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.
Optimization of antibody binding to gold surfaces
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.
Fluorescent markers
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.
Functionalisations of magnetic nanoparticles
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.
Characterization of nanoparticles
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.
Peptides on surfaces
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
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.
Electrochemistry
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.