Biosensor technology Flashcards
What is the difference between data and information?
Data can be raw figures and facts that are not put in a context, while information is data that is structured and put in a context
What is the definition of a biosensor?
A self-contained integrated device that can provide specific quantitative analytical data and uses a biorecognition element
What are the components of a biosensor?
Biorecognition element: enables specific recognition of the analyte
Interfacial component: facilitates immobilization of the bioreceptor and prevents unspecific binding of analyte
Transducer: converts the recognition event into a measurable signal
List some biorecognition elements
- Antibodies
- Enzymes
- DNA
- Aptamers
- Peptides
- Organelles
- Cells
List some transducer technologies
- Surface Plasmon Resonance
- Fluorescence
- Colorimetric
- Amperometric
- Potentiometric
- Piezoelectric
- Calorimetric
What are some differences between a biosensor and a bioassay?
An assay is an analytic procedure for quantitatively assessing the presence, amount or activity of a target. Uses BRE and is never an isolated process
What are some advantages of a biosensor compared to a clinical lab?
From a biosensor you can get real-time information and point of care. It is easier to obtain multi-time-point data.
What is a biorecognition element (BRE)?
Interactions between the recognition element and the analyte
Why does affinity matter for a affinity-based BRE?
A higher affinity, which means lower dissociation constant, of the bioregocnition for the analyte results in a larger sensor signal for a given analyte conc.
How does the turnover (kcat) affect the sensor signal?
The turnover is the maximum number of chemical conversions of substrate per second.
A higher kcat results in more product and therefore a larger sensor signal
What is unspecific binding?
It is binding of anything else than the analyte to the sensor or binding of the analyte to other parts of the signal generating components of the transducer.
What do you have to consider about the sample that you will use?
You must take the sample matrix and the analyte into consideration. Some properties that you have to consider are:
- Analyte size
- Chemical properties of analyte
- Range of analyte conc
- Which specificity and selectivity is required
- Complexity of the sample
What is the specificity of a sensor what is important to. consider?
The specificity of a sensor is the ability to explicitly assess the analyte in the presence of other components. The affinity for the analyte must be much higher than for binding other molecules that can bind to the BRE.
For detection of multiple different analyte we need multiple BRE for the specific analytes
What is selectivity?
A sensor array is selective if it can discriminate between different substances in the sample
What is the limit-of-detection (LOD)?
The lowest conc. of a substance that can be distinguished from the absence of that substance within a stated confidence limit.
Y(LOD) = y(blank) + k*σ(blank)
k is usually 3
What is the limit-of-quantification (LOQ)?
More reliable analytical results are obtained when the conc. is above a higher limit, LOQ.
Y(LOQ) = y(blank) + 10*σ(blank)
What is the dynamic range of the sensor?
The ratio between the upper and lower detection limits. The lower limit is the LOD/LOQ and the upper limit is where the response deviates from the assumed calibration function, due to saturation of BRE.
How can you decrease the LOD/LOQ?
Use a BRE with higher affinity for the analyte. For an enzymatic BRE you can use a BRE with higher turnover (kcat). You can also reduce noise-lever of the sensor, so you decrease the 3*σ
How do you prevent saturation of BRE?
Use BRE with lower affinity for the analyte (large KD) or dilute the sample or increase the sensor surface area
What is the sensitivity of a sensor?
The slope of the linear region.
The steeper slope the higher the sensitivity is. This means that there is a better possibility to distinguish small changes in the analyte conc = resolution.
The resolution is the smallest detectable change in analyte conc.
What is sensor noise and what can cause it?
The noise is random variation of sensor output that are unrelated to the sensor input. The noise can arise from random processes in the sensor function from electronic noise in the readout equipment.
It is important to look at the signal-to-noise ratio. A high sensitivity and low noise level gives a high resolution
What is accuracy, precision and reproducibility?
Accuracy: closeness of the measurements to a specific value
Precision: closeness of the measurements to each other
Reproducibility: ability if the sensor to produce identical results whenever the same sample is measured more than once
How can one improve the reproducibility?
The main reason for poor reproducibility is manual handling. So minimize manual handling.
Factors that affect the sensor robustness are environmental changes (temp and storage conditions) and sample interference
What are the different transducer categories and what do they measure?
- Electrochemical: detects redox reactions. Enzymatic reaction that generates electrochemical active species
- Optical: detects changes in wavelength or intensity. Analyte that are either colored or fluorescent or labeled or analyte-binding induces a change in refractive index.
- Gravimetric: detects changes in mass or viscoelastic properties. The change in the resonance frequency of an oscillating senor surface when the mass changes upon analyte binding
How can one use gold nanoparticles as a transducer?
