| 1. |
- Adler, Belinda, et al.
(author)
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Miniaturized and Automated High-Throughput Verification of Proteins in the ISET Platform with MALDI MS
- 2012
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In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 84:20, s. 8663-8669
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Journal article (peer-reviewed)abstract
- A major bottleneck in high-throughput protein production is the validation step, which is why parallel and automated sample processing methods are highly desirable. Also, a miniaturized sample preparation format is preferred, as the reduction of reagent volumes significantly decreases the analysis cost per sample. We have developed an automated and miniaturized protein sequence verification protocol for recombinant proteins utilizing peptide mass fingerprinting and MS/MS analysis. The integrated selective enrichment target (ISET) platform, previously developed in our group, with its dual functionality, being both a sample preparation platform and a MALDI target plate, is employed. All steps including immobilized metal ion affinity chromatography of protein on cobalt-loaded beads, tryptic digestion, and MALDI MS analysis are performed in an array format, without any sample transfers, on the same ISET chip. The automated configuration reduced the sample preparation time significantly. Starting with crude lysate, a full plate of 48 purified, digested samples prepared for MALDI-MS can be generated in 4 h, with only 30 min of operator involvement. This paper demonstrates the utility of the method by parallel analysis of 45 His-tagged human recombinant proteins.
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| 2. |
- Adler, Belinda, et al.
(author)
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Optimizing nanovial outlet designs for improved solid-phase extraction in the integrated selective enrichment target-ISET.
- 2012
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In: Electrophoresis. - : Wiley. - 0173-0835. ; 33:21, s. 3143-3150
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Journal article (peer-reviewed)abstract
- The integrated selective enrichment target is a microfluidic platform for SPE sample preparation with integrated nanocolumns, which simultaneously offers direct MALDI MS read-out. Here, we present a study on the importance of different nanocolumn outlet hole geometries and hole areas in relation to MS signal intensity and reproducibility. A design solution that provides the flow characteristics required for robust sample preparation using automated liquid handling is reported.
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| 3. |
- Adler, Belinda, et al.
(author)
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Porous silicon immunoaffinity microarrays
- 2018. - 2
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In: Handbook of Porous Silicon : Second Edition - Second Edition. - Cham : Springer International Publishing. - 9783319713793 - 9783319713816 ; 2-2, s. 1355-1367
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Book chapter (peer-reviewed)abstract
- Porous silicon with immobilized recognition biomolecules is an attractive platform for many microfluidic chip-based bioanalytical applications. We review the progress in the field since its earliest developments in the 1990s. An improved assay for early detection of prostate cancer has reached clinical evaluation, but there are also exciting developments in both aptamer-based biosensing and mass spectrometry-based biosensing.
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| 4. |
- Adler, Belinda
(author)
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Strategies for Miniaturized Biomarker Detection
- 2014
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Doctoral thesis (other academic/artistic)abstract
- The aim of this thesis is development and application of different miniaturized strategies for detection of biomarkers. The biomarker PSA (prostate specific antigen), which is prostate cancer specific, has been the main focus of the thesis. The papers present two miniaturized strategies for biomarker detection, developed at the department, an antibody microarray and the ISET platform. The antibody microarray, based on a micro- and nanoporous silicon surface, which increases the sensitivity of the assay, was utilized to quantitatively measure the prostate cancer biomarker PSA. The microarray was also developed to measure the two forms of PSA: total PSA and free PSA, which together gives a better indication of the prostate cancer disease. The second platform for proteomic analysis, the in-house developed platform ISET, which is a sample preparation platform for MALDI mass spectrometry, was first redesigned to be able to handle more viscous samples and larger volumes. Subsequent to the new configuration of the ISET platform, three new applications were developed and published within the framework of this thesis; digestion and detection of the biomarker PSA, protein validation of recombinant protein production, and aptamers as affinity ligand rather than antibody to reduce the background from the affinity probe when performing digestion of the captured protein. The ISET sample preparation was also automated using liquid handling robotics for faster analysis in for example screening procedures. In addition to the microarray, the porous silicon surface was utilized to capture PSA and analyse through the use of reporter mass tags ionized in the mass spectrometry.
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| 5. |
- Ahmad Tajudin, Asilah, et al.
(author)
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MALDI-target integrated platform for affinity-captured protein digestion.
- 2014
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In: Analytica Chimica Acta. - : Elsevier BV. - 1873-4324 .- 0003-2670. ; 807:Jan 7, s. 1-8
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Journal article (peer-reviewed)abstract
- 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 40fmol to 1pmol 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.
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| 6. |
- Järås, Kerstin, et al.
