Author's Accepted Manuscript
DNA-length-dependent fluorescent sensing based on energy transfer in self-assembled multilayers Xiang-Ying Sun, Bin Liu, Yan-Feng Sun, Yaming Yu
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Received date: 7 March 2014 Revised date: 5 May 2014 Accepted date: 7 May 2014 Cite this article as: Xiang-Ying Sun, Bin Liu, Yan-Feng Sun, Yaming Yu, DNAlength-dependent fluorescent sensing based on energy transfer in selfassembled multilayers, Biosensors and Bioelectronics, http://dx.doi.org/10.1016/j. bios.2014.05.055 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
DNA-length-dependent fluorescent sensing based on energy transfer in self-assembled multilayers Xiang-Ying Suna∗, Bin Liu, Yan-Feng Sun, Yaming Yu College of Materials Science and Engineering, Huaqiao University, Xiamen 361021, China
Abstract In this paper, a novel DNA-length-dependent fluorescent sensor was constructed based on the fluorescence
resonance
energy
transfer.
In
the
self-assembled
multilayers
(Quartz/GO/PDDA/Tx-DNA/PDDA/ZnO@CdS), ZnO@CdS and graphene oxide(GO) were employed as an energy donor and an energy acceptor, respectively. Single-stranded Tx-DNA (x represents different chain length of DNA) and poly(diallydimethylammonium) chloride (PDDA) were used as a linker.
In the presence of complementary Px-DNA, the formation of
double-stranded DNA leads to a change in chain length and achieves the purpose of changing the distance between ZnO@CdS and GO. Thereby, it enhances the efficiency of energy transfer between ZnO@CdS and GO resulting in the quench of fluorescence of ZnO@CdS, and thus different length DNA sequence was detected. Keywords: self-assembled multilayers; fluorescence resonance energy transfer; DNA; quantum dots; graphene oxide
1. Introduction Biomolecular detection has been world-widely used in many aspects, such as industrial and environmental monitoring, molecular diagnostics and civil defense (Hwang et al., 2009; Yapasan et al., 2010).
The specific sequences DNA detection is important for the applications in medical
diagnosis and the understanding of biomolecular mechanisms.
Therefore, a great deal of
attention has been paid to develop rapid, simple, and cost-effective biosensors for nucleic acids (Lubin et al., 2010; Li et al., 2004; Huang et al., 2009).
A variety of means have been developed
for the detection of DNA hybridization such as electrochemical (Zhang et al., 2008), colorimetry (Deng et al., 2008), quartz crystal microbalance (Aung et al., 2008) and fluorescence (He et al., 2010; Wang et al., 2010; Zhu et al., 2013) methods.
Among these methods, fluorescent sensing
based on fluorescence resonance energy transfer (FRET) has become a popular method for DNA sequence analysis due to its advantages of designing flexible program, also being done on the interface, and easily combined with other signal amplification technology (Hannestad et al., 2008). FRET is a process in which a donor in the excited state nonradiatively transfers energy to a proximal ground-state acceptor through a long-range dipole-dipole coupling. 1
It has become a
powerful technique to investigate molecular-level changes in the range of 1-10 nm via the dramatic change of FRET efficiency due to variations in the donor-acceptor distance (Jares-Erijman et al., 2003; Sapsford et al., 2006). Self-assembled multilayers (SAMs) have been extensively applied to the constructions of chemo- and bio-sensors. We previously showed that the SAMs-based fluorescent chemosensors exhibited extremely high sensitivity (Morozov et al., 2006; Wang et al., 2011), compared to those in the bulk solutions, as well as improved selectivity in some cases.
Since SAMs are easily
designed, it is easy to control the distance between donor and acceptor.
It is hence demanding to
investigate energy transfer in SAMs. We employed single-stranded DNA as a linker to control the distance between donor and acceptor in SAMs.
In the presence of the complementary DNA
to the single stranded DNA, the donor-to-acceptor distance changed and FRET enhanced, thus DNA sequence could be recognized easily. Graphene and graphene oxide (GO) are emerging materials capable of unique electronic, thermal and mechanical properties (He et al., 2012; Huang et al., 2012; Novoselov et al., 2005; Novoselov et al., 2005; Morozov et al., 2006; Yin et al., 2014; Yin et al., 2012; Zhang et al., 2005).
As a result, graphene and graphene oxide have attracted strong interest in biological
studies (He et al., 2011; He et al., 2010; Wang 2010; Wu et al., 2013).
Since GO possesses the
excellent quenching ability to the fluorophores and its quenching efficiency approaches 100% (He et al., 2010), GO as an energy acceptor has been highlighted by presenting a key role in designing the fluorescence sensor based on FRET. For example, Lu reported that GO could quench a dye-labeled single-stranded DNA probe and the fluorescence was recovered when the probe formed a duplex with its target, which kept the probe away from GO (Lu et al., 2009). Among the various FRET based fluorescent sensors, organic fluorescent dyes as fluorescence donors are conventionally employed. narrow excitation range.
However, they are prone to be photobleached and possess
To avoid the above-mentioned drawbacks, quantum dots (QDs) as ideal
candidates are currently used as fluorescence donors (Wang et al., 2010), since they have attractive physical and optical properties such as high quantum yield, broad absorption, narrow and tunable emission.
