Fabrication of multilayer systems combining microfluidic andmicrooptical elements for fluorescence detection

This paper presents the fabrication of a microchemical chip for the detection of fluorescence species in microfluidics. The microfluidic network is wet-etched in a Borofloat 33 (Pyrex) glass wafer and sealed by means of a second wafer. Unlike other similar chemical systems, the detection system is realized with the help of microfabrication techniques and directly deposited on both sides of the microchemical chip. The detection system is composed of the combination of refractive microlens arrays and chromium aperture arrays. The microfluidic channels are 60 μm wide and 25 μm deep. The utilization of elliptical microlens arrays to reduce aberration effects and the integration of an intermediate (between the two bonded wafers) aluminum aperture array are also presented. The elliptical microlenses have a major axis of 400 μm and a minor axis of 350 μm. The circular microlens diameters range from 280 to 300 μm. The apertures deposited on the outer chip surfaces are etched in a 3000-Å-thick chromium layer, whereas the intermediate aperture layer is etched in a 1000-Å-thick aluminum layer. The overall thickness of this microchemical system is less than 1.6 mm. The wet-etching process and new bonding procedures are discussed. Moreover, we present the successful detection of a 10-nM Cy5 solution with a signal-to-noise ratio (SNR) of 21 dB by means of this system     

Dielectric spectroscopy of Nano BaTiO/sub 3/ confined in MCM-41 mesoporous molecular sieve materials

Dielectric properties of barium titanate (BaTiO₃) particles, synthesized directly in the pores of MCM-41 materials, have been investigated in the frequency range from 20 Hz to 1 MHz for temperature intervals from 100 K to 500 K. The dielectric spectra of BaTiO₃ confined in these molecular sieves were compared with the results obtained from the investigation of pure MCM-41 materials. Obtained results confirmed successful incorporation of BaTiO₃ into porous matrix, but no phase transition from paraelectric to ferroelectric phase was observed due to the particle size being smaller than the critical size. Also, the overall dielectric response of investigated materials is strongly influenced by adsorbed water molecules     

Isolation from phage display libraries of lysine-deficient human epidermal growth factor variants for directional conjugation as targeting ligands

Ligand-targeted anticancer therapeutics represent an opportunityfor the selective and efficient delivery of drugs to tumours.The chemical coupling of ligands to drugs or drug carrier systemsis, however, often hampered by the presence of multiple reactivegroups within the ligand, for example, -NH₂ groups in lysineside chains. In this paper, we describe the isolation by phagedisplay of human epidermal growth factor (EGF) variants withoutlysine and a reduced number of arginine residues. The selectionon A431 carcinoma cells also revealed that R41 is indispensablefor EGF binding activity as all EGF variants contained an arginineresidue at this position. One EGF variant (EGFm1) with K28Q,R45S, K48S and R53S mutations was expressed in bacteria andshowed an identical binding activity as wild-type EGF. EGFm1could be labelled with fluorescein isothiocyanate demonstratingthe accessibility of the N-terminal amino group for couplingreagents. Furthermore, coupling of EGFm1 to PEGylated liposomesresulted in target cell-specific binding and internalizationof the liposomes. These human EGF variants should be advantageousfor the generation of anticancer therapeutics targeting theEGF receptor, which is overexpressed by a wide variety of differenttumours. Received July 8, 2003; revised October 23, 2003; accepted October 30, 2003     

Optimized linker sequences for the expression of monomeric and dimeric bispecific single-chain diabodies

Bispecific single-chain diabodies (scDb) consist of the variableheavy and light chain domains of two antibodies connected bythree linkers. The structure of an scDb in the V_H–V_L orientationis V_HA–linkerA–V_LB–linkerM–V_HB–linkerB–V_LA,with linkers A and B routinely chosen to be 5–6 residuesand linker M 15–20 residues. Here, we applied displayof scDb on filamentous phage to analyse the composition of optimallinker sequences. The three linkers were randomized in lengthand sequence using degenerated triplets coding for only sixhydrophilic or aliphatic amino acids (Thr, Ser, Asp, Asn, Gly,Ala). Antigen-binding clones were then isolated by one to tworounds of selection on the two different antigens recognizedby the bispecific scDb. Using an scDb directed against carcinoembryonicantigen (CEA) and ß-galactosidase (Gal), we foundthat monomeric scDb had a preferred length of 15 or more aminoacid residues for the middle linker M and of 3–6 residuesfor the linkers A and B. No obvious bias towards a preferredlinker sequence was observed. Reduction of the middle linkerbelow 13 residues led to the formation of dimeric scDb, whichmost likely results from interchain pairing between all theV_H and V_L domains. Dimeric scDb were also formed by fragmentspossessing a long linker M and linkers A and B of 0 or 1 residue.We assume that these dimeric scDb are formed by intrachain pairingof the central variable domains and interchain pairing of theflanking variable domains. Thus, the latter molecules representa novel format of bispecific and tetravalent molecules. Thedescribed strategy allows for the isolation of both optimizedand minimal linker sequences for the assembly of monomeric ordimeric single-chain diabodies.