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FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
PROVISIONAL SPECIFICATION
(See Section 10, rule 13)
“A METHOD FOR DETECTING AN ANALYTE”
Name and Address of the Applicant: INDIAN INSTITUTE OF SCIENCE,
Bangalore – 560 012, Karnataka, India.
Nationality: INDIAN
The following specification particularly describes the nature of the invention.
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FIELD OF INVENTION
The present invention relates to detection of analytes. In particular, the instant invention
relates to detection of analytes by the use of fluorescence from unfunctionalised dendritic
polymers.
BACKGROUND AND PRIOR ART OF INVENTION
Detection of analytes has enormous practical significance, the detrimental effects of
chemical compounds is a common knowledge. Detection of an analyte is being
performed currently by methods as disparate as, metal detectors (wherein the analyte
material is cased in a metal), canines and advanced analytical techniques, such as, mass
spectrometry, gas chromatography, cyclic voltammetry etc. Each of these techniques
have their short-falls. Metal detection requires that the analyte has a metal-casing,
canines can become fatigue, chromatographies and other physical methods, such as, Xray
screening and mass spectrometry, are expensive and are not easily adoptable. The
recently evolved conjugated aromatic polymers for detection of analytes are based on
fluorescent assays. However, severe requirements on the constitutions and compositions
of the polymers for their efficient functions are limitations, in addition to high costs of the
materials and possible carcinogenicities and health hazards associated with polyaromatics
in general. On the other hand, polymers, such as, polymetalloles use hazardous reagents
in their preparation. Immunosensors are also known, based on antibody-antigen
interactions, however, biochemical requirements to deal with antibodies and antigens are
still a demanding task. Whereas, detectors based on the above methods have been
explored, most of the detections are either non-trace level detections or highly specialized
techniques. The method of invention herein is based on the intensity changes of the
optical emissions of novel non-aromatic dendritic polymers.
OBJECTIVES OF INVENTION
The principal objective of invnetion is to develop a method for detecting an analyte.
Another objective of invention is to develop a sensor for detecting an analyte.
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STATEMENT OF INVENTION
Accordingly the present invention provides a method for detecting analyte comprising
step of measuring quenching of photoluminescence of dendritic polymer(s) exposed to
the analyte; a sensor comprising a non-aromatic dendritic polymer for detecting an
analyte.
BRIEF DESCRIPTION OF ACCOMPANYING FIGURES
Figure 1. Molecular structures of third and fourth generation PETIM dendrimers.
Figure 2. Structures, relative vapor pressure (vs trinitrotoluene, which is 8.02 x 10-6 mm
Hg or 10 ppb at 25 oC) and the redox potentials (vs standard calomel electrode) of the
analytes.
Figure 3. Photoluminescence quenching efficiencies of the third generation dendrimer,
upon addition of the analytes.
Figure 4. The relative quenching efficiency of non-aromatic dendritic polymers.
Figure 5. The time dependent emission spectra of a thin film of PETIM G3-amine, upon
exposure to chlorodinitrobenzene vapor (left) nitrotoluene vapor (right) (25 oC) after 0, 1,
5, 10, 15 minutes.
DETAILED DESCRIPTION OF INVENTION
The present invention is in relation to a method of detecting analyte comprising steps of
measuring quenching of photoluminescence of dendritic polymer(s) exposed to the
analyte.
In another embodiment of the present invention, the analyte is selected from a group
comprising chemical and biological analyte, preferably chemical analyte.
In yet another embodiment of the present invention, the dendritic polymer(s) is nonaromatic
dendritic polymer(s).
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In still another embodiment of the present invention, the non-aromatic dendritic polymer
is selected from a group comprising poly(propyl ether imine), poly(amido amine) and
poly(propylene imine).
The present invention is also in relation to a sensor comprising a non-aromatic dendritic
polymer for detecting an analyte.
The invention addresses detections of the analytes, with the aid of the inherent
fluorescence property of the PETIM series of dendrimers. With specific reference to the
application of fluorescence technique for analyte detections, the existing knowledge
would dictate that the detector chemical entities possess conjugated and aromatic
polymers. The fluorescence behavior of such polyaromatics is known widely in general.
