Fundamentals of Microfluidics with Applications in Biological Analysis and Discovery
Fundamentals of Microfluidics with Applications in Biological Analysis and Discovery
Harvard Extension School E155 Dr. Anas Chalah
03.29.16
1 Microfluidics
Microfluidics refers to the set of technologies used to control and manipulate the flow of few microliters of a fluid sample (liquid or gas) in a miniaturized microfluidic device
2 Microfluidic Devices by Defini on:
Architecture: 3D network of channels and other components (pumps, valves, heaters, reservoirs, ..etc)
Dimension: Has one or more flow channel component with at least one dimension less than 1mm
Function: Provide a high level of fluid control required for a certain application
Microarrays are broadly defined as tools for paralleled ligand binding assays
Biomolecules (oligonucleo des, protein fragments) are placed on a solid support (glass, PDMS slides) at high density
Goal: recognizing and analyzing a complex mixture of target molecules
4 Microarray Design
In general, A microarray is a DEVICE that consists of different PROBES chemically attached to a SUBSTARTE and designed to detect a biological target molecule
5 Microarray Technology
Microarray technology allows for:
(1) Estimation of target abundance (2) Detection of biological interactions
Both at the molecular or cellular level
6 Building a Microarray Chip Design Thinking
Application à Components à Fabrication
à
Redesign ß Testing
7 Design Factors
Device: Fabrica on - Design - Signal - Interface Probe: Bio materials (DNA, RNA, Protein, Cells, Tissue) Substrate: Chemical Pla orm (Polymers: Glass, Silicon, PDMS) Buffer: The fluid Surface Fabrica on: Binding Chemistry Signal: Test outcome Interface: Collec ng/analyzing the signal
8 Star ng Point: Think Components
Source: Paula Diez et al., (2012)
9 Types of Microarrays (Probes)
There are various types of microarrays based on the different probe materials:
• DNA/RNA and oligonucleo des • Soluble proteins • Membrane proteins • Pep des • Carbohydrates • Small molecules • Transfec on live cells • Tissue TMA
10 Microarray Substrate
The choice of Substrate on which biological Probes are printed is extremely important for chip design
Commercially available chips are made of: • Glass • Silicon • Plastic and Polymer membranes
11 Advantages of Glass Substrate
• Non-porous • Durable and high surface stability • Robust, resistant to solvents, acids and bases • Sustains high temperatures • Beneficial optical properties - Transparent across a wide spectrum • Negligible autofluorescence à fluorescence- based assay readouts • Rich silanol immobilization chemistries à Protein covalent binding
Disadvantages of Glass Substrate
• Can fracture - must be handled with a care • Glass microfabrication can be costly 12 Silicon
Well-characterized most used substrate, due to its historical role in the development of integrated circuit in the semiconductor industry
Suitable for high-resolution microfabrication technique
Ability to create very fine microfluidic channels and components (feature as fine as ~ 20nm)
Naturally or artificially grown oxide on silicon surfaces provides a silanol-based functionalization suitable for protein immobilization
13 Silicon Drawbacks for Microfluidic Design
(1) Opaque nature prevents the use of various optical imaging techniques
(2) Electric conductivity makes it incompatible with electro-based methods of analysis
(3) Cost associated with the sophisticated cleanroom microfabrication techniques
14 PDMS
• Elastomer: Rubber-like flexible polymer (elastomer)
• Excellent low cost material for rapid prototyping of microfluidic devices
• Rapid designàfabricateàtest soft lithography process
• Transparent - suitable for optical imaging
• Require low investment in infrastructure
15 PDMS Drawbacks for Microfluidic Design
• Limited resistance to organic solvents and gas permeability
• PDMS is hydrophobic in native form, so proteins tend to readily and nonspecifically bind to the surface
• Blocking of the adsorptive surface must be done before an assay is completed
• PDMS lacks functional groups for covalent functionalization
• Large numbers of microfluidic devices are made by sealing PDMS to glass à consideration of glass/PDMS surface properties
16 PDMS Surface Activation “Oxygen Plasma”
Oxygen Plasma Treatment = Introduction of functional groups
Silanol groups are introduced after PDMS oxygen plasma treatment
These groups do not offer long-term stability
After treating with Oxygen Plasma, PDMS could be irreversibly bonded/sealed with glass, plastic substrates, or PDMS surfaces to form enclosed microchannels
17 Plas c
Plastics such as PMMA (Polymethyl methacrylate), PS (Polystyrene), and COC (Cyclic Olefin Copolymer) are widely used
