An integrated microfluidic device for monitoring changes in nitric oxide production in single T-lymphocyte (Jurkat) cells.

A considerable amount of attention has been focused on the analysis of single cells in an effort to better understand cell heterogeneity in cancer and neurodegenerative diseases. Although microfluidic devices have several advantages for single cell analysis, few papers have actually demonstrated the ability of these devices to monitor chemical changes in perturbed biological systems. In this paper, a new microfluidic channel manifold is described that integrates cell transport, lysis, injection, electrophoretic separation, and fluorescence detection into a single device, making it possible to analyze individual cells at a rate of 10 cells/min in an automated fashion. The system was employed to measure nitric oxide (NO) production in single T-lymphocytes (Jurkat cells) using a fluorescent marker, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA). The cells were also labeled with 6-carboxyfluorescein diacetate (6-CFDA) as an internal standard. The NO production by control cells was compared to that of cells stimulated using lipopolysaccharide (LPS), which is known to cause the expression of inducible nitric oxide synthase (iNOS) in immune-type cells. Statistical analysis of the resulting electropherograms from a population of cells indicated a 2-fold increase in NO production in the induced cells. These results compare nicely to a recently published bulk cell analysis of NO.

CLIPB4 is a central node in the protease network that regulates humoral immunity in Anopheles gambiae mosquitoes.

Insect humoral immune responses are regulated in part by protease cascades, whose components circulate as zymogens in the hemolymph. In mosquitoes, these cascades consist of clip domain serine proteases (cSPs) and/or their non-catalytic homologs (cSPHs), which form a complex network, whose molecular make-up is not fully understood. Using a systems biology approach, based on a co-expression network of gene family members that function in melanization and co-immunoprecipitation using the serine protease inhibitor (SRPN)2, a key negative regulator of the melanization response in mosquitoes, we identify the cSP CLIPB4 from the African malaria mosquito Anopheles gambiae as a central node in this protease network. CLIPB4 is tightly co-expressed with SRPN2 and forms protein complexes with SRPN2 in the hemolymph of immune-challenged female mosquitoes. Genetic and biochemical approaches validate our network analysis and show that CLIPB4 is required for melanization and antibacterial immunity, acting as a prophenoloxidase (proPO)-activating protease, which is inhibited by SRPN2. In addition, we provide novel insight into the structural organization of the cSP network in An. gambiae, by demonstrating that CLIPB4 is able to activate proCLIPB8, a cSP upstream of the proPO-activating protease CLIPB9. These data provide the first evidence that, in mosquitoes, cSPs provide branching points in immune protease networks and deliver positive reinforcement in proPO activation cascades.

CLIPB4 Is a Central Node in the Protease Network that Regulates Humoral Immunity in Anopheles gambiae Mosquitoes.

Insect humoral immune responses are regulated in part by protease cascades, whose components circulate as zymogens in the hemolymph. In mosquitoes, these cascades consist of clip-domain serine proteases (cSPs) and/or their non-catalytic homologs, which form a complex network, whose molecular make-up is not fully understood. Using a systems biology approach, based on a co-expression network of gene family members that function in melanization and co-immunoprecipitation using the serine protease inhibitor (SRPN)2, a key negative regulator of the melanization response in mosquitoes, we identify the cSP CLIPB4 from the African malaria mosquito Anopheles gambiae as a central node in this protease network. CLIPB4 is tightly co-expressed with SRPN2 and forms protein complexes with SRPN2 in the hemolymph of immune-challenged female mosquitoes. Genetic and biochemical approaches validate our network analysis and show that CLIPB4 is required for melanization and antibacterial immunity, acting as a prophenoloxidase (proPO)-activating protease, which is inhibited by SRPN2. In addition, we provide novel insight into the structural organization of the cSP network in An. gambiae, by demonstrating that CLIPB4 is able to activate proCLIPB8, a cSP upstream of the proPO-activating protease CLIPB9. These data provide the first evidence that, in mosquitoes, cSPs provide branching points in immune protease networks and deliver positive reinforcement in proPO activation cascades.

Synthesis and characterization of a poly(dimethylsiloxane)-poly(ethylene oxide) block copolymer for fabrication of amphiphilic surfaces on microfluidic devices.

A poly(dimethylsiloxane)-poly(ethylene oxide) (PDMS-PEO) vinyl terminated block copolymer has been synthesized via a simple hydrosilylation reaction between hydride-terminated PDMS and PEO divinyl ether. This prepolymer can be subsequently cross-linked into an elastomer in a second hydrosilylation reaction involving a methylhydrosiloxane-dimethylsiloxane copolymer, forming a material suitable for the purposes of fabricating microfluidic devices. The presence of the PEO block in the prepolymer chain results in a much more hydrophilic material following cross-linking. The surface water contact angle of the PDMS-PEO material is 65 degrees +/- 3 (n = 6), as opposed to approximately 110 degrees for native PDMS. Droplets of water straddled by air within molded channels of the PDMS-PEO are concave in shape with contact angles where the fluid meets the side walls of 32 degrees +/- 4 (n = 8), while droplets in PDMS microchannels are more convex with contact angles of 95 degrees +/- 6 (n = 6). The length of the PDMS-PEO prepolymer chain and the multifunctional hydride cross-linker chains appear to dictate the durability of the elastomeric material. Young's modulus measurements yielded values of 0.94 +/- 0.08, 2.6 +/- 0.8, and 1.91 +/- 0.06 MPa for a [5% vinyl excess prepolymer and 10-fold excess of cross-linker], [10% vinyl excess prepolymer and 5-fold excess of cross-linker], and 10:1 PDMS, respectively, confirming that the elasticity of the cross-linked PDMS-PEO is similar to that of PDMS (Sylgard 184:10:1 mixture of elastomeric base to elastomer curing agent). The PDMS-PEO material still possesses enough PDMS character to allow molded channel architectures to be sealed between two pieces of the block copolymer by conformal contact. As a result of the more hydrophilic nature of the material, the channels of devices fabricated from this polymer are self-filling when using aqueous buffers, making it more user-friendly than PDMS for applications calling for background electrolytes void of organic modifiers. Different compositions of PDMS-PEO devices feature different electroosmotic flow values with the 5% vinyl excess prepolymer EOF values of 2.5 +/- 0.7 x 10(-4) and 5.7 +/- 0.8 x 10(-4) cm(2)/(V s) at pHs 6 and 9, respectively, and 1.2 +/- 0.3 x 10(-4) and 2.5 +/- 0.3 x 10(-4) cm(2)/(V s) for the 10% vinyl excess prepolymer device at pHs 6 and 9, respectively.