Bgm3039 Nanosensors

Front 1

What is a nanosensor?

Back 1

a

Front 2

What advantages do nanosensors offer over traditional assays?

Front 3

Why is ratiometric analysis important?

Front 4

What are the delivery systems into intracellular environments?

Front 5

What is importunate about the size of a biosensor/nanosensor?

Back 5

When penetrating individual live cells, even the introduction of a sub-micron sensor tip can cause biological damage and resultant biochemical consequences. So the sensor needs to be small enough to not change the biological function of the system.

Front 6

What are the complications of using fluorescent dyes when observing the structure of a cell?

Back 6

- The fluorescent dyes can be toxic to the cell.

- The fluorophore can cause damage to the cell and derange its normal behaviour

- So, we are not sure if the abnormal behaviour we are seeing is part of a pathological process or due to the toxicity of the introduced fluorophore.

Front 7

What are the 2 types of nanosensor?

Back 7

Active

Passive

Front 8

Give 7 advantages of magnetic nanosensing protein detection?

Front 9

What are 3 previous technologies for general diagnosis?

Back 9

- Biopsy

- Blood detection

- Clinical imaging

Front 10

Define biomarker?

Back 10

an indicator of a biological state or condition. It can be a protein, a fragment of a protein, DNA/RNA, or an organic chemical made by abnormal cells.

Front 11

What are the traditional diagnostic methods for cancer?

Back 11

- endoscopy,

- computed tomography,

- X-rays, positron emission tomography,

- mammography and

- magnetic resonance imaging

Front 12

What are some problems with traditional diagnostic cancer methods?

Back 12

- Not accessible to large populations

- Not practical for repeated screenings at early stages of disease

Front 13

Give a brief overview of ELISA as a high throughput screening method for detecting biomarkers.

Back 13

It involves specific antibodies for a particular biomarker and signals its detection by a chromogenic reporter and substrate.

Front 14

What are 3 disadvantages of ELISA?

Back 14

- time-consuming,

- technically burdensome,

- costly

Front 15

What are PEBBLEs?

Back 15

probes encapsulated by biologically localised embedding that allow for minimally intrusive sensing of ions in cellular environments due to their small size and protect the sensing elements (i.e. fluorescent dyes) by encapsulating them within an inert matrix.

Front 16

What size are PEBBLEs?

Back 16

20 to 600 nm in diameter

Front 17

What is an advantage of PEBBLEs?

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- The protective coating eliminates interference such as protein binding or membrane/organelle sequestration which alter dye response.

- The nanosensor matrix enables dyes that would usually be toxic to the cell to be used for intracellular sensing since the matrix provides protection to the cellular contents.

Front 18

What analytes have been measured with PEBBLEs?

Back 18

- calcium

- potassium

- NO

- oxygen

- chloride

- sodium

- glucose

Front 19

What was the first technology designed to measure intracellular space?

Back 19

Fibre-based optical nanosensors (optical fibre optodes)

Front 20

What is the size of an optical fibre optode?

Back 20

The tip was very small (100 nm) but they quickly become larger up the taper

Front 21

How did an optical fibre optodes work?

Back 21

The fibre optic tip was a pH responsive fluorophore coated with PVC. It measured the change in pH causing a change in fluorescence which was sent along the optical fibre, amplified and read.

Front 22

What were the problems with the optical fibre optode nanosensors?

Back 22

The size of the taper meant multiple insertions into the cell can damage it, so it was not responsive in its natural state.

Front 23

What nanosensor was developed from the optical fibre optodes?

Back 23

pulled fibre technology

Front 24

What nanosensor is derived from pulled fibre technology?

Back 24

PEBBLEs - fluorescent nanosensor

Front 25

What was the key developments of PEBBLE and where do they originate from?

Back 25

Derived from pulled fibre technology which is derived from optical fibre optodes

- PEBBLE enable a discrete observation platform

- the sensor can be integrated into the cell in large numbers

Front 26

What are the key characteristics of PEBBLEs?

Back 26

- 20 nm radius (1 parts per billion ppb of cell volume) making them easy to introduce into cells

- the cell viability remains high 97%

- the response time is <1 ms

- a wide number of analytes can be measured

Front 27

What materials can PEBBLEs matrices be made from?

Back 27

- polyacrylamide

- cross-linked decyl methacrylate,

- silica-based sol-gel - spacious, good for entrapping enzymes as it will not distort the structure of the enzyme

Each matrix can be combined with specific "free dyes", ionophores, or enzymes to produce sensors selective for the biological component of interest

Front 28

What is a ratiometric device?

Back 28

measures the ratio between two fluorescence intensity values that are taken at two excitation wavelength. It can correct for unequal dye loading

Front 29

What is Sol-gel PEBBLE used to measure and how does it do this?

Back 29

Intracellular oxygen

It is a ratiometric device

- The Ruthenium dye fluorescence is quenched very efficiently in the presence of oxygen, so its fluoressence intensity goes down with greater local oxygen concentration

- The Oregon green dye fluorescence is not sensitive to oxygen, so its intensity stays the same and is used as a reference.

Front 30

What changes the intensity of the fluorescence of the 2 dyes in a ratiometric PEBBLE device?

Back 30

- varying amount of PEBBLE present in solution

- changes in intensity of the excitation light

but the ratio between the active dye and the reference dye remain the same and this is what is being measured

Front 31

How has interference been compared between free fluorescent dyes and PEBBLEs?

