Fully electronic DNA hybridization detection by a standard CMOS biochip

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Abstract

A novel solid-state biosensor for label-free detection of DNA hybridization is presented. The new device is realized in a standard CMOS process, thus allowing the realization of low-cost, portable, fully integrated devices. The detection mechanism is based on the field-effect of the intrinsic negative electric charge of DNA molecules which modulates the threshold voltage of a floating-gate MOS transistor. A fluid cell was developed for delivering DNA samples on the active surface of the chip. The device has an integrated, individual counter-electrode, so dry measurements are possible increasing lifetime of the chip and speeding up the experiment. Successful measurements on a first prototype of the chip, hosting 16 sensors individually addressable, are provided as proof of concept.

Introduction

DNA hybridization detection is a key step in genomics studies and development of diagnostic tools [1]. The standard approach to its detection is based on optic or optic-like methods which make use of a passive substrate for DNA immobilization and indirect detection of labels bound to the target DNA molecule under investigation. A known single strand DNA (probe ssDNA) sequence is immobilized on a substrate (nylon, glass, silicon), the unknown target (target ssDNA) sequence is labelled with a fluorescent or radio label and injected on the substrate. If the two sequences are complementary they hybridize and form the double strand DNA (dsDNA) so the label is immobilized on the substrate as well. The passive substrate is then checked to verify the presence of the label rather than of the DNA molecule. Such technology is mature and established but presents some drawbacks such as the cost of the instrumentation, the necessity of labelling the molecules, the difficulty to integrate the different tools into a single, portable device. For these reasons, several new approaches have been proposed recently, to overcome such problems. These new methods make use of gravitometric measures based on microbalances (QCM, quartz crystal microbalance) which transduce the change of mass due to hybridization into a change in the resonant frequency of a crystal [2], or based on the use of micromachined cantilevers and their bending due to surface forces [3], [4]. Even if they are label-free, these methods are difficult to integrate with standard electronics and require sophisticated technology steps.

Fully electronic detection is the most promising approach to obtain really inexpensive, portable and low-cost devices [4]. Several devices have been proposed in the recent past, either making use of labels such as gold nanoparticles to measure resistance [5], [6] or capacitance [7] changes between two electrodes, or making use of electrochemical labels capable of generating a redox current [8], [9] detectable by electrodes. These approaches are not fully compatible with a CMOS process, so they cannot easily be integrated with the readout electronics.

In this paper we present a novel, solid-state CMOS device able to detect sequences of DNA by their intrinsic electric charge without the need for external components (counter-electrodes). The sensor is fully compatible with a standard CMOS process. With respect to other sensors for hybridization detection, which make use of capacitors [10], diodes [11] or transistors [12], [13], [14] implemented in silicon or with nanowires [15], this biosensor is attractive for its extreme simplicity. It allows direct detection of hybridization process, with no need for additional process steps. For this reason, it allows the realization of highly integrated arrays with thousands of active sites, direct detection and fully electronic readout capabilities. The electronic readout is also attractive for implementation of portable and/or disposable medical kits.

Section snippets

Device structure and working principle

The proposed device mixes the characteristics of the floating-gate MOS transistor and the gate-exposed FET sensors such as the ISFET or CHEMFET [16], [17], [18]. A cross-section of the sensor is shown in Fig. 1: it is made-up of a floating-gate transistor (M0), a control-gate with the role of reference electrode (CC) and an active area activated by charge induction (AS).

The single strand probe oligonucleotides are bound on top of the exposed floating-gate surface by an organic/inorganic

Test strategy

The sequence of steps required to test the hybridization process is given by the following procedure:

  • (a)

    START: activation of the exposed-gate surface by means of 3-MPTS.

  • (b)

    STEP_T1: immobilization of T1 (probe) oligonucleotide on sensors 0–7.

  • (c)

    STEP_T0: immobilization of T0 (probe) oligonucleotide on sensors 8–15.

  • (d)

    STEP_P1: injection, on the entire active surface (sensors 0–15) of P1 (target) oligonucleotide (complementary to T1).

  • (e)

    STEP_P0: injection, on the entire active surface (sensors 0–15) of P0

Discussion

The proposed device is able to detect an electric charge bound on the surface of its active area. With respect to many other devices developed to this purpose (such as the ISFET and its derivatives or other sensors for hybridization detection based on field-effect [6], [7]), this biosensor has the advantage of being completely integrated in a standard CMOS process. The transistor is a standard, native device of the process and no special manufacturing is needed to prepare the gate-oxide, as

Acknowledgement

The authors acknowledge the EU-IST-FET program (BEST) for partially funding the research.

Massimo Barbaro was born in Cagliari, Italy, in 1972. He received the PhD degree in electronic engineering and computer science from the University of Cagliari, Italy, in 2001. In 2002, he joined the Department of Electrical and Electronic Engineering, University of Cagliari, as an assistant professor in the field of microelectronics. His current research interests are the design of CMOS biosensors and CMOS imagers.

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    Massimo Barbaro was born in Cagliari, Italy, in 1972. He received the PhD degree in electronic engineering and computer science from the University of Cagliari, Italy, in 2001. In 2002, he joined the Department of Electrical and Electronic Engineering, University of Cagliari, as an assistant professor in the field of microelectronics. His current research interests are the design of CMOS biosensors and CMOS imagers.

    Annalisa Bonfiglio received the laurea degree in physics at the University of Genoa in 1991 and the PhD in bioengineering at the Politecnico di Milano, Italy in 1995. She is currently associate professor of electronics at the Electronic Engineering Faculty of the University of Cagliari, Italy. Her research interests concern innovative materials and devices for electronics and bioengineering.

    Luigi Raffo received the laurea degree in electronic engineering in 1989, and the PhD degree in electronic engineering and computer science in 1994 from University of Genoa, Italy. In 1994 he has joined the Microelectronic Laboratory of the Electronic Engineering Department of the University of Cagliari as assistant professor. Since 1998 he is professor at the same university. He is teacher of electronic and system design courses. His main research field is the design of digital/analog devices and systems. In this field he is author of more than 70 international publications, and patents. He has been coordinator of EU, Italian Research Ministry, Italian Space Agency, industrial projects.

    Andrea Alessandrini is a researcher at the Italian National Center S3 (Nanostructure and BioSystems at Surfaces) of CNR-INFM in Modena. His current research interests focus on bioelectronics, surface functionalization for biomolecules immobilization and scanning probe microscopy characterization of samples of biological interest.

    Paolo Facci is senior scientist at the Center “Nanostructures and Biosystems at Surface, S3” of the Italian National Research Council in Modena, Italy, where he leads the “nanobiolab”. His research interests range in protein and nucleic acid biophysics, bio-SPM, nanofabrication, and biomolecular nanoelectronics. He is graduated in Physics and got a PhD in Biophysics from the University of Genova, Italy. In 2002 he has been awarded “Campisano International Prize” for the physics of matter.

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