Third-Generation Biosensor using Semiconducting Polymers
1 – Applicant(s)
Francesco Maddalena (under the supervision of Bert de Boer).
Molecular Electronics, Physics of Organic Semiconductors, MSCplus
The project is part of a multidisciplinary project of the MSCplus: Biosensor/Bio-FET – project, which will comprise three different groups within the University of Groningen/MSCplus:
Physics of Organic Semiconductors (Prof. Dr. Ir. P. W. M. Blom, Dr. Bert de Boer, Francesco Maddalena)
Molecular Electronics (Prof. Dr. Kees Hummelen, Drs. Frank Brouwer)
Enzymology (Prof. Dr. Bert Poolman, Dr. Marion Kuipers)
The leader of the project is Prof. Dr. J. C. Hummelen
2 – Institute
Postal and visiting address:
MaterialScience Centerplus (MSCplus)
University of Groningen
Nijenborgh 4
NL-9747 AG
Groningen
The Netherlands
Telephone: +31 50 363 4843
Fax: +31 50 363 7732
Scientific Director:
Prof. Dr. J. Knoester
Electronic mail:
General Information:
3 – Abstract
Biosensors are here defined as sensors with an integrated biological component. The aim of this project is to combine polymer semiconductors and bio-molecules (enzymes, receptors, DNA, etc.) in thin film metal-oxide-semiconductor (MOS) field-effect transistors (FET) to obtain a biosensor. These novel biosensors will be solid-state sensors without the need for liquid electrochemical cells or reference electrodes. Initially the project focuses on the first- and second-generation biosensors and based on the knowledge obtain from these biosensors, we adjust the design and focus on the ultimate goal to create a third-generation biosensor, where there is direct charge or energy transfer between the biological component and the organic semiconductor. The project will focus both on fundamental studies of organic semiconductors and the biological components and on studies of devices with a real application in laboratory, industry and medicine.
4 - Duration of the project
The project will start in September 2005. The duration of the project will be 4 years (until 2009) for the designated PhD students, but the project itself might be prolonged after this term, with the hiring of other graduates to continue the work.
5 – Personnel
Molecular Electronics, Physics of Organic Semiconductors (P.O.S.), MSCplus
Prof. Dr. Ir. Paul W.M. Blom (Leader of the P.O.S. group)
Dr. Bert de Boer (Senior scientist)
Drs. Francesco Maddalena (PhD student)
Jan Harkema (Technician)
Molecular Electronics (M.E.)
Prof. Dr. Kees Hummelen (Leader of the M.E. group)
Drs. Frank Brouwer (PhD student)
Enzymology
Prof. Dr. Bert Poolman (Leader of the Enzymology group)
Dr. Marion Kuipers (Senior scientist)
The leader of the project is Prof. Dr. J. C. Hummelen
6 - Cost estimates
6.1 - Personnel positions
The personnel will be the same specified in paragraph 5 above, and funding is requested for:
2 PhD students
1 Post-doc
Each group will also have the possibility to eventually have undergraduate students aiding the PhD students or the post-doc during the project.
6.2 - Running budget
Chemicals and eventual small additions to the equipment that is already present (see paragraph 6.3) will be purchased using this budget.
6.3 - Equipment
The equipment needed for this project is already available within the participating groups. Tools needed are STM/AFM, SNOM, photolithography, (bio) chemistry labs, probe stations, clean room, metal and organic evaporators, UV-Vis spectrometer, fluorescence spectrometer, and electrophoresis equipment. Hence, there is no need for an investment budget.
6.4 - Other support
6.5 - Budget summary
2005 / 2006 / 2007 / 2008 / TOTAL(after 4 years)
personnel (posi-tions): / 3 / 3 / 2 / 2
PhD students / 2 / 2 / 2 / 2
Postdocs / 1 / 1 / - / -
technicians
quests
personnel (costs) / € 145,500 / € 145,500 / € 90,000 / € 90,000 / € 471,000
running budget / € 15,000 / € 15,000 / € 15,000 / € 15,000 / € 60,000
equipment FOM / - / - / - / - / -
TOTAL
(requested from FOM) / € 160,500 / € 160,500 / € 105,000 / € 105,000 / € 531,000
7 – Research programme
7.1 - Introduction
A biosensor is an analytical device which converts a biological response into an electrical signal. The term “biosensor” is often used broadly in literature, intended as a device which can determine the concentration of substances or other parameters in biological systems or of biological interest. We will not use such broad definition for biosensors. Here we define a ‘biosensor’ as a device with an integrated biological component such as an enzyme, a bio-receptor or DNA that measures an interaction of a (bio)molecule with a specific receptor. A schematic view is givenin figure 1.