Plastic is generally resistant to solvents, acids & bases
Optically transparent
Hydrophobic in native form making hydrophobic nonspecific protein adsorption a concern
Inert surfaces lack functional groups
Oxygen plasma or strong bases/oxidizers are commonly used to introduce surface functional groups
Mass fabrication and production (mold-based techniques) à Cost effective
18 Probe Immobiliza on
Surface chemistry strategies employed for attaching biomolecule (DNA/ protein) to the chip surface include:
(A) Non-specific interaction = Non-covalent interaction of proteins with hydrophobic (Nitrocellulose, Polystyrene) or positively charged (Polylysine, Aminosilane) surfaces
Polylysine Aminosilane Polyester Nitrocellulose
(B) Covalent cross-linking = Covalent attachment of proteins to chemically activated surfaces (Aldehydes, Epoxies, Active Esters)
19 Functionalized Microarrays Glass Substrates
“Clean slides” Amine substrates Nylon membrane
Aldehyde substrates Gold substrates Epoxy substrates
Source: www.arrayit.com
PVDF membrane Nitrocellulose 20 Design Considera on for Microchip 2D vs. 3D Substrates
Ideal immobilization surfaces should provide: • Large surface-to-volume ratios • Biomolecule-friendly environment • Minimal nonspecific adsorption • Mechanical and chemical stability
Random surface immobilization relying on multiple anchoring points can cause protein denaturing and lose of native activity
Active biomolecule sites could orient towards the immobilization surface, resulting in reduced activity
Single monolayer of biomolecules may not provide strong analytical signal
21 2D vs. 3D Substrates
3D substrate structures provide great increase in binding sites (100 ︎ - 1000 fold) compared to immobilization on 2D substrates (capillary or microchannel walls)
The diffusion length between reaction partners (antibody and antigen, or enzyme and substrate) is reduced in a 3D substrate structures
Introduction of nanoscale features allows for physical encapsulation of biomaterial without chemically activating substrate surfaces
Packed bead beds can be dynamically introduced and eluted from the microchannel for quick surface regeneration
22 041501-3 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
factors.63 We will cover the design and operation of these two canonical heterogeneous for- mats—the immunoassay and enzyme reactors—as we detail design and operational considera- tions for protein immobilization in microfluidic systems.
II. IMMOBILIZATION SURFACE Immobilization methods vary largely with immobilization surface, protein properties, and the goals of the immunoassay or enzyme reactor. A major factor to consider is immobilization surface properties. One of the simplest surfaces on which protein is immobilized is the inner surface of microfluidic channels (Figure 1(a)). Traditional inorganic microfluidic device sub- strates are glass and silicon, which originated from the semiconductor industry and benefit from mature microfabrication techniques. For specialized detection methods such as surface plasmon resonance (SPR),8,9,49 Raman spectroscopy,69 and electrochemical analysis,35,36,70 protein is im- mobilized on metal films deposited on a glass or silicon surface. Silicon and glass share a simi- lar surface chemistry, thus the route to immobilization is similar. Typically, the approach includes surface silanization followed by anchoring to a functional group of a silanizing agent. PDMS (polydimethylsiloxane), a silicon-based organic polymer, attained widespread use because of the low cost, rapid and prototype-friendly fabrication, as well as optical transpar- ency, malleability, and gas permeability (appropriate for some applications).10,71–73 Recently, plastic substrates such as PMMA [Poly(methyl methacrylate)], PS (polystyrene), and COC (cyclic olefin copolymer) have gained attention owing to low cost of fabrication (e.g., injection molding or hot embossing), a chemical resistance superior to PDMS, optical transparency, and low autofluorescence.74–76 PDMS and plastic surfaces are relatively inert and lack functional groups (i.e., sites for protein attachment). Thus, involved chemical surface preparation is gener- ally required to induce surface functional groups for protein immobilization.9,35,77–80 As immo- bilization on planar surfaces yields limited protein density, three dimensional (3-D) structures have been employed inside microfluidic channels for higher protein capture capacity,21,22,81 resulting2D vs. 3D Material in Microchips in improved immunoassay sensitivity or enzyme conversion rates (Figure 1(b)). 