Title: Biochip Technologies

Q: What is DNA compared to RNA
A: Nucleobases in DNA (Deoxyribonucleic acid):
- Thymine
- Cytosine
- Adenine
- Guanine
Double string helix form
In RNA (Ribonucleic acid):
- Uracil
- Cytosine
- Adenine
- Guanine
Single string
An Oligonucleotide is a short single stranded nucleic acid typically composed from RNA or DNA

Q: Definition of a Biochip
A: Biological reaction happens on this solid substrate (=Biochip).
Collection of miniaturized test sites
Microfluidic connections to other chips
Sampling, washing and detection happens seperatly.

Q: Definition of a Lab-on-a-Chip
A: Contains all steps for sample analysis.
Supplyed with microfluids for all steps.

Q: Definition of a microarray (Features, USP, advantages)
A: Miniaturized arrangement of multiple immobilized reagents in 2D Formation.
Tests are performed simultaneous
Many differefent datapoints on one array
Used in research and clincal diagnostics
USP: parallel approach, miniturization, automation, integration of functions
Advantages:
Conventional hybridization process
Automatic readout --> high throughput
Many sensors on many probes

Q: Explain the basic principle of a microarray and explain why they are used.
A: 1. Sample preparation
2. Sample labeling & injection
3. Washed off after incubation time
4. Analyzed with Reader
Intensity correlates to concentration information
Colour corresponds to gene expression
Determine which genes are active at which level
Check response to different test cases
Discovery of new genes

Q: How are Microarrays fabricated?
Name materials, coating methods and explain the different methods.
A: Materials: Glass, Silicon, Polymers (nowerdays: less silicon more polymers)
Depends on: chemical/thermal stabilities, optical properties, costs
Coating:
Directly: in situ synthesis
Chemical modified surfaces: activated glas plus graft coating
Fabrication methods:
- In situ sythesis: Oligonucleotide directly fabricated and sythesized on surface
Photolithography: UV light exposure with mask or programmable micromirrors
Short probes (20mer) only DNA microarray
Oligonucleotide is pre-synthesized
- Contact fabrication
Pin printing: (solid or split pin)
Very sensitive (precise temperature, speed, force settings)--> non-commericial
Microstamping: fast and rapid (direct or indirect stamping with linker)
- Non-contact fabrication
Spotting: deposition of microdroplets, no direct contace
Source activation: piezoelectric, thermal or valve
Piezoel. quite good, but donut shape

Q: Which 4 kinds of Labels can be used for Microarrays? Explain each.
A: Fluorescent labels
Commonly used: Cyanine 3/5
Efficient, moderate photostability, unique excitation
Detection with scanner
Enhanced fluorescent labels
DNA dendrimer technology (~250 fluorophores)
Higher detection sensitivity
Electrochemical detection labels
Target binds to capture probe --> report gene hybridizes to target complex in sandwich assay principle --> current which corresponds to amount of nucleic acid
+ Inexpensive, used optical sensing is difficult (f.e. blood)
- lower sensitivity (?)
Metal Nanoparticle labels
Use of uniques optical and electronic properties:
Scattered base detection, surface plasmon resonance, surface enhanced ramen scattering
- driect labels: Oligonucleotide covalently bound to nanoparticles
- indirect: labeling with antibody-nanoparticle

Q: Explain the immobilization in DNA microarrays.
A: Methods:
Physical Absorption: charge-charge or hydrophobic interaction
Simple, fast, but random orientation
Covalent binding: chemical bonding
Good stability, but slow and irreversible
Streptavidin-Biotin interaction:
Improved orientation, highly specific binding, well controlled,
but expansive, slow, crowding effect
Direct immobilization on metal surfaces
Strong affinity between thinol groups and gold --> chemisorption --> stable bond
Factors to consider when choosing a method:
Good accessibility for the target
Low non-specific binding
Surface functionality

Q: What is the crowding effect?
A: Closely packed probes --> low signal intensity --> high background noise due to non-specific interactions
Promoted through high temperature (but high temperature also used to enhance hybridzation) --> trade off in sensitivity

