biosensors lecture 11

fluorescence

limitations of spectrophotometric detection

• target molecules must exhibit absorption peaks at certain specific wavelengths

• such methods are not available for ALL biomolecules

Label-based optical biosensor

• geralized approach

• “label” the target molecule with a specific “dye” and quantify the concentration of the dye

• dye can be conjugated directly to the target molecule or to the secondary bioreceptor

2 major types of dye

radioisotope dye

fluorescent dye

radioisotope dye

• radioactive isotopes which have an unstable atomic nucleus

• emit energy and particles when it changes to a more stable form

• extremely powerful and sensitive

• strict regulation on the use

fluorescent dye

• less sensitive

• not toxic

• safe to use

• easy to conjugate

2 stage energy loss (fluorescent mechanism)

excitation occurs from high energy

stage 1: stokes shift

• a small amount of energy is lost in molecular rotation and/or vibration and heat production

stage 2:

• emission of light at lower energy

advantage of fluorescent dyes

• fluorescent photometry is superior in sensitivity compared to absorption photometry

• many solvents and solutes are transparent to fluorescent measurement therefore not generating background noise

FITC

fluorescein conjugated with isothiocyanate to facilitate chemical conjugation to proteins

TRITC

rhodamine conjugated with isothiocyanate to facilitate chemical conjugation to proteins

upright vs inverted microscope

upright: specimen on bottom, camera on top

inverted: specimen on top, camera on bottom

Dichroic filter

a filter with multiple layers that transmits one wavelength and reflects the others

advanced fluorescent dyes

do not require pre-assay conjugation to target molecule or bioreceptor

• green fluorescent protein (GFP)

• SYBR Green 1

what is Polymerase Chain Reaction (PCR) and what does it do

a means to amplify a particular piece of DNA

• makes numerous copies of a segment of DNA

laboratory version of DNA replication in cells

• can make billions of copies of a target sequence of DNA in a few hours

known disadvantage of fluorescent dyes

photo bleaching

what are quantum dots and how do they work

colloidal semiconductor nanocrystals

small size leads to confinement of excitons

quantum dot properties

• quantum dots are bandgap tunable by size.

• we can engineer their optical and electrical properties

• smaller QD’s have a large bandgap

what is related to QD size

absorption and emission occur at specific wavelengths, which are related to QD size

• wavelength range tunable by size

biological applications of QD’s

• excellent artificial fluorophores

• absorb over a broad spectrum and fluoresce over a narrow range of wavelengths

• high quantum yeild and photostability

• no photo bleaching

• much brighter

• can be toxic to cells!!

Autofluorescence

• different types of natural chemicals and biomolecules exhibit fluorescent characteristics

• can become background noise