# A New FRET-Based Sensitive DNA Sensor for Medical Diagnostics using

Authors: Nidhi Mathur; Anamika Aneja; P. K. Bhatnagar; P. C. Mathur
Journal: Journal of Sensors
 (2008)

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## Abstract

A reliable, fast, and low-cost biosensor for medical diagnostics using
DNA sequence detection has been developed and tested for the detection
of the bacterium “Bacillus anthracis.” In this sensor, Poly [9,9-di (6,6′- N, N′ trimethylammonium) hexylfluorenyl-2, 7-diyl)-alt-co- (1,4-phenylene)]
dibromide salt (PFP) has been taken as cationic conjugated polymer (CCP) and PNA
attached with fluorescein dye (PNAC∗) as a probe. The basic principle of this sensor is that when a PNAC∗ probe is hybridized with a single strand DNA (ssDNA) having
complementary sequence, Forster resonance energy transfer (FRET) may take place from PFP to the PNAC∗/DNA complex. If the FRET is efficient, the photoluminescence from the PFP will be highly quenched and that from PNAC∗ will be enhanced. On the other hand, if the DNA sequence is noncomplementary to PNA, FRET will not occur.


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## Body

## 1. Introduction

Conventional
methods of medical diagnostics are generally very expensive and time consuming
and are mostly based on antibody detection of the infected person. In these
methods, the structure of the antibodies and the antigens they bind to have to
be first determined. Sometimes the cost of diagnostics may become even higher
than that of the treatment, which makes the need for fast, cheaper, and
reliable medical diagnostic techniques indispensable. DNA sequence detection is
a topic of major scientific interest. Its application areas include clinical
diagnostics, single nucleotide polymorphism (SNP) genotyping, environmental
studies, antiterrorism, and forensic analysis [1]. This has motivated the
development of novel DNA sensors with optical [2–6], acoustic [7, 8], or
electronic [9–13]
readout. Among these, optical (fluorescent) detection methods have made their
place because of their high sensitivity, selectivity, and ease of operation
[14]. Homogeneous
DNA hybridization assays based on Forster resonance
energy transfer (FRET) between donor and acceptor molecules are
attractive due to their simplicity of operation and the use of standard optical
equipment. Conjugated polymers (CPs) possess a unique combination of
optoelectronic properties that have found use in a variety of areas [15]. In the present
biosensor application, CPs serve as light-harvesting molecules. The CPs exhibit
efficient energy migration along their delocalized backbone and the collected
energy can be efficiently transferred to acceptors via FRET [16–19]. The chemical
structures of conjugated polymers offer several advantages as the responsive
basis for chemical and biological detection schemes based on optical methods.
CPs may be viewed as a collection of short, conjugated (oligomeric) units kept
in close proximity by virtue of the polymer backbone. Their structure allows
effective electronic coupling and hence fast intra- and interchain energy
transfer [20, 21].In this paper,
we report the method of preparation, operation, and application of a biosensor
that provides fast, sensitive, and selective detection of DNA hybridization by
using water-soluble cationic conjugated polymer (CCP) and PNA probe. The three
components of the assay are a blue light emitting cationic conjugated polymer,
that is, polyfluorenephenylene (PFP), a peptide nucleic acid probe labeled with
fluorescent dye (PNAC*), C* being fluorescein dye, and the negatively charged
target single strand DNA (ssDNA), which may be complementary (cDNA) or
noncomplementary (nDNA) to the PNA sequence. FRET between the donor polymer and
the acceptor PNAC* is used to detect the presence of specific ssDNA sequence
when the sequence of DNA and PNA is complimentary to each other. PNA/DNA complexes hybridize more quickly and are tighter
and more specific than analogous DNA/DNA complexes [22]. This is
because of the fact that there is columbic repulsion between negatively charged
DNA strands, which is absent in the case of negatively charged DNA and neutral
PNA. However, PNA complexes are thus more thermally stable and, by
virtue of their backbone, less susceptible to biological degradation
by nucleases, proteases, and peptidases [23, 24]. Additionally, their
general insensitivity to ionic strength and pH during hybridization
provides a wider platform for DNA detection.When PFP is supplied with
sufficient quantum energy, it absorbs photons and reaches an electronically
excited state (excited singlet state) and emits blue PL. Once formed, the
excited singlet state immediately loses its excess energy and interacts with
the sourroundings. The exciton can lose its energy in a number of ways viz,
fluorescence, intersystem crossing, photobleaching, collisional interaction
with the quenching species, and radiationless transfer of energy (FRET) [25] to
a nearby molecule leading to the excitation of the neighbour. In this case,
exciton loses its energy (within few ps) by the radiationless transfer of
energy to the nearby acceptor PNAC* molecule leading to its excitation which
results in the emission of green light from the acceptor (fluorescent dye). In
the composite solution, hybridization of the neutral PNA probe to the
negatively charged ssDNA significantly creates an electrostatic attraction
between the negative PNAC*/DNA complex and the positive PFP, thus brings the
fluorophore and the polymer sufficiently close to allow efficient FRET, that is, well within the Forsters critical radius. The
time scale in which energy is transferred from donor to acceptor depends on the
distance between them. When this distance is close to Forster critical radius,
the energy transfer occurs within femtoseconds [26]. On the other hand, the
excited state decay of donor PFP occurs exponentially with an average lifetime
of ≈90 ps (≈30 ps in presence
of the acceptor) [27]. The electrostatic energy is, therefore, transferred from
donor to acceptor before the former can emit its photoluminescence (PL). The PL
emitted by the donor will be quenched significantly provided the distance
between the two is within the Forster critical radius.When the PFP is added to negatively charged
PNAC*/DNA complex, it binds only to the regions where there is a
complementation between DNA and PNAC*. The optical amplification due to FRET is
the result of optically exciting the polymer (PFP) which under favourable
conditions (appropriate distance between donor and acceptor in nm) transfers
energy to the dye, which emits amplified characteristic green light. On the
other hand, for noncomplementary ssDNA, no transfer of energy takes place and
hence there is no or very weak emission from the acceptor. A detailed schematic
diagram of the sensor is shown in Figure 1.Figure 1
Schematic representation
of a sensor using polymer and PNAC* probe to detect complementary DNA
sequence [28].