According to the theory of FRET, a series of QDs-based biosensors have
been developed (Sapsford et al., 2006; Liu et al., 2010). In this paper, we investigated a new type of fluorescent sensor based on fluorescence resonance energy transfer combined the self-assembled multilayers with the high sensitivity of fluorescence methods
to
detect
DNA.
Among
the
self-assembled
multilayers
films
(Quartz/GO/PDDA/Tx-DNA/PDDA/ZnO@CdS), ZnO@CdS was as an energy donor and GO was as an energy acceptor. Single-stranded Tx-DNA (x = 1, 2, 4, 6 represents chain length of 10 bp, 23 2
bp,
40
bp,
60
bp
of
DNA
chain,
respectively)
with
different
length
and
poly(diallydimethylammonium) chloride (PDDA) was as a linker.
When complementary
Px-DNA was added, DNA double helix structure was formed, which led to a change in chain length and achieved the purpose of shortening the distance between ZnO@CdS and GO (Scheme1).
Therefore, the efficiency of energy transfer between ZnO@CdS and GO was
enhanced with the quenching of fluorescence of ZnO@CdS.
Meanwhile, DNA sequences and
length can be detected in terms of FRET based SAMs system.
Scheme 1 Schematic diagram of Quartz/GO/PDDA/T4-DNA/PDDA/ZnO@CdS SAMs interaction with P4-DNA
2. Experimental 2.1 Chemicals and reagents ZnO@CdS QDs (Yang et al., 2012) and GO (Hummers et al., 1958) were synthesized according to the referred literatures. sequences
were
DNA was purchased from invitrogen (Shanghai, China) and the
listed
in
Table
S1(see
Supplementary
information).
Poly(diallydimethylammonium) chloride (PDDA, Mw 100000-200000) was obtained from J and K Chemical.
Other reagents were analytical grade and used without further purification.
All aqueous solutions were prepared in ultrapure water with a resistivity of 18.2 MΩ·cm (purified by Milli-Q academic purification set from Millipore).
2.2 Preparation of Quartz/GO/PDDA/Tx-DNA/PDDA/ZnO@CdS SAMs
The quartz substrates used for layer-by-layer deposition were cleaned by immersing into fresh piranha solution (1:3 (v/v) mixture of 30% H2O2 and 98% H2SO4) for 20 min (Caution: Piranha solution reacts violently with organic materials.). water and used immediately after cleaning.
After that, the substrates were rinsed with
Carefully cleaned quartz substrates were dipped into
0.12 mg/ml GO solution for 1.5 h, and dried with nitrogen flow after each deposition. Then the substrates were immersed into PDDA (1%, v/v), Tx-DNA, PDDA (1%, v/v), as well as ZnO@CdS(5.74×10-4mol/L) solution for 2 h, respectively and followed by washing with water and drying with nitrogen flow.
3
2.3 Apparatus for fluorescence and absorption measurements All fluorescence spectra were measured with a Varian Cary Eclipse luminescence spectrometer.
UV-Vis absorption spectra of QDs and GO were measured using the UV-2800H
UV-vis spectrometer (Unico).
The fluorescence images were captured by the use of the inverted
fluorescence microscope (Axio Observer A1, Zeiss).
The fluorescence lifetime measurements
were carried out by the FLS-920 steady-state/transient fluorescence spectrometer (Edinburgh Instruments).
The Scanning electron microscopy (SEM) is performed by field emission scanning
electron microscope (S-4800, Hitachi).
3. Results and discussions 3.1 FRET between ZnO@CdS and GO The necessary condition to the occurrence of FRET is that the acceptor can absorb energy from the donor so that there are overlaps between absorption spectrum of acceptor and emission spectrum of donor.
We chose ZnO@CdS QDs with the maximum emission at 481 nm as the
donor and GO as the acceptor.
It is shown in figure 1a that there is a sufficient spectral overlap
between emission spectrum of ZnO@CdS QDs and absorption spectrum of GO, which comply with the requirements of the occurrence of FRET.
Figure 1 (a) Normalized UV-Vis absorption spectrum I of GO and emission spectrum II of ZnO@CdS；(b) Fluorescence spectra of ZnO@CdS at different concentration of GO
As shown in Figure 1b, with adding of GO, the fluorescence intensity of ZnO@CdS at 481nm decreased sharply from nearly 1000 to 0, which can confirm the fact that GO can quench ZnO@CdS effectively.
Thus, Quartz/GO/PDDA/Tx-DNA/PDDA/ZnO@CdS self-assembled
multilayers films were constructed by using ZnO@CdS as energy donor, GO as energy acceptor as well as Tx-DNA with different length as a linker. hybridization on the FRET in SAMs were studied.
4
Meanwhile, the effects of DNA
3.2 SEM of GO and SAMs An apparent lamellar structure of GO was observed from SEM, as shown in Figure 2a.
The
Figure 2 SEM images of (a) GO; (b) Quartz/GO SAMs; (c) Quartz/GO/PDDA/T4-DNA/PDDA/ZnO@CdS SAMs
respective sheet layers of GO were bonded with each other by the van der Waals force and the surface was smooth. Firstly, GO was uniformly assembled onto the quartz glass surface to form quartz/GO film (Figure 2b). electrostatic interaction. adsorbed
onto
the
Then, PDDA was adsorbed onto the prepared quartz /GO layer via After that, T4-DNA, PDDA, and ZnO@CdS were alternatively
prepared
quartz/GO/PDDA
Quartz/GO/PDDA/T4-DNA/PDDA/ZnO@CdS SAMs. uniformly distributed with some gaps(Figure 2c).
multilayers
surface
to
achieve
The entire surface of the SAMs was
The hybridization of DNA can be conducted
with the inner layer through the gaps by adding pairing P4-DNA.
