Simon Ekström

Improved performance in silicon enzyme microreactors obtained by homogeneous porous silicon carrier matrix

The catalytic performance of porous silicon (PS) micro enzyme reactors (muIMER) is strongly dependent on the PS matrix morphology for enzyme immobilisation. PS was achieved in the muIMER by anodisation in a HF-ethanol mixture. PS etching of structured silicon surfaces commonly results in an inhomogeneous pore formation. The deep channel microreactors described herein have previously suffered from these phenomena, yielding non-optimised muIMERs. In order to obtain a homogeneous PS layer on the deep microreactor channel walls, different reactor geometries (channel wall thicknesses of 50 and 75 mum) were anodised at 10 and 50 mA cm(-2) for anodisation times ranging between 0 and 50 min. The muIMERs were evaluated by immobilising two types of enzymes, glucose oxidase (GOx) and trypsin, and the resulting catalytic turnover was monitored by a colorimetric assay. It was found that reactors with a homogeneous PS matrix displayed improved performance. The trypsin muIMERs were used to digest a protein, beta-casein, in an on-line format and the digest was analysed by MALDI-TOF MS. The importance of tailoring the muIMER geometry and the PS-matrix is crucial for the protein digestion. Successful protein identification after only 12 s. digestion was demonstrated for the best reactor, 75 mum channel wall, 25 mum channel width, anodised at 50 mA cm(-2) for 10 min.

On-chip microextraction for proteomic sample preparation of in-gel digests

Despite the high sensitivity and relatively high tolerance for contaminants of matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry (MALDI‐TOF MS) there is often a need to purify and concentrate the sample solution, especially after in‐gel digestion of proteins separated by two‐dimensional gel electrophoresis (2‐DE). A silicon microextraction chip (SMEC) for sample clean‐up and trace enrichment of peptides was manufactured and investigated. The microchip structure was used to trap reversed‐phase chromatography media (POROS™ R2 beads) that facilitates sample purification/enrichment of contaminated and dilute samples prior to the MALDI‐TOF MS analysis. The validity of the SMEC sample preparation technique was successfully investigated by performing analysis on a 10 nM peptide mixture containing 2 M urea in 0.1 M phosphate‐buffered saline with MALDI‐TOF MS. It is demonstrated that the microchip sample clean‐up and enrichment of peptides can facilitate identification of proteins from 2‐DE separations. The microchip structure was also used to trap beads immobilized with trypsin, thereby effectively becoming a microreactor for enzymatic digestion of proteins. This microreactor was used to generate a peptide map from a 100 nM bovine serum albumin sample.

Improved chip design for integrated solid-phase microextraction in on-line proteomic sample preparation.

A recently introduced silicon microextraction chip (SMEC), used for on‐line proteomic sample preparation, has proved to facilitate the process of protein identification by sample clean up and enrichment of peptides. It is demonstrated that a novel grid‐SMEC design improves the operating characteristics for solid‐phase microextraction, by reducing dispersion effects and thereby improving the sample preparation conditions. The structures investigated in this paper are treated both numerically and experimentally. The numerical approach is based on finite element analysis of the microfluidic flow in the microchip. The analysis is accomplished by use of the computational fluid dynamics‐module FLOTRAN in the ANSYS® software package. The modeling and analysis of the previously reported weir‐SMEC design indicates some severe drawbacks, that can be reduced by changing the microextraction chip geometry to the grid‐SMEC design. The overall analytical performance was thereby improved and also verified by experimental work. Matrix‐assisted laser desorption/ionization mass spectra of model peptides extracted from both the weir‐SMEC and the new grid‐SMEC support the numerical analysis results. Further use of numerical modeling and analysis of the SMEC structures is also discussed and suggested in this work.

Disposable polymeric high-density nanovial arrays for matrix assisted laser desorption/ionization-time of flight-mass spectrometry: I. Microstructure development and manufacturing.

