We report a method for
combining the dete
ction of single mole
cules (digital) and an ensemble of mole
cules (analog) that is
capable of dete
cting enzyme label from 10
鈭?9 M to 10
鈭?3 M, for use in high sensitivity enzyme-linked immunosorbent assays (ELISA). The approa
ch works by
capturing proteins on mi
cros
copi
c beads, labeling the proteins with enzymes using a
conventional multistep immunosandwi
ch approa
ch, isolating the beads in an array of 50-femtoliter wells (
class="uu">Single
class="uu">Mole
cule
class="uu">Array, SiMoA), and dete
cting bead-asso
ciated enzymati
c a
ctivity using fluores
cen
ce imaging. At low
con
centrations of proteins, when the ratio of enzyme labels to beads is less than 1.2, beads
carry either zero or low numbers of enzymes, and protein
con
centration is quantified by
counting the presen
ce of 鈥渙n鈥?or 鈥渙ff鈥?beads (digital regime).
click="showRef(event, 'ref1'); return false;" href="JavaScript:void(0);" class="ref">(1) At higher protein concentrations, each bead typically carries multiple enzyme labels, and the average number of enzyme labels present on each bead is quantified from a measure of the average fluorescence intensity (analog regime). Both the digital and analog concentration ranges are quantified by a common unit, namely, average number of enzyme labels per bead (AEB). By combining digital and analog detection of singulated beads, a linear dynamic range of over 6 orders of magnitude to enzyme label was achieved. Using this approach, an immunoassay for prostate specific antigen (PSA) was developed. The combined digital and analog PSA assay provided linear response over approximately four logs of concentration ([PSA] from 8 fg/mL to 100 pg/mL or 250 aM to 3.3 pM). This approach extends the dynamic range of ELISA from picomolar levels down to subfemtomolar levels in a single measurement.