## 2. Experimental

### 2.1. Materials

For developing a
typical sensor forBacillus anthracis detection, we have selected a blue light emitting, water-soluble, cationic
conjugated polymer (PFP), a known PNA probe corresponding to a segment of
Anthrax (Bacillus anthracis) attached to the fluorescent dye
(fluorescein), two DNA primers, one complementary and the other noncomplementary
to the PNA sequence. The light-emitting water-soluble cationic conjugated
polymer was purchased from American Dye Source, Inc., Canada. The PNA probe for Bacillus anthracis was purchased from Panagene, Korea.
A 20-mer strand labeled with Fluorescein dye corresponding to the following
sequence was used:
PNA 20 mer Flu-GTAAATGGTGTTAGGGTTGCN-termC-term.
For the PNA
sequence, “N” represents the free amino group, corresponding to the N-terminal
of a peptide, and “C” is the amidated carboxyl terminal, corresponding to the
C-terminal of a peptide. The PNA is known to display hydrophobic interactions,
leading to aggregation at high concentrations, suggesting that the experiments
must be carried out at very low concentrations (<10−5 M). The
ssDNA corresponding to the following sequences were obtained from Bangalore
Genei, India:ssDNA (Primer sequence 5′ to 3′) 20
merGCAACCCTAACACCATTTAC (cDNA),ssDNA  (Primer sequence 5′ to 3′) 20 merGACTCAATGGCGTTAGACTG (nDNA).