Fig. 1: Schematic diagram of a biosensors where the bioreceptor is integrated into the transducer. In the case of the Bio-FET, the transistor acts also as amplifier.
Since the first biosensor was invented1, many fundamental and application studies have been carried out. In the last few years biosensor development and use has spread in many scientific fields. Figure 2, below, gives a representation of the principles of the biosensors.
Fig. 2:The principles of the biosensor (from H. Nakamura, I. Karube, Anal. Bioanal. Chem., 377, 446–468 (2003) ).
Biosensor have an great variety of possible applications, from medical applications, such as quantitative analysis of chemicals in human blood, monitoring, in real time the glucose concentration in diabetes patients, to industrial applications such as in the food industry or even in the chemical industry, as sensors monitoring concentrations in chemical and bio-chemical reactors. The many possibilities offered by biosensors have greatly stimulated the research to develop new and improved biosensors. A handbook2 and several reviews3, 4, 5 have summarized biosensor development. Recent significant patents of biosensor technology published in the world are also listed in the literature6.
The research and development of biosensors has focused primarily on redox enzymes, which reduce or oxidize a substrate. The reason is that one can take advantage of the flux of electrons created in the redox reaction or keep track of the reducing or oxidizing agents. Non-redox enzymes can also be used in this setup if one of the analyte reaction products can subsequently undergo a redox reaction with an electrode-immobilized redox enzyme.Biosensors are divided in three generations. The first generation biosensor, proposed by Clark and Lyons and implemented by Updike and Hicks7, 8, use a biological component in solution or immobilized behind a dialysis or ion-selective (IS) membrane at the surface of an electrode and measure directly through electrodes (or sometimes though other methods) the production or disappearance of certain species. The enzyme, usually a redox enzyme, oxidizes the analyte, by reduction of oxygen into water peroxide. The electrode will then monitor the change in certain ionic species in water (for example the production of H2O2). First generation biosensors can vary the type of transducer they use. In Table 1 the type of electrochemical transducers are classified according to type of measurement depending on the species to be measured by the electrodes, which are not necessarily the analytes themselves but products of the reaction catalyzed by the enzymes. Non-electrochemical transducers such as piezoelectric, calorimetric and optical transducers are not listed in the table.
Table 1: Type of electrochemical transducers for classified type of measurements (from Thèvenot et al.; Biosensors & Bioelectronics 16 (2001) pp. 121-131)
A problem with this sensor arrangement is the loss in selectivity between the biorecognition event and the detection. Since the detection of the analyte is usually not direct, but goes trough the detection of other species this creates substantial interference since the electrodes (or other detection methods) are not always very selective or sensitive.
Second generation biosensors use an artificial electron mediator, which replaces O2 as the electron shuttle9. Ferrocene, quinones, quinoidlike dyes, organic conducting salts, and viologens have been used as mediators. This enhances the performance of the biosensors resolving the problem of the low solubility of O2 in water and the difficulty associated with controlling the O2 partial pressure. When a highly soluble artificial mediator is used, the enzyme turnover rate is not limited by the co-substrate concentration. These mediators also allow the exploitation of other oxidoreductase enzymes, including peroxidases and dehydrogenases, which cannot use molecular oxygen as a substrate.
First and second generation also make use of semiconductors in devices similar to MOS-FETs. There biosensors are liquid electrochemical cells, where the solution, In contact with the dielectic layer, acts as the gating potential instead of having a gate electrode. By covering the backside of the gate oxide with an ion-selective (IS) membrane, an ISFET is created10. Change in the gating potential of the solution is then measured, by keeping the drain-source current constants. In order to do this, the ISFET requires a SCE reference electrode in the solution (Figure 2). This design can be slightly improved by making it more selective by replacing the membrane with an active layer such as Persian Blue.