3.3 DNA hybridization enhancing FRET in SAM In
order
to
study
the
effect
of
DNA
hybridization
on
energy
transfer
in
Quartz/GO/PDDA/T4-DNA/PDDA/ZnO@CdS self-assembled multilayers, other two kinds of self-assembled
multilayers
were
assembled
and
compared.
One
was
Quartz/PDDA/T4-DNA/PDDA/ZnO@CdS SAMs without acceptor GO, and the other was Quartz/GO/PDDA/PSS/PDDA/ZnO@CdS SAMs without Tx-DNA.
Experimental results have
shown that with the increase in the concentration of P4-DNA, there was a gradual decline in the fluorescence intensity of the Quartz/GO/PDDA/T4-DNA/PDDA/ZnO@CdS SAMs.
Essentially,
there were no changes in the fluorescence intensity of Quartz/PDDA/T4-DNA/PDDA/ZnO@CdS SAMs and Quartz/GO/PDDA/PSS/PDDA/ZnO@CdS SAMs, which might be attributed to the absence of the energy transfer or invariability of energy transfer in the two films.
Figure 3 shows
the relationship curves of relative fluorescence intensity F0/F and P4-DNA concentration of three films, F and F0 were the fluorescence intensity of SAMs in the presence and absence of P4-DNA, respectively.
We found that the relative fluorescence intensity F0/F of the self-assembled
multilayers (a) increased with the increase of P4-DNA concentration, however, the relative fluorescence intensity of F0/F of self-assembled multilayers (b) and (c) essentially kept unchanged with increasing of P4-DNA concentration. 5
Figure 3 Relationship curves between F0/F of (b) (a)Quartz/GO/PDDA/T4-DNA/PDDA/ZnO@CdS; Quartz/GO/PDDA/PSS/PDDA/ZnO@CdS
different SAMs and DNA concentration Quartz/PDDA/T4-DNA/PDDA/ZnO@CdS; (c)
Compared with a and b, the fluorescence intensity of Quartz/GO/PDDA/T4-DNA /PDDA/ZnO@CdS SAMs decreased because DNA hybridization resulted in the enhanced energy transfer, energy was transferred from ZnO@CdS to the receptor of GO.
Film (b)
Quartz/PDDA/T4-DNA/PDDA/ZnO@CdS SAMs had no energy acceptor GO, so no energy transfer occurred.
Compared with a and c, T4-DNA chain was assembled in the film (a)
Quartz/GO/PDDA/T4-DNA/PDDA/ZnO@CdS SAMs and served as the bridge between GO and ZnO@CdS. When T4-DNA and P4-DNA hybridized, fluorescence resonance energy transfer in the self-assembled multilayers was enhanced and fluorescence intensity of ZnO@CdS quantum dots decreased.
The film (c) Quartz/GO/PDDA/PSS/PDDA/ZnO@CdS SAMs was prepared in
the absence of T4-DNA so that there was invariability of energy transfer in the self-assembled multilayers by adding the matching P4-DNA. In summary, it was observed that the hybridization of DNA could enhance energy transfer in self-assembled multilayers, which was confirmed by the following experiments as well.
The
fluorescence microscopy images of Quartz/GO/PDDA/T4-DNA/PDDA/ZnO@CdS were showed in Figure 4. It is observed that there are many blue light spots resulting from the emitting of the uniform distributed ZnO@CdS (Figure 4a).
After adding P4-DNA, most of the blue light spots
were vanished in figure 4b, because the fluorescence of SAMs was quenched with the addition of P4-DNA.
Therefore, energy transfer from ZnO@CdS to GO was significantly improved due to
the hybridization of DNA between P4-DNA and T4-DNA.
Figure 4 Fluorescence microscopy images of Quartz/GO/PDDA/T4-DNA/PDDA/ZnO@CdS in the (a) absence and (b) presence of P4-DNA
6
The fluorescence decay curves of Quartz/GO/PDDA/T4-DNA/PDDA/ZnO@CdS before and after adding P4-DNA were measured at 340 nm emission peak wavelengths. 14.97 ns and 12.97 ns, respectively.
The lifetime was
The reduction of lifetime was further evidences to prove
that the FRET occurred between ZnO@CdS and GO.
3.4 Investigation of Selectivity To study the selectivity of the Quartz/GO/PDDA/T4-DNA/PDDA/ZnO@CdS SAMs for DNA, we examined the change in fluorescence intensity of ZnO@CdS by adding different mismatched DNA, and obtained the F0/F-C(Px-DNA) relationship diagram， F and F0 were the fluorescence intensity of SAMs in the presence or absence of DNA, respectively. As shown in Figure S1 (see Supplementary information), when the perfectly matched DNA (P4-DNA) was added, the curves of F0/F-C(Px-DNA) was essentially linear. When totally mismatched P40-DNA was added, the curves of F0/F-C(Px-DNA) was substantially a straight line parallel to the X axis. While adding the other one-base mismatch and double nucleotide mismatches, there are little changes of fluorescence intensity of ZnO@CdS.
Thus, the sequence of DNA can be distinguished.
3.5 Effect of different chain lengths of DNA on energy transfer in SAMs It was known that energy transfer was distance-dependent.