### 2.2. Spectroscopic Measurements

The solutions of
PNAC* and DNA were prepared in deionized water obtained from TKA water
filtration system. For PFP, the solvent mixture of deionized water/ethanol (1:1
v/v) was used. The fluorescence measurements of PFP and PNAC* were made using Shimadzu
Spectrofluorophotometer model RF-5301-PC equipped with a xenon lamp excitation
source and absorption spectra were observed using Shimadzu UV Visible
Spectrophotometer model UV-2450 for different molar concentrations. The spectra were recorded in
the range 350 nm to 700 nm. The spectral overlap between the emission
spectrum of the donor PFP and the absorption spectrum of the acceptor PNAC* is
shown in Figure 2. PNAC* was used in the experiment
at pH 5.5 by using 0.1 N HCL and deionized water. After optimizing the
concentrations of the donor (2.3 × 10−7 M) and acceptor (2.5 × 10−8 M),
the PNA probe (pH = 5.5 at 10−8 M) was annealed at 70˚C, both
with its complementary ssDNA and the noncomplementary ssDNA (10−8 M)
separately. Annealing was done for about 35–40 minutes in order to improve the
hybridization between the ssDNA and PNAC* [28]. The solutions were allowed to cool
slowly up to the room temperature and PFP was added in small steps of 25 μL of 2.3 × 10−7 M to the above solution, that is, 150 μL each PNAC* and ssDNA in equimolar
concentrations of 2.5 × 10−8 M. Samples were placed in a vortex mixer
between each fluorescence measurement (after each addition of the
polymer) to mix the components well. The resulting fluorescence emission
spectra were compared and it was observed that the enhancement in the
Fluorescein emission was maximum, that is, FRET became saturated when the ratio
of the acceptor (PNAC*) to the donor (PFP) was about 1:1 after which no change
is observed in the fluorescence intensity obtained from PNAC* as shown in Figure 3.Figure 2
Absorption and emission
spectra of PFP (at 10−7 M) and that of PNAC* (at   10−8 M).Figure 3
Variation
of PL emission intensity of acceptor, PNAC* (at excitation wavelength
450 nm) for different Acceptor/Donor ratios (A:D) in the range from 1:0 to
1:2 in the solution of PNAC* and complementary ssDNA. At 1:1 ratio, the saturation
of FRET is observed, there is no further increase in PL emission for
higher A: D ratios.It may
be mentioned that we had to use excitation wavelength of 450 nm to obtain FRET
between donor and acceptor. As can be observed from Figure1, at this
wavelength, the absorbance of the donor is reduced by more than 90% as compared
to its peak absorbance. However, as compared to the donor, the acceptor has a
very poor absorbance. The peak absorbance of PFP measured by us in the present
experiment is 2.8 at 376 nm and that of PNAC*, 0.016 at 490 nm and we had to
select the excitation wavelength in such a manner that there is sufficient
absorbance in the acceptor though at the cost of absorption in the donor.When
the conditions for efficient FRET are satisfied, a significant enhancement in
the fluorescence intensity of the acceptor and a decrease in fluorescence
intensity of the donor are
observed because of the quenching of the fluorescence of the donor as shown in
Figure4.Figure 4
PL emission intensity of donor PFP and acceptor PNAC* (at
excitation wavelength 450 nm) corresponding to A:D ratios, 1:0.08 and 1:1.
When FRET is maximum, the donor is quenched by 7 times.A comparison of the emission spectra of PNAC* in the presence of
complementary and noncomplementary ssDNA has been shown in Figure 5. Intense Fluorescein emission from fluorescent
dye due to efficient FRET was observed in the presence of the complementary DNA
strand. The efficiency of FRET is dependent on the inverse sixth power of
distance between the donor and acceptor and is given by
(1)E=1{1+(R/R0)6},
where R0 is the distance at which
half of the energy is transferred. The Forster
critical radius R0 (in nanometers) is related to the spectral
overlap by the relation [29]:
(2)R0=2.11×10−2[K2⋅J(λ)⋅η−4⋅QD]1/6,
where K2 is a factor describing the relative orientation in space between
the transition dipoles of the donor and acceptor, J(λ) is the spectral overlap integral of donor
emission and acceptor absorbance (with wavelength in nanometers), η is the
refractive index of the medium, and QD is the quantum yield of the
donor. If the distance between donor and
acceptor is within Forster critical radius R0 which is calculated as 6.4 nm
between PFP and Fluorescein using (2), the FRET energy will enhance the
acceptor emission and quench the PL of the donor PFP. If the average distance
between the hybridized complex and the PFP molecules is more than Forster radius,
there will be no efficient transfer of energy from donor to acceptor and hence
no emission from the fluorophore. Therefore, this distance has been optimized
by varying the concentrations of the donor and acceptor in the solution as
shown in Figure 6. We
have also calculated the efficiency of energy transfer for different
concentrations of donor using (1) and it can be observed from Figure 6 that as the
concentration of donor increases, the efficiency of energy transfer also
increases till saturation of FRET is achieved and no change was observed beyond
the acceptor-donor ratio 1:1, that is, for 1:3 and further.Figure 5
PL emission spectra of PFP and PNAC*
in presence of complementary (as shown by (a) green colour) and
noncomplementary ssDNA (as shown by (b) blue colour).
Emission spectra of PNAC* on direct excitation in absence of polymer is
also shown (by (c) red colour).Figure 6
Efficiency of energy transfer,
that is, FRET from donor to acceptor with the variation in the
concentration of PFP. Inset shows the separation distanceR between donor
and acceptor corresponding to different A:D ratios.


