Applying optical spectroscopy for biomedical diagnostics at cellular and molecular level

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Arnold Baldwin
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1 Graduate School Vito Volterra XXXI Ciclo Applying optical spectroscopy for biomedical diagnostics at cellular and molecular level candidate: supervisor: prof. Paolo Postorino

2 Motivation & final aim develop experimental techniques for early cancer detection

3 Motivation & final aim develop experimental techniques for early cancer detection capability to reveal the presence of cancer at the first stage of its progression

4 Motivation & final aim develop experimental techniques for early cancer detection capability to reveal the presence of cancer at the first stage of its progression cellular level molecular level

Motivation & final aim develop experimental techniques for early cancer detection capability to reveal the presence of cancer at the first stage of its

5 Motivation & final aim develop experimental techniques for early cancer detection capability to reveal the presence of cancer at the first stage of its progression cellular level molecular level find specific properties of cancer cells (e.g. morphological, mechanical, etc..) to be experimentally revealed

6 Motivation & final aim develop experimental techniques for early cancer detection capability to reveal the presence of cancer at the first stage of its progression cellular level molecular level identify and quantify DNA oxidative damage

7 Motivation & final aim develop experimental techniques for early cancer detection capability to reveal the presence of cancer at the first stage of its progression cellular level molecular level identify and quantify DNA oxidative damage our approach is based on Raman spectroscopy! non-disruptive technique (visible light) molecular recognition structural information aqueous samples

8 Raman spectroscopy scattered light incident light w 0 + w V w 0 w 0 w V w 0 - w V

9 Raman spectroscopy scattered light incident light w 0 + w V w 0 w 0 w V w 0 - w V information on vibrational frequencies of the system chemical bonds

10 Raman spectroscopy scattered light incident light w 0 + w V w 0 w 0 w V w 0 - w V information on vibrational frequencies of the system chemical bonds but also structural conformation local environment guanosine monophosphate DNA nucleotides guanosine triphosphate

11 Raman spectroscopy scattered light incident light w 0 + w V w 0 information on vibrational frequencies of the system DNA nucleotides guanosine triphosphate w 0 w w chemical bonds V 0 - w V but also structural conformation local environment guanosine monophosphate I R σ R ( ) I 0 enhancement of Raman signal?

12 Surface Enhanced Raman Scattering 10 8 SERS phenomenon: enhancement of Raman signal coming from molecules bound/located very closed to metallic nanostructures I R σ R ( ) I 0

13 Surface Enhanced Raman Scattering I σ ( ) I R R 0 two enhancement mechanisms: SERS phenomenon: enhancement of Raman signal coming from molecules bound/located very closed to metallic nanostructures electromagnetic enhancement surface plasmons (collective oscillation of free electron gas in the metal) chemical enhancement structural modifications (metal-molecule bound) changing polarizability

Surface Enhanced Raman Scattering I σ ( ) I R R 0 two enhancement mechanisms: SERS phenomenon: enhancement of Raman signal coming from molecules bound/located very closed to metallic nanostructures

14 Surface Enhanced Raman Scattering I σ ( ) I R R 0 two enhancement mechanisms: SERS phenomenon: enhancement of Raman signal coming from molecules bound/located very closed to metallic nanostructures electromagnetic enhancement surface plasmons (collective oscillation of free electron gas in the metal) chemical enhancement structural modifications (metal-molecule bound) changing polarizability major contribution to enhancement

15 Surface Enhanced Raman Scattering I σ ( ) I R R 0 two enhancement mechanisms: SERS phenomenon: enhancement of Raman signal coming from molecules bound/located very closed to metallic nanostructures electromagnetic enhancement surface plasmons (collective oscillation of free electron gas in the metal) chemical enhancement structural modifications (metal-molecule bound) changing polarizability

16 biomedical diagnostics at cellular level

17 SERS-based diagnostics: idea 1 identify a specific property of cancer cells: folate receptor expression

18 SERS-based diagnostics: idea 1 identify a specific property of cancer cells: folate receptor expression 2 develop a SERS active nanovector based on this property and exploit it to treat different types of cells

SERS-based diagnostics: idea 1 identify a specific property of cancer cells: folate receptor expression 2 3 develop a SERS active nanovector based on

19 SERS-based diagnostics: idea 1 identify a specific property of cancer cells: folate receptor expression 2 3 develop a SERS active nanovector based on this property and exploit it to treat different types of cells use SERS signal to discriminate healthy/cancer cells in collaboration with F. Domenici F. Bordi

20 SERS results 4-aminothiophenol (4ATP) SERS reporter folic acid biointerface gold nanoparticles 60nm diameter C. Fasolato,, F. Ripanti,.. et al, Nanoscale, 2016

