Stick insects

But adhesives that work in water do exist. Many aquatic organisms stick themselves to wet, contaminated surfaces, or glue together protective shelters under water.

‘The problem of gluing things together underwater has been solved over and over in nature, in many different ways,’ says Russell Stewart, a bioengineer at the University of Utah, US. ‘Understanding the composition, packaging and chemical mechanisms of natural adhesives is a good starting point for engineering practical and cost-effective synthetic underwater adhesives,’ he says.

The marine sandcastle worm, for example, uses its own glue to stick together tubes from sand and bits of shells, creating reefs reminiscent of sandcastles. Freshwater caddisfly larvae, meanwhile, produce a sticky underwater silk to tape together similar structures.

Sandcastle worms (Phragmatopoma californica) live in the sea on the shoreline of California, US. Although only 2.5cm long, the worm builds tubes several times this length. By sticking its tentacles out from one end of the tube, the worm gathers building materials (and food). Tiny hairs on the tentacles brush the particles into the reach of the worm’s pincer-like ‘building organ’.

The worm secretes two dabs of glue onto a particle, explains Stewart, then the building organ puts the particle onto the end of the tube and holds it there. The glue hardens within 30s after the worm secretes it.

Stewart has discovered the worm glue consists primarily of oppositely charged components packed in ‘granules’ in at least four different cell types of an ‘adhesive gland’. For example, some granules contain sulfated macromolecules paired with polycationic proteins; others contain polyphosphorproteins paired with divalent cations such as calcium and magnesium.¹ All the granules also contain the enzyme catechol oxidase.

The contents of the granules appear to mix shortly after secretion; the enzyme catalysing the oxidative crosslinking of the amino acid L-DOPA dihydroxyphenylalanine to form a glue. Seawater has a significantly higher pH and ionic strength than the internal environment and it could be this jump in pH upon secretion that plays a key role in the setting and hardening reactions, he believes.

Stewart’s synthetic version of the glue uses water-soluble polyacrylates in place of the charged proteins as these are easier to synthesise. ‘We made polymers with side chains that mimicked the positive and negative charges in the worm glue,’ Stewart says. These side chains include the catecholic amino acid DOPA, found in high concentrations in mussel adhesive proteins.

When the polymers are mixed in a test tube, electrostatic charges between the polymers are neutralised, displacing small counterions in water. A dense fluid, called a ‘complex coacervate’, condenses out of the solution and sinks to the bottom of the test tube. As polymer side chains attach to each other, they form chemical bonds that make the ‘glue’ harden.

Complex coacervates are ideal as the basis for injectable, water-borne, non-toxic, wet tissue adhesives, says Stewart. They self-organise in water from pre-polymers so no toxic solvents, or exothermic polymerisation reactions, are required. Because they are denser than water, and blood, they sink onto the substrate and displace lighter fluids. They spread easily on submerged hydrophilic surfaces, which maximises adhesive contact. Just as the natural sandcastle glue does not dissolve into the sea before setting, complex coacervate glues do not mix with physiological fluids, including blood and amniotic fluid, before setting. And since the synthetic glue doesn’t mix with water, it doesn’t swell. This is critical for a tissue adhesive inside the body.

The synthetic glue is five to 10 times stronger than fibrin glues, but weaker than cyanoacrylates, and much weaker than metal pins and screws, says Stewart. But he believes this glue could have many applications, including use in foetal surgery. Ruptures of the sac containing the developing baby can occur when surgeons operate on a foetus, or check the foetus for problems. Leaks or ruptures often lead to premature births.

‘There are currently no effective treatments for preventing foetal membrane rupture caused by “in-utero” surgery,’ says Stewart. ‘Using glue in the fluid-filled uterus is challenging. Our adhesives have ideal properties for this challenging environment. The evidence so far – including preliminary animal experiments – suggests that our approach is promising. With further development, I am confident we will be able to seal foetal membranes after in-utero foetal surgeries.’

The synthetic glue could also be used to repair small bone fragments in fractured joints such as knees, wrists, elbows, ankles, and also in the face and skull. The glue could hold the pieces in alignment until they heal, which is difficult to do with screws and wires that require drilling.

Stewart’s team is also working with caddisflies, specifically the Brachycentrus echo larvae, which live around the world in waters, ranging from fast streams to quiet marshes. Like silkworm moths and spiders, caddisfly larvae spin silk, but they do so underwater.

The Brachycentrus echo larvae build a case, using the sticky silk and grains of sand or rock, and then drag it along underwater while foraging; other species build shelters glued to a rock, with a silk net to catch passing food. All caddisfly larvae, however, have an organ called a spinneret where the products of two silk glands converge.

This spinneret produces the silk adhesive, which the larvae weaves back and forth around sand grains, sticks or leaf pieces to create tubes, of the order of about one inch long. The adhesive can also bond to a wide range of surfaces underwater.

The researchers analysed the adhesive silk using several methods, including scanning electron microscopy,² and found it to be similar to that made by silkworm moths and spiders, ie comprising fibroin proteins, but differing in once crucial aspect. In caddisflies, the serines – amino acids that make up a fifth of the amino acids in fibroin – are phosphorylated. Phosphates are well-known adhesion promoters used in dental fixtures.