3-D structures have been created by patterning microstructures (e.g., microposts29,82,83 and micro- pits60) or through insertion of porous membranes67,84,85 before assembly of microfluidic chips. In post-assembly approaches, microbeads54,62,86–93 can be packed into enclosed channels or var- 3D surfaceious polymers formats such include as hydrogels, micro/nanostructures18–24,57,94–97 sol-gels,64,98 such,99 polymer as microposts monoliths,61, ormicrobeads mem- , hydrogelsbranes100 networks,can be polymerized and membranesin situ. For silica-based 3-D structures such as silica beads91 and 63 alkoxysilane-based sol-gels, a similar glass/silicon surface immobilization strategy can be used. For polymer-based 3-D structures like agarose beads, hydrogels, and polymer monoliths, vari- The design rationale behind 3D substrates stems from18– 20the,23,94 increased surface-area-101 ous immobilization methods including copolymerization of protein, graft polymerization, to-volumeand oxidative ratio activation, in comparison of functional groupsto 2D29 ,planar54 can be surfaces used. Among these 3-D structures, hydro- gels such as polyacrylamide gel and polyethyleneglycol(PEG)gelprovidehydrophilicenviron- Increasedments conducive effective to goodsurface protein in stability 3D substratesand retained proteinprovides activity. more102 Paper immobilization has recently gained sites momentum as a 3-D substrate material for POC (point-of-care) diagnostics for low-resource settings
FIG. 1. Role of surface geometrySource in binding: Dohyun site density. Kim Schematic & Amy drawing Herr of(2013) (a) planar and (b) high surface-area-to-vol- 23 ume ratio three-dimensional immobilization surfaces. Planar 2D Substrates
Example: Miniaturized in- parallel solid-phase chips
Capture molecules (substrates) are immobilized either (a) on the surface or (b) in microwells (diameter <250 µm)
Requires tiny amounts of biological samples & chemical reagents
Density of couple hundred spots per square centimeter (cm2) à Goal is to achieve maximum binding capacity Stefan Kramer et al., (2005)
24 Example: Protein Microarrays Preparation
Proteins/antibodies are immobilized in tiny Gel Pockets attached to a treated glass surface at high density using hand-spo ng devices or a microarray robot
20- 40% Glycerol solu on is added to each gel spot to prevent protein dehydra on
Advantages of glass slide microarrays: (a) Compatible with standard detection equipment (b) Inexpensive and efficient protein immobilization (c) Suitable for a wide range of immuno and enzymatic assays
Disadvantages of glass slide microarrays: (a) Large open surface = High evaporation rate (b) Susceptible to cross-contamination www.stratech.co.uk
25 PDMS Microwell
Micro/nanowell chips consist of an array of wells on a disposable PDMS (polydimethylsiloxane) or glass platform
Microwell arrays allow small volumes of different proteins to be densely packed on a single chip, yet remain physically separated
Proteins are covalently attached to the wells using a chemical cross-linker GPTS (3-glycidoxypropyltrimethoxysilane)
Captured molecules could be easily recovered from the nanowells
High-throughput mass spectrometry and surface plasmon resonance analyses could be performed on such chips
26 Advantages and Disadvantages of Microwell Chips
Advantages: (1) Compatible with standard microarray detection equipment (2) Limited evaporation (3) No cross-contamination (4) Relatively inexpensive (5) Possible recycling after removal of captured molecules
Disadvantages: The disadvantage of this technique is the need for specialized loading equipment for the nanowells
Automated Well Deposition System produce microarray slides of wells with precise biomoelcule concentration, density, and location
Planner vs. Bead-based Microarrays
Planar microarrays: Individual capture agents are immobilized on the surface in several hundreds spots/wells
The array is probed with a sample à the lignad of interest bind to capture agents
The binding reaction is verified by a fluorescence read out
Bead-based microarrays: Individual capture agents are bound to color- coded or size-coded microspheres
Flow cytometery is used to detect the fluorescent label (binding of labeled analytes)
28 Hydrogels
Widely used in protein microarray
Hydrophilic nature à capable of holding large amounts of water in their 3D networks provide a hydrated environment for proteins to sustain their native structure and activity
Flexible materials - well-ordered fibrous structure
Transparent à sensitive fluorescence imaging is possible
The swelling property of hydrogels allows integration of actuators such as valves, allowing integration of sophisticated fluid handling functions
29 Hydrogels Advantages & Disadvantages
• A wide range of immobilization methods are available to hydrogels, including copolymerization of proteins, activation for covalent linking of proteins or electrostatic capture on charged hydrogel