In this process, the distance from
QDs to GO was adjusted with DNA chain. To study the effect of chain lengths of DNA on energy transfer in self-assembled multilayers, DNA chains with different length were assembled in Quartz/GO/PDDA/Tx-DNA/PDDA/ZnO@CdS SAMs (x = 1, 2, 4, 6 represents chain length of 10 bp, 23 bp, 40 bp, 60 bp of the DNA chain, respectively). Comparing the relationship curves of F/F0 and C(Px- DNA) in different SAMs (Figure S2, see Supplementary information), it was found that with the increase in concentration of P1-DNA, F/F0 of T1-DNA with 10 bp basically does not change, so 10 bp DNA hybridization cannot change energy transfer from ZnO@CdS to GO in SAMs. With the increase of P6-DNA concentration, F/F0 of T6-DNA(60 bp) SAMs which is long basically ups and downs of volatility, no rules could be followed. T2-DNA (23 bp) and T4-DNA (40 bp) are corresponding completely paired DNA corresponding to their respective relative fluorescence intensity and the paired DNA concentration in a linear relationship, but we can clearly see that 40 bp of T4-DNA changes in the slope is larger, better response, greater change in fluorescence energy transfer. Therefore, according to the above experimental results, we speculated that if the chain length was too short and DNA hybridization had little effect on energy transfer, because this system itself had very strong energy transfer before DNA hybridization.
If chain length was too long, the
distance between the donor and acceptor was too large that the energy transfer in SAMs couldn't 7
take place. When the number of bases used was 23 bp and 40 bp, respectively, energy transfer was relatively large after DNA hybridization, which could be displayed by the dramatic decrease in fluorescence intensity of energy donor. Furthermore, according to the thickness of PDDA/PSS polyelectrolyte bilayer about 1.9-2.5 nm(Sapsford et al., 2006), we hypothesized that the thickness of the layer of PDDA approximately 0.95 to 1.25 nm.
Meanwhile, according to the distance of about 10 bases of DNA 3.4 nm, then a
40
chain
bp
DNA
length
of
13.6
nm.
Therefore,
we
speculate
that
the
Quartz/GO/PDDA/T4-DNA/PDDA/ZnO@CdS SAMs for receptor distance is about 16 nm, that is, greater than the limit distance of 10 nm in the traditional liquid phase energy transfer, which can be inferred from the assembly.
Hence, the energy transfer distance of the SAMs film can be
more than 10 nm.
3.6 Quantitative assay of DNA Fluorescence spectra of SAMs were measured after a sufficient time of hybridization reaction between Quartz/GO/PDDA/T4-DNA/PDDA/ZnO@CdS SAMs and P4-DNA.
The fluorescence
intensity of donor was gradually reduced according to the increase in the concentration of DNA (Figure 5), the dependence of F/F0 on the concentration of DNA is illustrated in inset. With the
Figure 5 Fluorescence spectra of Quartz/GO/PDDA/T4-DNA/PDDA/ZnO@CdS SAMs with different concentrations of P4-DNA in 10 mM Tris buffer (pH =8.0). Insert graph showed the ratio F/F0 of fluorescence intensity varied with negative logarithm of concentration of P4-DNA
DNA concentration ranging from 75.82 pM-15.28 nM, a good linear relationship between the relative fluorescence intensity ratio (F/F0) and the negative logarithm of DNA concentration is obtained with a correlation coefficient of 0.9920 and a detection limit of 8.289 pM, based upon which a quantitative DNA hybridization assay can be performed.
Comparing with the most of
DNA sensors based on FRET of graphene oxide (Gao et al., 2014), our proposed method has lower detect limit and wider linear range (Dong et al., 2010, Liu et al., 2010 and Wang et al., 2011).
8
3.7 Interfering components on the detect of DNA When the concentration of P4-DNA was 7.58 nM, a relative error was ± 10% range, the influence of common interfering substances on the determination of DNA were studied.
The
results were shown in Table S2(see Supplementary information), which showed that the method has good selectivity.
4. Conclusions In summary, with GO as energy acceptor, ZnO@CdS quantum dots as energy donor, using PDDA and DNA strand as a middle bridge, Quartz/GO/PDDA/Tx-DNA/PDDA/ZnO@CdS SAMs was built.
DNA hybridization can affect the energy transfer in self-assembled films, the
correlation of DNA chain length and the energy transfer in self-assembled multilayers was firstly explored.
On the basis of the above work, we have developed a novel sensor for the detection of
DNA sequence. A good linear relationship between the relative fluorescence intensity ratio (F/F0) and the negative logarithm of matching DNA concentration was obtained with a linear rage from 75.82 pM-15.28 nM and a detection limit of 8.289 pM.
The developed method was shown to be
a convenient and supplemental analytical tool for monitoring DNA.
Acknowledgements This work was supported by the National Natural Science Foundation of China (grant nos. 21275059 and 21175049), Natural Science Foundation of Fujian Province, China (grant no. Nos. 2011J01049 and 2012J01044) and Scientific Research Foundation for Talents from Huaqiao University (13BS102).
9
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DNA-length-dependent sensor was constructed based on energy transfer in SAMs.
Relation of
DNA hybridization, chain length and energy transfer in SAMs were explored. The developed method for detect DNA sequence was convenience and sensitive.
