Impact of membrane pore structure on protein detection sensitivity of affinity-based immunoassay

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Understanding a membrane’s morphology is important for controlling its final performance during protein immobilization. Porous, symmetric membranes were prepared from a polyvinylidene fluoride/N-methyl-2-pyrrolidinone solution by phase inversion process, to obtain membrane with various microsized pores. The concentration and surface area of aprotein dotted on the membrane surface were measured by staining with Ponceau S dye. The dotted protein was further scanned and analysed to perform quantitative measurements for relative comparison. The intensity of the red protein spot and its surface area varied depending on the membrane pore size, demonstrating the dependence of protein immobilization on this factor. The membrane with the smallest pore size (M3) showed the highest protein spot intensity and surface area when examined at different protein concentrations. An increase in the applied protein volume showed a linearity proportional trend to the total surface area, and an uneven round dot shape was observed at a large applied volume of protein solution.

1. Wasunna, A.A. (2007). Health Demands in Developing Country. Pennsylvania, USA: Elsevier B.V.

2. Yuan, Z., Chen, W., Zhang, J., Zhang, J., Xiang, T., Hu, J., Wu, Z., Du, X., Huang, A. & Zheng, J. (2012). Development of an immunoassay for differentiating human immunodeficiency virus infections-from vaccine-induced immune response in Tiantan vaccine trials in China. Clin Biochem. 45(15), 1219–1224. DOI: 10.1016/j.clinbiochem.2012.05.013.

3. Ivo dos Santos, J., Galvao-Castro, B., Mello, D.C., Pereira, H.G. & Pereira, M.S. (1987). Dot enzyme immunoassay. A simple, cheap and stable test for antibody to human immunodeficiency virus (HIV). J. Immunol. Methods 99(2), 191–194. DOI: 10.1016/0022-1759(87)90126-8.

4. Attallah, A.M., Osman, S., Saad, A., Omran, M., Ismail, H., Ibrahim, G. & Abo-Naglla, A. (2005). Application of a circulating antigen detection immunoassay for laboratory diagnosis of extra-pulmonary and pulmonary tuberculosis. Clin Chim Acta 356(1–2), 58–66. DOI: 10.1016/j.cccn.2004.11.036.

5. Olsen, S.J., Pruckler, J., Bibb, W., Thanh, N.T.M., Trinh, T.M., Minh, N.T., Sivapalasingam, S., Gupta, A., Phuong, P.T., Chinh, N.T., Chau, N.V., Cam, P.D. & D. Mintz, E. (2004). Evaluation of rapid diagnostic tests for typhoid fever. J Clin. Microbiol. 42(5), 1885–1889. DOI: 10.1128/JCM.42.5.1885-1889.2004.

6. Albertini, A., Ghielmi, S. & Belloli, S. (1982). Structure, immunochemical properties and immunoassay of human chorionic gonadotropin. Ric. Clin. Laborat. 12(1), 289–298. DOI: 10.1007/BF02909335.

7. Debnath, M., Prasad, G.B.K.S. & Bisen, P.S. (2010). Immunoassay. Berlin, Germany: Springer Science+Business Media.

8. O’Sullivan, M.J. (2005). Immunoassays. Berlin, Germany: Springer Science+Business Media.

9. Ciardelli, G., Silvestri, D., Barbani, N., Ionita, M., Redaelli, A. & Giusti, P. (2006). Bioartificial polymer membranes as innovative systems for biomedical or biotechnological uses. Desalination 200(1–3), 493–495. DOI: 10.1016/j.desal.2006.03.408.

10. Aizawa, K. & Gantt, E. (1998). Rapid method for assay of quantitative binding of soluble proteins and photosynthetic membrane proteins on poly(vinylidene difluoride) membranes Anal. Chim. Acta 365(1–3), 109–113. DOI: 10.1016/S0003-2670(97)00670-3.

11. Ebnesajjad, S. (2000). Fluoroplastics. Volume 1: Non-Melt Processible Fluoroplastics The Definitive User’s Guide and Databook. New York, USA: Plastics Design Library.

12. He, Q.H., Xu, Y., Wang, D., Kang, M., Huang, Z.B. & Li, Y.P. (2012). Simultaneous multiresidue determination of mycotoxins in cereal samples by polyvinylidene fluoride membrane based dot immunoassay. Food Chem. 134(1), 507–512. DOI: 10.1016/j.foodchem.2012.02.109.