• Structure fragility • Need for crosslinking • Application of shear forces or high electric fields can damage the gel
30 Paper
Cellulose membrane
Versatile material for biomolecule immobilization
More cost-effective in comparison to plastics or PDMS
Fluid and material transport in and through paper can be accomplished passively through using capillary action
Optically opaque à sensitive detection using fluorescence imaging is difficult
31 IMMOBILIZATION STRATEGY
32 Methods of Protein Surface Immobilization
From weakest to strongest:
Diffusion: Spontaneous net movement of protein molecules from an area of high concentration to an area of low concentration in a given volume of fluid
Adsorption: Protein molecules accumulate on the surface of the microchip and form a thin layer
Affinity Binding: Non-covalent interactions between the two molecules (hydrogen bonding, electrostatic interactions, hydrophobic or Van der Waals forces)
Covalent Cross-linking: Chemical reaction – Examples of functional groups that could take part in this binding: Amino, Carboxyl, Hydroxyl, and Thiol group Protein Microarray Technology (2007) David A. Hall, Jason Ptacek, and Michael Snyder
33 Microarray-prin ng Robo c System
(a) The robot X, Y, Z axes are labeled (1), (2) and (3)
The print-head containing the printing pens is marked (4)
Microscope glass slides are placed on the slide station - marked (5)
Protein samples are arrayed on 96-well plates - marked (6)
The pins are cleaned between sample injections at the washing station (7) and drying station (8)
(b) Table configuration of a microarray robot: contains 160 slides with four microtitre plates, two wash stations and the dryer
(c) The print-head with four of the possible twelve pen tips in use
(d) Laser scanner 34 Prin ng Heads “Arrayit” 946 Technology
946 style microarray spotting Pins Source: www.arrayit.com
(1) Pins are made of stainless steel
(2) Spacing between pins is plate related. For 384-well plates, space between pins is 4.5 mm center-to-center
(3) 946 style pins are available in different tip sizes (from 50 to 375 µm), and sample channel sizes that vary from 0.25 µl to 1.25 µl 384-well plate
35 Microarray Prin ng Time
The time needed to print 10,000 spots ranges based on the used pins
946PH4 printing time is 42 hours 946PH32 printing time is 5 hours 946PH48 printing time is 3.4 hours 946PH64 printing time is 2.6 hours
http://www.arrayit.com/Products/Microarray_Printing/Microarray_Pins_946_Technology/ microarray_pins_946_technology.html 36 Microarray Prin ng – 946 Technology
(a) (b) (c) Mechanism: The 946 printing mechanism depends on surface tension and adhesion to print proteins on microarrays
Printing occurs by a gentle “ink stamping” mechanism and does not require a tapping force to expel the sample
Printhead Preparation: The printhead pins are washed in buffer, sonicated, and dried before using them for loading protein samples Source: www.arrayit.com Loading: Sample (in blue) loads into the Pin (in gray) by capillary action
Printing: (a) The flat tip allows a thin layer of the biosample to form at the tip of the pin (b) Thin layer contacts the printing surface during the printing down-stroke, producing a droplet between the substrate and the Pin (c) As the Pin travels upward, the strong adhesive forces of the substrate pull the droplet off the end of the Pin, leaving behind the printed microarray spot 37 Microarrays
38 Microarray Biological Applica ons
For biological applica ons, the features (Probes) on the arrays could be:
ü DNA ü RNA ü Proteins ü Polysaccharides ü Lipids ü Small Organic Compounds ü Whole Cells ü Tissue Specimens
39 Microarray Experimental Design
A typical microarray experiment involves sample comparisons from various sources in an a empt to sort out the contribu on of a single factor as well as interac ons between mul ple factors
40 Microarray Data Analysis
A microarray typically consists of thousand of features
Provides a wealth of informa on leading to many interes ng discoveries
Data analyzing of the large amount of generated data generated could be a daun ng task
Sta s cal methods and commercial so ware have been developed for microarray data processing and analysis
41 Protein Microarrays
42 Protein Microarray v A protein microarray consists of: (1) Platform: Glass, silicon, or polymer base (2) Probes: Different sets protein immobilized (ordered) at separate locations (3) Substrate: Linker between the Platform and the probe v To build a protein microarray one needs to understand the building blocks of the probe v What is a protein? • A polymer made of building units/blocks called amino acids v DNA is made of repeating 4 nucleotides - Proteins are made of • repeating 20 amino acids v Each protein has a unique size, 3D structure, and amino acid sequence
