SNOTRA LLC.

Scanning probe nanotomography – a new technology for analysis and diagnostics

of biomaterials and polymers

The Problem We Address

Nanoscale structure analysis of majority (~80%) of biological and soft polymer materials require cryosectioning and investigation at low temperatures.

Existing approaches (cryoTEM) is very expensive and suffers from a number of issues (low EM contrast, beam damage, etc), especially for 3D analysis.

Thus a number of problems concerning nanoscale analysis of biological, polymer and other soft materials still do not have an adequate solution

SNOTRA LLC.The Goal:

Development and commercialization of methodology and combined analytical system for nanotomography – non-

destructive three-dimensional analysis of native nanoscale structures in a wide range of soft materials.

The Solution is based on combination of

scanning probe microscopy (nanoscale analysis of surface features)

and

cryoultramicrotomy (ultrathin sectioning of soft materials at temperatures down to -190°С).

SNOTRA LLC.A novel technology of scanning probe nanotomography

Is applicable to: biomaterials, biopolymers, other soft or hydrated polymers & materials (e.g. resins), nanoemulsions

Is ~ 10 times cheaper than currently available competing methods/devices (f.e.TEM electron tomography)

Provides access to unique information about nanostructure – not available by other existing methods.

How advantage is secured by the company IP protection (Russian patent filed, EU patent applied, license

agreements are under preparation), High-ranked and well-known scientists in the field are involved

(f.e. Dr. Prof. Ferdinand Hofer, Head of FELMI/ZFE Graz, Austria), Working prototype (proof-of-the-concept) of the system is

successfully built. Results are published in scientific journals.

SNOTRA LLC.The company

Is established by inventors personally, Federal Research Center for Transplantology and Artificial Organs, and Center of Innovative Enterprise (Moscow State University)

Is cooperating with one of the world leading scientific centers in the field of the project – Austrian Centre for Electron Microscopy (FELMI/ZFE Graz, Austria)

Is a participant of Skolkovo Foundation

Technology overwiew: AFM and TEM image contrast formation

Block face Ultrathing section10-90nm

AFM image TEM image

•Vacuum

•Electron beam

•Low contrast on polymer/bio unstained samples

Projection imaging

Vacuum OR

gas/liquidenvironment

Data of molecular (protein) content from the surface

and mechanical properties (phase imaging

of polymers)

Tuning fork-based AFM probe

Leica EM UC6/FC6 cryoultramicrotome performs ultrathin sectioning of soft materials at temperatures from -15 to -190°C. Section thickness ranges from 20 nm to 1 um.Cryo-AFM measuring head is installed directly into the cryochamber of the ultramicrotome

Technology overwiew: Prototype of the combined system

Techonolgy overwiew: Application results

The morphology of a cross-section of a nitrile butadiene-rubber latex sample characterized by cryo-AFM and TEM. (a) A topographical AFM image of an epoxy embedded latex stripe that was mounted in the cryo-chamber of SNOTRA, cryo-sectioned and immediately scanned at −120°C.(b) Immediately afterwards, the same sample was warmed to room temperature and then examined using the same AFM (c) A TEM image of the last thin section of the NBR latex sample. (d) A schematic description of the topographical change of the sample block phase that took place during sectioning and the following warming processes. The scale bars in (a, b, c) are 200 nm, and the topographical variations in (a) was 27.2 nm, and in (b) was

35.5 nm.

Techonolgy overwiew: Application results

Serial section 3D reconstructions of biological and polymer samples at room temperature and under cryo conditions. (a) An AFM image of the antennal sensilla Placodea of the parasitic wasp Cotesia glomerata. The sample was frozen under high pressure, epoxy freeze-substituted, embedded and observed at room temperature. (b) A bright-field TEM image of a thin section of a corresponding area of antenna that was stained and observed at room temperature. (c) 3D reconstructions of the antennal sensillas Placodea and Coeloconica obtained at room temperature. 12.5×13.0×0.7 µm3, 11 sections, section thickness 70 nm. (d) A 3D model of the chitin organization of Placodea and Coeloconica sensillas. The white arrows indicate the protrusion cavity of both sensillas. (e, f) Topographical AFM images of PA6/SAN polymer blend (30/70 w/w). The block face was cryo-sectioned and immediately scanned at −80°C (e) and again sectioned and immediately scanned at room temperature (f). (g, h) 3D reconstructions of PA6/SAN obtained at −80°C (g: 7.9×6.2×0.75 µm3, h: 2.0×2.0×0.75 µm3, 6 sections each, section thickness 125 nm). The scale bars in (a, b) are 500 nm and in (e, f) are 2000 nm; topographical variations are 230 nm in (e) and 820 nm in (f).

