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Colloidal/Solid Phase Extraction (C/S PE) Methods Based on Hydrogel Nanoparticles, Titanium dioxide microparticles and Empore Membranes Applied to Biological and Environmental Matrices
Stockholm University, Faculty of Science, Department of Analytical Chemistry.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The aim of the work described in this thesis was to create and develop novel technologies in order to overcome barriers and hurdles that analytical chemistry faces focusing on sample extraction. In paper I dye containing amine groups (e.g. Acid Black 48, Remazol Brilliant Blue R) were coupled to NIPA/Acrylic acid (AAc) particles by condensation of the amine group and the carboxylic group. The high affinity between dyes and proteins allow for fast kinetics and complete depletion of the supernatant and protection of the captured analyte from enzymatic degradation. The ability of particles to capture and concentrate analytes was tested against a panel of low abundance, labile tumor relevant biomarkers and in serum. Results indicate that the nanoparticles increased the sensitivity limit of mass spectrometry analysis and that the dye based baits have extremely high affinity for the target analytes so that particles capture all the analyte present in solution. Biomarker harvesting nanoparticles may be useful for discovery of novel diagnostic analytes, can increase the sensitivity of detection for analytical methods such as immunoassays and MS, and protect labile biomarkers from degradation during collection, shipment and storage. In paper II and paper III, applications of hydrogel nanoparticles to serum samples from cancer patients are reported. Hydrogel nanoparticles were integrated in a mass spectrometry based workflow for the discovery of candidate biomarkers. Lists of candidate biomarkers were identified that are under verification and validation. In paper IV and V hydrogel nanoparticles functionalized with dyes, were employed to increase the sensitivity of diagnostic test for Lyme disease and to detect human growth hormone (hGH) in urine samples. In paper VI, titanium dioxide (TiO2) microparticles were used to pack fused silica capillary column and used to capture and enrich phosphopeptides in vitreous samples. In paper VII, Empore disk membranes were used to capture organophosphates (OPEs) flame retardant from air samples. Empore disk membranes were used as on- line extraction followed by reverse phase liquid chromatography tandem mass spectrometry (RPLC-MS/MS) analysis. Optimized “geometry” settings were used to strip semi volatile and volatile compounds from C8 membrane. This novel design allowed for a better analyte focusing in the HPLC column, reduced the volume of the organic solvent employed for the extraction and the analysis time, and eliminated sample contamination, and loss of analyte.

Place, publisher, year, edition, pages
Stockholm: Department of Analytical Chemistry, Stockholms University , 2011. , 110 p.
Keyword [en]
biomarkers, nanogels, solid-phase extraction, organophosphate esters, peptides, mass spectrometry
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
URN: urn:nbn:se:su:diva-56873ISBN: 978-91-7447-231-8 (print)OAI: oai:DiVA.org:su-56873DiVA: diva2:413520
Public defence
2011-06-01, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 5: Manuscript. Paper 6: Manuscript.Available from: 2011-05-10 Created: 2011-04-28 Last updated: 2011-05-02Bibliographically approved
List of papers
1. Mass Spectrometry-based characterization of the vitreous phosphoproteome
Open this publication in new window or tab >>Mass Spectrometry-based characterization of the vitreous phosphoproteome
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2010 (English)In: Proteomics Clinical Applications, ISSN 1862-8346, Vol. 4, no 10-11, 839-846 p.Article in journal (Refereed) Published
Abstract [en]

Purpose: The vitreous gel is a highly hydrated extracellular matrix containing many proteins. These proteins are likely accumulated in the vitreous by local secretion, filtration from the blood, or diffusion from the surrounding tissues and vasculature, and may be altered in disease state. In the last several years, several reports of large-scale profiling of vitreous proteins have been published; however, there is little information on the characterization of the phosphoproteome of vitreous. Here, we sought to identify phosphopeptides and their phosphorylation sites from vitreous.

Experimental design: We used titanium dioxide (TiO2) to enrich phosphopeptides from vitreous and identified them by LC-MS/MS.

Results: We identified 85 unique phosphopeptides and the phosphorylation sites from 44 proteins.

Conclusions and clinical relevance: We present a method for characterization of phosphoproteome from vitreous samples using current MS technologies and yielded an initial assessment of the phosphoprotein/peptide content of human vitreous, thus providing important biological information toward further understanding of the post-translational modifications of vitreous proteins and their functional significance in disease.