43 Amino Acid Structure
• Chemically, an amino acid is a molecule that contains both amine and carboxyl functional groups
• -COOH is a carboxyl group (Acidic)
• -NH2 is an amino group (Basic) • -H is Hydrogen atom • -R is a residue which varies depending on the amino acid (mainly made of C & H)
• -R group is responsible for important characteristics of the amino acids such as chemical reactivity, ionic charge, and relative hydrophobicity
44 Degrees of Protein Structure
Primary Structure: Describes a long chain of amino acids with a specific sequence
Secondary Structure: Describes the hydrogen bonding interaction between adjacent amino acid residues leading to two distinct polypeptide chain arrangements: Alpha helices and Beta strands
Tertiary structure: Describes the bends and folds that allow the protein to adopt a globular shape which give the protein the lowest surface to volume ratio (Minimize protein interaction with surrounding environment)
Quaternary structure: Quaternary structure describes the kind, the number, and interactions between different polypeptide chains in a protein that consists of more than one amino acid chain (protein subunits) 45 Requirements for Protein Microchip Fabrication
A protein microchips must satisfy the following requirements:
Structure: All immobilized proteins should retain their active structure/ state
Orientation: Expose the protein active site to the surface
Coverage: Contain high protein densities (Why?) Eliminate non specific binding
Storage: Manintain moisturized environment = Controlled evaporation rate
Usage: Compatible with most commercial scanners = signal read-out
46 Chip Substrate
An important issue in the design of protein microarrays is the choice of substrate on which the protein microspots are be printed
Commercially available chips are made of glass, plastic and polymer membranes
Surface chemistry strategies employed for attaching proteins to the chip surface include:
(a) Non-specific interaction = Non-covalent interaction of proteins with hydrophobic (nitrocellulose, polystyrene) or positively charged (polylysine, aminosilane) surfaces
(b) Covalent cross-linking = Covalent attachment of proteins to chemically activated surfaces (aldehydes, epoxies, active esters)
47 Ques on
How do you immobilize membrane proteins on a microchip ?
48 Membrane Proteins
A membrane proteins are proteins attached or associated with a cell membrane
Most biomembranes contain two types of membrane proteins:
(a) Intrinsic: Proteins with domains embedded in the lipid bilayer membrane. Contain residues with hydrophobic side chains that interact with fatty acyl groups of the membrane phospholipids, thus anchoring the protein into the membrane
(b) Extrinsic: Proteins bound only to the membrane surface and do not interact with the hydrophobic core of the phospholipid bilayer
49 Phospholipids
Phospholipids are a class of lipids that contain a phosphate ester as part of the structure - PL are amphiphilic molecules
The general structure of a lipid molecule is: (1) Hydrophilic Head ( phosphate and amino groups) (loves water) (2) Hydrophobic hydrocarbon chain tail (hates water)
http://kvhs.nbed.nb.ca/gallant/biology/phospholipid_structure.jpg
When phospholipids are exposed to water they form a variety of structures
In all cases the hydrophilic phosphate region interacts with water molecules while the hydrophobic fatty acid regions are excluded from water and form hydrophobic interactions http://academic.brooklyn.cuny.edu 50 Func ons of Membrane Proteins
51 Membrane Proteins
Reconstitution of planar lipid bilayers:
(1) The microfluidic system is made of a PMMA (polymethyl methacrylate) plastic substrate (2) Planar lipid bilayers are formed at the apertures, 100µm in diameter, by alternating the flow of lipid solution and buffer into the integrated microfluidic channels (2) Once the bilayers are formed, membrane proteins will be incorporated by sending a flow of liposomes prepared with protein probes which will fuse with the formed bilayer 52 Diagram of forma on of a Membrane Protein Chip
Forma on Steps:
Sequence of the bilayer formation process for the channel device: The microfluidic apparatus has two channels (upper U and bottom B)
(a) Lipid and buffer are flown in the bottom channel while only air is in the upper channel. A lipid layer is formed spontaneously at the narrow side of the tapered aperture at the interface of buffer and air (b) Lipid and buffer are flown in the upper channel kicking air out of the upper channel (c) A thin bilayer of lipid is formed and sandwiched by the buffer in both channels ** Liposomes carrying proteins are flown in the channels are fused with the bilayer 53 Commercially Available Protein Microarrays