21 SERS results 4-aminothiophenol (4ATP) SERS reporter gold nanoparticles 60nm diameter folic acid biointerface spectroscopic imaging! nanovector alone treated cancer cell cell alone C. Fasolato,, F. Ripanti,.. et al, Nanoscale, 2016

22 SERS results 4-aminothiophenol (4ATP) SERS reporter gold nanoparticles 60nm diameter folic acid biointerface spectroscopic imaging! treated cancer cell optical image 5mm C. Fasolato,, F. Ripanti,.. et al, Nanoscale, 2016 nanovector alone treated cancer cell cell alone cell nanovector

23 SERS results 4-aminothiophenol (4ATP) SERS reporter gold nanoparticles 60nm diameter folic acid biointerface spectroscopic imaging! treated cancer cell optical image 5mm C. Fasolato,, F. Ripanti,.. et al, Nanoscale, 2016 nanovector alone treated cancer cell cell alone cell nanovector

24 different expression of folate receptors Cell treatment & SERS screening HaCaT normal keratinocyte cell line PC3 prostatic cancer cell line HeLa cervical cancer cell line C. Fasolato,, F. Ripanti,.. et al, Nanoscale, 2016

25 different expression of folate receptors Cell treatment & SERS screening HaCaT normal keratinocyte cell line PC3 prostatic cancer cell line low SERS expected high SERS expected HeLa cervical cancer cell line very high SERS expected C. Fasolato,, F. Ripanti,.. et al, Nanoscale, 2016

26 different expression of folate receptors Cell treatment & SERS screening HaCaT normal keratinocyte cell line PC3 prostatic cancer cell line low SERS expected high SERS expected HeLa cervical cancer cell line very high SERS expected C. Fasolato,, F. Ripanti,.. et al, Nanoscale, 2016

27 different expression of folate receptors Cell treatment & SERS screening HaCaT normal keratinocyte cell line low SERS expected PC3 prostatic cancer cell line HeLa cervical cancer cell line high SERS expected very high SERS expected diagnosis only based on SERS response! C. Fasolato,, F. Ripanti,.. et al, Nanoscale, 2016

28 biomedical diagnostics at molecular level

29 DNA oxidative damage Reactive Oxygen Species modification of DNA precursor pool incorporation into genome

30 DNA oxidative damage Reactive Oxygen Species modification of DNA precursor pool 8-oxo-deoxyguanosine is the prevalent oxidized nucleotide triphosphate goal: measurement of 8-oxo-dGTP/dGTP ratio in cellular pool incorporation into genome in collaboration with F. Mazzei, Istituto Superiore di Sanità dgtp: deoxyguanosine triphosphate 8-oxo-dGTP: 8-oxo-deoxyguanosine triphosphate

31 Raman results dgtp 8-oxo-dGTP

32 Raman results dgtp 8-oxo-dGTP mixture spectra log scale

33 Raman results dgtp 8-oxo-dGTP mixture spectra These spectroscopic markers effectively depend on 8-oxo-dGTP concentration! log scale 8-oxo-dGTP dgtp = 1%

34 Low-frequency Raman results high sensitivity at very low 8-oxo-dGTP/dGTP ratio! 8-oxo-dGTP dgtp = 0.1% frequency shift

35 Low-frequency Raman results Raman very low frequency in dgtp spectrum could be associated to a particular vibration of this tetrad structure high sensitivity at very low 8-oxo-dGTP/dGTP ratio! 8-oxo-dGTP dgtp = 0.1%

36 = Low-frequency Raman results Raman very low frequency in dgtp spectrum could be associated to a particular vibration of this tetrad structure With 8-oxo-dGTP: no hydrogen bond in tetrad disordered long range structure high sensitivity at very low 8-oxo-dGTP/dGTP ratio! 8-oxo-dGTP dgtp = 0.1% H O

37 @molecular level Conclusions correct functionalization of nanovector SERS imaging SERS screening protocol allows to distinguish different cell types Raman spectroscopy as a new method to quantify oxidized nucleotides: detection threshold of 1% in fingerprint spectral region detection threshold of low frequencies successful tests with both deoxy- and ribo-guanosine preliminary results also for 8-oxo-dATP (this method WORKS!)

38 Future outcomes quantification of oxidation level in real cellular pools: extraction of dntps and NTPs pool purification of guanosine compounds from other bases SERS of DNA nucleotides: adsorb or covalently bind nucleotides on metal nanostructures improve sensitivity in damage detection identify the percentage of oxidized bases in DNA/RNA chain side project: reveal formation and inhibition mechanisms of insulin fibrils combining microscopy and spectroscopy techniques

39 Acknowledgments & collaborations F. Domenici F. Bordi F. Mazzei M. Bignami A. Nucara and thank you for your kind attention! A. Filabozzi

CHAPTER 6. BLUE GREEN AND UV EMITTING ZnO NANOPARTICLES SYNTHESIZED THROUGH A NON AQUEOUS ROUTE

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