Biosensor integration on Si-based devices: Feasibility studies and examples
Introduction
The importance of integrated biosensors, especially miniaturized devices, has experienced a strong increase in the latest years, as witnessed by the almost exponential increase in the number of articles published on the subject in the last 20 years [1]. There is a growing interest in creating microbiosensors, fabricated in Si-compatible technologies, to be integrated within microelectronic circuits. The reason is that silicon-based devices would provide a lot of potential advantages such as small size and weight, fast response, high reliability, low output impedance, the possibility of automatic packaging at wafer level, on-chip integration of biosensor arrays and a signal processing scheme with the future perspective of low-cost mass production of portable microanalysis systems. Si-based micro-bio-sensors could actually benefit of: (i) the use of low cost and mature technology, fundamental for mass production; (ii) the possibility to shrink the devices, implying reduced molecular diffusion path, faster kinetics and an improvement of the device analytical performance and accuracy of the analysis [2], [3]; (iii) the possibility to create microstructured devices achieving complex functions, e.g. micro-total-analysis-systems; (iv) the integration on the same chip of the electrodes and/or photo-electronics needed for detection; (v) the integration of microelectronics circuitry that provides the so-called “intelligence on board” for system multifunctionalities. Moreover, biosensor miniaturization opens the application in field of in vivo physiological monitoring, since it allows lower reagent consumption, hence minimized sample volumes, lower energy consumption, and less space requirement (sensor portability). Now, conventional biosensors need extensive packaging, complex electronic interfaces and regular maintenance or reactivation.
To properly develop new effective miniaturized bio-sensors, the definition of an efficient transduction mechanism is one of the most important aspect to consider. It is strongly affected by both the immobilization method of the receptor (probe) and the label efficiency of the analyte to be detected. Nowadays, the most used approach is the optical trasduction, e.g. for DNA recognition [4], [5]. However, the need to manipulate the target, i.e. to label it with organic fluorescent dyes, limits the applications of fluorescence-based biosensors. Moreover, organic fluorescent dyes suffer from weak signal intensity, photo-bleaching and self-quenching [6]. Furthermore, the need of in vivo and/or in situ and real time measurements requires more versatile devices, and portable systems, to be implemented. To overcome the above mentioned limitations label-free detection platforms are needed.
In this context, an interesting solution is provided by electrochemical biosensors [7], [8], [9]. Electrical sensing could promote biosensor integration within complex electrical circuits. Due to their simple principle of measurement and the possibility to integrate the full signal processing on chip, biosensors which are based on electrochemical transducer principles are the most common sensor devices recently investigated [10], [11], [12], [13], [14], [15].
Among electrochemical biosensors, we focused our research interests on electrolyte-insulator-semiconductor (EIS) biosensors that vary their potential (or electric field) as a consequence of the recognition event occurring at, or nearby, the insulator/electrolyte interface [16], [17], [18], [19], [20], [21], [22], [23], [24], [25]. The size of the potential change can be measured as capacitance variation and it largely depends on the nature and the coverage of the modification material. If the insulator is covered with a metal the capacitance provides information on the metal-solution interface. The capacitance can be described as the built-up of three main capacitors in series: (i) the semiconductor/insulator capacitance (CS/I); (ii) the capacitance due to the recognition element and to any contribution from the Stern layer, which consists of hydrophobically bound water molecules between the recognition and the diffuse layers [26] (Cprobe); the capacitance due to concentration dependent diffuse layer (CInt), extending from the outer device surface into the bulk of the solution [27]. The recognition event will give a change in the capacitance of the recognition layer Cprobe. The total capacitance CTot can then be described by the following equation:
This equation clearly states that the lowest capacitance will dominate the total capacitance. It is therefore important to design the device in order to have the capacitance in the insulating layer as high as possible, otherwise it may dominate and analyte changes might not be detectable. Nevertheless, the oxide cannot be too thick, otherwise the device sensitivity will strongly reduce. Analyte recognition will cause a shift in the normalized capacitance curve that can be numerically defined by monitoring the flat band voltage (VFB) shift. Capacitance changes can be detected either as a change in the dielectric constant or as a change of the thickness of a layer immobilized on the transducer [28].
Another important aspect for the EIS device efficiency is the surface coverage with the receptor molecules. Actually, if these probes do not cover completely the electrodes, the measurements may be affected by the “holes” presence.
There are many drawbacks which prevent the commercialization of EIS biosensors. As an example, in this kind of structure there is a dependence of the sensor response to the on buffer capacity, there is a restricted dynamic measurement range and a non-linearity in the response. Other drawbacks are: the relatively slow response and recovery times; the operating and storage stability; the reproducibility; the dependence of the sensor signal on the biological probe immobilization method [14], [15]. Finally, the so-called counter-ion screening effect as well as the non-ideality of the molecular layer reduces the possibility to actually fabricate these devices for a direct electrostatic detection of charged macromolecules.
This work was focused on the study of probe immobilization methods that can overcome the main constrains currently limiting a wide application of EIS biosensor. According to the above reported discussion, the ideal immobilized probe had to correctly bind different biological molecules, as needed to foresee sensor multiplexing; it had to preserve the inorganic device functionality, i.e. the compatibility with the standard microelectronic front-end processing. Moreover, to foresee full integration in microelectronic circuits, we focused our efforts on Si-based miniaturized biosensor based on the EIS transduction mechanism. More in details, a particular attention was devoted to investigate the (a) device surface functionalization, (b) biological molecule functionality after immobilization and (c) biosensor working principle. These three topics are described in the three sub-sections of Section 3. A final sub-section is devoted to the perspectives for this kind of sensor.