13. Sulimenko, T. & Dráber, P. (2004). A fast and simple dot-immunobinding assay for quantification of mouse immunoglobulins in hybridoma culture supernatants. J. Immunol. Methods 289(1–2), 89–95. DOI: 10.1016/j.jim.2004.03.010.

14. Low, S.C., Ahmad, A.L., Ideris, N. & Ng, Q.H. (2011). Interaction of isothermal phase inversion and membrane formulation for pathogens detection in water. Biores. Technol 113, 219–224. DOI: 10.1016/j.biortech.2011.11.048.

15. Ahmad, A.L., Ideris, N., Ooi, B.S., Low, S.C. & Ismail, A. (2012). Synthesis of polyvinylidene fluoride (PVDF) membranes for protein binding: Effect of casting thickness. J. Appl. Polym. Sci. 128(5), 3438–3445. DOI: 10.1002/app.38522.

16. Kaur, S., Ma, Z., Gopal, R., Singh, G., Ramakrishna, S. & Matsuura, T. (2007). Plasma-induced graft copolymerization of Poly(methacrylic acid) on electrospun Poly(vinylidene fluoride) nanofiber membrane. Langmuir 23(26), 13085–13092. DOI: 10.1021/la701329r.

17. Nguyen, Q.T., Alaoui, O.T., Yang, H. & Mbareck, C. (2010). Dry-cast process for synthetic microporous membranes: Physico-chemical analyses through morphological studies. J. Mem. Sci. 358(1–2), 13–25. DOI: 10.1016/j.memsci.2010.04.022.

18. El-Sharif, H.F., Stevenson, D., Warriner, K. & Reddy, S.M. (2014) Hydrogel-based molecularly imprinted polymers for biological detection. Berlin, Germany: Springer-Verlag.

19. Morçöl, T. & Subramanian, A. (1999). A red-dot-blot protein assay technique in the low nanogram range. Anal. Biochem. 270(1), 75–82. DOI: 10.1006/abio.1999.4057.

20. Ming, Li, D.L.P., Yvonne Cosgrove-Sweeney, Deena Ratner, Lisa C. Rohan, Alexander M. Cole, Patrick M. Tarwater, Phalguni Gupta and Bharat Ramratnam (2011). Incorporation of the HIV-1 microbicide cyanovirin-N in a food product. J. Acquir. Immune. Defic. Syndr. 58(4), 379. DOI: 10.1097/QAI.0b013e31823643fe.

21. Bannur, S.V., Kulgod, S.V., Metkar, S.S., Mahajan, S.K. & Sainis, J.K. (1999). Protein determination by Ponceau S using digital color image analysis of protein spots on nitrocellulose membranes. Anal. Biochem. 267(2), 382–389. DOI:

22. Yunker, P.J., Still, T., Lohr, M.A. & Yodh, A.G. (2011). Suppression of the coffee-ring effect by shape-dependent capillary interactions. Nature 476(7360), 308–311. DOI: 10.1038/nature10344.

23. Gorr, H.M., Zueger, J.M. & Barnard, J.A. (2012). Characteristic size for onset of coffee-ring effect in evaporating lysozyme-water solution droplets. J. Phys. Chem. B 116(40), 12213–12220. DOI: 10.1021/jp307933a.

24. Norde, W. (1999). Proteins at Solid Surfaces. New York, USA: Marcel Dekker Inc.

25. Giacomelli, C.E. (2006). Adsorption of immunoglobulins at solid-liquid interfaces. Boca Raton, Fla: Taylor & Francis.

26. Nakanishi, K., Sakiyama, T. & Imamura, K. (2001). On the adsorption of proteins on solid surfaces, a common but very complicated phenomenon. J. Biosci. Bioeng. 91(3), 233–244. DOI: 10.1016/S1389-1723(01)80127-4.

27. Norde, W. (1998). Driving forces for protein adsorption at solid surfaces. New York, USA: Marcel Dekker Inc.

28. Liu, F., Awanis Hashim, N., Liu, Y., Moghareh Abed, M.R. & Li, K. (2011). Progress in production and modification of PVDF membranes. J. Mem. Sci. 375(1–2), 1–27. DOI: 10.1016/j.memsci.2011.03.014.

29. Baker, R.W. (2003). Membrane technology. New Jersey, USA: A John Wiley & Sons Publication.

Polish Journal of Chemical Technology

The Journal of West Pomeranian University of Technology, Szczecin

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