1. 1 TPD-MS for the Quantification of Protein Adsorption Capacity in porous Carbon M. Vijayaraj Institut de Sciences des Materiaux de Mulhouse – LRC CNRS 7228 Mulhouse (France)

2. 2 Acknowledgements C. Vix-Guterl, R. Gadiou, J. Dentzer., IS2M, France. T. Kyotani, H. Orikasa., IMRAM, University of Tohoku, Japan. CNRS-JSPS Collaborative Project Scheme.

3. 3 Introduction - Aim of the Study Protein Quantification - TPD-MS Analysis - Conventional and TPD Method (Validation) Results - BSA, Cytochrome C Adsorption - Adsorption inside CTs Conclusion Outline

4. 4 Process Significance – Proteins immobilization Bio-Catalysis – Separation processes Protein purification – Bio-Compatible materials Drug delivery devices Primary step: Quantification is as important as the process Detergent washing & analysis: Incomplete desorption from porous supports. Low precision with conventional depletion method (mg concentration). Direct quantification method would be better and interesting.

5. 5 TPD-MS Method Subjecting Support+Protein to Temperature Programmed Decomposition and analyzing the desorbed gases using MS. - analysis of specific molecule (H 2 S). - area under the desorption peak gives moles of H 2 S. - back calculate the amount of protein. CytC - 4 isolated sulfur atoms. BSA - 41 sulfur atoms involved in 20 disulfur bonds.

6. 6 TPD-MS  Custom made high vacuum device equipped with a furnace and MS.  Vacuum range 10 -5 to 10 -7 Torr.  Detected mass range 1-100 amu.  Calibration of MS with known amount of H 2, N 2, O 2, CO, CO 2, H 2 S, and NH 3.  Analysis of H 2 S, signals corresponding to m/z 33 and 34 (HS and H 2 S) are acquired.  Ratio between HS and H 2 S is 0.42  0.03, which is characteristics of pure H 2 S.

7. 7 TPD-MS of BSA Sulfur mostly evolved as H 2 S rather than SO 2 (<5%) because of reductive conditions. Fragment to molecular ions ratio is close to 0.42.

8. 8 TPD of BSA and H 2 S analysis Known amount of BSA in phosphate buffer solution (PBS). Moles of H 2 S linearly increases with BSA concentration. Sulfur Balance Alkyl sulfides, SO 2 and elemental sulfur formation and hence complete H 2 S analysis was not possible. Sulfur balance close to 65%.

9. 9 TPD of CytC and H 2 S analysis o CytC in bicarbonate buffer solution (BBS). o Moles of H 2 S linearly increases with CytC concentration. o 2 nd peak attributed to H 2 S from nonstoichiometric Fe sulfide decomposition. o Stable FeS formation and complete sulfur analysis not possible. o Sulfur balance 40%.

10. 10  0.2% Protein in buffer Adsorption Experiment Micro. Carbon  Protein Final C-Protein Washing Wash-outs C-Protein UV analysis TPD-MS C dep CwCw C tpd C dep ~ C tpd + C w

11. 11 SampleAnalysismg BSA /g carbon /mL C-BSATPD-MS1.06 Wash-outsUV0.46 TotalTPD+WO1.52 BSA Final UV1.62 Results BSA adsorption on Micro.carbon C dep ~ C tpd + C w C dep – Total adsorption C w – Reversible adsorption C tpd – Irreversible adsorption 30% reversible adsorption

12. 12 Ni-Fe alloy ECD inside CTs Kyotani et al, J. Mater.Chem. 17, 2007, 986. Somlak et al, Carbon, 46, 2008, 1361. 5s10s15s Like wise Drugs and Biomolecules adsorption are aimed and gold labelling helps to locate the biomolecules inside CTs. Specific adsorption inside the CTs. Protein quantification using TPD-MS. Adsorption inside Carbon Test tubes: A perspective

13. 13 Preparation of Carbon tubes Anodic oxidation AAO uniform channels CVD 600 o C C 2 H 2 CTs prepared by CVD of Aluminium oxide film Condition: Aq. Electrolyte, 20-120V. ~ 50  m Kyotani et al., Chem. Mater. 1996, 8, 2109.

14. 14 Morphology of AAO 1m1m 50 nm 30 nm 150 nm

15. 15 Morphology of AAO 1m1m CVD

16. 16 TPD of BSA-CTs  H 2 S desorption profile very similar to one from TPD of BSA.  H 2 S desorption onset is well below 200°C compared to one from free BSA.  BSA conformation change affects disulfur bond.  Calibration with known moles of gases allows precise quantification of desorbed gases.

17. 17 TPD of CytC-CTs  Very similar H 2 S desorption onset from adsorbed and free CytC.  Isolated sulfur atoms.  Second peak not so distinct possibly due to small amount of adsorbed CytC.

18. 18 CT-ProteinAmount of Protein (mg BSA /g MWCNT /mL) TPD-MSWash-outsTotal TPD+WO Depletion method C-CytC1.582.273.854.41 C-BSA1.022.643.664.00 Amount of Protein adsorbed in CTs  Decent agreement between the methods.  Very similar adsorption capacity irrespective of the protein size.  More than 65% reversible adsorption.

19. 19  Protein quantification was done directly in TPD-MS.  Good agreement obtained on comparing protein amounts from TPD, wash-outs and depletion method.  Wash-outs analysis revealed reversibly adsorbed protein.  Conformation change enhanced di-S bond decomposition as evident from the low temperature H 2 S desorption onset.  Other studies like Au labelling, influence of carbon surface property and tube size on the adsorption are underway. Conclusions

20. 20 Thank you