The present work demonstrates the possibilities of using macroporous silicon as a substrate for highly sensitive protein chip applications. The formation of 3D porous silicon structures was performed by electrochemical dissolution of monocrystalline silicon. The fabricated macroporous silicon network has a rigid spongelike structure showing high uniformity and mechanical stability. The microfluidic properties of the substrates were found to be essential for a good bioassay performance. Small spot area, good spot reproducibility, and homogeneous spot profiles were demonstrated on the substrates for immobilized aRIgG. Water contact angles were measured on the porous surface and compared to that of planar silicon, silanized glass, and ordinary microscope glass slides. The effect of the porous surface on the performance of a model IgG-binding immunoassay is presented. aRIgG was microdispensed onto the chip surface forming a microarray of spots with high affinity for the target analyte. The dispensing was performed using an in-house-developed piezoelectric flow-through dispenser. Each spot was formed by a single droplet (100 pL) at each position. The macroporous silicon allowed a high-density microarraying with spot densities up to 4400 spots/cm2 in human plasma samples without cross-talk and consumption of only 0.6 pmol of antibodies/1-cm2 array. Antigen levels down to 70 pM were detected.
A pore chip protein array (PCPA) concept based on a dual readout configuration, fluorescence imaging, and MALDI-TOF MS has been developed. Highly packed, (>4000 spots/cm2), antibody arrays were dispensed on the porous chip by using a piezo-electric microdispenser. Sandwich assay was made after blocking by addition of a secondary antibody either IgG-FITC-labeled or anti-Ang II. The antigen in the first system was a large protein (IgG), and in the other system, a FITC marked peptide Angiotensin II (Ang II) was used. Ang II antibodies showed specificity for Ang II, while the Ang I antibodies showed binding properties for Ang I, II, and Renin. Fluorescence and MALDI TOF MS read-out was made for IgG and Ang II. A major advantage of the dual read-out PCPA approach is that both affinity binding and mass identity are derived. Detection limits for Ang II on the chip is as low as 500 zmol (Ang II).
Speed and accuracy are crucial prerequisites in the application of proteomic methods to clinical medicine. We describe a microfluidic-based nanovial array for rapid proteolytic processing linked to MALDI-TOF MS. This microscale format consumes only minute amounts of sample, and it is compatible with rapid bioanalytical protocols and high-sensitivity readouts. Arrays of vials (300 microm in diameter and 25 microm deep), isotropically etched in silicon wafers were electrochemically porosified. Automated picoliter microdispensing was employed for precise fluid handling in the microarray format. Vials were prefilled with trypsin solution, which was allowed to dry. Porosified and nonporosified nanovials were compared for trypsin digestion and subsequent MS identification of three model proteins: lysozyme, alcohol dehydrogenase, and serum albumin at levels of 100 and 20 fmol. In an effort to assess the rapid digestion platform in a context of putative clinical applications, two prostate cancer biomarkers, prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2), were digested at levels of 100 fmol (PSA), 20 fmol (PSA) and 8 fmol (hK2). All biomarker digestions were completed in less than 30 s, with successful MS identification in the porous nanovial setting.
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