As gold nanoparticles aggrigate they change color - colorimetric detection.
They can absorb and scatter light very efficiently and are very stable (non-oxidizing) and they can be used to in gold-thiol-chemistry for functionalization for conjugation of AB
How can we optimize the naked eye detection of color change?
- The color change should appear within a narrow spectral range
- Produce a distint and sharp color transition
- Appear in the green ange where our eyes are the most sensitive
Describe the DNA hybridization triggered aggrigation using gold nanoparticles.
When the target DNA is present in the sample, it hybridizes with the probe on the gold nanoparticle surface, causing the nanoparticles to aggregate.
The melting temp is proportional to the number of interactions involved. More interactions result in a higher melting temp.
What are cantilever biosensors?
It is a microfabricated beam clamped at one and and at the other and, the surface is functionalized with bioreceptors (often affinity binding).
Deflection of the cantilever is caused by repulsive forces between bound analytes
What is a piezoelectric transducer?
The piezoelectric effect is the ability of a material to produce a voltage when mechanically stressed vice vera.
Used in static gravimetric sensor to transduce an analyte-induced bending of the cantilever load into an electric response
Used in dynamic gravimetric sensors to induce oscillations of the sensor surface (the resonance freq changes upon analyte binding)
What is a bulk acoustic wave (BAW) biosensor?
A type of biosensor that utilizes a thin piezoelectric material to detect changes in mass, viscosity, or elasticity caused by biomolecular interactions.
How does QCM-D work?
The driving voltage is intermittently switched off and the decay is monitored. Form the decay curve, the resonance freq and the energy dissipation are extracted. The disspation gives info about the energy loss in the system - how soft or viscoelastic the layer is
Why use enzyme-based recognition?
Possible to measure reaction products: pH, electrons, heat, and light
What is an allosteric enzyme?
An effector modulates the catalytic rate by inducing conformational change in protein structure -> activating or deactivting
Why use antibodies as BRE?
Perfect fit in terms of structure and chemical complementary towards the epitope of the antigen
What are aptamers?
Synthetic DNA/RNA receptors that exploits the ability of DNA and RNA to selectively recognize and bind to proteins or other molecules
Describe the SELEX
- Starts with library with random sequences
- Library is mixed with target
- Unbound sequences are removed
- Bound sequences are eluted and amplified using PCR
- Repeated 10-15X to narrow down the number of sequences and extract those with highest affinity
What are some advantages of using aptamers?
Adv:
- inexpensive and fast selection
- Uniform activity regardless of batch
- Affinity parameters can be controlled
- Unlimited shelf life
- Recover original conf. efter thermal shock
- Can diversify properties and function
What are some disadvantages of using antibodies?
- Screening monoclonal ab is expensive and time consuming
- Activity varies from batch to batch
- Difficult to modify
- Limited shelf life
- Temp causes irriversable denaturation
What are aptanzymes?
RNAzymes or DNAzymes have the potential to replace enzymes as BRE. Share the same properties as aptamers
What are molecular beacon aptamers?
The binding to analyte induces conformational changes that can open hairpin and “turn on” quenched fluorophore
What are peptide nucleic acid (PNA)?
Absense of phosphate groups results in less electrostatic repulsion. More resistant towards nucleases and poteases
Peptides as BRE
Natural and synthetic amino acids used in peptides
One bead one compound (OBOC)
Affibodies - peptides based on IgG z domain
Bacteriophages as BRE, what can they be used for?
Phage connects displayed peptide to encoding DNA. Can be used for B-cell and T-cell epitope mapping, disease-specific antigen mimics, peptides bound to non-protein target
Lectins as BRE
Glycan-binding proteins. Lectins can be found in all types of cells and viruses an have a crucial role in host pathogen recognition, cell-cell interaction. Binding strength is low. Can interact with multiple glycan epitopes
Using Molecular imprinted polymers (MIPS) as BRE?
- Very stable artificial BRE
- Selection of monomer is key
- Monomers interact with analyte
- Analyte can be removed and polymer contains chemical and sterical memory of analyte
What are the main approaches for immobilization?
- Adsorption/physisorption
- Entrapment
- Covalent binding/chemisorption
What are the requirements for immobilization?
- maintain activity and specificity
- provide a robust coupling
- facilitate a good signal transduction
- have an optimized packing density
- should be obtained with an easy and reproducible procedure
Describe adsorption.
Relies on nonspecific physical interaction.
+ Simple and cheap
+ less dissruptive to BRE than chemical adsorption
- randomized orientation - blocking of binding sites
- prone to desorption due to pH, temp, ionic strength
- hydrophobic surfaces may induce unfolding of proteins
Describe entrapment
Entrapment behind a semipermeable membrane. Allows for diffusion of small analytes.
