1. Laboratory course of biomedical engineering - Introduction
Vesa Vuorinen, Noora Isoaho, Tommi Palomäki, Antti Rautiainen
2016

2. Expectations?
1. Choose a card that reflects your expectations for the course.
2. Show the card to other students in your group and explain it.
3. Combine your separate expectations in one mutual expectation and tell about it to the other groups/teachers.

3. ELEC-D8723 Laboratory course of biomedical engineering (5 cr) Aims of the course
Course is aimed at both EST and BIO students
”Material choice for implantable sensor”
How to approach a research problem
Recognizing the relevant research questions
Selection criteria for research methods
Research plan
Laboratory practices
Safe laboratory work

4. ELEC-D8723 Laboratory course of biomedical engineering (5 cr) Main teachers and evaluation
Vesa Vuorinen and Emilia Peltola
Evaluation:
Scale 0-5
Research plan (group)
Report (group)
Laboratory notes (personal)
Essay on electrochemical sensors (personal)
Final grade = group ± 2

5. Material choice for implantable sensor
Objective is to choose the most suitable surface structure for an implantable sensor
Biocompatibility
Functionality

6. Sensors for neurotransmitters

7. Target: Detecting neurotransmitters in the CNS accurately, fast & reliably in vivo
Foreign body vs. delicate tissue
Small concentrations (nM-µM)
Small volumes
(µm3)
Organic and ionic interferents
(e.g. ascorbic acid)
Fast concentration changes
(ms-s)

8. Sensor biocompatibility
DLC
DLC-PDMS-h

9. Sensor functionality
DLC
DLC-PDMS-h

10. Course content and structure
Research plan
Modification of biocompatibility and functionality
Surface analysis
Biocompatibility
Essay
Analysis
Electrochemical characterization
Analysis
Summary – Presentation and report

11. Grouping Group 1 Michael Asplund Anastasiia Kravtcova Stefan Mocko
Anni Parkkila
Group 2
Anni Pyysing
Ari Ruotsalainen
Imaya Senthilnathan
Niko Silvonen

12. Available materials and treatments
Base materials:
Silicon
Tetrahedral amorphous carbon (ta-C)
Surface treatments:
Plasma (Ar, O2)
APTES SAMs (functionalizing)
Sputtering (Au, Pt, Cr, Ti, Ni, Al, TiW)
Evaporation [C(graphite)]
Acid/base treatments (no HF)
Mechanical coarsening
Anodic/cathodic treatment
Graphene flakes, carbon nanotubes
Lisää materiaalivaihtoehdot

13. Available analysis methods
Contact angle measurement
Electrochemical characterization
Cell culture (direct contact/extracts, MTT)
Microscopy (Optical, SEM+EDS)

14. Course contents and structure
Research plan
Modification of biocompatibility and functionality, surface treatments
Surface analysis, contact angle, SEM
Biocompatibility, cell culture
Analysis, optical microscopy, SEM, MTT
Electrochemical characterization, CV
Analysis
Summary – Presentation and report

15. Schedule
Wed
Introduction, aims, materials and methods, initializing research plan
week 12-14
Q&A session (16.3.) and finishing the research plan
week 15
Coatings, contact angle experiments
week 16-17
Cell culture, analysis
week 18-19
Electrochemical characterization
week 20/21
Summary, presentation and report

16. Research plan 3-4 reasonably chosen materials
What is going to be tested and why?
Description of methods
What is going to be measured and what is going to be analyzed?
Protocols for cell culture experiments (cell count, time points, sample preparation)
Parameters and setup of electrochemical measurements (solution, analytes, concentrations)
What is the experimental output?
What are the target conclusions?
How do the results reflect the real in vivo situation?

17. Introduction of available methods

18. Plasma etching What is plasma etching? Why do people use it?
What kind of process parameters can you find from the literature (for different materials)?
What happens to metals during the process?
How changes in the material are inspected after plasma treatment?
Parameters to be defined:
Gases available: O2, Ar (combinations possible)
Time, power (max 1000 W), flow (in ml/min),
Chamber ”cleaning” with N2 purge.

19. Sputtering
Atoms / molecules are removed from the target surface by ion bombardment → particles are transferred through the gas phase and condensate on the substrate surface as a film.
Uniform and thick coating
Applicable to complex compounds
Difficult to control the chemical composition of the surface
Line of sight technique

20. SAM coating
SAMs are formed spontaneously from the components of the system.
Uniform thin film that is (relatively) easy to make.
Coating of curved surfaces possible.
Limited amount of available molecules and substrates.
Hard to make thicker films than monolayers.
Mechanically and chemically frail.

