Screening of molluscan extrapallial proteins on CaCO3 crystallisation via microfluidics

Many living systems produce mineral materials via organic-inorganic interactions; through a process known as biomineralisation. The formation of all biomineral structures is under exquisite biological control, concerning crystal morphology, polymorph selection and crystal orientation. The common blue mussel Mytilus edulis produces a shell structure composed of heterogeneous calcium carbonate polymorphs: an outer layer of prismatic calcite and an inner layer of aragonite nacre. The extrapallial (EP) fluid, confined to the space between the organic mantle and inner shell, is considered to be a key participant during shell growth. The existence of both organic components i.e. proteins, glycoproteins and inorganic ions in the extrapallial fluid supports such a hypothesis of functional involvement. This study screens the influence of extrapallial (EP) proteins from M. edulis on in vitro crystallisation in the laminar flow microfluidic system. In laminar flow microfluidic systems, mass exchange between adjacent streams is driven by diffusion. This principle provides opportunities to simultaneously screen protein influences in a range of scenarios by mixing with different reagents. In addition, the combination of computational modelling and real-time crystallisation demonstrates the major influence of the microenvironment on crystal formation along microfluidic channels. The simulation of protein and ion concentration profiles, as well as the supersaturation ratio, contributes to our understanding of protein influence on crystal morphology and polymorph control. In order to identify the influence of EP proteins on crystallisation, the total wild-type extrapallial (TWEP) proteins were initially used to produce oval calcite crystals. For further investigation, individual purified proteins were used to modify crystallisation, including the wild-type proteins directly extracted from living mussels and the expressed proteins provided from an E.coli expression system. Novel lemon-shaped structures precipitated in the microfluidic channel when the main wild-type 28 kDa extrapallial protein was mixed with CaCl2 solution only. Similar structures were also generated in microfluidic channel with the expressed proteins in either CaCl2 solution only or both reagent solutions. Multilayer calcite structures were induced in the microfluidic channel in the presence of biomineral proteins, mixed with Na2CO3 solution only. All of these results suggest that the extrapallial proteins influence CaCO3 crystallisation. Microcontact printing (μCP) has been used to create two-dimensional protein and polymer patterns for in vitro crystallisation. Polyacrylic acid (PAA) has been used as polymer patterns to control CaCO3 crystal formation, including precipitation and morphology. Calcite crystals, composed of nano-blocks, are the only structures precipitated in the patterned regions while PAA and calcium ions are both printed on the substrates. Although encouraging, further work is required to fully establish the protocol for protein patterning as a means of screening biomineral protein function.