Multi-label

Luminescence has already been considered in general terms in Chapter 5. Luminescent POPAM dendrimers of various generations with peripheral dansyl units were studied by Balzani and Vogtle et al. as sensor model systems with regard to the fundamental suitability of dendritic structures for multiplication of signalling groups (multi-labelling) [48]. 

Thereafter, 200 pi of the doxorubicin containing triton-X 100 solution is added to at least three wells for each sample of a flat bottom 96 well cell culture plate. Only 1% triton-X 100 is taken as a blank. The fluorescence intensities are measured using a spectrofluorimeter (Wallac Victor 2 Multi-label Counter, PerkinElmer) using 485 nm excitation and 590 nm emission Alters. 

To quantify luciferase reporter gene expression in cell lysates, transfer 50 pi cell lysate from each well into a 96-well black flat-bottom microplate. Add 100 pi luciferase buffer per well, and optionally mix with a pipette. Measure the chemiluminescence intensity (count time 0.20 min with background correction) using a luminometer, e.g., a Microplate Scintillation Luminescence Counter (Canberra Packard) or a Wallac Victor 2 Multi-label Counter (PerkinElmer). 

Histamine was detected with a multi-label counter (Wallac 1420 ARVOsx, Perkin Elmer). Forty-/tL CH3CN solution containing 0.25 mM TCPO and 2.5 ftM DNS-Phe were added to 20 /tL histamine solutions diluted to appropriate concentrations. After addition of 20 juL water containing 200 mM H2O2 to each well, the luminescence produced was immediately measured. The calibration curve was conducted by CL intensity against added amounts of histamine (10 fmol 50 pmol). 

Since strong emission was observed in histamine, detection with a multi-label counter was also tried. In this method, 0.125 mM TCPO, 2.5 jiM DNS-Phe and 50 mM H2O2 were used. Figure 3 shows the calibration curve of histamine. Good linearity was observed in the range of 50 fmol 50 pmol. Under the conditions, the linearity was not obtained at concentration higher than 50 pmol. The detection limit of histamine was approximate 10 fmol. The method will be further optimised in our laboratory and applied to real specimens. 

Fig. 1.1 ELISA-like immunoarray strategy to detect proteins (PSA=prostate specific antigen) demonstrating some uses of nanoparticles. Gold nanoparticles on the spot areas are linked to primary antibodies that capture the protein analytes. After washing, a labeled secondary antibody, or as illustrated here a multi-labeled nanoparticle with attached secondary antibody, is added. This detection particle binds selectively to the captured analyte molecules. After additional washing with blocking agents to remove non-specific binding of the labeled species, electrical or optical detection is used to count the number of bound labels that is proprational to protein analyte concentration...

Fig. 1.7 Amperometric responses for AuNP immunosensors at -0.3 V and 3000 rpm in buffer containing 1 mM hydroquinone after injecting 0.04 mM H202 to develop the signal (a) using Ab2-magnetic bead-HRP with 7500 labels/bead at PSA concentrations shown. Controls a Immunosensors built on bare PG at 10 pg mL-1 PSA (b) Immunosensors built on PDDA coated PG surface at 10 pg mL-1 PSA b Influence of PSA concentration on steady state current for AuNP immunosensor using multi-label Ab2-Magnetic bead-HRP. Reproduced with permission from [46], copyright American Chemical Society 2009...

We also reported a AuNP immunosensor for detection of IL-6 with a DL of 10 pg mL in calf serum without using labeled magnetic particles [71]. A comparison under the same assay conditions using human IL-6 cancer biomarker in calf semm revealed that the AuNP immunosensor offers a threefold better detection limit than SWCNT forest immunosensors. In another strategy we used 0.5 pm multi-labeled polymeric beads (polybeads-HRP-Ab2) to achieve a DL of 10 pg mL for MMP-3 [72] in calf serum. 

Mansfield JR, Hoyt C, Lebenson RM (2008) Visualization of microscopy-based spectral imaging data from multi-label tissue sections. Curr Protoc Mol Biol 12 14-19... 

Hsu, D., S. Kakade, J. Langford, and T. Zhang. 2009. Multi-label prediction via compressed sensing. Advances in Neural Information Processing Systems 22 Proceedings of the 23rd Annual Conference on Neural Information Processing Systems, Vancouver, Canada, December 7-10,2009. 

Bai, L., Yuan, R., Chai, Y., Zhuo, Y, Yuan, Y, Wang, Y, 2012. Simultaneous electrochemical detection of multiple analytes based on dual signal amplification of single-walled carbon nanotubes and multi-labeled graphene sheets. Biomaterials 33, 1090-1096. 

Fluorescent secondary antibodies Alexa Fluor 488 (yellow-green) and/or Alexa Fluor 546 (orange-red) and/or Alexa Fluor 568 or 594 (red) can be obtained, e.g., from Molecular Probes , Life Technologies , Carlsbad, CA. According to necessity goat anti-rabbit or donkey anti-mouse IgG F(ab )2 should be used, cross-adsorbed for multi-labeling experiments. 

EnVision 2103 Multi-label Plate Reader (Perkin-Elmer Life Sciences). 

Measure GFP fluorescence with a Multi-label Plate Reader using the appropriate read mode, with excitation at 485 nm and emission at 535 nm (r Note 6). 

Measure the optical density (OD) of each well at 600 nm with a Multi-label Plate Reader. 

The top and bottom read mode could be chosen according to the corresponding function of the Multi-label Plate Reader. 

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