SERS
Intelligent and Ultrasensitive Analysis of Mercury Trace Contaminants via Plasmonic Metamaterial-Based Surface-Enhanced Raman Spectroscopy Cuong Cao, Jun Zhang, Shuzhou Li, and Qihua Xiong* Since the first introduction by Adleman in 1994,[1] the DNA logic gates have been considered as the future of computation technology where their small size is a distinct advantage over the conventional top-down semiconductor technology.[1–7] However, immediate interest has been particularly paid to diversifying applications of the logic circuits as smart sensing and diagnostic platforms owing to the unique properties of DNA such as self-assembly, specific recognition and conformation modulation upon exposing to external stimuli (i.e. metallic ions, proteins).[4,8–12] Along this line, DNA-based systems (e.g. DNAzymes,[4,8,9,13–16] molecular beacons,[3,17] Guanine-rich oligonucleotides (G-quadruplexes),[18,19] [20–22] have been devised on different nanometer aptamers scale carriers such as graphene,[23] graphene oxide,[20,23–25] solid-state nanochannels,[26] quantum dots,[27] and gold nanodisc arrays[28] for various logic gate operations and biosensing applications. Those DNA logic operations mostly rely on fluorescence and enzyme cascades to generate “ON” or “OFF” output signals which involve complex handling and analysis procedures, thus restricting the performance and applications of the sophisticated logic devices. In addition, it still remains very challenging to realize a label-free and switchable DNA logic gate-based biosensing platform that can selectively respond to extremely low concentration of the chemical and biological stimuli. To extend new dimensions towards this rapidly growing area, our work is motivated by the recent emergence of
Dr. C. Cao, Dr. J. Zhang, Prof. Q.-H. Xiong Division of Physics and Applied Physics School of Physical and Mathematical Sciences Nanyang Technological University Singapore 637371 E-mail: [email protected] Prof. Q.-H. Xiong NOVITAS, Nanoelectronics Centre of Excellence School of Electrical and Electronic Engineering Nanyang Technological University Singapore 639798 Prof. S. Li Division of Materials Sciences School of Materials Sciences and Engineering Nanyang Technological University Singapore 639798 DOI: 10.1002/smll.201400165 small 2014, DOI: 10.1002/smll.201400165
plasmonic metamaterials capable of providing high electromagnetic enhancement (hot-spots) and high reproducibility for surface enhanced Raman scattering (SERS).[29–31] By engineering the geometry of plasmonic metamaterials which consist of densely packed periodic arrays of artificial split-ring resonators (SRRs) made of Au or Ag, the resonant excitation of plasmons can be manipulated and confined into the subwavelength hot-spots on the SRRs’ surface, and their electromagnetic responses can be tuned over a broad range of frequency from microwave to optical regimes.[29,32–35] The high electromagnetic enhancements allow significantly increased Raman scattering signals from molecules in close proximity to the SRRs’ surface. In this study, we describe AND, INHIBIT and OR logic gate operations based upon the metallophilic properties of a guanine- and thymine-rich oligonucleotide sequence to K+ or Hg2+ ions, which can specifically trigger or interrupt the formation of Hoogsteen hydrogen bonding that could be monitored by means of SERS with high sensitivity and selectivity. Significantly different from many other fluorescence-based or DNAzymebased logic gate operations which involve complex handling and analysis procedures, SERS obtained from the metamaterials is a direct measurement and can be implemented without the need of any labelling fluorescent dyes or enzymatic activities. Moreover, the molecular logic enables the ultrasensitive detection of mercury ions at a concentration as low as 2×10−4 ppb, which is at least 3 orders of magnitude improvement compared to the literature.[15,17,24,27,36–39] As shown in Figure 1a, the principle of the logic operations is based on the sequentially coordinating effects of Hg2+ and K+ on the conformational modulation of a short guanine (G)- and thymine (T)-rich oligonucleotide sequence ((GGT)4TG(TGG)4). In the presence of K+ cations, G-rich oligonucleotides are known to fold into a specific and stable three-dimensional shape, namely G-quadruplex where four G can self-assemble to form a distinct Hoogsteen hydrogenbonded square (i.e. G-tetrad or G-quartet) via C8=N7-H2.[40] The Hoogsteen hydrogen bonding results a sharp and strong peak centered at ≈1485 ± 3 cm−1 as measured by Raman scattering spectroscopy.[41,42] On the other hand, Hg2+ cations have been demonstrated to bridge specifically with two thymines by labile covalent bonds via N–Hg2+ to form a hairpin T–Hg2+–T complex with a binding constant of ≈8.9 × 1017 M−1,[17] which is much higher than that of K+ cations stacking with the quadruplex structure (≈5 × 106 M−1).[43] Therefore, it
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Figure 1. Combinational logic gate operations (AND, INH, and OR) using metamaterialsgenerated SERS. a) Schematic illustration of the Hoogsteen hydrogen bonding activation using the GT-rich oligonucleotide, Hg2+, K+, and I− ions as inputs. The output is translated into “ON” or “OFF” states of SERS signal at ≈1485 cm−1, which is a diagnostic marker of the C8=N7−H2 Hoogsteen hydrogen bonding of the folded G-quadruplex structure. b) Network map schematically represents the combination of AND, INHIBIT and OR gates for the switchable logic operations. c) SEM micrograph represents uniform and reproducible fabrication of U45 SRRs used as the SERS substrate. Inset is a magnified image, average width of the SRRs w = 45.2 ± 2.6 nm.