Q: What are expression Arrays, what do they measure?
A: Most common: spotted glass, cartridge, electronic MA
Measures amount and type of mRNA transcripts
Checks if genes are up or down regulated in specific condition
Discovery of new genes (thousands of genes analyzed)
Higher mRNA levels --> higher protein expression
Cellular protein production to determine quantities of proteins measured by expression array:
- transcription from genes to mRNA
- translation or proteins from mRNA
- post-translation modifications

Q: Name the 6 steps of an microarray experiment.
A: 1. Manufacturing the microarray
2. Experimental design and reference choice
3. Target preparation and hybridization
4. Image acquisition and quantification
5. Database building, filtering and normalization
6. Bioninformstics (Data processing)

Q: How is mRNA transformed to cDNA?
A: 1. Starting material: high quality total RNA
2. Transcribed to single stranded cDNA
3. If neccessary amplification
4. Target labeling (of cDNA)

Q: Which imaging types exit for microarrays?
A: Both: fluorophore is attached to target DNA, if they hybridize with DNA probes, light is emitted
Single color hybridization
Intensity corresponds to signal strength
Two color hybridization
Cy3 and Cy5 --> red or green or yellow if both
--> 2 samples parallel

Q: What is a (DNA)probe and a target?
A: Target: Gene to be detected
Probe: DNA single strand
Probe set: set of Oligoprobes with as similar as possible hybridization properties

Q: Which factors can have an impact on raw mRNA concetration?
A: Biological:
Tissue contamination
RNA degradtion
Amplification or Reverse transcription efficiency
Technological:
Spotting efficiency
DNA support binding
Manufacturing issues
In general: raw data are not mRNA concentrations

Q: Name the steps of data analysis in microarrays.
Which factors have an impact on the quality?
How is background noise reduced?
A: Image scanned by laser
- Data pre-processing: Normalization (if differences in RNA quantity or labeling and detection efficiency), statistical classification
- Image analysis: find and determine spot, convert to numerical data
Data quality factors:
Irregular shap, size, placement, intensity, saturation
--> extra flags: bad or non-present spot
--> different spot identification methods for error correction
Background noise is measured for each spot to find systematic errors
Also some spots left empty --> measured signal just noise --> substract from regular signal

Q: Which methods are used for quality control and data validation in microarrays?
A: Quality control on multiple levels
Probe level: one spot on one array
Caused by contamination, spot quality --> optimization in fabrication procedure
Array level: one particular glass side
Array defect, fault in RNA extraction --> signal range adjustment, check error rate
Gene level: one probe across all arrays
Poor hybridization, printing problems --> error statistics
Validation by alternative second method
- technical replicates to validate instrument data
- biological replicates to validate biological phenomenon

Q: What are protein microarrays and what can be studied? Which technical challenges do they focus?
A: Evolved from DNA microarrays to analysis thousands of proteins simultaneously
Binding: 3D affinity
Lower stability than in 2D DNA microarrays
Proteins are immobilized on Chip
Various probes are used (protein, lipids, DNA, peptides,..)
Studies protein function and activity, post-translational modifications, protein-protein interaction, Antigen-antibody interaction
Advantages: protection of DNA, amplification of genetic information, efficient regulation of gene expression
Technical challenges:
Poor control of immobilized protein activity
High non-specific adsorption
Limited number of labels

Q: What is a proteomic? Name methods.
A: Catalog of all proteins expressed throughout life (under all conditions)
To catalog and understand their function and how they interact
Methods:
X-ray crystallography
Mass spectrometry
Protein microarrays

Q: Name different protein attachment methods.
A: - Adsorption (some proteins inactive, random orientation)
Aptes, Polylysine
And Absorption: larger molecules have more contact sites
- Covalent Binding (some proteins inactive, statistically orientated)
Crosslinker, Functional Groups of amino acids
- Affinity (orientation of protein precisely controlled, site specific)
Biotin, Protein A, G, L, Biotin-Streptavidin
Depends on protein structure and substrate

Q: Explain a sandwich assay
A: Sandwich assay = Detection technique
First antibodies are captured then labelled secondary antibody is used.
=An antibody that can recognize an epitope different from the one recognized by the capture antibody
--> used for detection purposes

Q: Which 3 kinds of protein microarrays do exist?
A: Analytical array
Probes on the surface recognize target proteins
For allergy tests
Functional array
Probes on surface react with target proteins
For characterization of protein functionality
Reverse phase array
Probes on surface interact with target proteins
For identification of protein interactions (high troughput)

