The PSA project was initiated to develop point of care diagnostics for
the detection of prostate cancer. However, this project has developed to
include the diagnosis of other types of the disease. In every case, early
detection and treatment is vital and the driving aim behind PSA was to
give cancer specialists a tool to meet these requirements. Prostate
cancer detection depends on determining the amount of PSA in the blood.
The same principle can be used for other types of cancer where the
specific antigen for the particular cancer type is detected.
Many other types of disease can be diagnosed in this way – in principle,
all diseases that produce antigens or specific proteins in the body.
Examples of the principle’s areas of application include the heart,
brain, allergies, rheumatism and infection. Working together, Imego
Institute and CanAg Diagnostics AB are developing a new technology
platform for point-of-care testing. We use PSA (prostate specific
antigen) as a model analyte, but the platform can be modified for
detection of other serum carried disease markers, i.e. other cancer or
allergy markers and also for multiple analyte detection. The concept is
based on an analysis instrument for quantitative detection and disposable
chips containing all bioreagents.
The principle of a sandwich assay
The detection principle of the blood carried prostate cancer marker
(prostate specific antigen, PSA) is a sandwich assay technique as shown
in figure 2. The patient’s serum sample is added to a credit card sized
chip in which the assay takes place. The function of the chip is
de¬scribed in more detail below. The assay chip defines a cavity where
the reaction takes place. The cavity is coated with an antibody (antibody
1) specific for PSA. If the patient’s serum sample contains PSA it will
bind to the antibodies on the walls of the cavity. Also present in the
cavity are fluorescent beads coated with another antibody (antibody 2)
specific for PSA. These will bind to PSA, which in turn, is bound to
antibody 1, forming a surface-anchored complex. The excess of reagent is
rinsed away, the cavity is illuminated with laser light and the
fluorescence intensity of the PSA surface complex is determined. The
light intensity is proportional to the number of bound fluorescent beads
and the concentration of PSA in the tested sample.
In our measurements we have used sub-micrometer sized fluorescent beads
called FluoSpheres. The FluoSpheres are coated with antibody 2 and used
as fluorescent probes in the PSA assay. The result of one measurement is
shown in figure 3. The FluoSpheres give a detection limit well below 0.1
ng/ml PSA, which shows that the system is sensitive enough for measuring
clinically relevant PSA levels. As seen in the figure, the plot of
fluorescent signal as a function of PSA concentration does not show a
linear dependence at high PSA concentration. This is due to the
relatively large size of the beads. Adding 60 ng/mL PSA to the cavity
makes the distances between PSA molecules attached to the surface small
compared to the size of the beads. A micrometer sized FluoSphere bead
that binds to a surface exposed to 60 ng/mL PSA covers several bound PSA
molecules and makes them invisible to other beads. This explains the
non-linear behaviour. Higher concentrations of PSA do not make more beads
bind to the surface when the saturation level is reached; it only makes
one bead cover more PSA molecules.
The work of optimizing the assay has involved coupling antibody 1 to the
chip cavity, coupling antibody 2 to the fluorescent beads, minimizing
non-specific binding of PSA and fluorescent beads, optimizing the size
and concentration of beads used in the assay, incubation times, etc. The
sensitivity of below 0.1 ng/ml and the dynamic range of the measurement
are sufficient at present. We have therefore focused on refinement of the
results towards better chip-to-chip stability of the assay. At Imego, we
also plan to expand an assay for the detection of multiple analytes, for
example to quantify the total and free PSA.
The chip
Our concept for point-of-care measurement divides the sensing system into
two parts: a measurement instrument and disposable chips. The instrument
performs automated control of the fluid transportation during the assay
procedure and performs the fluorescence measurements on the chip. A
crucial element of the point-of-care concept is that the instrument and
the chip must be easy for the user to handle. At the same time the
production costs must be low enough to make its application profitable.
The design and function of the disposable chips are key factors in the
point-of-care concept.