Porous silicon protein microarray technology and ultra-/superhydrophobic states for improved bioanalytical readout.

One attractive method for monitoring biomolecular interactions in a highly parallel fashion is the use of microarrays. Protein microarray technology is an emerging and promising tool for protein analysis, which ultimately may have a large impact in clinical diagnostics, drug discovery studies and basic protein research. This chapter is based upon several original papers presenting our effort in the development of new protein microarray chip technology. The work describes a novel 3D surface/platform for protein characterization based on porous silicon. The simple adjustment of pore morphology and geometry offers a convenient way to control wetting behavior of the microarray substrates. In this chapter, an interesting insight into the surface role in bioassays performance is made. The up-scaled fabrication of the novel porous chips is demonstrated and stability of the developed supports as well as the fluorescent bioassay reproducibility and data quality issues are addressed. We also describe the efforts made by our group to link protein microarrays to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), suggesting porous silicon as a convenient platform for fast on-surface protein digestion protocols linked to MS-readout. The fabrication of ultra- and superhydrophobic states on porous silicon is also described and the utilization of these water-repellent properties for a new microscaled approach to superhydrophobic MALDI-TOF MS target anchor chip is covered.

Bioelectrochemical studies of azurin and laccase confined in three-dimensional chips based on gold-modified nano-/microstructured silicon.

Double-sided three-dimensional porous silicon chips, 6 mm x 6 mm, covered with a 40 nm gold (nano)layer, were fabricated from a porous silicon wafer. Scanning electron microscopy along with electrochemical characterisation showed sample conductivity, mechanical stability, and high surface area of the thus fabricated devices, viz. 10 times higher electrochemically active surface area compared to the geometric area. The three-dimensional gold coated silicon chips were further modified with thiol layers, followed by immobilisation of a simple copper-containing redox protein, azurin, or a complex multicopper redox enzyme, laccase. The bioelectrochemical studies showed very high surface concentrations of azurin and laccase, i.e. close to the theoretical monolayer coverage. However, direct electron transfer reactions between the biomolecules and gold surfaces were observed only for a small percentage of the immobilised redox protein and enzyme, respectively. Thus, highly efficient oxygen-bioelectroreduction on laccase-modified 3D thiol-gold-porous silicon chips (as compared to planar laccase-modified gold electrodes, 42 microA/cm(2)vs. 7 microA/cm(2), respectively) was obtained only in the presence of an efficient soluble redox mediator.

ENSAM: Europium Nanoparticles for Signal enhancement of Antibody Microarrays on nanoporous silicon.

To improve the sensitivity of antibody microarray assays, we developed ENSAM (Europium Nanoparticles for Signal enhancement of Antibody Microarrays). ENSAM is based on two nanomaterials. The first is polystyrene nanoparticles incorporated with europium chelate (beta-diketone) and coated with streptavidin. The multiple fluorophores incorporated into each nanoparticle should increase signal obtained from a single binding event. The second nanomaterial is array surfaces of nanoporous silicon, which creates high capacity for antibody adsorption. Two antibody microarray assays were compared: ENSAM and use of streptavidin labeled with a nine-dentate europium chelate. Analyzing biotinylated prostate-specific antigen (PSA) spiked into human female serum, ENSAM yielded a 10-fold signal enhancement compared to the streptavidin-europium chelate. Similarly, we observed around 1 order of magnitude greater sensitivity for the ENSAM assay (limit of detection < or = 0.14 ng/mL, dynamic range > 10(5)) compared to the streptavidin-europium chelate assay (limit of detection < or = 0.7 ng/mL, dynamic range > 10(4)). Analysis of a titration series showed strong linearity of ENSAM ( R2 = 0.99 by linear regression). This work demonstrates the novel utility of nanoparticles with time-resolved fluorescence for signal enhancement of antibody microarrays, requiring as low as 100-200 zmol biotinylated PSA per microarray spot. In addition, proof of principle was shown for analyzing PSA in plasma obtained from patients undergoing clinical PSA-testing.

Porous silicon surfaces: a candidate substrate for reverse protein arrays in cancer biomarker detection.

This paper introduces a new substrate for reverse-phase protein microarray applications based on macroporous silicon. A key feature of the microarray substrate is the vastly surface enlarging properties of the porous silicon, which simultaneously offers highly confined microarray spots. The proof of principle of the reverse array concept was demonstrated in the detection of different levels of cyclin E, a possible cancer biomarker candidate which regulates G1-S transition and correlates with poor prognosis in different types of human cancers. The substrate properties were studied performing analysis of total cyclin E expression in human colon cancer cell lines Hct116 and SW480. The absence of unspecific binding and good microarray quality was demonstrated. In order to verify the performance of the 3-D textured macroporous surface for complex biological samples, lysates of the human tissue spiked to different levels with cell extract overproducing cyclin E (Hct116) were arrayed on the chip surface. The samples were spotted in a noncontact mode in 100 pL droplets with spots sizes ranged between 50 and 70 mum and spot-to-spot center distances 100 mum, allowing microarray spot densities up to 14 000 spots per cm(2). The different sample types of increasing complexities did not have any impact on the spot intensities recorded and the protein spots showed good homogeneity and reproducibility over the recorded microarrays. The data demonstrate the potential use of macroporous silicon as a substrate for quantitative determination of a cancer biomarker cyclin E in tissue lysates.

High-speed biomarker identification utilizing porous silicon nanovial arrays and MALDI-TOF mass spectrometry.

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.

Biocompatibility of surfaces for antibody microarrays: design of macroporous silicon substrates.

Major efforts to develop antibody microarray technology to enable global proteome analysis to be performed in a facile manner are under way. In this process, the design and the properties of the substrate will play crucial roles. In the present study, we have developed novel, highly biocompatible solid supports for microarrays, using adsorbed recombinant human single-framework antibody fragments as probes. Several silicon-based supports, including planar silicon, micro- and macroporous silicon, and nitrocellulose-coated variants thereof, were designed and evaluated in a stepwise procedure. The surfaces were scored based on biocompatibility and probe binding capacity as judged by spot morphology, signal intensities, signal to noise ratios, dynamic range, sensitivity, and reproducibility. A set of five commercially available substrates, selected to represent a set of supports providing different surface and coupling chemistries, was used as reference surfaces. The results showed that several well-performing silicon-based supports could be designed; in particular, a nitrocellulose-coated macroporous variant, MAP3-NC7, received the highest scores. In comparison, MAP3-NC7 displayed properties equal to or better than those of the reference substrates. Taken together, designed surfaces based on silicon can undoubtedly meet the requirements of the next generation of solid supports for antibody microarrays.

Macro-/nanoporous silicon as a support for high-performance protein microarrays.

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.