In order to meet the expected enormous demand for mass spectrometry (MS) throughput as a result of the current efforts to completely map the human proteome, this paper presents a new concept for low‐cost high‐throughput protein identification by matrix assisted laser desorption/ionization‐time of flight‐(MALDI‐TOF)‐MS peptide mapping using disposable polymeric high‐density nanovial MALDI target plates. By means of microfabrication technology precision engineered nanovial arrays are fabricated in polymer substrates such as polymethylmethacrylate (PMMA) and polycarbonate (PC). The target plate fabrication processes investigated were precision micromilling, cold embossing and injection moulding (work in progress). Nanovial dimensions were 300, 400 or 500 νm. Typical array densities were 165 nanovials/cm2, which corresponds to 3300 vials on a full Applied Biosystems MALDI target plate. Obtained MALDI data displayed equal mass resolution, accuracy, signal intensity for peptide standards as compared to high‐density silicon nanovial arrays previously reported by our group [7], as well as conventional stainless steel or gold targets.

Noncovalent antibody immobilization on porous silicon combined with miniaturized Solid-Phase Extraction (SPE) for array based immunoMALDI assays

This paper presents a new strategy to combine the power of antibody based capturing of target species in complex samples with the benefits of microfluidic reverse phase sample preparation on an integrated sample enrichment target (RP-ISET) and the analysis speed of MALDI MS. The immunoaffinity step is performed on an in-house developed 3D-structured high surface area porous silicon (PSi) matrix, which allows efficient antibody immobilization by surface adsorption without any coupling agents in 30-60 min. The hydrophilic nature of the porous silicon surface at the molecular level displays a low adsorption of background peptides when exposed to complex digests or plasma samples, improving the conditions for the antigen specific extraction and subsequent readout. At the same time, the hydrophobic behavior, due to the nanostructured surface, of the PSi material facilitates liquid confinement during the assay. Using a footprint conforming to the standard for 384 well microplates, direct adaption of the protocol into standard sample handling robots is possible. The performance of the proposed immunoaffinity PSi-ISET immunoMALDI (iMALDI) assay was evaluated by specific detection of angiotensin I at a 10 femtomol level in diluted plasma samples (10 μL, 1 nM).

Disposable polymeric high-density nanovial arrays for matrix assisted laser desorption/ionization- time of flight-mass spectrometry: II. Biological applications

A novel disposable high‐density matrix assisted laser desorption/ionization (MALDI) target plate made either from polymethylmethacrylate (PMMA) or polycarbonate (PC) is presented where thousands (1200–1600) of samples can be deposited and subsequently analyzed by MALDI‐time of flight (TOF) mass spectrometry. Good reproducibility was obtained across the plate regardless of position on the target plate with a relative standard deviation (RSD) on the peak intensity of typically 30% calculated from data generated by analysis of a 10 nM peptide mixture of angiotensin I, II, III and bradykinin. The nanovial array format combined with microdispensing technology makes it possible to carry out in‐vial chemistry on deposited samples. This is demonstrated by the analysis of peptides from β‐casein and subsequent in‐vial dephosphorylation of its phosphopeptides at 10 fmol levels by microdispensing of alkaline phosphatase, into the nanovial. The mass spectra obtained from these polymeric targets provides can also be used in high sensitivity applications as shown by peptide mass fingerprinting of human fibroblast proteins separated by two‐dimensional gel electrophoresis.

Acoustic trapping for bacteria identification in positive blood cultures with MALDI-TOF MS.

Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is currently changing the clinical routine for identification of microbial pathogens. One important application is the rapid identification of bacteria for the diagnosis of bloodstream infections (BSI). A novel approach based on acoustic trapping and an integrated selective enrichment target (ISET) microchip that improves the sample preparation step for this type of analysis is presented. The method is evaluated on clinically relevant samples in the form of Escherichia coli infected blood cultures. It is shown that noncontact acoustic trapping enables capture, enrichment, and washing of bacteria directly from the complex background of crude blood cultures. The technology replaces centrifugation-based separation with a faster and highly automated sample preparation method that minimizes manual handling of hazardous pathogens. The presented method includes a solid phase extraction step that was optimized for enrichment of the bacterial proteins and peptides that are used for bacterial identification. The acoustic trapping-based assay provided correct identification in 12 out 12 cases of E. coli positive blood cultures with an average score of 2.19 ± 0.09 compared to 1.98 ± 0.08 when using the standard assay. This new technology opens up the possibility to automate and speed up an important and widely used diagnostic assay for bloodstream infections.

