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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - RISH (Imaging of multiple mRNA targets using SERS nanoparticle labels and in situ hybridization in human cancer tissue sections)

Teaser

Summary

Immunohistochemistry and in situ hybridization techniques that employ fluorescence are routinely used as diagnosis tools, for the detection of several antigens and DNA/RNA in tissue sections. Surface-enhanced Raman scattering (SERS), can also be employed in biomedical diagnostics. In immuno-SERS (iSERS) microscopy, the corresponding antibodies are labelled by molecularly functionalized noble metal colloids (SERS labels or nanotags). i.e. metal nanoparticles (NPs) with organic Raman reporter molecules chemisorbed on their surface. The reporter molecules provide the characteristic â€œmolecular fingerprintâ€ for the identification of the label, while the metal NP provides the necessary signal enhancement upon. During this project, SERS was employed for the detection of DNA biomarkers on breast cancer tissue sections.
SERS applied for multiplex DNA and RNA detection for tissue diagnostics can have an impact on personalizing health and care. In situ hybridization utilizing Raman spectroscopy as an optical sensor has the potential of a quantitative multiplex in situ cancer diagnostic technique that can also provide spatial and cellular information. This level of insight can then lead to advances in early disease diagnosis, disease progression and response to therapy.
Objectives:
- Preparation and characterization of DNA targeting SERS probes.
- Multiplex detection of DNA in FFPE breast tumour tissue sections. Optimization of tissue pre-treatment.
- Detection of mRNAs in FFPE breast tumour tissue sections using Fluorescence and SERS microscopy

Work performed

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 FluorÂ® 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 â€“SERS microscopy was

Final results

It was possible using the methods described above to distinguish clinical samples of normal and cancer tissue received from patients of the University hospital of Essen. Further exploration of the multiplex capabilities of SERS microscopy for early cancer diagnosis can lead to advances in early disease diagnosis, disease progression and response to therapy.