Scientific publications on the matter of project

• 1) A. E. Efimov, H. Gnaegi, R. Schaller, W. Grogger, F. Hofer and N. B. Matsko, Analysis of native structure of soft materials by cryo scanning probe tomography, Soft Matter, 2012, Advance Article DOI:10.1039/c2sm26050f

• 2) V.Mittal and N.B.Matsko, Tomography of the Hydrated Materials, in Analytical Imaging Techniques for Soft Matter Characterization, EngineeringMaterials, Springer-Verlag Berlin Heidelberg, 2012, DOI:10.1007/978-3-642-30400-2_7, Ch. 7, pp. 85-93

• 3) A. Efimov; H. Gnaegi; V. Sevastyanov; W. Grogger; F. Hofer; N. Matsko, Combination of a cryo-AFM with an ultramicrotome for serial section cryo-tomography of soft materials - Proceedings 10th Multinational Congress on Microscopy 2011 SEP 4-9, 2011; Urbino, ITALY. pp.707-708;

• 4) N. B. Matsko, J. Wagner, A. Efimov, I. Haynl, S. Mitsche, W. Czapek, B. Matsko, W. Grogger, F. Hofer, Self-Sensing and –Actuating Probes for Tapping Mode AFM Measurements of Soft Polymers at a Wide Range of Temperatures, Journal of Modern Physics, 2011, 2, pp. 72-78

• 5) 4) A. Alekseev, A. Efimov, K. Lu, J. Loos. Three-dimensional electrical property reconstruction of conductive nanocomposites with nanometer resolution, Advanced Materials, Vol. 21, 48 (2009), рр. 4915 – 4919

• 6) A. Efimov, V. Sevastyanov, W. Grogger, F. Hofer, and N. Matsko. Integration of a cryo ultramicrotome and a specially designed cryo AFM to study soft polymers and biological systems, MC2009, Vol. 2: Life Sciences, p. 25, Verlag der TU Graz 2009.

• 7) Nemets, E.A., Markovtseva M.G., Efimov A.E., Egorova V. A., Tonevitsky A.G., Sevastyanov V.I., Micro- and nanostructural characteristics of three-dimensional porous matrixes Elastopob-3D , - Bulletin of experimental biology and medicine, 145(3):371-3, 2008

• 8) A. E. Efimov, A. G: Tonevitsky, M. Dittrich & N. B. Matsko. Atomic force microscope (AFM) combined with the ultramicrotome: a novel device for the serial section tomography and AFM/TEM complementary structural analysis of biological and polymer samples. - Journal of Microscopy, Vol. 226, Pt 3, June 2007, pp. 207–217

Revenue Generation

Company Revenue

Model

MarketsScientific & industry

customers specialized in:

1. Biomaterials

2. Polymers

3. Nanoemulsions and nanoliquids

4. Other soft/hydrated materials

Products:1. CryoUMT/AFM

system

2. CryoUMT/Raman/AFM system

(next stage of development)

Service:Analytical services with use of our nanotomography technology

World market estimation

* According to report by Future Markets, Inc. 2011

AFM/SEM/TEM (total), M$

AFM/SEM/TEM,Biomed+Polymers+Nanomaterials, M$

2010 328,44 97,6

2015 (prognosis)* 588,69 217,39

We develop unique product valuable for many users of AFM/SEM/TEM in Biomedical, Polymers and Nanomaterials market segments

We plan to achieve 10 M$ market share in 5 years

Companies and institutions expressed interest in the technology

• Dow Chemicals (Dow Benelux BV, polymers )

• FELMI/ZFE Graz (Austria)

• Anton Paar (Austria, analytical equipment)

• Boston Scientific (USA, drug translucent stents)

• Weizmann Institute (Israel, biomaterials)

• Carmel Olefins (Israel, polymers)

• Nike IHM (USA, multilayer polymers for the shoes)

• Cornell University (USA, nanofibers for textile)

• Shell International (Netherlands, Dr. R. Haswell, fuel membranes)

• General Motors, Rochester Lab (USA, fuel membranes)

• Xerox Inc. (USA, 3-D structure of ink and paint particles)

• Nova Nordisk (USA, polymers)

• South-Ural State University (Russia, nanocomposites)

• Bioplast LLC. (Russia, nanomaterials)

SNOTRA LLC.

SNOTRA LLC. is a participant of Skolkovo Foundation and is going to apply for a grant funding.

Terms of financing of R&D projects by Skolkovo Foundation (stage 1):

Total project budget: up to 1 Million Euro for 3 years Co-investment: 25% = 250 000 Euro

Granted financing: 750 000 Euro

SNOTRA LLC. is looking for co-investors for a grant application.