Entrapment in hydrogel. Can entrap large molecules or whole cells. Small BRE, which can escape from matrix
Describe covalent binding
Formation of covalent bonds. Very stable towards changes in temp, ion strength, pH. Coupling can often be preformed using mild conditions. May react with active/binding site
What are possible solutions for unspecific binding?
- Block uncovered surface with known biomolecule (BSA or HSA)
- Functionalize surface with hydrophilic matrix/polymer: dextran, PEG, PC
- Change surface properties using surface chem., make more hydrophilic by oxidize surface, introducing self assembled monolayers
What is a working electrode?
The electrode in an e.c. system on which the reaction of interest is occuring: metals, carbons, semiconductors
What is a counter electrode?
The electrode in an e.c. system on which electric current is expected to flow: inert metals, carbons, semiconductors
What is a reference electrode?
An electrode which has a stable and well-known electrode potential: Ag/AgCl (KCl), saturated calomel electrode (Hg/HgCl, KCl)
What is potentiometry?
Electrochemical technique where the potential between 2 electrodes is measured using a high impedance voltmeter without net current flow
Application: ion-selective electrode
What is voltammetry?
Electrochemical technique where current is measured as a function of the applied potential
Application: analyte redox peaks, reversibility, electrode kinetics
What is amperometry?
Electrochemical technique where current is measured at a fixed level. Non faradic reaction
Application: chemical-/biosensing
Enzymatic electrodes
Enzyme layer + electrode (transducer)
What are some important enzyme immobilization aspects?
- Loading amount
- Biological activity and structure retention after
- Long-term storage and operation stability
- Immobilization stability to minimize the leakage during operation
- Reproducibility, cost, difficulty of process
Electrochemical immunosensors. To label or not?
Retain binding affinity, real-time binding. Affinity binding is transduced into changes in electrical properties of the surface from the presence of analyte.
Electrochemical immunosensor, to use direct or indirect?
Enhanced sensitivity with indirect but no direct binding kinetics. Affinity binding is transduced into changes in the electroactive label
What is a direct DNA biosensor?
It involves the measurement of physicochemical changes occuring on the surface of the transducer device due to DNA hybridization
What are some indirect DNA biosensors?
- DNA-specific electron-mediator detection
- DNA-specific redox-indicator detection
- Enzyme-amplified electrochemical
What happens in a SPR?
The biomolecular recognition event is coupled to a change in refractive index, detected by optical method.
By changing the refractive index of the ambient (by analyte binding), the angle where SPR occurs will also change.
The sensor signal is proportional to the mass of the molecule on the surface
What is a surface plasmon?
An electron density wave that propagates along the metal-ambient interface
What is the resonance condition?
kx = ksp
The horisontal component of the wavenumber is equal to that of the plasmons
SPR only happens if this condition is fulfilled
What are the 2 modes if operation in SPR?
- 2D monolayer:
all immobilized molecules are at the same distance from the surface. Possible to control the surface conc. of immobilized molecules - 3D assembly “hydrogel”
The hydrogel used in SPR is usually a dextran-based matrix. The sensor signal is increased, avoid non-specific binding, proteins are more likely to stay native
Steps of the covalent immobilization chemistry
- Activation with EDC/NHS for amine coupling
- Immobilization of R’ via amine coupling to lysine residues
- Activation with PDEA for thiol coupling
- Immobilization of R’’ via thiol coupling to cysteine residues
What can you do with an SPR instrument?
- Binding parameters - drug delivery
- Kinetics and affinity
- Thermodynamics
- Screen for inhibitor specificity
- Map the epitopes for Mabs
- Test culture lines for expression of protein
- Look for activity after purification
- Detect and quantify analytes
What is Localized Surface Plasmon Resonance (LSPR)?
Excitation of surface plasmons confined in nanostructures. Can be monitored in an absorbance spectrum. LSPR depends on the shape, size, and material of the nanostructure and the refractive index of the surrounding medium
What are som adv/disadv with LSPR?
+ High local RI sensitivity
+ Low background signal
+ Less temp dependent
+ Simple instrumentation
+ Flexible design
- Low bulk RI sensitivity
What are some adv/disadv with SPR?
+ High buk RI sensitivity
+ Versatile surface chem
+ Established sensing technique
- High background signal
- Advanced instrumentation
- Requires temp control
Why microfluidics?
- Small sample volume and reduced reagents
- New functionality, new detection methods
- Speed, faster response
- Small systems, integrated systems: PoC
- Cost and single use: massproduction
What are some effects that become dominant when down scaling?