12
DNA-length-dependent fluorescent sensing based on energy transfer in self-assembled multilayers Xiang-Ying Sun, Bin Liu, Yan-Feng Sun, Yaming Yu
www.elsevier.com/locate/bios
PII: DOI: Reference:
S0956-5663(14)00396-0 http://dx.doi.org/10.1016/j.bios.2014.05.055 BIOS6817
To appear in:
Biosensors and Bioelectronics
Received date: 7 March 2014 Revised date: 5 May 2014 Accepted date: 7 May 2014 Cite this article as: Xiang-Ying Sun, Bin Liu, Yan-Feng Sun, Yaming Yu, DNAlength-dependent fluorescent sensing based on energy transfer in selfassembled multilayers, Biosensors and Bioelectronics, http://dx.doi.org/10.1016/j. bios.2014.05.055 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
DNA-length-dependent fluorescent sensing based on energy transfer in self-assembled multilayers Xiang-Ying Suna∗, Bin Liu, Yan-Feng Sun, Yaming Yu College of Materials Science and Engineering, Huaqiao University, Xiamen 361021, China
Abstract In this paper, a novel DNA-length-dependent fluorescent sensor was constructed based on the fluorescence
resonance
energy
transfer.
In
the
self-assembled
multilayers
(Quartz/GO/PDDA/Tx-DNA/PDDA/ZnO@CdS), ZnO@CdS and graphene oxide(GO) were employed as an energy donor and an energy acceptor, respectively. Single-stranded Tx-DNA (x represents different chain length of DNA) and poly(diallydimethylammonium) chloride (PDDA) were used as a linker.
In the presence of complementary Px-DNA, the formation of
double-stranded DNA leads to a change in chain length and achieves the purpose of changing the distance between ZnO@CdS and GO. Thereby, it enhances the efficiency of energy transfer between ZnO@CdS and GO resulting in the quench of fluorescence of ZnO@CdS, and thus different length DNA sequence was detected. Keywords: self-assembled multilayers; fluorescence resonance energy transfer; DNA; quantum dots; graphene oxide
1. Introduction Biomolecular detection has been world-widely used in many aspects, such as industrial and environmental monitoring, molecular diagnostics and civil defense (Hwang et al., 2009; Yapasan et al., 2010).
The specific sequences DNA detection is important for the applications in medical
diagnosis and the understanding of biomolecular mechanisms.
Therefore, a great deal of
attention has been paid to develop rapid, simple, and cost-effective biosensors for nucleic acids (Lubin et al., 2010; Li et al., 2004; Huang et al., 2009).
A variety of means have been developed
for the detection of DNA hybridization such as electrochemical (Zhang et al., 2008), colorimetry (Deng et al., 2008), quartz crystal microbalance (Aung et al., 2008) and fluorescence (He et al., 2010; Wang et al., 2010; Zhu et al., 2013) methods.
Among these methods, fluorescent sensing
based on fluorescence resonance energy transfer (FRET) has become a popular method for DNA sequence analysis due to its advantages of designing flexible program, also being done on the interface, and easily combined with other signal amplification technology (Hannestad et al., 2008). FRET is a process in which a donor in the excited state nonradiatively transfers energy to a proximal ground-state acceptor through a long-range dipole-dipole coupling. 1
It has become a
powerful technique to investigate molecular-level changes in the range of 1-10 nm via the dramatic change of FRET efficiency due to variations in the donor-acceptor distance (Jares-Erijman et al., 2003; Sapsford et al., 2006). Self-assembled multilayers (SAMs) have been extensively applied to the constructions of chemo- and bio-sensors. We previously showed that the SAMs-based fluorescent chemosensors exhibited extremely high sensitivity (Morozov et al., 2006; Wang et al., 2011), compared to those in the bulk solutions, as well as improved selectivity in some cases.
Since SAMs are easily
designed, it is easy to control the distance between donor and acceptor.
It is hence demanding to
investigate energy transfer in SAMs. We employed single-stranded DNA as a linker to control the distance between donor and acceptor in SAMs.
In the presence of the complementary DNA
to the single stranded DNA, the donor-to-acceptor distance changed and FRET enhanced, thus DNA sequence could be recognized easily. Graphene and graphene oxide (GO) are emerging materials capable of unique electronic, thermal and mechanical properties (He et al., 2012; Huang et al., 2012; Novoselov et al., 2005; Novoselov et al., 2005; Morozov et al., 2006; Yin et al., 2014; Yin et al., 2012; Zhang et al., 2005).
As a result, graphene and graphene oxide have attracted strong interest in biological
studies (He et al., 2011; He et al., 2010; Wang 2010; Wu et al., 2013).
Since GO possesses the
excellent quenching ability to the fluorophores and its quenching efficiency approaches 100% (He et al., 2010), GO as an energy acceptor has been highlighted by presenting a key role in designing the fluorescence sensor based on FRET. For example, Lu reported that GO could quench a dye-labeled single-stranded DNA probe and the fluorescence was recovered when the probe formed a duplex with its target, which kept the probe away from GO (Lu et al., 2009). Among the various FRET based fluorescent sensors, organic fluorescent dyes as fluorescence donors are conventionally employed. narrow excitation range.
However, they are prone to be photobleached and possess
To avoid the above-mentioned drawbacks, quantum dots (QDs) as ideal
candidates are currently used as fluorescence donors (Wang et al., 2010), since they have attractive physical and optical properties such as high quantum yield, broad absorption, narrow and tunable emission.
