Microchip Electrophoresis for Glycoprotein Separation. Analytical Strategy, September 30th, 2014 Karina Hasler & Adrian Müller

Transcription

1 Microchip Electrophoresis for Glycoprotein Separation Analytical Strategy, September 30th, 2014 Karina Hasler & Adrian Müller Karina Hasler & Adrian Müller

2 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

3 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) Karina Hasler & Adrian Müller

4 Glycoproteins Functions & Examples Important function as integral membrane proteins especially in the cell-cell, hormone-cell, bacterium-cell and virus-cell interactions Karina Hasler & Adrian Müller

5 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

6 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

7 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

8 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

9 Available techniques Capillary electrophoresis (CE) with laser-induced fluorescence detection Group of electrokinetic separation methods which are performed in submillimeter capillaries. Karina Hasler & Adrian Müller

10 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

11 Microchip - Microfluidics High-throughput Low cost Reduced sample volumes ( 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), Whitesides, Nature 442 (2006), Karina Hasler & Adrian Müller

12 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), Zhuang et. al., Anal. Chem. 79 (20107) Karina Hasler & Adrian Müller

13 Technical Issues of Channels in Microchip Electrophoresis «Racetrack» effect Channel length Coating Sample introduction Detection Karina Hasler & Adrian Müller

14 «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 Karina Hasler & Adrian Müller

15 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

16 Coating with Linear Polyacrylamide (PAM) Minimize electroosmotic flow Prevent analyte adsorption Karina Hasler & Adrian Müller

17 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) Karina Hasler & Adrian Müller

18 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), Dittrich and Manz, Nature Drug Disc 5 (2006), Karina Hasler & Adrian Müller

19 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), Vanderschaeghe et. al., Anal. Chem. 82 (2010), Karina Hasler & Adrian Müller

20 Patients serum Collection of blood samples Take 3µL of blood serum Vanderschaeghe et. al., Anal. Chem. 82 (2010), Karina Hasler & Adrian Müller

21 Protein Denaturation Dilution of samples Denaturation of proteins Vanderschaeghe et. al., Anal. Chem. 82 (2010), Karina Hasler & Adrian Müller

22 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), Karina Hasler & Adrian Müller

23 Desialylation Remove sialyl group for simplifying the capillary electrophoresis output. Vanderschaeghe et. al., Anal. Chem. 82 (2010), Karina Hasler & Adrian Müller

24 Evaporate to Dryness The samples were then evaporated to dryness in a thermocycler at 80 C. Vanderschaeghe et. al., Anal. Chem. 82 (2010), Karina Hasler & Adrian Müller

25 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), Karina Hasler & Adrian Müller

26 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), Karina Hasler & Adrian Müller

27 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

28 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

29 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 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

31 Thank you for your attention! Karina Hasler & Adrian Müller