Keyword
Crystallin; LTQ-Orbitrap; Phosphopeptide; Titanium dioxide; Vitreous
National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-56867 (URN)10.1002/prca.201000032 (DOI)
Available from: 2011-04-28 Created: 2011-04-28 Last updated: 2011-05-03Bibliographically approved
2. Nanoparticles technology: Amplifying the effective sensitivity of biomarker detection to create a urine test for hGH
Open this publication in new window or tab >>Nanoparticles technology: Amplifying the effective sensitivity of biomarker detection to create a urine test for hGH
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2009 (English)In: Drug Test Analysis, ISSN 1942-7611, Vol. 1, no 9-10, 447-454 p.Article in journal (Refereed) Published
Abstract [en]

Several clinical-grade immunoassays exist for the specific measurement of hGH or its isoforms in blood but there is an urgent need to apply these same reliable assays to the measurement of hGH in urine as a preferred 'non-invasive' biofluid. Unfortunately, conventional hGH immunoassays cannot attain the sensitivity required to detect the low concentrations of hGH in urine. The lowest limit of sensitivity for existing hGH immunoassays is >50 pg/mL, while the estimated concentration of urinary hGH is about 1 pg/m-50 times lower than the sensitivity threshold. We have created novel N-isopropylacrylamide (NIPAm)-based hydrogel nanoparticles functionalized with an affinity bait. When introduced into an analyte-containing solution, the nanoparticles can perform, in one step, (1) complete harvesting of all solution phase target analytes, (2) full protection of the captured analyte from degradation and (3) sequestration of the analyte, effectively increasing the analyte concentration up to a hundredfold. N-isopropylacrylamide nanoparticles functionalized with Cibacron Blue F3GA bait have been applied to raise the concentration of urinary hGH into the linear range of clinical grade immunoassays. This technology now provides an opportunity to evaluate the concentration of hGH in urine with high precision and accuracy

National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-56868 (URN)20355230 (PubMedID)
Available from: 2011-04-28 Created: 2011-04-28 Last updated: 2011-05-02Bibliographically approved
3. The use of hydrogel microparticles to sequester and concentrate bacterial antigens in a urine test for Lyme disease
Open this publication in new window or tab >>The use of hydrogel microparticles to sequester and concentrate bacterial antigens in a urine test for Lyme disease
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2011 (English)In: Biomaterials, ISSN 0142-9612, E-ISSN 1878-5905, Vol. 32, no 4, 1157-1166 p.Article in journal (Refereed) Published
Abstract [en]

Hydrogel biomarker capturing microparticles were evaluated as a biomaterial to amplify the sensitivity of urine testing for infectious disease proteins. Lyme disease is a bacterial infection transmitted by ticks. Early diagnosis and prompt treatment of Lyme disease reduces complications including arthritis and cardiac involvement. While a urine test is highly desirable for Lyme disease screening, this has been difficult to accomplish because the antigen is present at extremely low concentrations, below the detection limit of clinical immunoassays. N-isopropylacrylamide (NIPAm) - acrylic acid (AAc) microparticles were covalently functionalized with amine containing dyes via arnidation of carboxylic groups present in the microparticles. The dyes act as affinity baits towards protein analytes in solution. NIPAm/AAc microparticles functionalized with acid black 48 (AB48) mixed with human urine, achieved close to one hundred percent capture and 100 percent extraction yield of the target antigen. In urine, microparticles sequestered and concentrated Lyme disease antigens 100 fold, compared to the absence of microparticles, achieving an immunoassay detection sensitivity of 700 pg/mL in 10 mL urine. Antigen present in a single infected tick could be readily detected following microparticle sequestration. Hydrogel microparticles functionalized with high affinity baits can dramatically increase the sensitivity of urinary antigen tests for infectious diseases such as Lyme disease. These findings justify controlled clinical studies evaluating the sensitivity and precision of Lyme antigen testing in urine.

Keyword
Hydrogel, Microparticle, Affinity, Bacteria, Protein
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-56869 (URN)10.1016/j.biomaterials.2010.10.004 (DOI)000285675200023 ()
Available from: 2011-04-28 Created: 2011-04-28 Last updated: 2017-12-11Bibliographically approved
4. Investigation of the Ovarian and Prostate Cancer Peptidome for Candidate Early Detection Markers Using a Novel Nanoparticle Biomarker Capture Technology 
Open this publication in new window or tab >>Investigation of the Ovarian and Prostate Cancer Peptidome for Candidate Early Detection Markers Using a Novel Nanoparticle Biomarker Capture Technology 
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2010 (English)In: AAPS Journal, ISSN 1550-7416, E-ISSN 1550-7416, Vol. 2, no 4, 504-518 p.Article in journal (Refereed) Published
Abstract [en]

