1.
Végvári, Ákos, et al.
(author)
Bioinformatic strategies for unambiguous identification of prostate specific antigen in clinical samples
2011
In: Journal of Proteomics. - : Elsevier BV. - 1874-3919. ; 75:1, s. 202-210
Journal article (peer-reviewed) abstract
Prostate specific antigen (PSA), as a widely used clinical biomarker in prostate cancer diagnostics, exists in multiple molecular forms. However, all of these forms might not be recognized in a given sample by the standard immunoassays. Therefore, we have investigated PSA isoforms separated by size using mass spectrometric analyses. The objective of these developments was to identify and specify the various forms of PSA. To optimize successful identification of different PSA forms, we have developed a bioinformatic strategy, consisting of high resolution MALDI-MS PMF and sequencing MS/MS data searches. To improve sequence-based identification, the recently introduced Proteios software environment was employed, allowing the combination of multiple database search engines in an automated manner. We could unambiguously identify PSA in clinical samples by all detectable tryptic peptides, which were found to be common in several isoforms.
2.
Ahmad Tajudin, Asilah, et al.
(author)
Integrated acoustic immunoaffinity-capture (IAI) platform for detection of PSA from whole blood samples.
2013
In: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0189 .- 1473-0197. ; 13:9, s. 1790-1796
Journal article (peer-reviewed) abstract
On-chip detection of low abundant protein biomarkers is of interest to enable point-of-care diagnostics. Using a simple form of integration, we have realized an integrated microfluidic platform for the detection of prostate specific antigen (PSA), directly in anti-coagulated whole blood. We combine acoustophoresis-based separation of plasma from undiluted whole blood with a miniaturized immunoassay system in a polymer manifold, demonstrating improved assay speed on our Integrated Acoustic Immunoaffinity-capture (IAI) platform. The IAI platform separates plasma from undiluted whole blood by means of acoustophoresis and provides cell free plasma of clinical quality at a rate of 10 uL/min for an online immunoaffinity-capture of PSA on a porous silicon antibody microarray. The whole blood input (hematocrit 38-40%) rate was 50 μl min(-1) giving a plasma volume fraction yield of ≈33%. PSA was immunoaffinity-captured directly from spiked female whole blood samples at clinically significant levels of 1.7-100 ng ml(-1) within 15 min and was subsequently detected via fluorescence readout, showing a linear response over the entire range with a coefficient of variation of 13%.
3.
Antfolk, Maria, et al.
(author)
A single inlet two-stage acoustophoresis chip enabling tumor cell enrichment from white blood cells
2015
In: Lab on a Chip. - 1473-0189. ; 15:9, s. 2102-2109
Journal article (peer-reviewed) abstract
Metastatic disease is responsible for most cancer deaths, and hematogenous spread through circulating tumor cells (CTC) is a prerequisite for tumor dissemination. CTCs may undergo epithelial–mesenchymal transition where many epithelial cell characteristics are lost. Therefore, CTC isolation systems relying on epithelial cell markers are at risk of losing important subpopulations of cells. Here, a simple acoustophoresis-based cell separation instrument is presented. Cells are uniquely separated while maintained in their initial suspending medium, thus eliminating the need for a secondary cell-free medium to hydrodynamically pre-position them before the separation. When characterizing the system using polystyrene particles, 99.6 ± 0.2% of 7 μm diameter particles were collected through one outlet while 98.8 ± 0.5% of 5 μm particles were recovered through a second outlet. Prostate cancer cells (DU145) spiked into blood were enriched from white blood cells at a sample flow rate of 100 μL min−1 providing 86.5 ± 6.7% recovery of the cancer cells with 1.1 ± 0.2% contamination of white blood cells. By increasing the acoustic intensity a recovery of 94.8 ± 2.8% of cancer cells was achieved with 2.2 ± 0.6% contamination of white blood cells. The single inlet approach makes this instrument insensitive to acoustic impedance mismatch; a phenomenon reported to importantly affect accuracy in multi-laminar flow stream acoustophoresis. It also offers a possibility of concentrating the recovered cells in the chip, as opposed to systems relying on hydrodynamic pre-positioning which commonly dilute the target cells.
4.
Antfolk, Maria, et al.