Section snippets
Biological species
The biological molecules used in this work are: the enzyme Glucose oxidase (GOx) from Aspergillus niger (type X-S, Aspergillus niger, 179,000 U g−1 solid, from Sigma), The proteine metallothyoneine (MT) from rabbit liver (from Sigma), bovine serum albumin (BSA) and short synthetic oligonucleotides (from Operon Biotechnologies GmbH).
GOx is 160 kDa homodimeric globular protein, with a tightly bound (Ka = 1 × 10−10) flavin adenine dinucleotide (FAD) per monomer. The overall dimer dimensions are 6.0 nm × 5.2
Results and discussion
In order to properly fabricate a biosensor three main issues must be addressed: appropriate device functionalization; testing of the biological molecule functionality; transduction effectiveness. These three points will now be separately addressed.
Conclusions
In this paper we review the main results obtained by our team in the fabrication of Si-based miniaturized biosensor based on the EIS transduction mechanisms. We investigated three main issues: (i) device surface functionalization, (ii) biological molecule functionality after immobilization and (iii) biosensor working principle.
We optimized and tested two different immobilization protocols on SiO2 surfaces. Contact angle, XPS and TEM measurements were used to study and monitor the immobilization
Acknowledgments
The authors acknowledge A. Spada and N. Parasole, C. Bongiorno of CNR – IMM and G. F. Indelli of Consorzio Catania Ricerche (SUPERLAB) for the expert technical assistance. Dott. S. Lombardo of CNR – IMM is also acknowledged for the helpful discussions.
This work has been partially founded by the national project PON “Hyppocrates – Sviluppo di Micro e Nano-Tecnologie e Sistemi Avanzati per la Salute dell’uomo” (PON02_00355).
Sebania Libertino got her bachelor in Physics and her Ph.D. at the University of Catania in Italy. Part of her Ph.D. was spent in Bell Labs Laboratories, Lucent Technology (Murray Hill – NJ). She works as Senior Researcher at the Microelectronic and Microsystems institute (IMM) of the Italian National Council of Research (CNR), in Catania (ex IMETEM). Her research interests are oriented to the design, fabrication and testing of Si-based devices for microelectronic, optoelectronic and sensor
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Sebania Libertino got her bachelor in Physics and her Ph.D. at the University of Catania in Italy. Part of her Ph.D. was spent in Bell Labs Laboratories, Lucent Technology (Murray Hill – NJ). She works as Senior Researcher at the Microelectronic and Microsystems institute (IMM) of the Italian National Council of Research (CNR), in Catania (ex IMETEM). Her research interests are oriented to the design, fabrication and testing of Si-based devices for microelectronic, optoelectronic and sensor applications. A further interest is the study of defects formation, evolution and effects on device performances. Dr. Libertino has co-authored about 100 papers published in international journals and conference proceedings. She holds 3 European patents, all extended to USA.
Sabrina Conoci received her bachelor in Chemistry at the University of Bologna. She worked at Eniricherche (Milan, Italy) as Manager of Research. She received the Ph.D. in Engineering of Materials spending 1 year working at the University of Ottawa (Canada). From 1999 she has been working in STMicroelectronics covering several R&D positions in the field of Nanomolecular Devices and Biotechnologies. Particularly, she accomplished the qualification of the first Biotechnology of STMicroelectronics and recently she has been selected into the international experts panel to edit the CLSI MM22 technical rule ‘Microarrays for Diagnosis and Monitoring of Infectious Diseases’ recognized from FDA as Consensus Standard for Molecular Diagnostic. She is co-author of 55 publications in international journals, six international patents.
Antonino Scandurra received the degree in chemistry from University of Catania (Italy) in 1988. He takes qualification to practise profession as Chemist in 1988. He worked for STMicroelectronics, National Research Council (CNR) as fellow, and he is currently senior research scientist at Consorzio Catania Ricerche. Dr. Scandurra is co-author of 2 patents, more than 60 papers in international journals, more than 50 international talks. He currently works mainly on aspects related to surfaces, interfaces and thin films of materials employed in semiconductors technologies as well as in power electronics packaging technology.
Corrado Spinella received the degree in physics from the University of Catania, Catania, Italy, in 1985. He joined the National Institute of Methodologies and Technologies for Microelectronics (IMETEM), Italian National Council of Research (CNR), Catania, in 1989, as a Researcher. He is currently a Senior Researcher and, since 2008, the Director of the Institute of Microelectronics and Microsystems, CNR. He is the coauthor of more than 200 scientific papers. His research activity is in the field of new material and processes for micro-e nanosystems based on silicon technology, such as (1) front-end processing in the ultralarge-scale Si technology, (2) Si-based optoelectronic, (3) science and technology of silicon carbide for RF or power applications, (4) silicon-based microfuel cells, (5) memory devices or light-emitting diodes based on silicon nanocrystals, and (6) advanced techniques for the synthesis and characterization of nanostructured materials.