Microchip Electrophoresis for Glycoprotein Separation Analytical Strategy, September 30th, 2014 Karina Hasler & Adrian Müller Karina Hasler & Adrian Müller 30.09.2014 1
Content Glycoproteins Available techniques Electrophoresis microchip design Technical issues of channels in microchip electrophoresis Workflow from sample to analysis Microchip electrophoresis and related microfluidic methods promising solution for point-of-care diagnostics? Karina Hasler & Adrian Müller 30.09.2014 2
Glycoproteins Structure & Synthesis Protein that contains an poly-saccharide chain which is covalently attached to a polypeptide side chain Glycosylation takes place in the ER and is a co- or post-translational modification N-linked (e.g. asparagine) or O-linked (e.g. serine, threonine) http://www.foodnetworksolution.com/wiki/word/1595/glycoprotein Karina Hasler & Adrian Müller 30.09.2014 3
Glycoproteins Functions & Examples Important function as integral membrane proteins especially in the cell-cell, hormone-cell, bacterium-cell and virus-cell interactions http://de.wikipedia.org/wiki/zellmembran Karina Hasler & Adrian Müller 30.09.2014 4
Glycoproteins Functions & Examples Function Structural Molecule Hormone Enzyme Receptor Glycoprotein Collagens HCG (Human chorionic gonadotropin) TSH (thyroid-stimulating hormone) e.g. alkaline phosphatase, patatin Various proteins involved in hormone and drug action Karina Hasler & Adrian Müller 30.09.2014 5
Glycoproteins Biomakers Definition: A biomarker is a molecule that allows the detection and isolation of a particular cell type. In this case, glycoproteins are used to detect different diseases in the blood serum. Diseases: Ovarian cancer, chronic liver disease, esophageal adenocarcinoma Karina Hasler & Adrian Müller 30.09.2014 6
Glycoproteins Analytical challenges Structural information must be obtained from small quantities of isolated glycoproteins. Resolution of different glycan isomers They do not contain good chromophores. A chromophore is the part of a molecule which is responsible for its colour. To solve this problem, a glycan can be labelled. Karina Hasler & Adrian Müller 30.09.2014 7
Available techniques - MS e.g. MALDI-TOF-MS A powerful tool for the identification of cancer-linked markers by comparative glycomic analysis Capable for structural characterization of unknown glycans through the molecular mass High-throughput analysis of known glycan structure + small sample volumes and quantities - resolution of structural isomers + sensitive - expensive + effective in glycan profiling - too sophisticated for routine clinical work Karina Hasler & Adrian Müller 30.09.2014 8
Available techniques Capillary electrophoresis (CE) with laser-induced fluorescence detection Group of electrokinetic separation methods which are performed in submillimeter capillaries. http://en.wikipedia.org/wiki/capillary_electrophoresis Karina Hasler & Adrian Müller 30.09.2014 9
Available techniques Capillary electrophoresis (CE) with laser-induced fluorescence detection The analytes migrate through the electrolyte solution under the influence of an electric field. The separation of the compounds is dependent on the different migrations of the analytes in the applied electrical field. E.g. capillary gel electrophoresis + highly efficient in isomers separation (e.g. positional and linkage) + practical for clinical analysis + tremendous detection selectivity - capability of structural identification Karina Hasler & Adrian Müller 30.09.2014 10
Microchip - Microfluidics High-throughput Low cost Reduced sample volumes (10-9 -10-18 L) Increased efficiency in separation methods Improved heat transfer Simplicity of the planar design parallelization Separation time Serial processing Materials: Mostly polymers: PDMS Or glass, silicon etc. Dittrich and Manz, Nature Drug Disc 5 (2006), 210-218 Whitesides, Nature 442 (2006), 368-373 http://de.wikipedia.org/wiki/datei:pmdsstructure.png Karina Hasler & Adrian Müller 30.09.2014 11
Electrophoresis Microchip Designs Serpentine channel Spiral channel Incorporate low dispersion turns Minimized racetrack effect Potentials are applied simultaneously to sample buffer and waste volumes Mitra et al. Anal. Chem. 84 (2012), 3621-3627 Zhuang et. al., Anal. Chem. 79 (20107) 7170-7175 Karina Hasler & Adrian Müller 30.09.2014 12
Technical Issues of Channels in Microchip Electrophoresis «Racetrack» effect Channel length Coating Sample introduction Detection Karina Hasler & Adrian Müller 30.09.2014 13
«Racetrack» Effect While passing the U-turn, the fluid changes its velocity distribution to non-homogeneous The change in fluid arrangement changes the results of the measurements http://www.google.com/patents/us6186660 Karina Hasler & Adrian Müller 30.09.2014 14