21. Contact angle measurement
Principle:
A drop of liquid (polar/nonpolar) is dropped on the surface. The contact angle is measured and defined with a computer.
Fairly simple experiment.
Surface energy (min. 2 different liquids, preferably more)
Information on protein adsorption (some)
Several sources of error (surface roughness, debris, experimenter…)
Different models for defining surface energy
Hydrophilic
Hydrophobic
Young’s equation
𝛾 𝑆𝐿 + 𝛾 𝐿𝑉 cos 𝜃 = 𝛾 𝑆𝑉
[Yuan and Lee, 2013]

22. SEM+EDS Field emission scanning electron microscope (FESEM)
JEOL JSM-6335F
Magnification
10- 
The optimal resolution
is 1.5nm.
Imaging modes SEI, BEI
LINK x-ray microanalyzer (EDS)

23. Surface characterization
Microscopy
Optical microscopy
SEM
Surface structure and chemical analysis
XRD (X-Ray Diffraction)
XPS (X-ray Photoelectron Spectroscopy)
EDS (Energy-dispersive X-ray spectroscopy)
Not available on this course

24. Cyclic voltammetry (CV)
Electrochemical method
Potential is varied linearly as a function of time
Oxidation and reduction of the analyte cause a current peak (exchange of e-)
Current is proportional to the amount of reacted analyte
Oxidation: A → B + ne-
Switching potential
Potential E (V)
Slope = scan rate v (V/s)
Peak potential separation ΔEp
Time t (s)
Reduction: B + ne- → A

25. Cyclic voltammetry (CV)
+ Qualitative analysis of an electrochemical system
+ Information on the reaction mechanism
(adsorption, diffusion, reaction rate constant)
+ Good selectivity
+ Excellent response time
- Quantitative analysis (amount of reaction)
Requires knowledge on the electrode material
and analyte in advance

26. Cyclic voltammetry (CV)
Experimental setup:
Three-electrode cell
Working electrode is the electrode under investigation
Potential is measured against a reference electrode
Current passes through the working and counter electrodes
The electrodes are connected to a potentiostat that sets V and measures I
Ar / N2
Reference electrode (Ag/AgCl)
Working electrode
Counter electrode
(graphite rod)
Analyte-containing solution
Ar / N2

27. For the research plan
Possible measurements:
Width of the potential window
Selectivity: measurement with an analyte and an interferent
Sensitivity: lowest concentration that can be detected
Electron transfer rate for determining how fast the electrode is
Choose 2-3 analytes and appropriate solutions (H2SO4, KCl, PBS…):
Dopamine
Ascorbic acid
Uric acid
Ferrocenemethanol
Ruthenium Ru(NH3)63+
Ferrocyanide Fe(CN)64-
Also choose:
Potentiostat parameters:
Potential limits
Scan rate
Solutions and analytes:
Concentration
Purging (Ar or N2)

28. Biocompatibility
Standards define several experiments to be conducted when testing for biocompatibility.
In general, when designing biomedical devices it is best to try to avoid toxic or unknown materials.
Testing is both time consuming and expensive!

29. Primary / secondary cell line?
Choosing the cell line
Other properties
Growth properties
Recognition
Type
Stability
Cells available in this course are mouse 3T3 fibroblasts.
Availability
Primary / secondary cell line?

30. Passage

31. Detaching the cells Method Equipment Type of cells Shaking
Careful shaking or intense pipetting
Loosely adherent cells, mitotic cells
Scraping
Cell scraper
Protease sensitive cells
Enzymatic
Trypsine
Adherent cells
Dispase
Detaching epidermal cells as ”sheets”
Temperature
Temperature responsive culture dish

32. Passage steps 1/2 Inspect cells Remove media Rinse cells Incubate
3 – 10 min
Add trypsine
Inspect cells

33. Passage steps 2/2 Centrifugate cells Add media Suspense cells
Count cells
Distribute cells

34. SEM Electron microscopy Preparations for biological sample Dehydration
Coating with conducting material

35. Optical microscopy Histological staining
Stainings available in this course:
Actin
Nucleus

36. MTT e.g. (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
MTT assay
Color change of yellow MTT to purple is related to (live) cell metabolic activity. Adsorbance can be measured with a spectrometer.
MTT e.g. (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

37. People as contamination sources
People generate particles continously (≥ 0.3µm)
Standing still particles / min
Small hand gesture particles / min.
Normal hand/body movement particles / min.
Walking particles / min
Walking fast particles / min

38. Aceptic technique Washing hands Labcoat with long sleeves Gloves
Spraying surfaces, gloves and equipment with EtOH
Reagents and equipment used only for cell culture
Working in laminar flow cabinet

39. Biological contamination
Rendered images of different types of contamination.
No contamination
Bacillus contamination
Coccus contamination
Fungal contamination
Yeast contamination
Cross-contamination or mycoplasma contamination
Note that F) resembles culture with no contamination!
Need other means to detect this type of contamination
Bacillus = sauvabakteeri (esim. E. Coli & salmonella)
Coccus = kokkibakteerit (Streptokokki jne)
Fungal contamination = sieni
Yeast = hiiva
Mycoplasma can be detected by fluorescent staining.
Sandell L, Sakai D. Mammalian cell culture. Current Protocols Essential Laboratory Techniques Unit 4.3, 2011.

40. Cross contamination 246 cell lines were inspected in 1976.
14% were contaminated with cells from different species
25% of human cells were HeLa cells
→ almost 30% were labeled wrongly

41. Most common mistakes in aceptic technique I

42. Most common mistakes in aceptic technique II

43. Most common mistakes in aceptic technique III

44. Hazardous situations
Watch video on aceptic technique from MyCourses!

45. Final report What did your group do? What kind of results did you get?
Did you deviate from your plan? Why?
What kind of results did you get?
How would you continue the study?

46. Materials etc. MyCourses Use the forum for questions
Other groups might have been wondering the same!