Synthesis, characterization and modification of Gold Nanoparticles.
Different types of gold nanoparticles have been synthesized and characterized such as gold nanoshells, gold nanostars and gold superstructures. Gold nanostars (AuNS) were used throughout the project because of their high plasmonic activity.
In order to combine a fast global tissue analysis utilizing fluorescence with a subsequent local SERS analysis the AuNS were modified with both a Raman reporter and a fluorophore. Two different meth-ods for targeting DNA and mRNA were developed. For DNA targeting, an antigen-antibody reaction was employed. DNA sequences modified with a suitable antigen were used to target the HER2 gene and chromosome 17 centromere on normal and tumor tissue sections. In that case, the AuNS were modified with a suitable fluorophore-labelled antibody to recognize the corresponding antigen. Hydrophilic stabilization of the metal colloid was achieved by coating the nanoparticles with carboxylic acid functionalized, PEG 3000. A double layer of PEG and a Raman reporter molecule was achieved. For antibody conjugation, the COOH group at the terminus of the PEG molecule was activated for covalent binding to primary amines (lysine residues) of the fluorophore-labelled secondary antibody. The re-sulting AuNS retained both fluorescent and Raman activity. Figure 1 shows a SEM photo of gold nano-particle covered with a PEG layer.
For mRNA recognition, three short DNA sequences were designed to target directly the mRNA se-quence at different positions of the sequence. In this case the AuNS were modified with thiolated DNA sequences and a thiolated Raman reporter. The double layer was optimized by measuring fluorescence and Raman spectroscopy.
Optimization of the conditions for the detection of a specific DNA sequence in FFPE human breast carcinoma tissue sections.
Initially, similar pretreatment method to conventional fluorescence in situ hybridization procedures was followed but was not adequate to allow AnNS permeation and SERS detection. The tissue pretreatment was optimized with respect to the heat pretreatment, the enzymatic antigen retrieval using pepsin, blocking reagent and washing buffer. HER2 gene was successfully detected on tissue sections using both fluorescence and Raman microscopy and by employing AuNS with dual activity i.e. SERS and fluorescence.
To validate the assay, conventional FISH was employed with the new optimized conditions. Figure 2(a) shows fluorescence images of normal breast tissue section. It is obvious that the HER2 gene expression is increased for the cancer tissue. In addition, DAPI staining was carried out to show that the HER2 gene is localized inside the cells. Dual mode fluorescence-SERS microscopy was then carried out after staining with antibody conjugated Au nanostars. Figures 3 and 4 show that it is possible to successfully localize the HER2 gene with both fluorescence (big areas) and SERS (smaller areas) microscopy.
Application of the methodology for multiplex DNA analysis.
After the tissue pretreatment was optimized, two DNA sequences, HER2 gene and Chromosome 17 centromere, were detected simultaneously using dual mode fluorescence/Raman imaging approach. For this work the HER2 probe was labeled with DIG antigen, while the probe for the chromosome 17 centromere was labeled with DNP antigen. Correspondingly AuNS were modified with a monoclonal goat anti-DIG Alexa-647 and the raman reporter 4-Nitrophenol (4NTP) for HER2 gene localization. For chromosome 17 centromere localization, AuNS were modified with goat DNP monoclonal anti-rat sec-ondary antibody, Alexa Fluor® 488 conjugate and the raman reporter 7-mercapto-4-methylcoumarin (mmc).
The method was first validated with fluorescence. For normal and cancer tissue the expression of chromosome 17 centromere is similar, as expected. For the cancer tissue the HER2 gene expression is increased, as expected.
Dual mode fluorescence –SERS microscopy was followed only for the normal breast, to demonstrate the multiplexing capability of the assay. In the case, the fluorescence images obtained by wide field
Fluorescence microscopy served as a starting point for selecting smaller regions on the tissue for anal-ysis by SERS microscopy in the mapping mode (figure 5). Similar number of spots for can be seen for both HER2 gene and chromosome 17 centromere, as expected for normal tissue.
Optimization of the conditions for the detection of a specific mRNA sequence in FFPE human breast carcinoma tissue sections from patients using wide-field fluorescence microscopy.
A design for the detection of HER2 mRNA sequence was achieved. Three small (~25 DNA bases) DNA sequences were designed to target the mRNA sequence in the tissue. Modification of gold nanoparti-cles with small DNA sequences via a thiol bond is well documented in the literature. The specificity of the sequences to recognize the mRNA sequence was validated using commercially available software such as BLAST (
https://www.ncbi.nlm.nih.gov/blast/(se abrirá en una nueva ventana)). The specificity was then tested utilizing FISH. In this case breast cancer tissue specimens were first incubated with the in house-designed DIG modified probes followed by addition of Fab fragments from polyclonal anti-digoxigenin antibodies, conjugated to horseradish peroxidase (HRP). Tyramide signal amplification was then followed. Figure 6 shows the IF images. It is obvious that mRNA expression is increased in the tumor tissue. The DNA sequences can be used for SERS microscopy.