Finally we have third generation biosensors, where the reaction itself causes the response and no product or mediator diffusion is directly involved. Several examples of third generations biosensors have been constructed such ad a superoxide bismutase11 biosensor or glucose biosensors and other. These biosensors still use a liquid electrochemical cell with a reference electrode. In the case of a FET biosensors, usually inorganic semiconductors are used, although polyaniline12 is also often used in this type of devices.
Fig. 2: Schematic view of current biosensors with a liquid electrochemical cell separated by an oxide layer (gray layer above the electrodes and the semiconductor).
7.2 – Third generation biosensors using Polymer FETs
The aim of this project is to create a solid-state first-, second- and ultimately third-generation biosensor based on thin film (TF) MOS-FET technology (Bio-FET), where the semiconductor is a semiconducting polymer.The device proposed here will be the first solid-state biosensor on which one can place a drop of analyte solution and a reference electrode is not needed. The structure of this device will be based on FET-geometry, where the bio-receptor will be immobilized on top of the semiconducting layer ad seen in Figure 3.
Fig. 3: Biosensor based on TF-MOS-FET technology.
This design will directly improve biosensor technology. The Bio-FET will be a solid state device without a liquid electrochemical cell and no reference electrode will be needed.
The transducer of a (bio)sensor has to be able respond selectively and reversibly to biological compounds and yield signals that depend on the concentration of the analyte. In order to improve the signal-to-noise ratio, often a third and amplifying component is incorporated in the sensor. In a FET, such signal amplification is obtained by applying a voltage on the gate electrode. The gate, acting as a capacitor, will increase the charge carrier density in the semiconductor creating a ‘channel’ of relatively high density charge carriers.
To understand how a MIS-FET operates it is important to keep focus on what happens at the semiconductor-insulator interface when different biases are applied to the metal, respect to a ground. We will discuss the case for a p-type semiconductor since most conjugated polymers are of this type. There are three possible cases. The first case is when the bias on the gate electrode (VGS) is equal to the flat-band bias (VFB). Then we will have a flat-band condition, where, as the name says it, there is no band bending and the Fermi levels of the semiconductor and the gate electrodes are aligned. The only charge carriers present in the semiconductor are the carriers that occur by ‘natural’ means such as thermal excitation or doping (Figure 4a.).
Fig. 4:Energy band diagram of an ideal Metal-Insulator-Semiconductor device with p-type semiconductor, similar to the gate-oxide-semiconductor structure in a MIS-FET. The picture depicts three situations: (a.) The Flat-band condition where there is no applied bias, hence no band-bending. (b.) The Accumulation condition where a negative bias is applied on the metal causing the bands of the semiconductor to bend ‘upwards’ which causes accumulation of holes at the semiconductor-insulatorinterface. (c.) Depletion condition where a positive bias is applied to the metal which causes a depletion of holes in the semiconductor.
The second case is when we apply a negative bias on the gate electrode (or more negative of VFB), then its Fermi energy level will be raised. In consequence the bands of the semiconductor will bend upwards in energy causing and accumulation of positive charges in the valence band at the interface to compensate for the negative charges on the gate (keeping in mind that the device is similar to a capacitor). This is the accumulation condition or regime (see Figure4b.). In this regime the accumulation of charges present at the interface with insulator (the S/I-interface) forms a channel between the source and drain electrode, allowing (hole) current to flow between them. This is called the on-current.
The last case is when we apply a bias (or more positive of VFB, Figure 4c.) to the gate electrode. The Fermi level of the electrode will then be lowered, the consequence is the reverse of the previous case: the bands of the semiconductor will be bent downwards causing a depletion of positive charges in the valence band and a slight accumulation of negative charges in the conduction band. This is called the depletion condition or regime. In this regime there will be a very low charge density, hence the current flow, called the off-current, between the source and the drain will be very low, usually several orders of magnitude lower than the on-current.
The majority of the charge carriers are located close to the semiconductor/dielectric interface, in the first 1-3 nm, where the charge carriers also have the highest mobility13 as it can be seen from Figure 5.
