensor.

Si cantilever Si cantilever

Fig.1 Principle of working of a nanomechanical biosensor.

The main tasks performed in order to obtain the biosensor microsystem are the fabrication of arrays of 20 microcantilevers, the development of a self- assembly chemistry for the DNA probe immobilization, the fabrication of a polymer microfluidic header integrated with the cantilever array, the development of an optical detection subsystem with a monolithic array of 20 monomode oxide-confined VCSELs (Vertical Cavity Surface Emission Lasers) and a coupling element, and finally the integration of an array of 20 segmented photodetectors and CMOS circuits for the detection and processing of the laser beam displacement.

In the following, we will describe in detail each of the above tasks

2. Fabrication of microcantilever array

The design of arrays of microcantilevers for application as highly sensitive biosensors have to be done carefully according with the dimensions and the mechanical material properties. The cantilever geometry, dimensions and materials have been modelled in order to obtain a highly sensitive nanomechanical response and a small sensor area. A technology of fabrication of single-crystalline-silicon

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thin cantilevers using commercially available Silicon-On-Insulator (SOI) wafers has been developed and optimised to achieve 100% yield of cantilevers per wafer. The technology allows the fabrication of arrays composed of 20 cantilevers, each being 200 or 100 µm long, 40 µm wide, and 340 nm thick (see Figure 2).

Figure 2. (up) Array of 20 microcantilevers (bottom) details of same of the fabricated cantilevers.

The mechanical properties of the cantilevers have been experimentally determined (see Table 1). The spring constant of cantilevers were evaluated using a calibrated AFM probe. In addition, the harmonic characterisation of the cantilevers was also done. The experimental results match with the simulated ones when the overetching of the cantilevers is considered. The measured resonance frequencies are also in good agreement with the calculated ones. The Q factors are a little smaller than the Q factor of commercial ones, however this is in good agreement with the results obtained in other works for very thin cantilevers.

Table 1 Comparatives values of the mechanical properties for fabricated and commercial cantilevers.

L = 200 µm

Materials properties E (10¹¹ N/m)

ν

Thick ness x

with (µm)

K (N/m)

Z (nm)

∆σ = 1mN/m

F (kHz)

(air) Q(air)

Veeco (SiN) (V-shaped)

1.75 0.25 0.6 x

40 0.12 1.4 20.9 30 Olympus

(SiN) 1.75 0.25 0.8 x

40 0.10 0.8 23.9 35 Micromasch

(silicon) 1.69 0.20 1 x 50 0.26 0.6 38.4 45 CNM

(silicon) 1.69 0.20 0.33 x

40 0.009 5.1 7.8 6

For the silicon nitride cantilever a young’s modulus of E =1.75 N/m2 has been used, and for the silicon, E =1.49 N/m2

3. Immobilization and regeneration protocols.

Development of a self-assembly chemistry which allows the addressing of DNA immobilization at the nanometer-scale level only on one side of the cantilever array. The optimised immobilisation procedure is based on thiolated self-assembled

monolayers. Figure 4 shows the bending of the cantilevers observed during the immobilisation of the oligonucleotide receptors. Due to the attractive interaction between the nucleotide chain and the gold, the most favourable structure for adsorbed thiolated DNA monolayer is the DNA strongly anchored at the surface by the thiol linker, and with the nucleotide chain in contact with the surface. To enhance the accessibility of the DNA probes to the target and assures that the DNA probes are only attached to the gold surface through the terminal sulphur atom of the thiol linker, the surface was exposed to 6-mercapto-1-hexanol (MCH) after DNA immobilization (see Figure 3). MCH adsorption produces a large compressive surface stress, giving a downwards bending of about 30-35% of the cantilever deflection resulting from the thiolated DNA immobilisation [3].

0 20 40

0 20 40 60

MCH 1mM

12t15SH 2µM

Deflection (mV)

time (min)

Figure 3. Real time simultaneously monitoring of the oligonucleotide receptors immobilisation over five

microcantilevers.

To understand the effects of the MCH treatment, we performed radiolabelling experiments, (see Figure 4) using the thiol modified DNA radioactively labelled.

0 1 2 3 4 5 6 7 8 9

0.00E+000 1.00E+012 2.00E+012 3.00E+012 4.00E+012

molecules/cm2

DNA concentration (µM)

without MCH treatment with MCH treatment

Figure 4. Radiolabelling data of the thiolated DNA coverage on gold before and after the MCH treatment

The quantification of the immobilized DNA after and before of the MCH treatment was carried out

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with a scintillation counting. The results indicate that the MCH molecules displace about 60% of the immobilized DNA, suggesting that only the 40% of the immobilized chains are covalently attached to the gold surface of the cantilever [3].

