Transcript Fluorimetric analysis

NOVEL FLUORESCENT SENSORS FOR THE
DETECTION OF ORGANIC MOLECULES IN
EXTRA-TERRESTRIAL SAMPLES
Roy C. Adkin, James I. Bruce and Victoria K. Pearson
Twitter: @RCAdkin
Aim of the Research
Development of a fluorescent
lanthanide complex which will
interact with meteoritic organic
species in situ and/or in the
aqueous phase
Background
• Organic material is found in carbonaceous chondrite (CC) meteorites: <5% by
mass (~14000 different molecules (Schmitt-Kopplin et al., 2010))
• Although (most) confirmed as extra-terrestrial in origin due to:
– Isotope ratios (H/D, C, N, O)
– Structural isomerisation and diversity e.g. racemic ratio, branching
– Compounds present in higher concentrations but rare on Earth, e.g.,
isovaline, pseudoleucine etc. (Kvenvolden et al., 1970)
• No defined environment of formation for what is seen in meteorites although
several possible cosmological provinces suggested
• BUT, minerals are formed under distinct chemical and physical conditions so
can be used as environmental indicators (Velde, 2000)
• Understanding mineral/organic associations more could help clarify organic
compound source regions and formation processes
The problem
• We know a relationship exists;
– Amount of matrix vs bulk organic material indicated by C
and N content (e.g. Anders et al., 1973)
– Removal of minerals by dissolution releases more organic
material (e.g. Sephton and Gilmour, 2001)
– Basic labelling reveals organic material predominately
associated with matrix (e.g. Pearson et al., 2007)
• Organic molecular inventory and concept of
mineral/organic material associations elucidated by
destructive analysis of carbonaceous chondrites
Development of a new, non-destructive, in situ
analytical tool is required…
Fluorescence - Overview
Emitted light < energy
and > λ than the light
absorbed
Usually, emission ceases almost
instantaneously as irradiation is
terminated (ns to μs timescale)
The sensor – Introducing the lanthanides
•
Lanthanides (Ln) are elements, e.g. europium (Eu) and
terbium (Tb) - amongst the most luminescent elements
in the Periodic Table
•
Extensively used in biomedical imaging techniques
•
Lanthanide metal ion coupled to an organic ligand
•
•
•
‘Fingerprint’ emission spectrum consisting of line-like
peaks or bands – indicative of the element
Have long fluorescent lifetimes – the time between
termination of irradiation and cessation of emission (ms)
Ln must be stable, chemically inert yet subject to physical
interactions
The sensor – ligand: DOTA
Commercially available
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraethanoic acid
The sensor – EuDOTA and TbDOTA
Preliminary research
Sources of intrinsic CC fluorescence
• Some minerals exhibit fluorescent properties
• Presence of Eu or Tb can activate, enhance or intensify that
fluorescence
• Identify organic and inorganic CC components whose excitation
and emission λ may be similar to Eu and Tb
• Organic excitation below that of Eu and Tb so not a concern
• Mineral fluorescence, activated and intrinsic, can be background
corrected
Preliminary research – DOTA experimentation
1) DOTA was synthesised
2) Suitable analytes were chosen representative of
all classes of organic molecules identified in CCs
taking into consideration:
– The number and type of reactive sites and functional
groups
– Likelihood of interaction with the sensor – structure/size
– Whether they are terrestrially rare or potentially
prebiotic
– Solubility in water
Results of DOTA experiments and discussion
• 1 mM LnDOTA solution mixed with a range of meteoritic
organic molecules at concentrations expected in CCs
• Spectra showed no peak shifts but a slight, yet trendless,
variation in intensity
• Lack of spectral deviation
• No interaction with metal centre?
• Lanthanide/analyte interaction but fluorescence not altered by
presence of analyte?
• Limit of detection?
• Concentrations consistent with chondritic organic matter
(µM, 10-6 mol dm-3, to nM, 10-9 mol dm-3) may be too low
for detection by this sensor
Fluorimetric analysis – Equimolar (1 mM) EuDOTA/analyte
analysis
Fluorimetric analysis – Equimolar (1 mM) TbDOTA/analyte
analysis
DOTA Fluorimetric analysis – Conclusion
• Would expect analytes to increase fluorescent intensity
due to displacement of water molecules
• No discernible trend regarding analyte structure;
• It was expected that conjugated and aromatic analytes could
increase fluorescent intensity by absorption of excitation
energy
• Hypothesis: DOTA ligand does not afford interactions
• Steric hindrance
• Ln atom is too well enveloped
• Cannot be sure of limit of detection
• Solution? – Use DO3A ligand…one less pendant arm
The new ligand – DO3A
Fluorimetric analysis – EuDOTA/TbDO3A comparison
200000
180000
160000
Fluorescent intensisty
140000
120000
TbDO3A
TbDOTA
100000
80000
60000
40000
20000
0
450
500
550
Fluorescent emission wavelength, nm
600
650
Fluorimetric analysis – Eu3+(aq)/EuDOTA/EuDO3A
comparison
EuDO3A fluorimetric analysis – EuDO3A and all analytes
EuDO3A fluorimetric analysis – EuDO3A and all analytes
(L)-serine
(L)-tyrosine
(L)-threonine
EuDO3A/EuDOTA fluorimetric analysis - conclusions
• EuDOTA and EuDO3A have shown intensity
increase with certain structures or chemical
classes only
• Identification of structures or functional groups
is feasible
• Individual molecular specificity may not be
achievable
Future work
• Produce standards for mixtures of:
– Similar compound classes (e.g. all amino acids or all
carboxylic acids etc.)
– Similar or analogous structures (e.g. hypoxanthine
and cytosine or adenine and 2,4-diaminopyrimidine
etc.)
– Complex mixtures of classes and structures
• Introduce LnDOTA and LnDO3A complexes to
these mixtures
• Measure the effects on Ln fluorescent properties
Future work (continued)
•
Development of other ligand molecules
• change nature of the pendant arms
- facile ligand modifications
- broaden the scope of
interactions with analytes
- selectivity and sensitivity
•
Development of
methodology for future
solid sample analysis
Thank you for listening.
Any questions?
Twitter: @RCAdkin
Email: [email protected]
Fluorimetric analysis – Analyte structures
(L)-serine
(L)-threonine
(L)-ornithine
(L)-tyrosine
(L)-aspartic acid
Benzoic acid
Fluorimetric analysis – Analyte structures
Maleic Acid
Fumaric acid
Cytosine
Adenine
Itaconic acid
Hypoxanthine
N-guanylurea
2,4-diaminopyrimidine