According to the theory of FRET, a series of QDs-based biosensors have
been developed (Sapsford et al., 2006; Liu et al., 2010). In this paper, we investigated a new type of fluorescent sensor based on fluorescence resonance energy transfer combined the self-assembled multilayers with the high sensitivity of fluorescence methods
to
detect
DNA.
Among
the
self-assembled
multilayers
films
(Quartz/GO/PDDA/Tx-DNA/PDDA/ZnO@CdS), ZnO@CdS was as an energy donor and GO was as an energy acceptor. Single-stranded Tx-DNA (x = 1, 2, 4, 6 represents chain length of 10 bp, 23 2
bp,
40
bp,
60
bp
of
DNA
chain,
respectively)
with
different
length
and
poly(diallydimethylammonium) chloride (PDDA) was as a linker.
When complementary
Px-DNA was added, DNA double helix structure was formed, which led to a change in chain length and achieved the purpose of shortening the distance between ZnO@CdS and GO (Scheme1).
Therefore, the efficiency of energy transfer between ZnO@CdS and GO was
enhanced with the quenching of fluorescence of ZnO@CdS.
Meanwhile, DNA sequences and
length can be detected in terms of FRET based SAMs system.
Scheme 1 Schematic diagram of Quartz/GO/PDDA/T4-DNA/PDDA/ZnO@CdS SAMs interaction with P4-DNA
2. Experimental 2.1 Chemicals and reagents ZnO@CdS QDs (Yang et al., 2012) and GO (Hummers et al., 1958) were synthesized according to the referred literatures. sequences
were
DNA was purchased from invitrogen (Shanghai, China) and the
listed
in
Table
S1(see
Supplementary
information).
Poly(diallydimethylammonium) chloride (PDDA, Mw 100000-200000) was obtained from J and K Chemical.
Other reagents were analytical grade and used without further purification.
All aqueous solutions were prepared in ultrapure water with a resistivity of 18.2 MΩ·cm (purified by Milli-Q academic purification set from Millipore).
2.2 Preparation of Quartz/GO/PDDA/Tx-DNA/PDDA/ZnO@CdS SAMs
The quartz substrates used for layer-by-layer deposition were cleaned by immersing into fresh piranha solution (1:3 (v/v) mixture of 30% H2O2 and 98% H2SO4) for 20 min (Caution: Piranha solution reacts violently with organic materials.). water and used immediately after cleaning.
After that, the substrates were rinsed with
Carefully cleaned quartz substrates were dipped into
0.12 mg/ml GO solution for 1.5 h, and dried with nitrogen flow after each deposition. Then the substrates were immersed into PDDA (1%, v/v), Tx-DNA, PDDA (1%, v/v), as well as ZnO@CdS(5.74×10-4mol/L) solution for 2 h, respectively and followed by washing with water and drying with nitrogen flow.
3
2.3 Apparatus for fluorescence and absorption measurements All fluorescence spectra were measured with a Varian Cary Eclipse luminescence spectrometer.
UV-Vis absorption spectra of QDs and GO were measured using the UV-2800H
UV-vis spectrometer (Unico).
The fluorescence images were captured by the use of the inverted
fluorescence microscope (Axio Observer A1, Zeiss).
The fluorescence lifetime measurements
were carried out by the FLS-920 steady-state/transient fluorescence spectrometer (Edinburgh Instruments).
The Scanning electron microscopy (SEM) is performed by field emission scanning
electron microscope (S-4800, Hitachi).
3. Results and discussions 3.1 FRET between ZnO@CdS and GO The necessary condition to the occurrence of FRET is that the acceptor can absorb energy from the donor so that there are overlaps between absorption spectrum of acceptor and emission spectrum of donor.
We chose ZnO@CdS QDs with the maximum emission at 481 nm as the
donor and GO as the acceptor.
It is shown in figure 1a that there is a sufficient spectral overlap
between emission spectrum of ZnO@CdS QDs and absorption spectrum of GO, which comply with the requirements of the occurrence of FRET.
Figure 1 (a) Normalized UV-Vis absorption spectrum I of GO and emission spectrum II of ZnO@CdS；(b) Fluorescence spectra of ZnO@CdS at different concentration of GO
As shown in Figure 1b, with adding of GO, the fluorescence intensity of ZnO@CdS at 481nm decreased sharply from nearly 1000 to 0, which can confirm the fact that GO can quench ZnO@CdS effectively.
Thus, Quartz/GO/PDDA/Tx-DNA/PDDA/ZnO@CdS self-assembled
multilayers films were constructed by using ZnO@CdS as energy donor, GO as energy acceptor as well as Tx-DNA with different length as a linker. hybridization on the FRET in SAMs were studied.
4
Meanwhile, the effects of DNA
3.2 SEM of GO and SAMs An apparent lamellar structure of GO was observed from SEM, as shown in Figure 2a.
The
Figure 2 SEM images of (a) GO; (b) Quartz/GO SAMs; (c) Quartz/GO/PDDA/T4-DNA/PDDA/ZnO@CdS SAMs
respective sheet layers of GO were bonded with each other by the van der Waals force and the surface was smooth. Firstly, GO was uniformly assembled onto the quartz glass surface to form quartz/GO film (Figure 2b). electrostatic interaction. adsorbed
onto
the
Then, PDDA was adsorbed onto the prepared quartz /GO layer via After that, T4-DNA, PDDA, and ZnO@CdS were alternatively
prepared
quartz/GO/PDDA
Quartz/GO/PDDA/T4-DNA/PDDA/ZnO@CdS SAMs. uniformly distributed with some gaps(Figure 2c).
multilayers
surface
to
achieve
The entire surface of the SAMs was
The hybridization of DNA can be conducted
with the inner layer through the gaps by adding pairing P4-DNA.