## 2.1. Materials

For developing a
typical sensor forBacillus anthracis detection, we have selected a blue light emitting, water-soluble, cationic
conjugated polymer (PFP), a known PNA probe corresponding to a segment of
Anthrax (Bacillus anthracis) attached to the fluorescent dye
(fluorescein), two DNA primers, one complementary and the other noncomplementary
to the PNA sequence. The light-emitting water-soluble cationic conjugated
polymer was purchased from American Dye Source, Inc., Canada. The PNA probe for Bacillus anthracis was purchased from Panagene, Korea.
A 20-mer strand labeled with Fluorescein dye corresponding to the following
sequence was used:
PNA 20 mer Flu-GTAAATGGTGTTAGGGTTGCN-termC-term.
For the PNA
sequence, “N” represents the free amino group, corresponding to the N-terminal
of a peptide, and “C” is the amidated carboxyl terminal, corresponding to the
C-terminal of a peptide. The PNA is known to display hydrophobic interactions,
leading to aggregation at high concentrations, suggesting that the experiments
must be carried out at very low concentrations (<10−5 M). The
ssDNA corresponding to the following sequences were obtained from Bangalore
Genei, India:ssDNA (Primer sequence 5′ to 3′) 20
merGCAACCCTAACACCATTTAC (cDNA),ssDNA  (Primer sequence 5′ to 3′) 20 merGACTCAATGGCGTTAGACTG (nDNA).


## 2.2. Spectroscopic Measurements

The solutions of
PNAC* and DNA were prepared in deionized water obtained from TKA water
filtration system. For PFP, the solvent mixture of deionized water/ethanol (1:1
v/v) was used. The fluorescence measurements of PFP and PNAC* were made using Shimadzu
Spectrofluorophotometer model RF-5301-PC equipped with a xenon lamp excitation
source and absorption spectra were observed using Shimadzu UV Visible
Spectrophotometer model UV-2450 for different molar concentrations. The spectra were recorded in
the range 350 nm to 700 nm. The spectral overlap between the emission
spectrum of the donor PFP and the absorption spectrum of the acceptor PNAC* is
shown in Figure 2. PNAC* was used in the experiment
at pH 5.5 by using 0.1 N HCL and deionized water. After optimizing the
concentrations of the donor (2.3 × 10−7 M) and acceptor (2.5 × 10−8 M),
the PNA probe (pH = 5.5 at 10−8 M) was annealed at 70˚C, both
with its complementary ssDNA and the noncomplementary ssDNA (10−8 M)
separately. Annealing was done for about 35–40 minutes in order to improve the
hybridization between the ssDNA and PNAC* [28]. The solutions were allowed to cool
slowly up to the room temperature and PFP was added in small steps of 25 μL of 2.3 × 10−7 M to the above solution, that is, 150 μL each PNAC* and ssDNA in equimolar
concentrations of 2.5 × 10−8 M. Samples were placed in a vortex mixer
between each fluorescence measurement (after each addition of the
polymer) to mix the components well. The resulting fluorescence emission
spectra were compared and it was observed that the enhancement in the
Fluorescein emission was maximum, that is, FRET became saturated when the ratio
of the acceptor (PNAC*) to the donor (PFP) was about 1:1 after which no change
is observed in the fluorescence intensity obtained from PNAC* as shown in Figure 3.Figure 2
Absorption and emission
spectra of PFP (at 10−7 M) and that of PNAC* (at   10−8 M).Figure 3
Variation
of PL emission intensity of acceptor, PNAC* (at excitation wavelength
450 nm) for different Acceptor/Donor ratios (A:D) in the range from 1:0 to
1:2 in the solution of PNAC* and complementary ssDNA. At 1:1 ratio, the saturation
of FRET is observed, there is no further increase in PL emission for
higher A: D ratios.It may
be mentioned that we had to use excitation wavelength of 450 nm to obtain FRET
between donor and acceptor. As can be observed from Figure1, at this
wavelength, the absorbance of the donor is reduced by more than 90% as compared
to its peak absorbance. However, as compared to the donor, the acceptor has a
very poor absorbance. The peak absorbance of PFP measured by us in the present
experiment is 2.8 at 376 nm and that of PNAC*, 0.016 at 490 nm and we had to
select the excitation wavelength in such a manner that there is sufficient
absorbance in the acceptor though at the cost of absorption in the donor.When
the conditions for efficient FRET are satisfied, a significant enhancement in
the fluorescence intensity of the acceptor and a decrease in fluorescence
intensity of the donor are
observed because of the quenching of the fluorescence of the donor as shown in
Figure4.Figure 4
PL emission intensity of donor PFP and acceptor PNAC* (at
excitation wavelength 450 nm) corresponding to A:D ratios, 1:0.08 and 1:1.
When FRET is maximum, the donor is quenched by 7 times.A comparison of the emission spectra of PNAC* in the presence of
complementary and noncomplementary ssDNA has been shown in Figure 5. Intense Fluorescein emission from fluorescent
dye due to efficient FRET was observed in the presence of the complementary DNA
strand. The efficiency of FRET is dependent on the inverse sixth power of
distance between the donor and acceptor and is given by
(1)E=1{1+(R/R0)6},
where R0 is the distance at which
half of the energy is transferred. The Forster
critical radius R0 (in nanometers) is related to the spectral
overlap by the relation [29]:
(2)R0=2.11×10−2[K2⋅J(λ)⋅η−4⋅QD]1/6,
where K2 is a factor describing the relative orientation in space between
the transition dipoles of the donor and acceptor, J(λ) is the spectral overlap integral of donor
emission and acceptor absorbance (with wavelength in nanometers), η is the
refractive index of the medium, and QD is the quantum yield of the
donor. If the distance between donor and
acceptor is within Forster critical radius R0 which is calculated as 6.4 nm
between PFP and Fluorescein using (2), the FRET energy will enhance the
acceptor emission and quench the PL of the donor PFP. If the average distance
between the hybridized complex and the PFP molecules is more than Forster radius,
there will be no efficient transfer of energy from donor to acceptor and hence
no emission from the fluorophore. Therefore, this distance has been optimized
by varying the concentrations of the donor and acceptor in the solution as
shown in Figure 6. We
have also calculated the efficiency of energy transfer for different
concentrations of donor using (1) and it can be observed from Figure 6 that as the
concentration of donor increases, the efficiency of energy transfer also
increases till saturation of FRET is achieved and no change was observed beyond
the acceptor-donor ratio 1:1, that is, for 1:3 and further.Figure 5
PL emission spectra of PFP and PNAC*
in presence of complementary (as shown by (a) green colour) and
noncomplementary ssDNA (as shown by (b) blue colour).
Emission spectra of PNAC* on direct excitation in absence of polymer is
also shown (by (c) red colour).Figure 6
Efficiency of energy transfer,
that is, FRET from donor to acceptor with the variation in the
concentration of PFP. Inset shows the separation distanceR between donor
and acceptor corresponding to different A:D ratios.