Back 31

When measuring Ruthenium:Oregon green fluorescence, the ratio of Ru:OG goes up in the free dye solution with increasing concentration of BSA, whereas the ratio stays the same on the Sol-gel PEBBLEs this is the desired result since the amount of oxygen in the solution is not changing.

- the ratio has been maintained because the dye is encapsulated in the porous shell, protecting it from the effect of protein. The analyte can still get in and out through the pores of the nanosensor and interact with the fluorophore, but because the protein is large, with a high MW the BSA cannot.

Front 32

What is the most important consideration when choosing a delivery methods for PEBBLE sensor insertion?

Back 32

Methods of sensor insertion can be specific to the cell type used for experimentation and many factors play a role in the choice, most importantly whether the cells in culture are adherent.

Front 33

Who developed pulled fibre technology?

Back 33

Tan et al.

Front 34

What are the disadvantages of optical fibre optodes?

Back 34

- Invasive to the cellular system (taper size)

- Technique requires highly skilled operators to position the probe in the cell

- Even though the optode has submicron diameters the penetrations are still >1% of the cell volume, which could induce severe perturbations, limiting cellular viability

Front 35

What are the disadvantages of fluorescent probes?

Back 35

- easier to use than optical fibre optodes but still perturb the cell.

- can be cytotoxic

- they have variations in signal intensity due to the non-specific protein binding, organelle sequestration, efflux of dye by membrane proteins

- fluorescent dyes have limited experimental time once the fluorophore is loaded in the cell

Front 36

What are the advantages of the sensor platform in PEBBLEs?

Back 36

- it can provide a convenient cage that facilitates the entrapment of a number of sensing components, and access to just small analytes, offering the ability to fabricate ratiometric sensors from complementary probes, the senors can be complex and they will not get interference as other substances with big MW eg proteines etc cant enter the cage.

Front 37

Why is the delivery method of PEBBLE sensors so important?

Back 37

- important to retain cellular viability

- suitable amount of sensor must be loaded into the cell to ensure an adequate fluorescent signal

- critical in a long term experimentation is the cells are still dividing as this will effectively reduce the amount of sensors within the cell at each division.

Front 38

What are some methods of PEBBLE delivery into cells?

Back 38

- Gene gun offers high loading of PEBBLE sensors for adherent cells and is the best method

- CPP functionalization has proved effective for cell cultures generally with varying levels of sensor loading, depending on the cell type

- Lipid transfection gives good results once conc of transfection reagent and sensor loading are established

- Picoinjection allows location specific PEBBLE delivery but the loading is not as high so it is more suited to larger cells

Front 39

What is the consideration regarding positioning with the Gene gun method?

Back 39

- there is no control over the positioning of thesensors although once the correct pressure and particle concentrationsare found, this method is very effective for the delivery ofmany sensors in a single blast with a fairly even spread ofsensors throughout the cell

Front 40

What is the cell viability when using the Gene gun method?

Back 40

98%

Front 41

Why is the Gene gun only suitable for adherent cell cultures?

Back 41

The media must be removed prior to sensor bombardment

Front 42

Give some examples of cells that the Gene gun has been used in for inserting PEBBLE?

Back 42

A range of nanosensors have been inserted into many different adherent cell types (with high sensor loading, and good cell viability):

- astrocytes

- hepatocytes

- CHO cells

- several epithelial cell lines

Front 43

What is a way that free radicals could be measured using nanosensors?

Back 43

Phagocytosis

When a macrophage comes into contact with a foreign compound, it will engulf it. Groups of nanosensors can be internalised into macrophage vacuoles via routine cell processes. The nanosensors are then encapsulated in phagosomes, they are then bombarded with free radicals, dyes that are sensitive to ROS measure the amount.

Front 44

What are polyacrylamide sensors synthesised in?

Back 44

Water-in-oil microemulsion system

Front 45

What are the properties of a water-in-oil microemulsion system?

Back 45

- transparent

- isotropic

- thermodynamically stable liquid media

- continuous oil domain

- aqueous domain thermodynamically compartmentalised by a surfactant

Front 46

What is the water-in-oil microemulsion sometimes referred to as?

Back 46

revers micellar solution/droplet

Front 47

What main 2 events take place in the aqueous core of the micellar droplet?

Back 47

- The polymerisation reaction

- Formation of nanoparticles

Front 48

What is the size of the synthesised polyacrylamide PEBBLEs and why?

Back 48

nanometer range

The PEBBLE components are hydrophilic so stay in the aqueous core where both the reaction and formation occurs.

Front 49

How can the size of the polyacrylamide nanospheres be varied?

Back 49

By changing the amount of

- surfactant

- solvent

- monomer

- temperature of the reaction

Front 50

What is the reproducibility of polyacrylamide matrix PEBBLEs?

Back 50

Very good, once a procedure is optimised the protocol will be reliable for producing the required sensors

Inter and intra batch reproducibility is consistent but each batch should be calibrated to ensure this

Front 51

What applications have been used by polyacrylamide sensors so far?

Back 51

- pH

- calcium

- oxygen

- glucose

- zinc

- magnesium

Front 52

What determines the size of the PEBBLE and how?

Back 52

The initial size of the aqueous core of the micellar droplet by encapsulating the reactions and preventing the sphere from becoming too large during polymerisation