3.3 DNA hybridization enhancing FRET in SAM In
order
to
study
the
effect
of
DNA
hybridization
on
energy
transfer
in
Quartz/GO/PDDA/T4-DNA/PDDA/ZnO@CdS self-assembled multilayers, other two kinds of self-assembled
multilayers
were
assembled
and
compared.
One
was
Quartz/PDDA/T4-DNA/PDDA/ZnO@CdS SAMs without acceptor GO, and the other was Quartz/GO/PDDA/PSS/PDDA/ZnO@CdS SAMs without Tx-DNA.
Experimental results have
shown that with the increase in the concentration of P4-DNA, there was a gradual decline in the fluorescence intensity of the Quartz/GO/PDDA/T4-DNA/PDDA/ZnO@CdS SAMs.
Essentially,
there were no changes in the fluorescence intensity of Quartz/PDDA/T4-DNA/PDDA/ZnO@CdS SAMs and Quartz/GO/PDDA/PSS/PDDA/ZnO@CdS SAMs, which might be attributed to the absence of the energy transfer or invariability of energy transfer in the two films.
Figure 3 shows
the relationship curves of relative fluorescence intensity F0/F and P4-DNA concentration of three films, F and F0 were the fluorescence intensity of SAMs in the presence and absence of P4-DNA, respectively.
We found that the relative fluorescence intensity F0/F of the self-assembled
multilayers (a) increased with the increase of P4-DNA concentration, however, the relative fluorescence intensity of F0/F of self-assembled multilayers (b) and (c) essentially kept unchanged with increasing of P4-DNA concentration. 5
Figure 3 Relationship curves between F0/F of (b) (a)Quartz/GO/PDDA/T4-DNA/PDDA/ZnO@CdS; Quartz/GO/PDDA/PSS/PDDA/ZnO@CdS
different SAMs and DNA concentration Quartz/PDDA/T4-DNA/PDDA/ZnO@CdS; (c)
Compared with a and b, the fluorescence intensity of Quartz/GO/PDDA/T4-DNA /PDDA/ZnO@CdS SAMs decreased because DNA hybridization resulted in the enhanced energy transfer, energy was transferred from ZnO@CdS to the receptor of GO.
Film (b)
Quartz/PDDA/T4-DNA/PDDA/ZnO@CdS SAMs had no energy acceptor GO, so no energy transfer occurred.
Compared with a and c, T4-DNA chain was assembled in the film (a)
Quartz/GO/PDDA/T4-DNA/PDDA/ZnO@CdS SAMs and served as the bridge between GO and ZnO@CdS. When T4-DNA and P4-DNA hybridized, fluorescence resonance energy transfer in the self-assembled multilayers was enhanced and fluorescence intensity of ZnO@CdS quantum dots decreased.
The film (c) Quartz/GO/PDDA/PSS/PDDA/ZnO@CdS SAMs was prepared in
the absence of T4-DNA so that there was invariability of energy transfer in the self-assembled multilayers by adding the matching P4-DNA. In summary, it was observed that the hybridization of DNA could enhance energy transfer in self-assembled multilayers, which was confirmed by the following experiments as well.
The
fluorescence microscopy images of Quartz/GO/PDDA/T4-DNA/PDDA/ZnO@CdS were showed in Figure 4. It is observed that there are many blue light spots resulting from the emitting of the uniform distributed ZnO@CdS (Figure 4a).
After adding P4-DNA, most of the blue light spots
were vanished in figure 4b, because the fluorescence of SAMs was quenched with the addition of P4-DNA.
Therefore, energy transfer from ZnO@CdS to GO was significantly improved due to
the hybridization of DNA between P4-DNA and T4-DNA.
Figure 4 Fluorescence microscopy images of Quartz/GO/PDDA/T4-DNA/PDDA/ZnO@CdS in the (a) absence and (b) presence of P4-DNA
6
The fluorescence decay curves of Quartz/GO/PDDA/T4-DNA/PDDA/ZnO@CdS before and after adding P4-DNA were measured at 340 nm emission peak wavelengths. 14.97 ns and 12.97 ns, respectively.
The lifetime was
The reduction of lifetime was further evidences to prove
that the FRET occurred between ZnO@CdS and GO.
3.4 Investigation of Selectivity To study the selectivity of the Quartz/GO/PDDA/T4-DNA/PDDA/ZnO@CdS SAMs for DNA, we examined the change in fluorescence intensity of ZnO@CdS by adding different mismatched DNA, and obtained the F0/F-C(Px-DNA) relationship diagram， F and F0 were the fluorescence intensity of SAMs in the presence or absence of DNA, respectively. As shown in Figure S1 (see Supplementary information), when the perfectly matched DNA (P4-DNA) was added, the curves of F0/F-C(Px-DNA) was essentially linear. When totally mismatched P40-DNA was added, the curves of F0/F-C(Px-DNA) was substantially a straight line parallel to the X axis. While adding the other one-base mismatch and double nucleotide mismatches, there are little changes of fluorescence intensity of ZnO@CdS.
Thus, the sequence of DNA can be distinguished.
