Bio & Chemical Sensors

Commercial applications of tomorrow, and also many of them of today, require biosensor systems that respond so quickly and are so easy to use that analyses can be performed “in field” by an unskilled personnel. For many applications you have much to gain from having fast and simple read-out units that enable diagnostics to be performed at local healthcare centers or by the patients themselves. For people suffering from heart attacks, fast diagnostics is of utter importance. For cancer patients the number of follow up visits can be reduced if the samples are analysed while the patient is waiting, instead of beeing sent to a central laboratory. Less severe afflictions can be appropriate for self diagnosis.

There are three main technical challenges encountered when designing these systems: (i) the high degree of automation needed, (ii) sufficient detection sensitivity achieved in a short time (few minutes), and (iii) reliability and robustness of the performance. These three demands are the main reasons why construction of a biosensor system requires a truly multidisciplinary approach. The core expertise of the bio- and chemical sensor group is focused on optimizing the various interactions between the (bio)molecules and between the (bio)molecules and the substrate surfaces to optimize the system with respect to the sensitivity and reliability. The specific examples chosen below illustrate the breadth of our approach. The complexity of the sensing system and the variety of possible applications require use of advanced instrumentation when doing a design work. Therefore Imego has acquired a range of top-line commercially available instrumentations that are utilized during system development and optimization.

With our expertise complemented by the other expertise available at Imego we are able to develop commercially viable biosensing systems.
 

We have been studying means to optimize antibody binding to surfaces as well as the ability of different chemicals to protect the substrate from adsorption of unwanted biomolecules. The characterization of antibody binding to gold surfaces has been perfomed using both intact IgG antibodies and modified antibodies made in-house. The kinetics of antibody binding to gold has been followed using a QCM-D (Quartz Crystal Microbalance with Dissipation) equipment from Q-Sense available at our laboratory. We have also compared the efficiency of different molecules, for example bovine serum albumin, casein and polyethylene glycol, to block the surface against non-specific (unwanted) binding when the substrate surface is exposed to serum or plasma.

We have gained understanding of how the antibodies bind to surfaces and how to increase the specific binding and to decrease the nonspecific one. This knowledge is essential when designing sensor systems for medical diagnostics as well as for the detection of microorganisms and cells.
 

A commonly used technique to quantify concentrations of a certain biological agent, for example a disease marker (an antigen), is a fluorescent based sandwich assay. In the sandwich assay antibodies specific for the antigen are attached on a substrate. After that the sample is added and the antigen binds to the antibodies on the substrate. Then you allow other, fluorescently labeled, antibodies also specific for this antigen to bind to the coupled antigen. After washing away excess antibodies one measures fluorescence intensity which is directly related to the concentration of the marker in the original sample.

To enhance fluorescent signal one can also use fluorescent nanoparticles covered with antibodies. We have studied means to improve functionalizations of such particles by suitable antibodies. We have worked both with commercially available nanoparticles that have embedded molecular fluorescent probes (FluoSpheres) as well as with the so called Quantum Dots. The emission spectra of the latter particles are much narrower then the emission from the FluoSpheres. Another large advantage of the Quantum Dots is that particles with different emission spectra can be excited with the same laser. This makes Quantum Dots suitable for an assay where one targets to detect several antigens at one and the same time using different fluorescent markers for different antigens. Mastering the Quantum Dots functionalisation is a necessary prerequisit to construct sensor systems for simultaneous detection of several disease markers and to improve the diagnostic precision.
 

We develop functionalizations of magnetic nanoparticles with different biomolecules such as Protein G and L, Streptavidin and NiNTA, respectively. These functionalized nanoparticles can be used in assays where antibodies and proteins are quantified using a proprietary detection scheme.
A dedicated portable instrument has been built which enables fast analyses of the size changes of magnetic nanoparticles upon different functionalizations.  We have developed a software package for quantification of these changes. The technique allows to follow and quantify every step in an assay; for example it enables to quantify the amount of protein G and of the blocker attached to particle surface, and subsequent adsorption of  proteins and antibodies, respectively.
 

In our laboratory we have instruments to characterize different properties of magnetic nanoparticles of importance for their use as substrates for sensing chemical reaction. The magnetic properties of the nanoparticles are determined using instruments that quantify the static and dynamic magnetization.

The particle size distribution can be deducted from the magnetic properties as well as from light scattering measurements. The latter measurements can be performed on magnetic as well as on non-magnetic particle suspensions and are made using Malvern Instruments, Zeta Sizer Nanoseries, available at our laboratory.

Another important factor when working with nanoparticles is the stability of suspensions towards agglutination. The latter is strongly related to particle charges which may change during functionalisation(s). The Zeta Sizer enables us to measure the so called Z-potential of the particles after every functionalisation step. The Z-potential is directly related to particle charge.
 

Although great progress has been made concerning development of artificial replacements for body parts, many problems still remain. One of the problems is the rejection by the human immune system. A step towards the development of materials more bio-compatible than those used today, can be to tailor implant surfaces for attachment of particular cells by modifying the surfaces using cell-adhesion promoting peptides.
The goal of one of the Master´s thesises performed at Imego was to investigate if it was possible to tailor one of these cell-adhesion promoting peptides´ architecture on a gold surface. Each end of the pepide was anchored to the gold surface using thiol chemistry, making it form a bent structure on the surface. The bent structure of this particular peptide is especially prone to cell adhesion.

The experiments were performed using a Quartz Crystal Microbalance (QCM) instrument with dissipation monitoring. This is a surface sensitive technique that allows to follow quantitatively time evolution of chemical reactions. It is used in many of the bio-projects at Imego for evaluation of surface reaction.
 

Molecularly Imprinted Polymers (MIPs) are used to specifically bind and enrich medium and small sized molecules. The MIPs can be used to detect a wide variety of compounds, most molecules below about 5 kDa are suitable for imprinting.
A specific MIP is designed and prepared for each molecule of interest, and the MIP can be used as a selective sensing material integrated in a sensoric system. The MIPs can be designed to be used in a sensor system for the detection of for example toxins that can appear in drinking water, agricultural products and food.

The detection part of the sensor system is quite generic so similar detection techniques can be used to sense a variety of different compounds. Imego´s strategy is to evaluate and optimise several detection techniques using model imprint systems. After identifying the optimal detection technique, we will produce molecular imprints of target analytes of commercial interest or environmental importance and use them in our sensor system.
 

Imego has developed a sensor chip that can be used for monitoring surface reactions by measuring the conductivity and capacitance (complex impedance) as a function of frequency of thin film layers coated on the chip. Depending on the properties of the coating the chip can be used for a variety of applications: for example deposition of a pH sensitive polymer would enable to monitor the pH of a solution.
The sensor chip is at present mainly used to follow the uptake of target molecules by Molecularly Imprinted Polymer films deposited onto microelectrodes.

When the target molecule is absorbed by the Molecularly Imprinted Polymer layer, the impedance vs. frequency characteristics of the layer changes, which can be measured by the Imego chip.

We use a commercial Electrochemical Impedance Analyzer and the Potentiostat / Galvanostat from Solartron Instruments available at Imego for these measurements. These instruments allow not only to evaluate the properties of imprinted polymers but can also be used to electrochemically polymerize suitable Molecularly Imprinted Polymers. The latter is done using so called Cyclic Voltammetry. When performing cyclic voltammetry measurements, an alternating voltage is applied to a sample electrode, and the current resulting from surface reactions on the electrode is measured.