Channel Length & Electric Field Strength Long channels >20 cm Higher electric fields >1000 V/cm Rapidly and efficiently separate N-glycans Analysis time < 100s Short channels <10 cm Modest electric field strengths <500 V/cm Karina Hasler & Adrian Müller 30.09.2014 15
Coating with Linear Polyacrylamide (PAM) Minimize electroosmotic flow Prevent analyte adsorption http://upload.wikimedia.org/wikipedia/commons/6/6b/polyacrylamide.png Karina Hasler & Adrian Müller 30.09.2014 16
Sample Introduction Standard or modified pinched injection a) Transmitted light image of the cross intersection b) Sample loading c) Injection d) Analysis Zhuang et al., Anal. Chem. 79 (20107) 7170-7175 Karina Hasler & Adrian Müller 30.09.2014 17
Labelling & Detection Label with APTS fluorophor to permit fluorescence detection Most popular approach is fluorescence analysis for detection Fluorescence detection with the use of an inverted optical microscope is usual Vanderschaeghe et. al., Anal. Chem. 82 (2010), 7408-7415 Dittrich and Manz, Nature Drug Disc 5 (2006), 210-218 Karina Hasler & Adrian Müller 30.09.2014 18
Workflow from Sample to Analysis µtas Sampling, sample pre-treatment & transport, chemical reactions, analyte separation, product isolation and detection Dittrich and Manz, Nature Drug Disc 5 (2006), 210-218 Vanderschaeghe et. al., Anal. Chem. 82 (2010), 7408-7415 Karina Hasler & Adrian Müller 30.09.2014 19
Patients serum Collection of blood samples Take 3µL of blood serum Vanderschaeghe et. al., Anal. Chem. 82 (2010), 7408-7415 Karina Hasler & Adrian Müller 30.09.2014 20
Protein Denaturation Dilution of samples Denaturation of proteins Vanderschaeghe et. al., Anal. Chem. 82 (2010), 7408-7415 Karina Hasler & Adrian Müller 30.09.2014 21
N-Glycan Release PNGase F (reagent B) specifically removes the N-glycans from the denatured proteins to which they are attached. Vanderschaeghe et. al., Anal. Chem. 82 (2010), 7408-7415 Karina Hasler & Adrian Müller 30.09.2014 22
Desialylation Remove sialyl group for simplifying the capillary electrophoresis output. Vanderschaeghe et. al., Anal. Chem. 82 (2010), 7408-7415 Karina Hasler & Adrian Müller 30.09.2014 23
Evaporate to Dryness The samples were then evaporated to dryness in a thermocycler at 80 C. Vanderschaeghe et. al., Anal. Chem. 82 (2010), 7408-7415 Karina Hasler & Adrian Müller 30.09.2014 24
APTS Labelling Labelling of the N-glycans with APTS: Labelling by reductive amination Increase of sensitivity Provides the analytes with a charge and fluorescence 8-aminopyrene-1,3,6-trisulfonic acid Vanderschaeghe et. al., Anal. Chem. 82 (2010), 7408-7415 Karina Hasler & Adrian Müller 30.09.2014 25
Electrophoretic Separation Electrophoresis profile: Nomenclature and structures of the most abundant N-glycans in the glycome profile: Mmitra et. al. J. Proteome Res. 12 (2013), 4490-4496 Karina Hasler & Adrian Müller 30.09.2014 26
Microchip electrophoresis & related microfluidics methods positive features Potential to be used as a simple and robust method for routine analysis of clinical samples Low costs of mass producing microchips (1 CHF) Short times for analysis Simplicity of the set-up & easy to bring to field Ability to use very small quantities of samples and reagents Carry out separations and detections with high resolution and sensitivity Physiologically relevant environments can be achieved Parallel reactions on one chip Karina Hasler & Adrian Müller 30.09.2014 27
Microchip electrophoresis & related microfluidics methods current challenges Ease of handling and robustness of systems have to be refined Process complex biological samples without the sophisticated sample preconditioning capabilities available in centralized labs All steps of their use should be simple and as culturally independent as possible The reaction are influenced by temperature, ph, ionic concentration etc. and should be able to be used in the whole world, so it has to be stable Karina Hasler & Adrian Müller 30.09.2014 28
Microchip electrophoresis & related microfluidics methods Point-of-care diagnostics? Definition: Point-of-care testing or short POCT refers to the medical diagnostic tests that are performed directly in the hospital, in practice of a medical practitioner or in a pharmacy and not in a laboratory. Examples: Electrochemical sensors (e.g. blood chemistry, urinalysis and small molecules analytes) and lateralflow assays (LFAs) (e.g. pregnancy test) Karina Hasler & Adrian Müller 30.09.2014 29
Microchip electrophoresis & related microfluidics methods Point-of-care diagnostics? Electrochemical sensor (e.g. diabetes test) Lateral-flow assays (LFAs) (e.g. pregnancy test) Karina Hasler & Adrian Müller 30.09.2014 30
Thank you for your attention! Karina Hasler & Adrian Müller 30.09.2014 31