3.5 Effect of different chain lengths of DNA on energy transfer in SAMs It was known that energy transfer was distance-dependent.
In this process, the distance from
QDs to GO was adjusted with DNA chain. To study the effect of chain lengths of DNA on energy transfer in self-assembled multilayers, DNA chains with different length were assembled in Quartz/GO/PDDA/Tx-DNA/PDDA/ZnO@CdS SAMs (x = 1, 2, 4, 6 represents chain length of 10 bp, 23 bp, 40 bp, 60 bp of the DNA chain, respectively). Comparing the relationship curves of F/F0 and C(Px- DNA) in different SAMs (Figure S2, see Supplementary information), it was found that with the increase in concentration of P1-DNA, F/F0 of T1-DNA with 10 bp basically does not change, so 10 bp DNA hybridization cannot change energy transfer from ZnO@CdS to GO in SAMs. With the increase of P6-DNA concentration, F/F0 of T6-DNA(60 bp) SAMs which is long basically ups and downs of volatility, no rules could be followed. T2-DNA (23 bp) and T4-DNA (40 bp) are corresponding completely paired DNA corresponding to their respective relative fluorescence intensity and the paired DNA concentration in a linear relationship, but we can clearly see that 40 bp of T4-DNA changes in the slope is larger, better response, greater change in fluorescence energy transfer. Therefore, according to the above experimental results, we speculated that if the chain length was too short and DNA hybridization had little effect on energy transfer, because this system itself had very strong energy transfer before DNA hybridization.
If chain length was too long, the
distance between the donor and acceptor was too large that the energy transfer in SAMs couldn't 7
take place. When the number of bases used was 23 bp and 40 bp, respectively, energy transfer was relatively large after DNA hybridization, which could be displayed by the dramatic decrease in fluorescence intensity of energy donor. Furthermore, according to the thickness of PDDA/PSS polyelectrolyte bilayer about 1.9-2.5 nm(Sapsford et al., 2006), we hypothesized that the thickness of the layer of PDDA approximately 0.95 to 1.25 nm.
Meanwhile, according to the distance of about 10 bases of DNA 3.4 nm, then a
40
chain
bp
DNA
length
of
13.6
nm.
Therefore,
we
speculate
that
the
Quartz/GO/PDDA/T4-DNA/PDDA/ZnO@CdS SAMs for receptor distance is about 16 nm, that is, greater than the limit distance of 10 nm in the traditional liquid phase energy transfer, which can be inferred from the assembly.
Hence, the energy transfer distance of the SAMs film can be
more than 10 nm.
3.6 Quantitative assay of DNA Fluorescence spectra of SAMs were measured after a sufficient time of hybridization reaction between Quartz/GO/PDDA/T4-DNA/PDDA/ZnO@CdS SAMs and P4-DNA.
The fluorescence
intensity of donor was gradually reduced according to the increase in the concentration of DNA (Figure 5), the dependence of F/F0 on the concentration of DNA is illustrated in inset. With the
Figure 5 Fluorescence spectra of Quartz/GO/PDDA/T4-DNA/PDDA/ZnO@CdS SAMs with different concentrations of P4-DNA in 10 mM Tris buffer (pH =8.0). Insert graph showed the ratio F/F0 of fluorescence intensity varied with negative logarithm of concentration of P4-DNA
DNA concentration ranging from 75.82 pM-15.28 nM, a good linear relationship between the relative fluorescence intensity ratio (F/F0) and the negative logarithm of DNA concentration is obtained with a correlation coefficient of 0.9920 and a detection limit of 8.289 pM, based upon which a quantitative DNA hybridization assay can be performed.
Comparing with the most of
DNA sensors based on FRET of graphene oxide (Gao et al., 2014), our proposed method has lower detect limit and wider linear range (Dong et al., 2010, Liu et al., 2010 and Wang et al., 2011).
8
3.7 Interfering components on the detect of DNA When the concentration of P4-DNA was 7.58 nM, a relative error was ± 10% range, the influence of common interfering substances on the determination of DNA were studied.
The
results were shown in Table S2(see Supplementary information), which showed that the method has good selectivity.
4. Conclusions In summary, with GO as energy acceptor, ZnO@CdS quantum dots as energy donor, using PDDA and DNA strand as a middle bridge, Quartz/GO/PDDA/Tx-DNA/PDDA/ZnO@CdS SAMs was built.
DNA hybridization can affect the energy transfer in self-assembled films, the
correlation of DNA chain length and the energy transfer in self-assembled multilayers was firstly explored.
On the basis of the above work, we have developed a novel sensor for the detection of
DNA sequence. A good linear relationship between the relative fluorescence intensity ratio (F/F0) and the negative logarithm of matching DNA concentration was obtained with a linear rage from 75.82 pM-15.28 nM and a detection limit of 8.289 pM.
The developed method was shown to be
a convenient and supplemental analytical tool for monitoring DNA.
Acknowledgements This work was supported by the National Natural Science Foundation of China (grant nos. 21275059 and 21175049), Natural Science Foundation of Fujian Province, China (grant no. Nos. 2011J01049 and 2012J01044) and Scientific Research Foundation for Talents from Huaqiao University (13BS102).
9
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DNA-length-dependent sensor was constructed based on energy transfer in SAMs.
Relation of
DNA hybridization, chain length and energy transfer in SAMs were explored. The developed method for detect DNA sequence was convenience and sensitive.
12
