PARTICLE BASED IMMUNOASSAY WITH ALTERNATING CURRENT ELECTROKINETICS
Disclosed are methods, devices and systems of an immunoassay using an alternate current electrokinetic platform. Also disclosed are methods of separating and detecting analytes from a sample using the disclosed methods.
1 . An immunoassay device for detecting an analyte in a sample, the device comprising:
(a) a microelectrode array, the array capable of establishing an AC electrokinetic field region and isolating a bead complex in a high conductivity buffer, wherein the bead complex comprises a functionalized bead bound to a labelled-primary antibody;
(b) a fluidic cartridge, the fluidic cartridge capable of housing the microelectrode array and further comprising at least one port for addition and removal of buffers and reagents; and
(c) a fluorescent or luminescent detection system, whereby the presence, absence and/or amount of said analyte in the sample is determined by assessing fluorescence or luminescence from the isolated bead complex bound to a secondary antibody labelled with a fluorescent or luminescent probe.
2 . The device of claim 1 , wherein the microelectrode array comprises an array of alternating current (AC) electrodes.
3 . The device of claim 1 , wherein the microelectrode array comprises an array of direct current (DC) electrodes.
4 . The device of claim 1 , wherein the microelectrode array is a planar electrode array.
5 . The device of claim 1 , wherein the AC electrokinetic field is produced using an alternating current having a voltage of 1 volt to 40 volts peak-peak, and/or a frequency of 5 Hz to 5,000,000 Hz and duty cycles from 5% to 50%.
6 . The device of claim 1 , wherein the microelectrode array further comprises a passivation layer with a relative electrical permittivity from about 2.0 to about 4.0.
7 . The device of claim 1 , wherein the conductivity of the fluid is greater than 100 mS/m.
8 . The device of claim 1 , wherein the microelectrode array is spin-coated with a hydrogel having a thickness between about 0.1 microns to about 1 micron.
9 . The device of claim 1 , wherein the bead is a hydrophilic bead.
10 . The device of claim 1 , wherein the bead is a polystyrene, poly(methacrylate) or polyacrylate bead.
11 . The device of claim 1 , wherein the bead is functionalized with streptavidin and the primary antibody is labelled with biotin.
12 . The device of claim 1 , wherein the bead is functionalized with biotin and the primary antibody is labelled with streptavidin.
13 . The device of claim 1 , wherein the sample is a bodily fluid, blood, serum, plasma, urine, saliva, a food, a beverage, a growth medium, an environmental sample, a liquid, water, clonal cells, or a combination thereof.
14 . The device of claim 1 , wherein the fluorescent tag is green fluorescent protein (GFP), cyan fluorescent protein, or yellow fluorescent protein.
15 . The device of claim 1 , wherein the luminescent tag is luciferin.
16 . The device of claim 1 , wherein the analyte is chosen from the group consisting of cellular material, particulate material, cellular particles, exosomes, nucleosomes, liposomes, chromosomes, a protein aggregate, a protein, a peptide, a nucleic acid, fragments thereof and combinations thereof.
17 . A method of detecting a target analyte in a sample, comprising,
a. functionalizing a bead in a buffer;
b. contacting the functionalized bead with a primary antibody-labelled conjugate;
c. introducing the functionalized bead-antibody-labelled conjugate into a device comprising a sample;
d. introducing a secondary antibody labeled with a fluorescent tag into the device;
e. applying an alternating current (AC) electrokinetic field; and
f. detecting bound analyte.
18 . The method of claim 17 , wherein the bead is a hydrophilic bead.
19 . The method of claim 17 , wherein the bead is a polystyrene, poly(methacrylate) or polyacrylate bead.
20 . The method of claim 17 , wherein the bead is functionalized with streptavidin and the primary antibody is labelled with biotin.
21 . The method of claim 17 , wherein the bead is functionalized with biotin and the primary antibody is labelled with streptavidin.
22 . The method of claim 17 , wherein the sample is a bodily fluid, blood, serum, plasma, urine, saliva, a food, a beverage, a growth medium, an environmental sample, a liquid, water, clonal cells, or a combination thereof.
23 . The method of claim 17 , wherein the fluorescent tag is green fluorescent protein (GFP), cyan fluorescent protein, or yellow fluorescent protein.
24 . The method of claim 17 , wherein the device is a device of claim 1 .
25 . The method of claim 17 , wherein applying the AC electrokinetic field comprises dielectrophoresis.
26 . The method of claim 17 , wherein applying the AC electrokinetic field creates areas of low and high dielectrophoresis.
27 . The method of claim 26 , wherein applying the AC electrokinetic field separates analytes by size.
28 . The method of claim 17 , wherein the analyte is chosen from the group consisting of cellular material, particulate material, cellular particles, exosomes, nucleosomes, liposomes, chromosomes, a protein aggregate, a protein, a peptide, a nucleic acid, fragments thereof and combinations thereof.
29 . The method of claim 17 , wherein the analyte is an exosome.
30 . The method of claim 17 , wherein the analyte is a nucleosome.
31 . The method of claim 17 , wherein the analyte is a liposome.
32 . The method of claim 17 , wherein the analyte is a protein.
33 . The method of claim 17 , wherein AC electrokinetic field separates the bound and unbound beads according to charge and size across a platform using dielectrophoresis.