provides a rationale for a DNA-based detection of Hg2+ in which the formation of T–Hg2+–T complex in the presence of Hg2+ will inhibit the formation of G-quadruplex structure that in turn leads to the diminishment of the diagnostic Hoogsteen hydrogen bonding at ≈1485 cm−1. In addition, it has been previously reported that the binding constant of Hg2+ ions and iodide (I−) is as high as 5.6 × 1029,[44] therefore the introduction of I− could competitively disrupt the T–Hg2+–T bonding and lead to the reversible formation of the Hoogsteen band under the presence of K+. This will generate reversibly the combinational AND, INHIBIT and OR logic gates schematically represented in Figure 1b. This study illustrates the underlying metamaterial nanostructures of which we have systematically studied and engineered geometrical parameters of the SRRs.[29,32] Our previous results have shown that Au U-shaped SRRs with a line width of 45 nm (U45) give the strongest Raman signal for DNA samples as excited by a 785-nm diode laser with a parallel polarization to the gap of the SRRs.[32] Figure 1c shows representative SEM images of the U45 SRRs with uniformity of device width of 45.2 ± 2.6 nm across the arrays. Figure 2a and 2b depict the interaction of GT-rich DNA and K+ inputs for the development of a SERS-based AND
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logic gate operation using the U45 SRR as a substrate. The presence and absence of GT-rich DNA or K+ input are defined as 1 and 0, respectively. The intensity of Raman band at ≈1485 cm−1 normalized by its full width at half maximum (FWHM) is represented for the output 1 or 0. The combinations of four possible inputs are listed in the truth table (Figure 2c). The bare U45 SRRs (0,0) and the addition of K+ buffer (0,1) do not generate any noticeable SERS modes. When the GTrich DNA prepared in K+-free buffer is introduced (1,0), strong Raman bands at 860, 1005, 1128, 1274, 1237, 1374 cm−1 have been observed, and only weak hydrogen bonding of the Guanine N7 centered at 1492 cm−1 is present. However, in the presence of both GT-rich DNA and K+ buffer (1,1) the strong Hoogsteen band at ≈1485 cm−1 is generated, giving rise to the true value of output. This leads to the formation of AND logic operation as shown in the truth table (Figure 2c). The assignment of a few other strong peaks in the SERS spectra is discussed in detail in another work.[32] The INHIBIT molecular logic gate is presented in Figure 3a and 3b, where the DNA concentration is kept constantly, and the presence and absence of Hg2+ or K+ input are respectively defined as 1 and 0. The combinations of four possible inputs are listed in the truth table (Figure 3c). In the absence of inputs (0,0) or with the
Figure 2. a) SERS spectroscopy using U45 SRRs surface for the monitoring of Hoogsteen band formation at 1485 cm−1 of the GT-rich DNA (2 µM) under coordinating effects of 20 mM K+. b) and c) are normalized Raman intensity changes (ISERS/FWHM) at 1485 cm−1 and a truth table for the AND logic gate, respectively.
