Dielectrophoretic label-free immunoassay for rare-analyte quantification in biological samples

The current gold standard for detecting or quantifying target analytes from blood samples is the ELISA (enzyme-linked immunosorbent assay). The detection limit of ELISA is about 250 pg/ml. However, to quantify analytes that are related to various stages of tumors including early detection requires detecting well below the current limit of the ELISA test. For example, Interleukin 6 (IL-6) levels of early oral cancer patients are <100 pg/ml and the prostate specific antigen level of the early stage of prostate cancer is about 1 ng/ml. Further, it has been reported that there are significantly less than $1\mathrm{pg}/\mathrm{mL}$ of analytes in the early stage of tumors. Therefore, depending on the tumor type and the stage of the tumors, it is required to quantify various levels of analytes ranging from ng/ml to pg/ml. To accommodate these critical needs in the current diagnosis, there is a need for a technique that has a large dynamic range with an ability to detect extremely low levels of target analytes ($\sim \mathrm{zmoles}$).

Figure 1

Pictures of the PIDE electrodes utilized in crossover frequency measurement experiments. (a) A picture of a clean PIDE structure with connecting pads (A and B) to connect the electrodes to external function generator. (b) Section of PIDE structures showing how individual bead electrodes are placed, a gap between the electrodes and a connection between individual bead electrodes. Scale bar indicates 500 μm. (c) Closeup of the single-bead electrode showing a hollow interior with uneven outer boundaries to generate large electric field gradients. Scale bar indicates 250 μm.

Figure 2

comsol simulations results. (a) Variation of the electric field at 120 kHz over PIDE electrodes. These electric fields are sufficient to polarize the polystyrene beads and generate DEP forces. (b) Calculated electric field gradients ($\mathbf{\nabla }{E}^2$) at 120 kHz over the PIDE electrodes. Both electric field and electric field gradients are necessary to set up DEP forces on the beads. (c) Closeup view of the electric field gradient ($\mathbf{\nabla }{E}^2$) showing high and low electric field gradient regions in PIDE structures. When polystyrene beads are experiencing attaching or positive DEP, they are attracted to the high field gradient regions. Polystyrene beads move to the lowest field gradient regions when they experience negative DEP. (d) Variation of the average electric field and electric field gradients ($\mathbf{\nabla }{E}^2$) with frequency. Scale bars indicate 500 μm in (a,b), 250 μm in (c).

Figure 3

Experimental scheme used to find the crossover frequency of a sample of polystyrene beads. The moving directions of polystyrene beads under various frequencies are indicated by arrows. (a) Experimental observation of positive DEP. During the positive DEP, polystyrene beads are moving to the highest electric field gradient ($\mathbf{\nabla }{E}^2$) regions. (b) Observation of negative DEP. Note that the polystyrene beads are moving to the lowest electric field gradient ($\mathbf{\nabla }{E}^2$) regions (away from the electrodes). (c) Polystyrene beads are transitioning from positive DEP to crossover DEP. Note that at crossover frequency, polystyrene beads are gradually scattering over the PIDE electrodes. Scale bars indicate 100 µm.

Figure 4

Experimental and theoretical results. (a) Variation of the fluorescence intensity of polystyrene beads with a number of fluorescently labeled avidin molecules on their surfaces. (b) Experimentally measured crossover frequency of the polystyrene beads with avidin molecules on the surfaces. (c) Calculated relative dielectric constant of polystyrene beads with varying number of avidin molecules. This calculation was performed using the experimental data in (b).