2 research outputs found
Surface Enhanced Laser Desorption Ionization of Phospholipids on Gold Nanoparticles for Mass Spectrometric Immunoassay
High-throughput
and sensitive detection of proteins are essential
for clinical diagnostics and biomarker discovery. We develop a novel
high-throughput, multiplexed, sensitive mass spectrometric (MS) immunoassay
method, which utilizes antibody-modified phospholipid bilayer coated
gold nanoparticles (PBL-AuNPs) as the detection label and antibody-immobilized
magnetic beads as the capture reagent. This method enables magnetic
enrichment of the PBL-AuNPs label specific to target protein, allowing
sensitive surface enhanced laser desorption ionization (SELDI)-TOF
MS detection of the protein via its specific label. AuNPs act as not
only the support but also the matrix for the phospholipids in SELDI
TOF MS detection. Moreover, with phospholipids with varying molecular
weights as the encoded MS reporters, this method allows multiplexed
detection of multiple proteins. With the use of a predefined phospholipids
internal standard, this method also affords excellent reproducibility
in protein quantification. We have demonstrated this method using
the assays of two tumor biomarkers, and the results reveal that it
provides a sensitive platform for multiplexed protein detection with
detection limits in the picomolar ranges. This method may provide
a useful platform for high-throughput and sensitive detection of protein
biomarkers for clinical diagnostics
Branched Hybridization Chain Reaction Circuit for Ultrasensitive Localizable Imaging of mRNA in Living Cells
Hybridization chain reaction (HCR)
circuits are valuable approaches
to monitor low-abundance mRNA, and current HCR is still subjected
to issues such as limited amplification efficiency, compromised localization
resolution, or complicated designs. We report a novel branched HCR
(bHCR) circuit for efficient signal-amplified imaging of mRNA in living
cells. The bHCR can be realized using a simplified design by hierarchically
coupling two HCR circuits with two split initiator fragments of the
secondary HCR circuit incorporated in the probes for the primary HCR
circuit. The bHCR circuit enables one to generate a hyperbranched
assembly seeded from a single target initiator, affording the potential
for localizing single target molecules in live cells. In vitro assays
show that bHCR offers very high amplification efficiency and specificity
in single mismatch discrimination with a detection limit of 500 fM.
Live cell studies reveal that bHCR displays intense fluorescence spots
indicating mRNA localization in living cells with improved contrast.
The bHCR method can provide a useful platform for low-abundance biomarker
detection and imaging for cell biology and diagnostics