Bioimprint – Photo nanoimprint lithography of biological samples


BNN Members involved:

Maan Alkaisi
John Evans
Volker Nock

Lynn Murray

Fahmi Samsuri
James Muys


Detection of subtle dff erences in cell surfaces and morphology  that  may  be  indicative  of  diseases  and  abnormalities such as cancer [1] has potential for the early diagnosis and treatment of disease and for use in biological studies. Accurate discrimination of such changes by optical microscopy remains a major challenge [2]. High-resolution microscopic techniques such as atomic force microscopy (AFM)  are much better suited to such analyses as they enable three-dimensional (3D) nanometer resolution. However, they have the major disadvantage of requiring high scanning tip forces that can damage the sensitive soft biological tissue. Blunter tips have been used to minimize the damage but they result in greatly diminished resolution [3]. Furthermore, scanning using a tip in an aqueous environment can cause structural movement and loss of resolution due to damping effects [4].

However, it has been predicted that AFM imaging of cells in liquid should be capable of resolutions of 50–500 nm regardless of whether the cells are living or fixed [5,6].  Time-consuming preparation procedures when using either air or vacuum environments associated with TEM have been a further limitation which is exacerbated by the need for dehydration and  fixation, which can deform the cells and introduce artifacts in the imaging process [7].  Such nanoscale imaging tools have therefore not been widely used in life science applications. Whilst polymers have been used in the imprinting of yeasts for quartz crystal microbalance sensors  [8,9], they have only recently been applied to nanoscale imaging.

To overcome these imaging challenges, a polymer-based approach has been recently developed whereby the cells are coated with a monomer mixture, which is then polymerized over the cell surfaces [10–12] in a positive soft lithography technique [13].  By careful selection  of  the  monomers  it is  possible  to  obtain  an  imprint  of  the  cellular  surface features in the polymer once the cellular material has been washed away from the replica. This imprint replica can then be imaged using high-resolution techniques such as AFM without the concerns of cellular damage. This approach has come to be known as a “Bioimprint” technique [11,14]. When combined with AFM imaging, continuous sampling to capture snapshots of biological events and monitoring of cell conditions can be achieved to allow studies of cellular structure and cell response to physiological and noxious stimuli. Furthermore, such techniques have potential as biomedical procedures to form 3D biocompatible and bioactive scaffolds for tissue culture.

Previous work on obtaining Bioimprints has utilized the nonbiohazardous poly(dimethylsilxoane) (PDMS) polymer composite [10,11] and applied this to endometrial cancer cells [14]. This has allowed high-resolution imaging of membrane morphological  structures consistent with exocytosis. This technique utilized thermal setting of the polymer [11] and so had the disadvantage of exposing cells to very high temperatures  for  some  minutes, followed by another curing step taking hours to complete. A UV-cured Bioimprint technique was then adopted by photocuring a siloxane copolymer to visualize pituitary cells [12]. However, there were some significant challenges to overcome with this approach, namely, distorted or permeation artifacts resulting from prolonged curing time, cell  dehydration effects, an irradiation time of several minutes which had the potential to  induce  alterations in cell characteristics, and the large number of curing steps that were required to complete the process. Use of methacrylic acid/ethylene glycol dimethacrylate copolymer has the advantages that the polymer will set under UV irradiation within seconds [15] if applied at appropriate volumes and polymerizes smoothly around surface features, making it potentially useful for application in Bioimprinting of nanoscale features. Furthermore, it can be spin-coated onto immobilized cells to produce polymer layers of various thicknesses.

While previous work has investigated the Bioimprint for high-resolution imaging and cell analysis applications, recent investigations center on using the Bioimprint as an independent cell culture substrate.

