ABSTRACT
The design and characterization of two kinds of poly(dimethylsiloxane)(PDMS) microfluidic enzymatic-reactors along with their analytical utility coupled to MALDI TOF and ESI MS were reported. Microfluidic devices integrated with microchannel and stainless steel tubing (SST) was fabricated using a PDMS casting technique, and was used for the preparation of the enzymatic-reactor. The chemical modification was performed by introducing carboxyl groups to PDMS surface based on ultraviolet graft polymerization of acrylic acid. The covalent and physical immobilization of trypsin was carried out with the use of the activation reagents 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide(EDC)/N-hydroxysuccinimide (NHS) and a coupling reagent poly(diallyldimethylammonium chloride)(PDDA), respectively. The properties and success of processes of trypsin immobilization were investigated by measuring contact angle, infrared absorption by attenuated total reflection spectra, AFM imaging and electropherograms. An innovative feature of the microfluidic enzymatic-reactors is the feasibility of performing on-line protein analysis by embedded SST electrode and replaceable tip. The lab-made devices provide an excellent extent of digestion of several model proteins even at the fast flow rate of 3.5 microL min(-1) for the EDC/NHS-made device and 0.8 microL min(-1) for the PDDA-made device, which afford very short residence times of 5 s and 20 s, respectively. In addition, the lab-made devices are less susceptive to memory effect and can be used for at least 50 runs in one week without noticeable loss of activity. Moreover, the degraded PDDA-made device can be regenerated by simple treatment of a HCl solution. These features are the most required for microfluidic devices used for protein analysis.
Subject(s)
Dimethylpolysiloxanes/chemistry , Enzyme-Linked Immunosorbent Assay/instrumentation , Microfluidic Analytical Techniques/instrumentation , Nylons/chemistry , Peptide Mapping/instrumentation , Protein Interaction Mapping/instrumentation , Spectrometry, Mass, Electrospray Ionization/instrumentation , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/instrumentation , Enzyme-Linked Immunosorbent Assay/methods , Equipment Design , Equipment Failure Analysis , Microfluidic Analytical Techniques/methods , Peptide Mapping/methods , Protein Interaction Mapping/methods , Reproducibility of Results , Sensitivity and Specificity , Spectrometry, Mass, Electrospray Ionization/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methodsABSTRACT
The design and characterization of titania-based and alumina-based Poly(dimethylsiloxane) (PDMS) microfluidics enzymatic-reactors along with their analytical features in coupling with MALDI-TOF and ESI-MS were reported. Microfluidics with microchannel and stainless steel tubing (SST) were fabricated using PDMS casting and O(2)-plasma techniques, and were used for the preparation of an enzymatic-reactor. Plasma oxidation for the PDMS microfluidic system enabled the channel wall of the microfluidics to present a layer of silanol (SiOH) groups. These SiOH groups act as anchors onto the microchannel wall linked covalently with the hydroxyl groups of trypsin-encapsulated sol matrix. As a result, the trypsin-encapsulated gel matrix was anchored onto the wall of the microchannel, and the leakage of gel matrix from the microchannel was effectively prevented. A feature of the microfluidic enzymatic-reactors is the feasibility of performing on-line protein analysis by attached SST electrode and replaceable tip. The success of trypsin encapsulation was investigated by AFM imaging, assay of enzymatic activity, CE detection, and MALDI-TOF and ESI-MS analysis. The lab-made devices provide an excellent extent of digestion even at a fast flow rate of 7.0 microL/min, which affords the very short residence time of ca. 2 s. With the present device, the digestion time was significantly shortened compared to conventional tryptic reaction schemes. In addition, the encapsulated trypsin exhibits increased stability even after continuous use. These features are required for high-throughput protein identification.