(author)
Continuous flow microfluidic separation and processing of rare cells and bioparticles found in blood – A review
2017
In: Analytica Chimica Acta. - : Elsevier BV. - 0003-2670. ; 965, s. 9-35
Research review (peer-reviewed) abstract
Rare cells in blood, such as circulating tumor cells or fetal cells in the maternal circulation, posses a great prognostic or diagnostic value, or for the development of personalized medicine, where the study of rare cells could provide information to more specifically targeted treatments. When conventional cell separation methods, such as flow cytometry or magnetic activated cell sorting, have fallen short other methods are desperately sought for. Microfluidics have been extensively used towards isolating and processing rare cells as it offers possibilities not present in the conventional systems. Furthermore, microfluidic methods offer new possibilities for cell separation as they often rely on non-traditional biomarkers and intrinsic cell properties. This offers the possibility to isolate cell populations that would otherwise not be targeted using conventional methods. Here, we provide an extensive review of the latest advances in continuous flow microfluidic rare cell separation and processing with each cell's specific characteristics and separation challenges as a point of view.
5.
Ekström, Simon, et al.
(author)
Integrated selective enrichment target - a microtechnology platform for matrix-assisted laser desorption/ionization-mass spectrometry applied on protein biomarkers in prostate diseases
2004
In: Electrophoresis. - : Wiley. - 0173-0835. ; 25:21-22, s. 3769-3777
Journal article (peer-reviewed) abstract
The performance of a miniaturized sample processing platform for matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), manufactured by silicon microfabrication, called integrated selective enrichment target (ISET) technology was evaluated in a biological context. The ISET serves as both sample treatment device and MALDI-MS target, and contains an array of 96 perforated nanovials, which each can be filled with 40 nL of reversed-phase beads. This methodology minimizes the number of sample transfers and the total surface area available for undesired adsorption of the analytes in order to provide high-sensitivity analysis. ISET technology was successfully applied for characterization of proteins coisolated by affinity chromatography of prostate-specific antigen (PSA) from human seminal fluid. The application of ISET sample preparation enabled multiple analyses to be performed on a limited sample volume, which resulted in the discovery that prolactin inducible protein (PIP) was coisolated from the samples.
6.
Finnskog, David, et al.
(author)
High-speed biomarker identification utilizing porous silicon nanovial arrays and MALDI-TOF mass spectrometry
2006
In: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 27:5-6, s. 1093-1103
Journal article (peer-reviewed) 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.
7.
Ku, Anson, et al.
(author)
Acoustic Enrichment of Extracellular Vesicles from Biological Fluids
2018
In: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 90:13, s. 8011-8019
Journal article (peer-reviewed) abstract
Extracellular vesicles (EVs) have emerged as a rich source of biomarkers providing diagnostic and prognostic information in diseases such as cancer. Large-scale investigations into the contents of EVs in clinical cohorts are warranted, but a major obstacle is the lack of a rapid, reproducible, efficient, and low-cost methodology to enrich EVs. Here, we demonstrate the applicability of an automated acoustic-based technique to enrich EVs, termed acoustic trapping. Using this technology, we have successfully enriched EVs from cell culture conditioned media and urine and blood plasma from healthy volunteers. The acoustically trapped samples contained EVs ranging from exosomes to microvesicles in size and contained detectable levels of intravesicular microRNAs. Importantly, this method showed high reproducibility and yielded sufficient quantities of vesicles for downstream analysis. The enrichment could be obtained from a sample volume of 300 μL or less, an equivalent to 30 min of enrichment time, depending on the sensitivity of downstream analysis. Taken together, acoustic trapping provides a rapid, automated, low-volume compatible, and robust method to enrich EVs from biofluids. Thus, it may serve as a novel tool for EV enrichment from large number of samples in a clinical setting with minimum sample preparation.
8.
Lee, Sang Wook, et al.
(author)
A Highly Sensitive Porous Silicon (P-Si)-Based Human Kallikrein 2 (hK2) Immunoassay Platform toward Accurate Diagnosis of Prostate Cancer.
