Cancer Diagnostics

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 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.
 
 

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.