The phosphates attached to the serines are negatively charged while other amino acids in the protein are positively charged. They are all water soluble, but when the chains of proteins – each with alternating regions of positive and negative charges – line up in parallel with opposite charges attracting each other, they become insoluble, the team says.

According to Stewart: ‘Both the worm glue and the caddisfly silk comprise polyelectrolytic macromolecules. Both are processed as complex fluids that may be something like complex coacervates into their final form. But the processes involved are different.’

Stewart and his group plan to study the strength of the caddisfly silk next. ‘Individual threads aren’t very strong, but it lays down dozens of them. If we can copy this material and make tape out of it, the bond strength would go up dramatically,’ he says, reasoning that a synthetic version would be used like a pressure-sensitive tape instead of dabs of glue. ‘I picture a sort of a wet Band-Aid [plaster], maybe used internally in surgery – like using a piece of tape to close an incision as opposed to sutures.’

Barnacles stick to pretty much anything – crabs, whales, boats, rocks – in shallow and tidal waters all over the world. Once a barnacle larva has fixed onto something, it’s generally there for the rest of its life, about five to 10 years on average, despite varied and often hostile conditions.

‘It’s over 150 years since Darwin first described the cement glands of barnacle larvae,’ says Nick Aldred of the School of Marine Science and Technology at Newcastle University, UK. But while researchers have known for some time that the barnacle glue contains two components, until now they had thought it behaved like some synthetic glues, ie they mix first and then harden. Now they believe this is not the case.

Advances in imaging techniques, such as 2-photon microscopy, have allowed the Newcastle team, working with others from Clemson University, South Carolina, US; the National Institutes of Standards and Technology in Maryland, US; and Duke University, US, to observe the adhesion process in real time and characterise the two components. ‘In the past, the strong lasers used for optically sectioning biological samples have typically killed the samples, but with these new tools we are able to study processes in living tissues as they happen,’ Aldred explains.

The team observed two materials of differing composition in the cement glands and after release to the surface, reports Aldred.³ ‘That is, the two components are kept separate in the body of the organism and remain separate post-release’, he says. The researchers believe the two substances play very different roles: the first to be secreted clears water from the surface and the second cements the barnacle down.

The final-stage larva – the cyprid, which is unique to barnacles – selects a surface and then releases the first substance, a lipid-rich droplet. Their theory is that the oily material displaces water from the surface, making it easier for the second substance, a phosphoprotein adhesive, to latch on. The lipid material may also help the protein spread out, and protect it from bacterial degradation. Aldred believes the lipid in the cyprid cement has not been observed anywhere previously.

This discovery will change the way researchers think about developing bio-inspired adhesives, he insists. ‘What makes barnacles particularly interesting is that their adhesive mechanisms seem to be very different to other organisms, such as sandworms, caddis flies and mussels. They therefore have potential to provide inspiration beyond the DOPA and or phosphoserine-based glues that have been described from mussels and tube worms. Barnacle adhesive is also among the strongest, if not the strongest.’

Ultimately, the researchers want to make a synthetic version of the barnacle glue, but it is unlikely that a final product would be a direct mimic, says Aldred. ‘In the end, a bio-inspired glue will likely take on elements from the glues of multiple organisms. What makes this easier is that many unrelated organisms have evolved similar solutions. There seems to be significant convergent evolution in the “design” of biological adhesives, for example, the ubiquitous presence of DOPA and/or phosphoserine.’

A team from the Massachusetts Institute of Technology (MIT) in the US claims to have made the strongest biologically-inspired, protein-based, underwater adhesive to date, using mussel foot proteins as its starting point.⁴ The first adhesive protein was isolated from the foot of the common blue mussel by Herbert Waite at the University of Delaware, US, in the 1980s. Since then other scientists have engineered E. coli bacteria to produce individual mussel foot proteins, but these materials do not capture the complexity of the natural adhesives, says MIT’s Timothy’s Lu.

Instead, the MIT team engineered bacteria to produce a hybrid material that incorporates one of two mussel proteins as well as a bacterial protein found in biofilms (curli fibres). When combined, these proteins form dense, fibrous meshes that bind strongly to dry and wet surfaces, even more strongly than the real thing, the team says.

Using this technique, the researchers can produce only small amounts of the adhesive, so they are now trying to improve the process and generate larger quantities. They also plan to experiment with adding some of the other mussel foot proteins. ‘We’re trying to figure out if by adding other mussel foot proteins, we can increase the adhesive strength even more and improve the material’s robustness,’ Lu says.

The prospect of putting a plaster on wet skin could soon be in place and no longer be a mere pipedream.

1 C. S. Wang and R. J. Stewart, Biomacromolecules, 2013, 14(5),1607
2 R. J. Stewart et al, Insect Biochem. Mol. Biol., 2014, 54C, 69
3 N. V. Gohad et al, Nature Communications, doi:10.1038/ncomms5414
4 Timothy Lu et al, Nature Nanotechnology, doi:10.1038/nnano.2014.199