Use of Ice-Nucleating Proteins To Improve the Performance of Freeze–Thaw Valves in Microfluidic Devices

Abstract

Currently, reliable valving on integrated microfluidic devices fabricated from rigid materials is confined to expensive and complex methods. Freeze-thaw valves (FTVs) can provide a low cost, low complexity valving mechanism, but reliable implementation of them has been greatly hindered by the lack of ice nucleation sites within the valve body's small volume. Work to date has required very low temperatures (on the order of -40 °C or colder) to induce freezing without nucleation sites, making FTVs impractical due to instrument engineering challenges. Here, we report the use of ice-nucleating proteins (INPs) to induce ice formation at relatively warm temperatures in microfluidic devices. Microfluidic channels were filled with buffers containing femtomolar INP concentrations from Pseudomonas syringae. The channels were cooled externally with simple, small-footprint Peltier thermoelectric coolers (TECs), and the times required for channel freezing (valve closure) and thawing (valve opening) were measured. Under optimized conditions in plastic chips, INPs made sub-10 s actuations possible at TEC temperatures as warm as -13 °C. Additionally, INPs were found to have no discernible inhibitory effects in model enzyme-linked immunosorbent assays or polymerase chain reactions, indicating their compatibility with microfluidic systems that incorporate these widely used bioassays. FTVs with INPs provide a much needed reliable valving scheme for rigid plastic devices with low complexity, low cost, and no moving parts on the device or instrument. The reduction in freeze time, accessible actuation temperatures, chemical compatibility, and low complexity make the implementation of compact INP-based FTV arrays practical and attractive for the control of integrated biochemical assays.

Psychrophilic lifestyles: mechanisms of adaptation and biotechnological tools

“…INPs are presently being commercialised for artificial snow production (Cochet and Widehem 2000) but are believed to have a number of other potential applications. These include, reducing freezing energy costs in the food industry (Li et al 1997), freeze-concentrating beverages, in freeze-thaw valves of microfluidic devices (Gaiteri et al 2017), as anchoring motifs for cell surface display applications (Jung et al 1998), as well as in cloud seeding for climate control (Pummer et al 2015).…”

Overcoming bottlenecks for in vitro synthesis and initial structural insight of ice nucleating protein InaZ

“…For industry, the bacterium Pseudomonas syringae that possesses InaZ on its 188 outer layer has been utilized and is known as the best ice-inducing agent for decades in the snow-making 189 industry [28]. Partially purified InaZ from native sources has also been more recently demonstrated to 190 perform reliable freeze-thaw valving in the microfluidic devices, providing also a reduction in cost and 191 design complexity of these elements [29].…”

Recent advances and challenges in temperature monitoring and control in microfluidic devices

“…To achieve this, microfluidic devices also require temperature control capabilities, which can be obtained through heating and cooling instruments [43]. Temperature control-described in terms of field profile (i.e., temporal and spatial distribution) or range (i.e., all temperature values between the minimum and maximum exhibited by the field)-is mostly necessary in three specific cases: (1) to lower the temperature of the liquid when it rises unwantedly as a consequence of the phenomena taking place during a microfluidic experiment [7], (2) when the application requires to cool down the sample [44], and (3) when the application requires to heat up the sample [45]. The control of temperature gradients is also crucial for the performance of microfluidic devices [46,47].…”

“…Temperature control in microfluidics has been explored in applications, including cell culture [ 49 , 50 , 51 , 52 ], cell imaging [ 53 ], cell analysis [ 54 , 55 , 56 ], PCR [ 22 , 57 – 60 ], nucleic acid amplification test assays [ 61 ], microreactors [ 62 ], transparent electrodes [ 63 , 64 ], enzyme‐linked immunosorbent assay [ 44 ], biochemical synthesis [ 65 ], particle trapping [ 66 ], and microelectronic device cooling [ 67 ], among others [ 68 , 69 ]. For this, the use of microheaters [ 52 , 55 – 57 , 62 , 70 , 71 ], Peltier elements [ 25 , 58 , 59 , 72 ], two‐phase materials [ 73 ], laser beams [ 74 ], and cement power resistors [ 60 ] has been reported.…”

“…A similar method was used by Vloemans et al, by exploiting the exothermic crystallization reaction caused by a mixture of supercooled SAT with water to modulate the temperature between 37 and 42 • C[73]. Furthermore, Gaiteri et al utilized femtomolar concentrations from Pseudomonas syringae (PSy) proteins to control the temperature in a freeze-thaw valves microfluidic system[44]. The PSy produces ice-nucleating proteins (INPs) that mimic the lattice structure of ice and, therefore, trigger freezing at higher temperatures.…”