The concept of fluorescence exhibited by polyaromatics was thus applied in the detection
of analytes. For example, analytes constituted with nitro-groups and the conventional
conjugated aromatic polymers are able to form a charge-transfer complex, thereby
causing a reduction in the emission intensities of the aromatic polymers. A nonfluorescent
dendrimer, upon functionalization with fluorescence emitting moieties also
quenches the fluorescence. However, it is noted that the intentional incorporation of a
fluorescence-capable aromatic moiety has to be exercised, and the dendrimer alone plays
the role of passive carrier of the fluorescent moiety. The invention presented in this
application relates to establishing the analyte detection through quenching the inherent
fluorescence of the non-aromatic dendritic polymers, as a result of the interaction
between the dendritic polymers and the analytes. The method is distinct and unique, and
do not have an overlap with any of the known detection systems.
The present invention discloses a method for detecting nitroaromatic compounds, using
an organic dendritic polymer as a sensor. The inherent photoluminescence of dendritic
polymers, namely, poly(propyl ether imine) (PETIM) dendrimers, is affected
significantly in the presence of nanomolar concentrations of analytes, thereby forming the
first instance where-in a non-aromatic dendritic polymer is employed to detect analytes.
Even sub-nanomolar or sub-picomolar concentrations of the analyte may be detected,
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when either very high concentrations or just the thin film of the dendritic polymers are
used.
The PETIM dendrimers are constituted with tertiary amines as the branching points,
propyl groups as the linkers and ethers that connect the branch points and the linkers.
The dendrimers are non-aromatic, do not possess a conventional chromophore nor
fluorophore, nor an un-saturation in their chemical constitutions. Sustained studies have
shown that these dendrimers are inherently fluorescent, emitting light at around 390 nm,
when excited at 330 nm. The tertiary amines have the imminent role in the un-usual and
anomalous fluorescence behavior of these dendrimers. It is also seen that the
fluorescence is attenuated under acidic conditions, as also under highly viscous solvents.
The fluorescence behavior can be studied in the virgin compounds, without the presence
of a solvent. Studies have also shown that certain inorganic anions can quench the
fluorescence of the dendrimers. Following these exploratory studies that helped to
establish the un-usual and anomalous fluorescence behavior of the PETIM series of
dendrimers, efforts were focused to the study the effect of the nitroaromatics on the
fluorescence of the PETIM dendrimers. Intense studies have shown that the PETIM
series of dendrimers are, in essence, respond to chemical analytes, particularly,
nitroaromatics, with response time in seconds. Thus, the invention is directed to the use
of photoluminescent non-aromatic dendritic polymers for the detection of chemical
analytes based on photoluminescence quenching. The invention includes an inexpensive
and highly efficient organic polymer sensor that can detect chemical analytes,
particularly, nitroaromatic compounds, such as, picric acid (trinitrophenol), nitrobenzene,
2,4,6-trinitrotoluene etc in air, water, or other complex aqueous media.
The photoluminescent non-aromatic dendritic polymers of generations 1 to 9 are stable in
air, water, bases and common organic solvents. The fluorescence intensities of the nonaromatic
dendritic polymers could be increased in the presence of acids. In addition to
PETIM series of dendrimers, the non-aromatic, fluorescent dendrimers also includes
poly(amido amine) and poly(propylene imine). The molecular structures of the various
functional groups terminated third and fourth generation PETIM dendrimers are
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presented in Figure 1. These structures are representative to the series of several
generations of the PETIM dendrimers.
The invention is further elaborated with the help of following examples. However, these
examples should not be construed to limit the scope of invention.
EXAMPLE 1
Detection of the analytes is accompanied by the measurement of the quenching of the
photoluminescence of non-aromatic dendritic polymers by the analyte. Fluorimetric
titration of third generation dendrimer by various nitroaromatic analytes, such as,
nitrobenzene, nitrotoluene, dinitrobenzoic acid, chlorodinitrobenzene, dinitrotoluene,
trinitrotoluene, nitrophenol, dinitrophenol and trinitrophenol in methanol showed a
dramatic decrease in fluorescence intensity. The structures, relative vapor pressure and
the redox potentials (vs standard calomel electrode) of the analytes investigated are given
in Figure 2. Figure 3 displays the photoluminescence quenching efficiencies of the third
generation dendrimer, upon addition of the analytes. Sensitivity of non-aromatic
dendritic polymers to the analytes picric acid, trinitrotoluene, dinitrotoluene and
nitrobenzene was as follows: trinitrophenol > trinitrotoluene > dinitrotoluene >
nitrobenzene. Further, the quenching constants were linear with the reduction potentials
of analytes, which would indicate the charge-transfer complex formation between the
analyte and the dendritic polymer.
EXAMPLE 2
Solution Fluorescence Quenching Studies with Nitroaromatic Analytes.