3. Microfluidics system.

A polymer microfluidic header has been developed and integrated with the cantilever array. The microfluidic header incorporates flow channels to allow the delivery of reagents to the cantilevers, and subsequent removal of waste. Two different types of flow cells have been implemented: (i) a common flow cell for the whole array (one channel) (ii) a discrete flow cell for independent measurement of each cantilever in the array. Some features are common across these designs: all swept volume with a flow path through the window in the cantilever chip to avoid dead areas and to aid removal of air bubbles during priming, thin cover layer to reduce the optical path to the cantilever, and alignment pins to facilitate accurate assembly of the planar structure.

4. Optical subsystem for excitation.

Development of an optical detection subsystem that follows the parallelism of the cantilevers and the microfluidics sub-units. We have developed an original optical subsystem, which enables to target accurately each cantilever following the parallelism of the cantilevers and the microfluidics sub-units and amplify the cantilever deflection without any resonant measurement. The optical subsystem consists of a 20 single-mode oxide-confined 850nm emitting VCSEL (Vertical Cavity Surface Emission Lasers) array emitting at 850 nm that have been fabricated and characterised (see Figure 5). A microlens array have been used to collimate the optical beams and a coupling element for guiding the beams to the detection array has been developed aimed to amplify deflection of the laser beams reflected onto the cantilevers upon hybridization.

a)

b) c)

Figure 5. a) photograph of the array of 20 single mode VCSELs (3.5µm oxide aperture); b) top view of a single device; c) zoom

on the emission surface

The originality lies in the simplicity of the alignment control of the VCSEL beams with the 20 cantilever array, and in the compactness of the guiding and amplifying element. Moreover, the system amplification can easily be tuned owing to a single

tilt setting on VCSEL array and makes it possible nanometric cantilever deflection to be detected.

Integration in the final packaging is now being carried out

5. Optical subsystem for detection and data acquisition.

For the detection of the displacement of the laser beam reflected off the cantilever backside, an array of 20 segmented photodetectors, integrated with their respective amplifier and signal CMOS processing circuits have been designed and fabricated using IC silicon technology. The optical detection system has been employed to measure changes on the position of a light spot emitted by a VCSEL cell. The changes in spot positions are caused by the deflection of cantilever nano-structures used to sense the hybridisation process of biological material (DNA) for gene identification. The process is illustrated in Figure 6; when gene detection takes place, the cantilever deflection (d) goes from the initial value (d0) to other (d1), provoking a displacement of the Spot Center (SC) from its initial location (SC0) to the new one (SC1). Detection of displacement in the sub-pixel range are required for this application. The photodetectors and the proposed detection algorithm have been designed and optimized to obtain this resolution. To validate the design, a AHDL model for photodetector cell developed is used. This allows us to perform complex system characterizations including optical and electrical parts using the same simulation environment (SPECTRE in our case).

Figure 6. Illustrating the DNA sensing process: when gene detection takes place, the cantilever deflection (from d0 to d1)

produces a Spot Center (SC) displacement from SC0 to SC1.

6. Final integration and packaging.

The final integration of the whole system is being performed by incorporating the microfluidic header into an optical sub-system which includes the optical sources, coupling element, position sensing photodetectors and mechanical alignment stages. The sensor with the microfluidics unit will be the disposable part of the microsystem which will be replaced after each measurement cycle. This sub-

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assembly, shown in Figure 7, is mounted in an instrument that incorporates fluid components, power supplies, temperature control, data acquisition and CMOS control electronics.

Figure 7 Base unit for flow cell.

Acknowledgements

This work has been supported by the EU Contract IST-2001-37239. Authors would like to thank the work of the rest of the OPTONANOGEN partners.

References

[1] R. Mckendry, et al., Multiple label-free biodetection and quantitative DNA-binding on a nanomechanical cantilever array, Proc. Natl. Acad. Sci. 99 (15) (2002) 9783–9788.

[2] Y. Arntz, et al., Label-free protein assay based on a nanomechanical cantilever array. Nanotechnology 14 (1) (2003) 86–90.

[3] M. Álvarez, A. Calle, J. Tamayo, A. Abad, A.

Montoya and L.M. Lechuga. Biosensors &

Bioelectronics 18, 649 (2003)

[4] M. Alvarez, L.G. Carrascosa, A. Calle, M. Moreno, A.

Zaballos, L.M. Lechuga, C. Martínez-A & J. Tamayo.

Langmuir, 20, 9663, 2004.

[5]www.optonanogen.com