Former PhD student of Prof Paunov Pietro graduated in 2005 and was last known to work as post-doctoral researcher at the University of Leeds. PhD supervisors: Prof Vesselin Paunov (PI) and Prof Paul Fletcher (Co-PI)

Research project 1 (PhD thesis):

Fabrication of monosisperse giant liposomes on solid substrates by microcontact printing

We have designed a novel method for the preparation of relatively monodisperse giant liposomes using a combination of microcontact printing of lipid ink on electrode surfaces with the well-known electroformation procedure (see Fig. 1). The mechanism of producing liposomes of similar size relies on multiple coalescence of growing liposomes within the patches of lipid deposit on the electrode surface produced by microcontact printing technique. The method was found to work well for DMPC but we expect that it is also applicable for the production of monodisperse vesicles of other materials, including polymerosomes [1,2].

Fig. 1 The preparation of monodisperse giant liposomes by micro-contact printing technology involves the following stages: (A) and (B) the lipid (stained with a fluorescent dye) is deposited on an ITO glass by using a PDMS stamp of square grid pattern. (C) The liposomes are grown on the ITO slide in water by electroformation in AC field. (D) The monodisperse liposomes are released from the ITO slide after application of ultrasound (ultrasonic bath) 2].

The mechanism of formation of liposomes of similar size on each of the square patches of lipid deposit was revealed by observing the growth of the liposomes. If was found that initially a number of smaller polydisperse liposomes sprout on each square patch of lipid and they coalesce with each other as they grow within this limited space. Finally, only one giant liposome per lipid patch (square) survives, and since all patches of lipid on the patterned surface grow vesicles at the same conditions, this results in an array of attached liposomes of relatively narrow size distribution. Since the coalescence between liposomes happen when they are compressed against each other within a limited space, it is essential that the lipid patches are spaced from each other by a distance sufficient to prevent the coalescence of neighbouring liposomes. The latter mechanism becomes possible since the liposomes do not detach spontaneously from the lipid patches on the surface. However, by raising the pH and/or adding charged lipid (e.g. phosphatidylglycerol) to the lipid ink we encountered conditions where liposomes detach early in the process of their electroformation. This does not produce samples of relatively narrow size distribution. In a separate experiment, we also left the liposomes on the patterned surface to grow to a size bigger than the distance between the squares which lead to coalescence between liposomes originating from different lipid patches and the formation of a polydisperse sample of liposomes. As such extended coalescence progresses, the biggest liposomes reach a size of over 100 μm at which they become unstable and break up. These observations serve to emphasise that the growth process should be stopped well before such overlapping occurs in order to produce a liposome population of narrow size distribution (Fig. 2).

Fig. 2 (A) Microcontact printing of ITO glass surface with a hydrophilised PDMS stamp. The array of square phospholipid patches is imaged by fluorescent microscopy. (B) ITO surface during AC field application in 0.1865 M glucose solution (pH 5.76). Liposomes are seen to sprout on the square pattern after 1.5 h of application of AC field. (C) Liposomes detach after application of ultrasound [2].

References

1. Taylor, P., Xu, C., Fletcher, P.D.I., Paunov, V.N., “Fabrication of 2D Arrays of Giant Liposomes on Solid Substrates by Microcontact Printing”, PCCP, 5 (2003) 4918-4922.
2. Taylor, P., Xu, C., Fletcher, P.D.I., Paunov, V.N., “A Novel Technique for Preparation of Monodisperse Giant Liposomes”, Chem. Comm., 14 (2003) 1732-1733.

  

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 Research project 2:

Micro-contact printing of DNA-surfactant arrays on solid substrates

We have designed a novel method for fabrication of DNA arrays based on microcontact printing of DNA surfactants on solid substrates [1]. DNA-surfactants were prepared by covalent attachment of a hydrophobic anchoring group to the (3’- or 5’-) end of DNA oligonucleotides (see Fig. 1). This anchoring group allows DNA-strands to be immobilised on hydrophobic surfaces by hydrophobic interactions. The microcontact printing method was adapted for aqueous ‘‘inks’’ containing DNA-surfactants. Special attention was paid to the wetting properties of the ink with respect to the stamp and the solid substrates (see Fig. 2). The method allows for efficient attachment of DNA strands to solid surfaces and hybridisation with complementary DNA strands. This new technology could be utilised for rapid preparation of DNA-assays and genetic biochips.

Fig.1 A DNA-surfactant consists of a hydrophobic chain and hydrophilic oligonucleotide [1].

Fig. 2 Scheme of the preparation ofDNA micro-arrays bymicrocontact printing with DNA-surfactant ink on solid substrates. (A) Synthesis of DNA-surfactant. (B) Inking of the PDMS stamp with an aqueous solution of DNA-surfactant. (C) Printing of the pattern on to solid substrate. (D) Anchoring of the DNA strand by hydrophobic interaction of the surfactant ‘‘hydrophobic tail’’ with the solid substrate in water [1].

The major achievement of this work is the demonstration that DNA-surfactants can be deposited and immobilised on suitable solid surfaces by hydrophobic interactions and the produced pattern is stable under the conditions of hybridisation with complementary TAMRA-DNA, thus producing a readable signal upon fluorescence (Fig.3). Hence, only the way of dispensing the DNA-surfactant ink (in this paper—microcontact printing) determines whether the DNA array will consist of similar or different DNA spots on the solid surface.

Fig. 3 Fluorescent micrograph of cholesteryl-5’-TTTTTTCCCCCC-3’ (from 1.5 mM aqueous solution) printed on a glass cover slip, blocked with BSA and exposed to (A) complementary TAMRA-5’-GGGGGGAAAAAA-3’ and (B) non-complementary TAMRA-DNA (TAMRA-5’-GAATCCTACC-3’). The samples are washed with buffer before imaging with an exposure time of 156 ms and binning 4 x 4. Bars are 100 mm [1].

References

1. Xu, C., Taylor, P., Ersoz, M., Fletcher, P.D.I., Paunov, V.N., “Microcontact Printing of DNA-Surfactant Arrays of Solid Substrates”, J. Mater. Chem., 10 (2003) 3044-3048.