(author)
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Porous silicon antibody microarrays for quantitative analysis: Measurement of free and total PSA in clinical plasma samples.
- 2012
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In: Clinica Chimica Acta. - : Elsevier BV. - 0009-8981.
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Journal article (peer-reviewed)abstract
- The antibody microarrays have become widespread, but their use for quantitative analyses in clinical samples has not yet been established. We investigated an immunoassay based on nanoporous silicon antibody microarrays for quantification of total prostate-specific-antigen (PSA) in 80 clinical plasma samples, and provide quantitative data from a duplex microarray assay that simultaneously quantifies free and total PSA in plasma. To further develop the assay the porous silicon chips was placed into a standard 96-well microtiter plate for higher throughput analysis. The samples analyzed by this quantitative microarray were 80 plasma samples obtained from men undergoing clinical PSA testing (dynamic range: 0.14-44ng/ml, LOD: 0.14ng/ml). The second dataset, measuring free PSA (dynamic range: 0.40-74.9ng/ml, LOD: 0.47ng/ml) and total PSA (dynamic range: 0.87-295ng/ml, LOD: 0.76ng/ml), was also obtained from the clinical routine. The reference for the quantification was a commercially available assay, the ProStatus PSA Free/Total DELFIA. In an analysis of 80 plasma samples the microarray platform performs well across the range of total PSA levels. This assay might have the potential to substitute for the large-scale microtiter plate format in diagnostic applications. The duplex assay paves the way for a future quantitative multiplex assay, which analyzes several prostate cancer biomarkers simultaneously.
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| 7. |
- Lee, Sujin, et al.
(author)
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Aptamer/ISET-MS: A New Affinity-Based MALDI Technique for Improved Detection of Biomarkers
- 2014
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In: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 86:15, s. 7627-7634
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Journal article (peer-reviewed)abstract
- 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.
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| 8. |
- Lorey, Martina, et al.
(author)
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Mass-Tag Enhanced Immuno-Laser Desorption/Ionization Mass Spectrometry for Sensitive Detection of Intact Protein Antigens
- 2015
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In: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 87:10, s. 5255-5262
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Journal article (peer-reviewed)abstract
- A new read-out method for antibody arrays using laser desorption/ionization-mass spectrometry (LDI-MS) is presented. Small, photocleavable reporter molecules with a defined mass called "mass-tags" are used for detection of immunocaptured proteins from human plasma. Using prostate specific antigen (PSA), a biomarker for prostate cancer, as a model antigen, a high sensitivity generic detection methodology based immunocapture with a primary antibody and with a biotin labeled secondary antibody coupled to mass-tagged avidin is demonstrated. As each secondary antibody can bind several avidin molecules, each having a large number of mass-tags, signal amplification can be achieved. The developed PSA sandwich mass-tag analysis method provided a limit of detection below 200 pg/mL (6 pM) for a 10 mu L plasma sample, Well below the clinically relevant cutoff value of 3-4 ng/mL. This brings the limit of detection (LOD) for detection of intact antigens with matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) down to levels comparable to capture by anti-peptide antibodies selected reaction monitoring (SISCAPA SRM) and enzyme linked immunosorbent assay (ELISA), as 6 pM corresponds to a maximal amount of 60 amol PSA captured on-spot. We :propose the potential use Of LDI (laser desorption/ionization) with mass-tag read-out implemented in a sandwich assay format for low abundant and/or early disease biomarker detection.
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| 9. |
- Zhang, Chengdong, et al.
(author)
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Exophiala macquariensis sp. nov., a cold adapted black yeast species recovered from a hydrocarbon contaminated sub-Antarctic soil
- 2019
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In: Fungal Biology. - : Elsevier BV. - 1878-6146. ; 123:2, s. 151-158
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Journal article (peer-reviewed)abstract
- A new black yeast species, Exophiala macquariensis is described that is a member of the ascomycete family Herpotrichiellaceae, order Chaetothyriales. The genus Exophiala is comprised of opportunistic pathogens isolated from clinical specimens as well as species recovered from hydrocarbon contaminated environments. Several species have been reported to be able to degrade benzene, toluene, ethylbenzene and xylenes. Here, a novel species of Exophiala (CZ06) previously isolated from a Sub-Antarctic, Macquarie Island soil that was spiked with Special Antarctic Blend diesel fuel (SAB) is described. This isolate has the capacity of toluene biodegradation at cold temperatures. Multilocus sequence typing showed that this fungus was closely related to the pathogenic species Exophiala salmonis and Exophiala equina. With the capacity to utilise hydrocarbons as a sole carbon source at 10 °C, this fungus has great potential for future bioremediation applications.
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