Q: Compare tissue microarrays to conventional tissue analysation. Name advantages.
A: Faster
Consume less tissue
Spatially ordered
Miniaturized arrangement

Q: List the steps for analysation in microarrays.
A: 1. Sample collection
(Most important: base for whole process --> clean & precise, manual steps, scale?, costs)
2. DNA extraction
3. PCR
4. Hybridization
5. Washing
6. Evaluation

Q: Explain briefly a brain microarray.
A: Advantages: less time and resources, standardization, high throughput
Disadvantages: does not represent whole tissue, sample loss due to adherence problems
Use: target identification for drugs
For neurodegenerative disease treatment
Problems: tissues cores not properly annealed to parafin, insufficient washing steps

Q: What is are cell microarrays? Name cell capturing methods.
A: Similar to tissue microarrays (fixed in parafin)
Replaces expansive tissue probes
Used to study effect of drugs, gene slicing experiments
Cell capturing methods: coating, affinity-based, 3D microstructured cavities, microfluidic devices

Q: What is the definition and use of live-cell microarrays?
A: Used for
Cell-cell/-surface/-capture/-substance interaction
--> monitoring complex, functional responses in realtime
Label free detection methods: Electrochemical methods, surface plasmon resonance

Q: Name and explain fabrication methods for live-cells microarrays.
A: Photolithography
Replica molding (like microstamping)
Microcontact printing
Microfluidic patterning

Q: Explain the dilemma and challenges with live-cell microarrays.
A: Good Quality and reliababilty, High Throughput and physiological relevance at the same time
Challenges:
Biosensors: non-invasive, time-resolved, multi-parametric
Microfluidics: fluid flow, micropumps, gradients
Tissue engineering: 3D constructs, spheroids, organoids

Q: Explain spheroid and organoid formation in live-cell microarrays.
A: Geometical form depends on shape of microwell
--> round, rectangular

Q: Name examples and types of Microarrays.
A: Examples:
DNA microarray, Protein MA, Peptide MA, Tissue MA
Types:
- 2D Microarray
Most common method, main application genomics, 5um
Fluorescent markers & detection software used
- Spotted glas microarray
For cDNA, Oligonucleotides
Printed to f.e. glass surface
- Cartridge-based microarray
Oligonucleotide arrays: Sets of DNA-25mers instead of entire genes
Fluorescent labeling
--> more expansive
- High density bead array
3D Array, each well has a 3um microsphere
Fluorescence excitation
- Electronic microarray
F.e. 400 electrode test sites
Electric field for acceleration and surface modification

Q: Explain the function of electronic microarrays.
A: Individual spots are activated with a current --> positively charged
Negatively charged DNA is attracted (Electrode spots are coated)
Probes are immobilized through f.e. streptavidin-biotin binding
Afterwards deactivated and process can be repeated

Q: How is a fabrication method choosen?
A: Throughput
Spot quality (size, uniformity)
Influence through sample or substrate surface
Environmental factors: Temperature, Humidity
Pin printing has highest throughput

Q: Briefly explain the workflow in a DNA microarray and name applications.
A: 1. Sample preparation: RNA is extracted and via reverse transcribtion cDNA is created, which gets fluorcence labeling
2. Each spot of the array is coatted with multiple identical probes (=Oligonucleotides that cover the sequence of specific gene)
3. Adding samples --> hybridize with probe, if they contain complementary cDNA, others are washed away.
4. Colored read out(with laser and detector), if gene is expressed in sample (A, B or both can be expressed)
Gene discovery
Protein-DNA interaction
Microbial infection detection (Disease diagnoses)
Drug discovery

Q: Describe the workflow of a tissue microarray.
A: A hallow needle takes tissue from sample and embedds it in parafin
Multiple samples are placed in block
Block cut into thin sections by microtome
(Microtome = cutting instrument to get thin slices f.e. rotary, made from steel or glass)
Placed on adhesive-coated glass

Q: Explain on chip spheroid formation
A: Shape and size depends on the geometry of the device
Used for drug testing
--> cavity & size optimization
--> seeding density optimization
Viability test for optimization