| 1. |
- Finnskog, David, et al.
(författare)
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High-speed biomarker identification utilizing porous silicon nanovial arrays and MALDI-TOF mass spectrometry
- 2006
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 27:5-6, s. 1093-1103
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Tidskriftsartikel (refereegranskat)abstract
- 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 mu m in diameter and 25 mu m 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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| 2. |
- Järås, Kerstin, et al.
(författare)
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ENSAM: Europium Nanoparticles for Signal Enhancement of Antibody Microarrays on Nanoporous Silicon.
- 2008
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 7, s. 1308-1314
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Tidskriftsartikel (refereegranskat)abstract
- 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 10 (5)) compared to the streptavidin-europium chelate assay (limit of detection 10 (4)). Analysis of a titration series showed strong linearity of ENSAM ( R (2) = 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.
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| 3. |
- Järås, Kerstin
(författare)
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Miniaturised and Quantitative Techniques Targeting Prostate Cancer
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The prostate-specific antigen (PSA) test used in the clinic today has been subjected to suspicions regarding specificity and sensitivity, since cases of benign prostatic hyperplasia or prostatitis are hard to distinguish from those with prostate cancer. This thesis is based upon five original papers describing efforts to develop antibody microarrays enabling improved diagnostic prostate cancer analysis. All assay developments were performed on our in-house developed and microstructured silicon surfaces, which generate increased spot signals and rapid analysis. On these surfaces, two common techniques, sandwich and reverse phase antibody microarrays were compared from a clinical perspective by analysing PSA. Using FITC-labeled detection we found the sandwich format to outperform the reversed phase assay; the sandwich assay gave five orders of magnitude better limit of detection. In an attempt to also address low abundant markers in blood serum or plasma, signal amplification was developed using europium-incorporated nanoparticles coated by streptavidin. The nanoparticles generated a signal amplification of one order of magnitude as compared to europium-labeled streptavidin. Furthermore, we validated our antibody microarray platform using 80 blood plasma samples from the clinical routine; we developed a true quantitative sandwich antibody microarray for PSA analysis in crude EDTA-plasma samples, and benchmarked the assay to a well-documented DELFIA. The first steps to a duplex assay were also taken by analysing total and free PSA on the same chip. In addition, proof-of-principle for a future lab-on-a-chip approach was shown by combining plasmapheresesis, and subsequent PSA analysis by a sandwich antibody microarray assay. The final work describes a nanovial array for rapid protein digestion and Maldi-TOF analysis; two prostate cancer biomarkers were successfully identified after 30 seconds of tryptic digestion. In conclusion, we have now developed protein chip technology to better suite the clinic within the field of prostate cancer diagnostics.
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| 4. |
- Järås, Kerstin, et al.
(författare)
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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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Ingår i: Clinica Chimica Acta. - : Elsevier BV. - 0009-8981.
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Tidskriftsartikel (refereegranskat)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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| 5. |
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| 6. |
- Järås, Kerstin, et al.
(författare)
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PSA quantification of 80 plasma samples from the clinical routine using antibody microarrays
- 2009
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Ingår i: Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - 9780979806421 ; , s. 457-459
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Konferensbidrag (refereegranskat)abstract
- Antibody microarrays are becoming increasingly established in clinical studies. However, the arrays are seldom used in a quantitative approach but rather for detecting up or down regulated proteins. In this study we describe a microarray procedure being standardized by placing the in-house developed porous silicon surfaces into a commercially available 96 well microtiter plate for analyzing the PSA concentrationsof 80 EDTA-plasma samples obtained from patients undergoing clinical PSAtesting.
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| 7. |
- Järås, Kerstin, et al.
(författare)
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Reverse-phase versus sandwich antibody microarray, technical comparison from a clinical perspective
- 2007
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 79:15, s. 5817-5825
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Tidskriftsartikel (refereegranskat)abstract
- Protein microarrays are powerful tools to quantify and characterize proteins in multiplex assays. They have great potential within clinical diagnostics and prognostics, as they minimize consumption of both analyte and biological sample. Assays that do not require labeling of the biological specimen, henceforth called label-free, are vital for ease of clinical sample processing. Here, we evaluate two label-free techniques, reverse-phase and sandwich antibody assays, using microarrays on high-performance porous silicon surfaces and fluorescence detection. In view of increasing interest in reverse microarrays, this paper focuses on analytical sensitivity of the reverse assays compared to the more complex but highly sensitive sandwich assay. Sensitivity, linear range, and reproducibility of the two assays were compared using prostate-specific antigen (PSA) in buffer. The sandwich assay displayed 5 orders of magnitude lower detection limit (0.7 ng/mL) compared to the reverse assay (70 mu g/mL). PSA at 50 nM (1.5,mu g/mL) in cell lysates was detected by the sandwich assay but not by the reverse assay, demonstrating again a far lower detection limit for sandwich microarrays. In independent assay runs of PSA spiked in female serum, the sandwich assay had good linearity (R-2 > 0.99) and reproducibility (coefficient of variation <= 15%), and the detection limit could be improved to 0. 14 ng/mL. Without further signal amplification, the sandwich assay would be our choice for PSA analysis of clinical samples using a microarray technology platform.
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| 8. |
- Lenshof, Andreas, et al.
(författare)
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Acoustic Whole Blood Plasmapheresis Chip for Prostate Specific Antigen Microarray Diagnostics.
- 2009
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 81:Jul 13, s. 6030-6037
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Tidskriftsartikel (refereegranskat)abstract
- The generation of high quality plasma from whole blood is of major interest for many biomedical analyses and clinical diagnostic methods. However, it has proven to be a major challenge to make use of microfluidic separation devices to process fluids with high cell content, such as whole blood. Here, we report on an acoustophoresis based separation chip that prepares diagnostic plasma from whole blood linked to a clinical application. This acoustic separator has the capacity to sequentially remove enriched blood cells in multiple steps to yield high quality plasma of low cellular content. The generated plasma fulfills the standard requirements (<6.0 x 10(9) erythrocytes/L) recommended by the Council of Europe. Further, we successfully linked the plasmapheresis microchip to our previously developed porous silicon sandwich antibody microarray chip for prostate specific antigen (PSA) detection. PSA was detected by good linearity (R(2) > 0.99) in the generated plasma via fluorescence readout without any signal amplification at clinically relevant levels (0.19-21.8 ng/mL).
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| 9. |
- Lenshof, Andreas, et al.
(författare)
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Acoustic whole blood plasmapheresis chip for PSA microarray diagnostics
- 2009
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Ingår i: Proceedings of Conference, MicroTAS 2009 : The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - 9780979806421 ; , s. 1330-1332
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Konferensbidrag (refereegranskat)abstract
- An acoustic device for generating high quality blood plasma for PSA microarray diagnostics is presented.
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| 10. |
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