Direct and multiplex quantification of protein biomarkers in serum samples using an immuno-magnetic platform† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5sc04115e

A direct and ultrasensitive multiplex assay using an immuno-magnetic platform has been developed for the quantification of trace amounts of circulating cancer-associated antigens in serum.


Synthesis and NMR characterization of cyanine fluorophores
Scheme S1. Synthesis of cyanine fluorophores.
General Procedure All the solvents were dried by the standard methods whenever needed. 1 H NMR spectra were recorded using a Bruker-400 NMR spectrometer and referenced to the residue CDCl 3 7.26 ppm or DMSO-d 6 2.5 ppm. 13 C NMR spectra were recorded using a Bruker-400 NMR spectrometer and reference to the CDCl 3 77 ppm or DMSO-d 6 39.5 ppm. Mass Spectroscopy (MS) measurements were carried out by using either fast atom bombardment on the API ASTER Pulser I Hybrid Mass Spectrometer or matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) technique.

Synthesis of the silica coated iron oxide nanoparticles
Aqueous dispersions of magnetic iron oxide nanoparticles were prepared according to

Preparation of the capture antibody conjugated nanoparticles
Amino-functionalized particles were prepared by using APTES and silylation agent.
The functionalization was prepared according to previous reported method. 4 Generally, the APTES was added to the ethanolic solution that contained the silica

Selectivity of the assay
To study the selectivity of the nanoparticles/Ab 1 nanocomposite, four protein samples:

Enzyme-linked immunosorbent assay (ELISA)
Prostate specific antigen (PSA) ELISA kit was purchased Sigma-Aldrich (USA). The detection of PSA in serum sample was performed as the manufacture's instruction.
Briefly, 25 μL of PSA standards and serum sample were added into the streptavidincoated micro-wells and followed by 100 μL anti-PSA conjugate reagent. Solution mixtures were then incubated at room temperature for 1 hr. The liquid was removed from each well and washed with 300 μL of 1 X wash buffer. Then, 100 μL TMB substrate was added to the wells and incubated at room temperature for 15 min.
Finally, 50 μL of stop solution was added into all wells and shaken gently for 15 min.
The absorbance at 450 nm was recorded by Benchmark Plus Microplate Reader.

Multiplicity of the assay
To demonstrate the multiplicity of the detection assay 10 pM of AFP, CEA, and PSA were incubated with the optimal amount of detection probe conjugated with the corresponding capturing antibody and labeling antibody at 37 °C for 1 h. For the quantification of AFP, CEA, and PSA in serum, 2 μL of serum with optimal amount of corresponding detection probe and 200 pM detection antibody at 37 °C for 1 h. The immno-assemblies of AFP, CEA and PSA were then labeled with 100 μM SPAce, VLAce and SLAce respectively and visualized under the TIRFM imaging system. A transmission grating with 70 lines/mm was placed in front of the EMCCD to capture the fluorescence signal from each nano-assembly. The distance between the grating and the EMCCD was set as 23.5 mm, so that the zero and first order image would not overlap.

Imaging system and data analysis
The prism-type total internal reflection fluorescence was setup as mentioned elsewhere. 26,27 Generally, the flow cell was placed between a fused-silica isosceles prism (CVI, laser USA) and a 60 × oil-type objective that equipped on an Olympus