Downsizing proteolytic digestion and analysis using dispenser‐aided sample handling and nanovial matrix‐assisted laser/desorption ionization‐target arrays

An efficient technique for enzymatic digestion of proteins in nanovial arrays and identification by peptide mass fingerprinting using matrix‐assisted laser desorption/ionization (MALDI‐MS) is presented in this work. Through dispensing of a protein solution with simultaneous evaporation the protein (substrate) is concentrated up to 300 times in‐vial. At higher substrate concentrations the catalytic turnover numbers increase according to the Michaelis‐Menten kinetics. Therefore, the dispenser‐aided nanodigestion is valuable for identification of low‐level proteins (10 nM–500 nM) as well as for automatic high efficiency digestions performed in 0.2–10 min. As an example of low‐level protein identification, a 10 nM solution of lysozyme C was unambiguously identified after 5 min of nanodigestion. Moreover, only 30 s nanodigestion was sufficient to identify hemoglobin (10 νM), exemplifying the fast catalysis of the nanodigestion technique. The developed silicon flow‐through piezoelectric dispenser is adapted for low‐volume and preconcentrated samples in the nL‐νL range and provides fast, accurate and contact‐free sample positioning into the nanovials. In this work, the properties of the nanodigestion concept regarding proteins of different characteristics are explored. Furthermore, the potential of automated protein identification using precoated proteolytic nanovial‐arrays is demonstrated.

Aptamer/ISET-MS: A New Affinity-Based MALDI Technique for Improved Detection of Biomarkers

With the rapid progress in the development of new clinical biomarkers there is an unmet need of fast and sensitive multiplex analysis methods for disease specific protein monitoring. Immunoaffinity extraction integrated with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis offers a route to rapid and sensitive protein analysis and potentially multiplex biomarker analysis. In this study, the previously reported integrated selective enrichment target (ISET)-MALDI-MS analysis was implemented with ssDNA aptamer functionalized microbeads to address the specific capturing of thrombin in complex samples. The main objective for using an aptamer as the capturing ligand was to avoid the inherently high background components, which are produced during the digestion step following the target extraction when antibodies are used. By applying a thrombin specific aptamer linked to ISET-MALDI-MS detection, a proof of concept of antibody fragment background reduction in the ISET-MALDI-MS readout is presented. Detection sensitivity was significantly increased compared to the corresponding system based on antibody-specific binding as the aptamer ligand does not induce any interfering background residues from the antibodies. The limit of detection for thrombin was 10 fmol in buffer using the aptamer/ISET-MALDI-MS configuration as confirmed by MS/MS fragmentation. The aptamer/ISET-MALDI-MS platform also displayed a limit of detection of 10 fmol for thrombin in five different human serum samples (1/10 diluted), demonstrating the applicability of the aptamer/ISET-MALDI-MS analysis in clinical samples.

MALDI-target integrated platform for affinity-captured protein digestion.

To address immunocapture of proteins in large cohorts of clinical samples high throughput sample processing is required. Here a method using the proteomic sample platform, ISET (integrated selective enrichment target) that integrates highly specific immunoaffinity capture of protein biomarker, digestion and sample cleanup with a direct interface to mass spectrometry is presented. The robustness of the on-ISET protein digestion protocol was validated by MALDI MS analysis of model proteins, ranging from 40 fmol to 1 pmol per nanovial. On-ISET digestion and MALDI MS/MS analysis of immunoaffinity captured disease-associated biomarker PSA (prostate specific antigen) from human seminal plasma are presented.