54 Commercial Microarrays
Example (1): Protein Capture Chip
Surface: Libraries of expressed/purified proteins are printed onto the microarray surface with the NanoPrint Microarrayer
Chip is used for the analysis of samples in the flow applied directly to the chip
Detection: Labeled or a non-label detection techniques could be used
Applications: protein-protein, protein-drug, and protein-small biomolecule/ fragment interactions
Source: Arrayit® http://www.arrayit.com/Services/Protein_Microarrays/protein_microarrays.html 55 Commercial Microarrays
Example (2): Sandwich Chip
Surface: This chip uses both antigens and antibodies
Implement MicroSpot ELISA (micro enzyme linked immunosorbent assay) and antibody microarrays
Detection: Methods include fluorescent or colorimetric using secondary antibodies labeled with AP, HRP, biotinylated 2oAB that bind labeled streptavidin or direct fluorescent labeling
Applications: Variety of sandwich assays can be implemented
Source: Arrayit® http://www.arrayit.com/Services/Protein_Microarrays/protein_microarrays.html 56 Commercial Microarrays
Example (3): Engineered Chip
Surface: Synthetic proteins, peptides and engineered proteins
Detection: Fluorescence labeled ABs
Application: Detect the presence of proteins in complex samples
Detect a unique protein binding events, epitope mapping studies, small molecule binding events or protein/protein interactions
Source: Arrayit® http://www.arrayit.com/Services/Protein_Microarrays/protein_microarrays.html 57 Commercial Microarrays
Example (4): Reverse Phase Microarrays
Surface: Cell lysate, protein mix
Detection: Fluorescent labeled secondary antibodies
Applications: Profile bound complex mixture of proteins from cell lysates
The bound samples can be probed with antibodies for the detection of antigens present in the printed samples
Source: Arrayit® http://www.arrayit.com/Services/Protein_Microarrays/protein_microarrays.html 58 Two Color Antibody Microarray
Goal: compare two biological samples and measure the absolute and relative differences in protein expression
The procedure is fluorescence-based and compatible with the current microarray scanners
Extracted proteins are labeled differently with two Cyanine dyes (Cy3 and Cy5 Dyes) and mixed
The covalently immobilized antibodies capture fluorescently labeled antigens during the reaction step
The raw data provide a measure of proteins from samples A & B
www.arrayit.com Antibody MicroChip - Data Analysis
Scanned fluorescence image of an antibody microarray detected by two- color analysis 84 antibodies were spotted onto microscope slides coated with nitrocellulose
Each antibody was printed in triplicate on each array
A control samples labeled with biotin used as reference samples were immobilized on the microarray - bound proteins were detected
The microarray was scanned for Cy3 Green and Cy5 red Gao et al. BMC Cancer (2005) fluorescence Microarray Slide Scanner
Scanning Parameters:
(1) Fast scanning time ~ 4 minutes per slide scan time
(2) High resolution data acquisition: scans spot sizes from 5 micron and higher based on scanned side
(3) Real-time image acquisition – HTS
(4) Uniform scanning across the entire microarray surface regardless of substrate type (glass, membrane and plastic)
(5) Easy to use data acquisition and image analysis software
(6) Small footprint (desktop)
Source: www.arrayit.com 61 Protein Microarray Applications v Identify protein-protein interactions (proteomics research) v Identify the substrates of protein kinases v Identify the targets of biologically active small molecules (protein– drug interactions) v Detect protein misfolding v Detect presence and/or amount of target proteins in biological samples, e.g. blood v HTS High-throughput screening
Protein Microarray Technology David A. Hall, Jason Ptacek, and Michael Snyder (2007) 62 Hepatitis C Virus Diagnosis by Protein Microarray
In 2004 Yuk et al developed a kit for the diagnosis of hepatitis C virus (HCV) – Especially useful in third world countries
Chip was designed for low-density protein analysis (Why?) as well as multiple sample screening
The kit is based on protein chip platform and the immuno- concentration method
The chip was evaluated using 96 blood specimens and the results were compared by anti-HCV enzyme immunoassay (EIA) test
Chul-Soo Yuk et al., (2004) Protein Microchip for Hepatitis C Virus Diagnosis
Serum specimens were collected from 3260 patients
Four HCV antigens (Core, NS3, NS4, and NS5) were chosen as ligands immobilized on the surface of the protein microchip
What is the chip detecting for then?
The test is based on the diagnosis of HCV antibodies in patient blood samples
Chul-Soo Yuk et al., (2004)