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Weiner, J. Register your product at informit. Items related to Microfluidics for Biotechnology, Second Edition. Molecular Biotechnology: Principles and Applications of Recombinant DNA Completely revised and updated, this second edition of the best-selling Molecular Biotechnology covers both the underlying scientific principles and the wide-ranging industrial, agricultural, pharmaceutical, and biomedical applications of recombinant DNA. Quick links.
Micro-Drops and Digital Microfluidics 2nd ed. Has little wear to the cover and pages. Contains some markings such as highlighting and writing.
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May be ex-library. Buy with confidence, excellent customer service!. Items related to Microfluidics for Biotechnology, Second Edition. Microfluidics for Biotechnology, Second Edition. The switching time of the valves can in fact be inferred from Figure 2b iv. In all these experiments, the lower green valve is maintained open. Using an exponential fit, we find a time constant equal to 0.
One may speculate that these switching times can favorably be compared with the characteristic time h 2 D gel Ref. From a practical prospective, the sub-second switching times we find are well adapted to biotechnological applications. Caging is a new functionality that is achieved by patterning hydrogel in the shape of square enclosures, as shown in Figure 3.
The movie presented in Supplementary Information 5 shows the dynamics of opening and closing of such cages. The system is washed by water while fluorescein is isolated in the closed chambers iii. The right figure shows fluorescein white trapped in the circular walls black in water gray. The density is 44 cages per mm 2. Cages can be used to isolate entities, such as molecules, cells see the following section , particles, and deliver them on demand them. For the sake of visualization, this functionality can be illustrated with fluorescein and rhodamine.
The cage walls appearing as red in the figure due to rhodamine labelling are in a low position. Then, the flow is maintained while the temperature is decreased: because the cages are thicker, they appear as dark zones, whereas the premises of photobleaching appear inside the cages as a result of the isolation and immobilization of the fluorescein Figure 3a ii. Finally, Figure 3a iii shows that, after flushing the system, the fluorescein is trapped in the cages. Isolation is efficient because, as shown in Figure 3b , there is no observable fluorescein exchange between the inside and the outside of the cages within the time scale we have considered minutes.
The fluorescein can later be released with further heating shown in Supplementary Information 6. The question of the permeability of the gel barrier is addressed in Figure 3b and developed in Supplementary Information 7. We performed diffusion of fluorescein experiments through the walls of a single rectangular trap. In these experiments, fluorescein is initially outside the wall and we measure the kinetics of diffusion of this solute through the four walls of the cage by tracking the fluorescence intensity level of the fluorescein in the cage as a function of time, averaged over the cage area.
By fitting the curve with a diffusion model Supplementary Information 7 , we find an effective diffusion constant of 2. This experiment shows that barriers can be used as membranes with controlled transport properties, a property potentially interesting for microbiology applications.
The concept of high density, that is, the capacity of the hydrogel technology to operate with a large number of cages, is illustrated in Figure 3c see Supplementary Information 8 for details. Here, we produced a device with circular chambers and perform a series of experiments identical to those in Figure 3a. This density is comparable to the state-of-the-art achieved with PDMS valves and droplet technology.
The figure on the right shows the details of the system: the fluorescein is trapped in 23 cages similarly to Figure a iii. This demonstrates that hydrogel caging technology enables the fabrication of functional high-density devices. In this section, we illustrate two applications of our hydrogel technology.
The first, shown in Figure 4a , is the trapping and release of individual cells. In Figure 4a i , the cage is at an expanded state and several cells accumulate at the wall side exposed to the upstream current near the stagnation point. As the system heats up, the walls collapse and a flow above the walls is generated, freeing the accumulated cells. By lowering the temperature, a single cell can be trapped in the cage Figure 4a ii. This cell is released upon further heating Figure 4a iii.
Although the protocol is manual, it nonetheless illustrates the potential of the technology regarding capture and release of individual cells.
In i , the cage is closed and a suspension of circulating B cells is introduced into the device. In ii , the cage walls are lowered, then raised, to capture an individual cell noted by the white arrow In iii , the cage is lowered again to release the cell. In practice, the fluorescence intensity is measured in two situations: cages filled with the mix and the DNA sample positive , and cages filled with the mix and water instead of DNA control , keeping the same mix concentration for the two experiments.
The signal is provided by a single cage with the intensity being integrated over the cage area. We checked that all the cages behave the same. The application of our hydrogel technology to isothermal amplification and NAAT is illustrated in Figure 4b. The goal of the experiment illustrated here is to detect its presence.
The image shows that the gene is amplified homogeneously in each cage. The presence of the hydrogel is not detrimental to RCA amplification. Figure 4b ii shows a typical amplification curve along with a negative control containing no DNA. In the control experiment, a small fluorescence signal is produced by the probes. However, the signal over noise ratio remains important.
From a NAAT prospective, one can conclude from the inspection of Figure 4b that the gene is present in the sample. This system illustrates the diagnostic capability of our technology. Two applications of this technology were illustrated in the domain of single cell handling and NAAT for the Human Synaptojanin 1 gene. In this case, we can reach a density of 1 million valves or cages per cm 2. With cages actuated independently, the situation is more challenging, but densities of thousands of cages per cm 2 —still a high throughput—can certainly be envisioned.
Caging functionality allows the trapping of entities under high throughput conditions similar to droplet-based technology. However, since gels are permeable, hydrogel walls can also operate as membranes, allowing the exchange of small molecules between the sequestered entity for example, cells and the external medium. From a general perspective, there is room to extend or diversify the range of functionalities of our hydrogel technology by varying the gel chemistry, using different stimuli for example, light, pH , developing different behaviors LCST or UCST , or grafting molecules, exploiting specific affinities for analytical purposes.
The biotechnological applications of our responsive hydrogel-based technology are numerous. Digital amplification is an example. In our case, thousands of cages can be integrated on a single system so that high throughput screening or high sensitivity analyses of samples are feasible and with direct readout. Single-cell manipulation, stimulated by a growing recognition of the important role that play genomic heterogeneities in biological systems, provides another example of the applications of our technology. Microbiology is another field where thermo-sensitive hydrogels with well-controlled chemistry offer potential advantages.
In this case, the permeability of the gel can be harnessed to deliver therapeutic molecules to cells trapped in cages to screen antibiotics.
In addition, most importantly, these applications can be developed under interesting cost conditions because the materials are inexpensive and actuation requires low voltage sources or standard hot plates. A gas chromatographic air analyzer fabricated on a silicon wafer. A review of microvalves.
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