ProteinSimple eBook - 24

Simplifying Protein Analysis * Multi-Analyte, Automated, Microﬂuidic Immunoassay Platform

analyte ELISAs. Simple Plex assays run on Ella™ enabled simultaneous quantitation of four analytes, in discrete channels, from 16 individual samples in a
single disposable microﬂuidic cartridge in an hour. This eliminated potential
negative interactions or interference from the antibody pairs for other assays,
while simultaneously providing the beneﬁts of a multiplexed antigen analysis
and rapid microﬂuidic reaction kinetics. Glass nano reactors (GNRs) were
developed as a solid-phase support for the capture antibodies, and were
shown to possess enabling physical, optical, and chemical properties. GNRs
were functionalized to allow antibody immobilization on the GNRs internal,
but not external, surfaces (Figure 1). The composition of the GNR provided
a highly uniform surface, supporting multiple well-characterized, stable
immobilization chemistries, and exhibited low intrinsic ﬂuorescence.

A
A

~70 µm

B
B

C
C

D
D

~250 µm

CARTRIDGE CONSTRUCTION

Cartridges were assembled with three analyte-speciﬁc GNRs, composed of
glass capillary 250±25 µm (length) by150±6 µm (outer diameter) with an
inner diameter of 75 ±3 µm, per channel in every circuit (Figure 1).
DAILY REAGENTS

Calibration curves were made daily over the course of ﬁve days with freshly
thawed multi-analyte standard spiked and serially diluted into freshly
thawed calibrator buffer. Curves consisted of eight points, including zero,
spanning the biologically relevant range. The limit of detection (LOD)
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Figure 1. The Glass Nano Reactor (GNR). (A) Physical dimensions of a GNR.
(B) Photograph of a GNR embedded in the channel of the Simple Plex cartridge.
(C) Fluorescent image of a GNR revealing exclusive localization of antibodies in
the center of the GNR, with no detectable signal on the outer surfaces.
(D) Cartoon depicting placement of GNRs in discrete channels of one of the
eight circuits on an 8-sample 4-plex cartridge. Different colors indicate
GNRs specific for different analytes.

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