The non-aromatic dendritic polymers with various functional groups at their peripheries
and dendrimer generation 1-9 may be used to detect analytes, so as not to limit the
detection with respect to the dendritic polymer functionalizations at their peripheries, and
the dendritic polymer generations. Detection is achieved by measuring the quenching of
the photoluminescence of the dendrimer by the analyte. Accordingly, the invention
discloses the use of the non-aromatic dendritic polymers susceptible to measurement of
photoluminescence quenching.
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The detection method involves measurement of the quenching of photoluminescence of
the non-aromatic dendritic polymers at ~390 nm, by the analyte in, for example, a
methanolic solution. Fluorescence spectra of methanol solutions of the dendrimer were
obtained by successive addition of aliquots of trinitrophenol, dinitrophenol, nitrophenol,
trinitrotoluene, dinitrotoluene, nitrotoluene, chloro-dinitrobenzene, dinitrobenzoic acid,
nitrobenzene and nitromethane. Figure 3 shows the photoluminescence quenching
efficiencies of the third generation dendrimer, upon addition of trinitrophenol,
dinitrophenol, nitrophenol, trinitrotoluene, dinitrotoluene, nitrotoluene, chlorodinitrobenzene,
dinitrobenzoic acid, nitrobenzene and nitromethane. Fluorescence
quenching efficiencies of the dendrimers was measured and the order was
trinitrophenol>> dinitrophenol>> nitrophenol>> trinitrotoluene> dinitrotoluene>
nitrotoluene> chloro-dinitrobenzene> dinitrobenzoic acid> nitrobenzene>>nitromethane.
The observed trend is correlated with values of reduction potentials of the tested analytes.
Higher reduction potential of the analyte provided higher quenching efficiency.
Quenching the fluorescence of dendrimers with varying functional groups at their
peripheries were also measured with chlorodinitrobenzene and nitrobenzene. The
relative efficiency of the photoluminescence quenching of non-aromatic dendritic
polymers is unique for trinitrophenol, trinitrotoluene, dinitrotoluene and nitrobenzene, as
shown in Figure 4.
Additionally, it has been confirmed that the fluorescence quenching properties of the
dendrimers are dependent on the concentrations and dependent on the dendrimer
generation. In addition to the sensitivity of third generation G3-amine dendrimer, even
more sensitivity was observed for the fourth generation G4-amine dendrimer.
EXAMPLE 3
Fluorescence Quenching in Thin Films:
Studies of the thin film quenching properties of third generation dendrimer were focused
on chlorodinitrobenzene and nitrotoluene, as they provide rapid and large-amplitude
responses that are convenient to monitor. A solution of G3 dendrimer 1 in methanol was
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drop-cast on the side wall of the quartz cuvette and air dried. The analyte (chlorodinitrobenzene,
nitrotoluene) was placed in a vial (10 mL) and kept at 25 oC for a period
of time. Representative data of chloro-dinitrobenzene and nitrotoluene are plotted in
Figure 5. chlorodinitrobenzene and nitrotoluene quenched the dendrimer fluorescence in
a similar fashion.
An important aspect of the PETIM dendrimer fluorescence is their relative insensitivity to
common interferents. Control experiments, including those aromatic compounds that do
not have the nitro-group in their molecular structure, did not show any change in the
photoluminescence spectrum, in both solutions and thin films of dendrimers. Similarly,
exposure of dendrimers both as solutions and thin films to organic solvents such as
toluene, tetrahydrofuran and methanol produced no change in the photoluminescence
intensity.
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WE CLAIM:
1. A method for detecting analyte comprising step of measuring quenching of
photoluminescence of dendritic polymer(s) exposed to the analyte.
2. The method as claimed in claim 1, wherein the analyte is selected from a group
comprising chemical and biological analyte, preferably chemical analyte.
3. The method as claimed in claim 1, wherein the dendritic polymer(s) is nonaromatic
dendritic polymer(s).
4. The method as claimed in claim 1, wherein the non-aromatic dendritic polymer is
selected from a group comprising poly(propyl ether imine), poly(amido amine)
and poly(propylene imine).
5. A sensor comprising a non-aromatic dendritic polymer for detecting an analyte.
6. The sensor as claimed in claim 5, wherein the analyte is selected from a group
comprising chemical and biological analyte, preferably chemical analyte.
Dated this 11th day of September, 2009
K. RAMA
OF K&S PARTNERES
AGENT FOR THE APPLICANT