Current efforts to identify protein biomarkers of disease use mainly mass spectrometry (MS) to analyze tissue and blood specimens. The low-molecular-weight "peptidome" is an attractive information archive because of the facile nature by which the low-molecular-weight information freely crosses the endothelial cell barrier of the vasculature, which provides opportunity to measure disease microenvironment-associated protein analytes secreted or shed into the extracellular interstitium and from there into the circulation. However, identifying useful protein biomarkers (peptidomic or not) which could be useful to detect early detection/monitoring of disease, toxicity, doping, or drug abuse has been severely hampered because even the most sophisticated, high-resolution MS technologies have lower sensitivities than those of the immunoassays technologies now routinely used in clinical practice. Identification of novel low abundance biomarkers that are indicative of early-stage events that likely exist in the sub-nanogram per milliliter concentration range of known markers, such as prostate-specific antigen, cannot be readily detected by current MS technologies. We have developed a new nanoparticle technology that can, in one step, capture, concentrate, and separate the peptidome from high-abundance blood proteins. Herein, we describe an initial pilot study whereby the peptidome content of ovarian and prostate cancer patients is investigated with this method. Differentially abundant candidate peptidome biomarkers that appear to be specific for early-stage ovarian and prostate cancer have been identified and reveal the potential utility for this new methodology

Keyword
Peptidome; Cancer; Biomarker; Nanoparticle; Mass spectrometry
National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-56870 (URN)10.1208/s12248-010-9211-3 (DOI)
Available from: 2011-04-28 Created: 2011-04-28 Last updated: 2017-12-11Bibliographically approved
5. Candidate Serum Biomarkers of Doxorubicin-Induced Cardiotoxicity in Pediatric Acute Lymphoblastic Leukemia: Results of Nanoparticle-Capture Mass Spectrometry
Open this publication in new window or tab >>Candidate Serum Biomarkers of Doxorubicin-Induced Cardiotoxicity in Pediatric Acute Lymphoblastic Leukemia: Results of Nanoparticle-Capture Mass Spectrometry
(English)Manuscript (preprint) (Other academic)
National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-56871 (URN)
Available from: 2011-04-28 Created: 2011-04-28 Last updated: 2011-05-02Bibliographically approved
6. Multifunctional core-shell nanoparticles amplify sensitivity for low abundance biomarkers.
Open this publication in new window or tab >>Multifunctional core-shell nanoparticles amplify sensitivity for low abundance biomarkers.
(English)Manuscript (preprint) (Other academic)
National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-56872 (URN)
Available from: 2011-04-28 Created: 2011-04-28 Last updated: 2011-05-02Bibliographically approved
7. Air sampling with Empore solid phase extraction membranes and online single-channel desorption/liquid chromatography/mass spectrometry analysis: Determination of volatile and semi-volatile organophosphate esters
Open this publication in new window or tab >>Air sampling with Empore solid phase extraction membranes and online single-channel desorption/liquid chromatography/mass spectrometry analysis: Determination of volatile and semi-volatile organophosphate esters
2006 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1129, no 1, 1-8 p.Article in journal (Refereed) Published
Abstract [en]

A method for determining organophosphate esters in air samples using C8 Empore solid phase extraction (SPE) membranes has been developed. After the sampling the analytes trapped in the membrane are completely desorbed with methanol, using an extraction cell connected online to the organic modifier channel of a HPLC gradient pump. The addition of water to the mobile phase prior to analytical chromatography ensures that the analytes are refocused and efficiently separated. Sampling with Empore SPE membranes enables the collection of analytes in both the vapour phase and particulate matter. During the air sampling procedure no losses were observed after 24 h of sampling, yielding a total volume of 14.4 m3, even for the most volatile compound used in this investigation (trimethylphosphate). Complete desorption was observed for all the organophosphate esters and recoveries were greater than 95%, with a relative standard deviation of less than 8%. The limits of detection ranged between 0.4 and 19 pg/m3. The effect of particulate matter on the extraction efficiency was investigated in detail by spiking the membranes with reference standard material. It was also found that the SPE membranes could be stored for at least 5 days at room temperature without any evidence of loss. The efficacy of the method was verified using real samples from different common indoor environments. Interestingly, significant quantities of several phosphate esters were found in a NIST standard reference material (urban dust, SRM 1649a).

Keyword
Air sampling; Air analysis; Online; Single-channel desorption; Dynamic solvent extraction; Extraction methods; LC-MS; Volatile; Semi-volatile; Organophosphate esters; Empore disk; Solid phase extraction; SPE; Membranes; Particulate matter; Urban dust; Environmental analysis; Organophosphorus compounds; Phosphate
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-26595 (URN)10.1016/j.chroma.2006.05.086 (DOI)16934277 (PubMedID)
Available from: 2009-04-07 Created: 2009-04-02 Last updated: 2017-12-13Bibliographically approved

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  • Other locale
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Output format
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