Rectangular cross-section 200 × 2000 μm2 trapping capillary and transducer set-up (a) used like a pipette in aspirate/dispense-mode, allowing trapping of a controlled amount of beads (b) above the millimeter sized ultrasonic transducer. The schematic side view (c) shows how the beads are retained in a thin layer by ultrasonic forces. These forces allow retention against fluid flow for buffer exchange and washing purposes.
(a) Photo of the acoustic trapping capillary over the ISET RP-SPE chip in its vacuum fixture (made in a 384 microtitre plate). (b) Schematic work-flow of immunocapture using the proposed acoustic trapping iMALDI method: (1) Antibody beads are trapped and sample solution is aspirated and incubated in the rectangular acoustic trapping capillary (blue). (2) The washed beads with bound antigen are released on top of an ISET nanovial packed with SPE beads and the antigen is transferred from the Ab-beads to the SPE. (3) Buffer salts are washed away and antigen displaced from the antibody beads and captured on the SPE beads. (4) The antigen is eluted from the SPE beads onto the analysis zone on the backside of the ISET using matrix. (5) The ISET is inserted up-side down in the MALDI MS instrument and analyzed.
Investigation of dispersion in the system: Peptide 2 at 1 pmol/μl was aspirated and incubated in the trapping capillary for 60 min. After collection of 20 μl fractions, the samples were mixed 1:1 with an isotope of the same peptide (peptide 1) and spotted on a MALDI target for analysis. Note that in the 3rd fraction (spectra) no more peptide 2 can be observed which implies that the levels of peptide 2 are at least 1000 times lower than that of peptide 1.
Efficient wash of detergents. The lower spectrum results from an acoustic trapping iMALDI analysis of a 15 μl sample containing 5 nM Ang I in 1:10 diluted human plasma. The top spectra show the results of a standard assay of the same sample amount using the magnetic beads according to manufacturer specifications. Note the intense background ladder from the tween 20, which hampers analysis at low concentrations.
Immunocapture from a complex sample. Top spectra (A) results after a direct RP-SPE sample preparation, i.e., no immunocapture (reference sample). Spectra (B) results after acoustic trapping iMALDI analysis of a 15 μl sample containing 1 nM Ang I in 200 fold excess of BSA digest (peptides). Note the lack of unspecific binding observed in spectra B.
Fluorescent image of the acoustic streaming phenomena visualized using 230 nm tracer particles. Dashed line shows trapping site and arrows the streaming vortexes.
Ang I (1296 Da) spiked in 1:10 human plasma together with a standard isotope of Ang I (1309 Da) at 8:10 ratio and falling concentration from 400 nM to 400 pM. It can be noted that the intensity increase levels out when processing concentrations higher than 4 nM. Also at the lowest analyzed concentration no isotope was added.
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