Fig. 5: Numerically calculated distribution of charge carriers in the accumulation channel perpendicular to the S/I interface for Vg =−19 V and Vg = −10 V. Picture taken from the PhD Thesis“Charge transport in disordered organic field-effect transistors” by E. Meijer(ISBN 90-6734-306-4)
The binding of eventual charged analytes to the bio-receptor (or formation of charged species by chemical reaction) will alter the charge carrier distribution due to electrostatic force similar to a gate electrode, and consequently, change the charge density distribution at the interface and altering the mobility of the charge carriers. This can be seen in Figure 6. In Figure 6a. the charge density for a p-type semiconductor at the S/I-interface of a FET in accumulation mode is depicted. If a negative charge is formed the interface between the analyte solution and the Bio-FET (by for example binding of a charges species by the bio-receptor or by formation through chemical reaction) it will influence the charges at the S/I-interface drawing them away from the interface, hence spreading the channel width and reducing the charge density, since no extra charges are created. The mobility will then also be reduced since it is density-dependent (Figure 6b.).
Fig. 6:Schematic pictorial representation of charge density and conduction channel-width dependence in a p-type semiconductor FET in accumulation mode due to the effect of additional charges at the interface with the analyte solution.
The opposite will occur if a positive charge at the solution/Bio-FET interface is present. In that case the charges in the conduction channel will be repelled towards the S/I-interface, making the channel width smaller (see Figure 6c.), hence increasing the charge carrier density and, consequently, the mobility.
This will be the operation of a first generation biosensor.The ultimate goal is to achieve direct charge or energy transfer between the bio-receptor and the polymer layer, creating a third generation biosensor14. The receptor will act as a donor while the polymer layer as an acceptor, or vice versa, directly influencing the charge carrier conduction channel. When an analyte binds to the receptor then charge (or energy) transfer will occur between the donor and acceptor, creating a shift in the current between the source and the drain. This charge transfer or the energy transfer might also be aided by excitation of the donor with light.
7.3 – Objectives
Collaborating closely with a PhD student in organic/polymer chemistry, which will supply the needed polymer with stable properties in vacuum, air and water and perhaps have some special features (such as donor or acceptor groups for charge transfer) the first objective will be to make a solid state thin film FET by spin-coating the polymer from solution. The I/V-characteristics of the polymer in the FET will be analyzed in vacuum, in air and in presence of pure water.The second objective, after established the stability of the polymer, is to investigate the effect of different ions in water and of different pH values on the operation of the polymer FET, such as shifts in switch-on and threshold voltages.
After the completion of the second objective, the third objective will be to immobilize a protein, either an enzyme or a receptor, which will be obtained from a chemistry Post-Doc, to the surface of the polymer thin film in the FET. The objective is to immobilize the protein by chemically bind it to the polymer surface without activity loss of the protein. Part of this objective will be the characterization and quantifications of the proteins residing on the polymer film and the characterization of the interaction between the protein and the analyte. After characterizing the bio-receptor itself and how it binds to the polymer film the main task will be preparing Bio-FETs, FETs with a semiconducting polymer as active layer with a protein (enzyme or receptor) fixed onto its surface by chemical bonding. First the base of the transistors, electrodes and insulating oxide, will be made by photolithography and evaporation techniques. Afterwards the polymer will be spincoated on the FET substrate, searching for the right parameters to create of very thin films (10-20 nm). The achievement of very thin films is crucial since it is desired that the biological part and the analyte present on the film is as close to the conduction channel of the FET as possible. The final step in the Bio-FET processing will be to attach the protein to the polymer surface by chemical bonding.
After the Bio-FET is made, current-voltage characteristics will be measured in presence of different analytes. In order to determine the specificity and performance of the device and for a specific analyte (or a range of analytes), control and reference experiments have to be performed. This type of characterization is crucial for latter technological applications of the Bio-FET as Biosensor. Also the theoretical models already available for semiconducting polymers for charge carrier density changes, density of states and I/V-characteristics must be extended and applied to Bio-FETs, by incorporating in the theory the presence of the bio-molecules, the watery environment, and the presence of the analytes.