2015
In: Sensors. - : MDPI AG. - 1424-8220. ; 15:5, s. 11972-11987
Journal article (peer-reviewed) abstract
Levels of total human kallikrein 2 (hK2), a protein involved the pathology of prostate cancer (PCa), could be used as a biomarker to aid in the diagnosis of this disease. In this study, we report on a porous silicon antibody immunoassay platform for the detection of serum levels of total hK2. The surface of porous silicon has a 3-dimensional macro- and nanoporous structure, which offers a large binding capacity for capturing probe molecules. The tailored pore size of the porous silicon also allows efficient immobilization of antibodies by surface adsorption, and does not require chemical immobilization. Monoclonal hK2 capture antibody (6B7) was dispensed onto P-Si chip using a piezoelectric dispenser. In total 13 × 13 arrays (169 spots) were spotted on the chip with its single spot volume of 300 pL. For an optimization of capture antibody condition, we firstly performed an immunoassay of the P-Si microarray under a titration series of hK2 in pure buffer (PBS) at three different antibody densities (75, 100 and 145 µg/mL). The best performance of the microarray platform was seen at 100 µg/mL of the capture antibody concentration (LOD was 100 fg/mL). The platform then was subsequently evaluated for a titration series of serum-spiked hK2 samples. The developed platform utilizes only 15 µL of serum per test and the total assay time is about 3 h, including immobilization of the capture antibody. The detection limit of the hK2 assay was 100 fg/mL in PBS buffer and 1 pg/mL in serum with a dynamic range of 106 (10-4 to 102 ng/mL).
9.
Lee, Sang Wook, et al.
(author)
Porous silicon microarray for simultaneous fluorometric immunoassay of the biomarkers prostate-specific antigen and human glandular kallikrein 2
2016
In: Microchimica Acta. - : Springer Science and Business Media LLC. - 0026-3672 .- 1436-5073. ; 183:12, s. 3321-3327
Journal article (peer-reviewed) abstract
The authors have developed a porous silicon (P-Si) based duplex antibody microarray platform for simultaneous quantitation of the biomarkers prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2) in serum. Pore size-controlled P-Si surfaces have an extremely enlarged surface area that enables high-density immobilization of fluorescently labeled antibodies by physical adsorption. Automated microarraying of the antibodies provides a fast and reproducible duplex format of antibody arrays on the P-Si chips placed in the wells of a microtiter plate. The assay platform showed a 100 fg·mL−1 limit of detection for both PSA and hK2, and a dynamic range that extends over five orders of magnitude. After optimization of the density of both capture antibodies, neither the PSA nor the hK2 array showed cross-sensitivity to non-target proteins or other plasma proteins. The microarray was evaluated by titration of PSA and hK2, respectively, in the same serum samples. In our perception, this highly sensitive and selective platform holds promise for improved detection of tumor markers in an early diagnostic stage, but also to monitor the recurrence of prostate cancer. [Figure not available: see fulltext.]
10.
Lenshof, Andreas, et al.
(author)
Acoustofluidics 8: Applications of acoustophoresis in continuous flow microsystems
2012
In: Lab on a Chip. - 1473-0189. ; 12:7, s. 1210-1223
Research review (peer-reviewed) abstract
This acoustofluidics tutorial focuses on continuous flow-based half wavelength resonator systems operated in the transversal mode, where the direction of the primary acoustic force acts in plane with the microchip. The transversal actuation mode facilitates integration with up- and downstream microchannel networks as well as visual control of the acoustic focusing experiment. Applications of particle enrichment in an acoustic half wavelength resonator are discussed as well as clarification of the carrier fluid from undesired particles. Binary separation of particle/vesicle/cell mixtures into two subpopulations is outlined based on the different polarities of the acoustic contrast factor. Furthermore, continuous flow separation of different particle/cell types is described where both Free Flow Acoustophoresis (FFA) and binary acoustophoresis are utilized. By capitalizing on the laminar flow regime, acoustophoresis has proven especially successful in performing bead/cell translations between different buffer systems. Likewise, the ability to controllably translate particulate matter across streamlines has opened a route to valving of cells/particles without any moving parts, where event triggered cell sorting is becoming an increasing area of activity. Recent developments now also enable measurements of fundamental cell properties such as density and compressibility by means of acoustophoresis. General aspects on working with live cells in acoustophoresis systems are discussed as well as available means to quantify the outcome of cell and particle separation experiments performed by acoustophoresis.