[1]  G. N. Papanicolaou and H. F. Traut, “The diagnostic value of vaginal smears in carcinoma of the uterus,” American Journal of Obstetrics & Gynecology, vol. 42, no. 2, pp. 193–206, 1941.
[2]  L.  Hamby,  “Gene  expression  patterns  and  breast  cancer,” Cancer Genetics News, vol. 4, p. 1, 2002.
[3]  H. X. You and L. Yu, “Atomic force microscopy imaging of living cells: progress, problems and prospects,” Methods in Cell Science, vol. 21, no. 1, pp. 1–17, 1999.
[4]  W. H¨aberle, J. K. H. H¨orber, and G. Binnig, “Force microscopy on living cells,” The Journal of Vacuum Science & Technology B, vol. 9, no. 2, pp. 1210–1213, 1990.
[5]  M. Fritz, M. Radmacher, and H. E. Gaub, “Granula motion and membrane spreading during activation of human platelets imaged by atomic force microscopy,” Biophysical Journal, vol. 66, no. 5, pp. 1328–1334, 1994.
[6]  M. Radmacher, “Measuring the elastic properties of biological samples  with  the  AFM,”  IEEE Engineering in Medicine and Biology Magazine, vol. 16, no. 2, pp. 47–57, 1997.
[7]  A.  Garg  and  E.  Kokkoli,  “Characterizing  particulate  drug-delivery carriers with atomic force microscopy,” IEEE Engineering in Medicine and Biology Magazine, vol. 24, no. 1, pp. 87–95, 2005.
[8]  F. L. Dickert and O. Hayden, “Bioimprinting of polymers and sol-gel  phases.  Selective  detection  of  yeasts  with  imprinted polymers,” Analytical Chemistry, vol. 74, no. 6, pp. 1302–1306, 2002.
[9]  K.  Seidler,  P.  A.  Lieberzeit,  and  F.  L.  Dickert,  “Application of  yeast  imprinting  in  biotechnology  and  process  control,” Analyst, vol. 134, no. 2, pp. 361–366, 2009.
[10]  M.  M.  Alkaisi,  J.  J.  Muys,  and  J.  J.  Evans,  “Bioimprint replication  of  single  cells  on  a  biochip,”  in  BioMEMS and Nanotechnology III, vol. 6799 of Proceedings of SPIE, Canberra, Australia, December 2007.
[11]  J. J. Muys, M. M. Alkaisi, and J. J. Evans, “Nanoscale analysis by replication of cellular topography using soft lithography,” Journal of Biomedical Nanotechnology, vol. 2, no. 1, pp. 11–15, 2006.
[12]  J. J. Muys, M. M. Alkaisi, and J. J. Evans, “Cellular replication and atomic force microscope imaging using a UV-bioimprint technique,” Nanomedicine, vol. 2, no. 3, pp. 169–176, 2006.
[13]  G. M. Whitesides, E. Ostuni, S. Takayama, X. Jiang, and D. E.  Ingber,  “Soft  lithography  in  biology  and  biochemistry,” Annual Review of Biomedical Engineering, vol. 3, pp. 335–373, 2001.
[14]  J.  J.  Muys,  M.  M.  Alkaisi,  D.  O.  Melville,  et  al.,  “Cellular transfer and AFM imaging of cancer cells using Bioimprint,” Journal of Nanobiotechnology, vol. 4, article 1, 2006.
[15]  R. H. Schmidt, K. Mosbach, and K. Haupt, “A simple method for  spin-coating  molecularly  imprinted  polymer  films  of controlled thickness and porosity,” Advanced Materials, vol. 16, no. 8, pp. 719–722, 2004.


Relevant Publications:


L.M. Murray, V. Nock, M.M. Alkaisi, J.J.M. Lee, and T.B.F. WoodfieldFabrication of polymeric substrates with micro- and nanoscale topography bioimprinted at progressive cell morphologiesJournal of Vacuum Science and Technology B: Microelectronics and Nanometer Structures: Processing, Measurement, and Phenomena3006F9022012paper
V. Nock, L. Murray, F. Samsuri, M. M. Alkaisi, and J. J. EvansMicrofluidic arrays for bioimprint of cancer cellsMicroelectronic Engineering88 (8)814-8162011paper
F. Samsuri, M. M. Alkaisi, J. J. Evans, K. Chitcholtan, and J. S. MitchellDetection of changes in cell membrane structures using the Bioimprint techniqueMicroelectronic Engineering88 (8)1871-18742011paper
V. Nock, L. Murray, F. Samsuri, M. M. Alkaisi and J. J. EvansMicrofluidics-assisted photo nanoimprint lithography for the formation of cellular bioimprintsJournal of Vacuum Science and Technology B28 (6)C6K17-C16K222010paper
M. M. Alkaisi, J. J. Muys, and J. J. EvansSingle cell imaging with AFM using Biochip/Bioimprint TechnologyInternational Journal of Nanotechnology6 (3-4)355-3682009paper
F. Samsuri, J. S. Mitchell, M. M. Alkaisi, and J. J. EvansFormation of Nanoscale Bioimprints of Muscle Cells Using UV-Cured Spin-Coated PolymersJournal of Nanotechnology5934102009paper
J. J. Muys, M. M. Alkaisi, and J. J. EvansCellular replication and atomic force microscope imaging using a UV-Bioimprint techniqueNanomedicine: Nanotechnology, Biology and Medicine2 (3)169-1762006paper
J. J. Muys, M. M. Alkaisi, D. O. S. Melville, J. Nagase, P. Sykes, G. M. Parquez, and J. J. EvansCellular transfer and AFM imaging of cancer cells using BioimprintJournal of Nanobiotechnology4 (1)2006paper
J. J. Muys, M. M. Alkaisi, and J. J. EvansBioimprint: Nanoscale Analysis by Replication of Cellular Topography Using Soft LithographyJournal of Biomedical Nanotechnology2 (1)11-152006paper