- Laminar flow
- Diffusion
- Fluidic restance
- Surface area to volume ratio
- Surface tension and capillary flow
What are the basic principles of microfabrication?
- Design using CAD
- Patterning
- Subtractive technique or deposition/additive
- Modify material
- Repeat for multiple layers
- Back-end processing
What are the steps of photolithography?
- Si substrate surface preparation
- Spin-coating: photoresist applied to the surface
- Soft bake
- Align and expose mask in close contact to the photoresist covered surface
- Develop
- Hard bake
- Dry etch. RIE (or wet etch)
- Remove photoresist
That are the steps of soft lithogrphy?
Fabricate template
1. Pour on PDMS on master
2. Cure and peel off
Pattern and contact printing
1. Printing
2. Wet etch
Direct patterning methods
- Inject printing
- Drop on demand printing
- Laser ablation/cutting
- Screen printing
Additive techniques
- Physical vapor deposition: thermal evaporation, sputtering
- Spin coating
- Bar coating
- Electroplating
- Electroless plating
Subractive techniques
- Wet etch: Si, SiO2, HF, metal, oxides
- Dry etch: Reactive ion etching (RIE), sputter etching
What is the difference between isotropic vs anisotropic etching?
Isotropic gives an overetch.
Anisotrpoic, underetch
What are 2 replication methods?
- Hot embossing
- Injection molding
What are some sealing methods?
- wafer/glass bonding
- PDMS/PDMS bonding
- Glass/Si bonding
What components do you need for a microfluidic chip?
- Sample injection
- Channels
- Propulsion: pumps, capillary, centrifugal
- Valves
- Mixing
- Detectors
What is fluorescence?
The property of some atoms and molecules to absorb light at a specific wavelength and emit light at a long wavelength
What is Stokes shift?
The difference between positions of the band maxima of the absorption and emission spectra.
As it increases it becomes easier to seperate excitation from emission light
What is Förster resonance energy transfer?
Phenomenon that occurs when an excited chromophore transfer energy to an acceptor.
- need to be close
- require spectral overlap
- relative orientation between dipole moments
What is fluorescence lifetime?
The average time between the excitation and its return to the ground state
What is fluorescence polarization?
Analyses as output the emission intensity of different polarization planes. When plane-polarized light excites a small and unbound fluorescent molecule, this mainly emits unpolarized light
What are some advantages of quantifying fluorescence polaization?
- Enable direct monitoring of the free/bound ratio
- Does not require the separation of bound and free species
- Eliminates the negative influence of compound absorbance or quenching
What are some disadvantages of quantifying fluorescence polaization?
- Requires large change in molecular volume for the generation of maximum signal
- Require label smallest interaction partner with a fluorophore (not suited for interaction between 2 large proteins)
What are some fluorescent materials?
- Fluorescent proteins
- Synthetic fluorophores
- Unconversion nanoparticles
- Quantum dots
What are some advantages with fluorescence detection?
- Sensitivity (much better than absorption)
- Specificity (only specific fluorophore detected)
- Wide conc range
- Accurate results
What is the difference between fluorescent labels and probes?
Fluorescent probes are expected to be inert to other chemical species present in the environment, while probes are not
How can fluorescent probes be used for DNA assay?
Can be used to detect and characterize nucleotide sequences using a particular nucleotide sequence as a probe
What is a DNA microarray?
Collection of microscopic DNA spots attached to a solid surface to identify specific sequences and probes the genome of an organism.
RNA is extracted and cDNA is synthesized + fluorescence label. Amplified through PCR
Surface is covered with oligonucleotide probes.
Add sample, it hybridized with the probe. In not, it’s not complementay to probe (and is washed away)
Green - sample hybridizes
Red - control hybridizes
No color - no hybridization
Yellow - both hybridizes
What is Affymetrix?
Collection of oligonucleotide on Si chip
1. Isolate mRNA
2. Sample amplification and labelling
3. Inject sample in microarray
4. Probe array hybridization, staining and washing
5. Probe array scanning and intensity quantification
6. Intensity translated into nuclei acid abundance
What are some criteria for the material of wearable biosensors?
- Intrinsic properties
- Stretchability
- Transparency
- Permeability
- Flexibility
- Adhesion
- Biocompatibility
- Scalability
What are some advantages of using body fluids other than blood for analysis? What are some disadvantages?
+ Minimum invasive or non-invasive
+ Proof-of-concepts
+ Minimal risk of harm or infection
- Accuracy (need better understanding of the correlations between blood and biofluids)
How does the glucose sensor work?
An enzyme (GOx) is immobilized on an electrode. The recognition of the analyte by GOx generates a current, this current is proportional to the glucose conc