## 3. Results and Discussion

The
condition of maximum overlap between the emission spectrum of the donor PFP and
the absorption spectrum of the acceptor PNAC* for efficient FRET to occur is
satisfied as is clear from Figure 2. Fluorescence studies performed for the
composite solution PFP/PNAC*/ssDNA (both complementary and noncomplementary at
excitation wavelength 450 nm) show that the fluorescence emission was about 12
times higher for PFP/PNAC*/ssDNA (complementary) relative to PFP/PNAC*/ssDNA
(noncomplementary) complex for which only a weak signal is observed (Figure 5). The experiment
was repeated four times to get consistent results within 5%. This large
difference between the emission intensities (for complementary and
noncomplementary ssDNA) validates the technique used by us for detection of the
DNA sequence where it has been possible to take advantage of the optical
amplification of the conjugated polymers to detect DNA hybridization. It can be
observed from Figure 6 that the FRET saturates beyond A: D ratio 1:1, and also
the separation distance calculated using (1) between donor and acceptor
decreases upto this ratio. The technique is not only simple but requires very
low (10−7-10−8 M) concentrations of PFP, PNA,
and ssDNA thereby making the sensor cost effective. The reporter emission
(PNAC* here) “turns on” only when the target ssDNA is present in the solution.
The technique does not require multiple probes and complex DNA structures. It
will also be interesting to determine the detection limit of the sensor, that
is, the minimum concentration of PNAC* which can be used for the present
experiment. We have tried to excite PL in a PNAC* solution having a
concentration of 10−9 M. As will be seen in Figure 7, the instrument
does not detect PL emission and only noise is observed. We, therefore, conclude
that detection limit of PNAC* is ~10−8 M.Figure 7
PL emission of PNAC* at molar
concentration 2.5 × 10−8 M and 2.5 × 10−9 M (at excitation
wavelength 492 nm).


## 4. Conclusions

FRET-based sensor for a
typical PNA probe forBacillus anthracis has been tested successfully
and presented in the paper. Similarly, PNA probes for various bacteria, such as Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus
aureus, will be used to standardize the sensor for other unknown DNA sequences.
It may be mentioned that we have proposed a low-cost portable sensor [30] based
on the above-mentioned technique, where a small UV-Vis lamp is used as the
source of exciting the polymer PFP. Under the condition of efficient FRET, the
PL from the PFP is quenched, energy is transferred to the dye and significant
change in emission intensity of the acceptor is observed. This technique is fast and cost-effective over other
conventional diagnostic techniques, which are not only expensive but also
time-consuming and less reliable.



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*Source: 270475-2008-07-20.xml*