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Intelligent and Ultrasensitive Analysis of Mercury Trace Contaminants via Plasmonic Metamaterial-Based SERS
Figure 3. a) SERS spectroscopy using U45 SRRs surface for the monitoring of Hoogsteen band formation at 1485 cm−1 of the GT-rich DNA (2 µM) under coordinating effects of 1 mM Hg2+ and 20 mM K+. b) and c) are normalized Raman intensity changes (ISERS/FWHM) at 1485 cm−1 and a truth table for the INHIBIT logic gate, respectively.
Hg2+ alone (1,0), the GT-rich oligonucleotide is respectively in its unfolded state or in complexed form with T–Hg2+–T, and therefore no strong Hoogsteen band is observed. The intensity of the Hoogsteen hydrogen band is significantly increased when the K+ ions are introduced (0,1), giving rise to the output of 1. However, the band at ≈1485 cm−1 is completely diminished in the presence of both inputs (1,1), meaning that the coordination of Hg2+ in the T–Hg2+–T complex inhibits the formation of the Hoogsteen hydrogen bonding. As a means to evaluate the reversibility of the Hoogsteen hydrogen bonding, I− ions are subsequently introduced to the logic operation (Figure 4). In this case, the output
Figure 4. a) SERS spectra show that formation of the Hoogsteen band is switchable upon the introduction of 50 mM iodide anion (I−). The normalized Raman intensities at 1485 cm−1 are plotted in e), and f) is a truth table for the OR logic gate operation. small 2014, DOI: 10.1002/smll.201400165
of the INHIBIT logic gate (output 2) is used as one of the inputs. Figure 4a and 4b show the SERS spectra and the normalized Raman intensities at ≈1485 cm−1 for monitoring the reformation of the Hoogsteen band. It should be noted that K+ has been already introduced in the buffer solution, thus G-quadruplex formation will be generated as long as the free GT-rich DNA is present. I− ions strongly bind with Hg2+ to break the bridge between thymine and Hg2+ and to liberate the GT-rich oligonucleotide in such a way that the G-quadruplex could be reformed by stacking with K+. Therefore, in the absence of inputs (0,0) the T–Hg2+–T complex could not be disrupted, resulting in the output 3 of 0. However, the output 3 is true if either the output 2 or I− ion is true ((0,1), (1,0), or (1,1)), leading to an OR logic operation as shown in the truth table (Figure 4c). Bivalent mercury ions Hg2+ are the most stable inorganic forms of mercury contaminant in environment and food products and are responsible for a number of life-long fatal effects in human health such as kidney damage, brain damage, and other chronic diseases.[45–47] According to the United States Environmental Protection Agency (EPA), the maximum amount of mercury should be lower than ppb and ppm levels for drinking water and food products, respectively.[45,46] However, because mercury has a strong bioaccumulation effect through the food chain,[47] therefore there exists a great demand and also a significant challenge for development of a method that allows facilely monitoring the concentration of mercury below the defined exposure limit level. The principle of molecular logic gates discussed in Figure 1 presents a rationale for ultrahigh sensitive detection of Hg2+ ions: the trace amount of Hg2+ ions bind to the GTrich oligonucleotides to form hairpin structures thus strongly inhibiting the formation of the quadruplex structures. As a result, the 1485 cm−1 Raman fingerprint of the Hoogsteen hydrogen bonding diminishes. This suggests that lower Hg2+ ion concentration actually leads to a stronger the Raman fingerprint band, which is reminiscent of the recent “inverse sensitivity” mechanism discussed in Au nanostructures.[48] Figure 5a shows the representative SERS spectra of the GT-rich oligonucleotide under coordination of various concentrations of Hg2+ ranging from 0 to 4 × 106 ppb, where the Hoogsteen bands at ≈1485 cm−1 are inversely proportional to the Hg2+ concentrations. The intensities were normalized and plotted statistically as shown in Figure 5b, where a threshold level defined as three times of standard deviation from the blank sample is used to identify detection limit of the assay (L.O.D). The bar graph indicates that concentrations of Hg2+ ranging from 2 × 10−4 to 4 × 106 ppb could be detected, and the lowest detectable concentration (2 × 10−4 ppb) is four orders of magnitude lower than the exposure limit allowed by EPA. Strikingly, the detection limit of the assay far exceeds all reported sensitivity of Hg2+ detections, including the L.O.D of 20 ppb[36] or 2 ppb[37] for colorimetric detections using DNA-functionalized gold nanoparticles, 0.2 ppb for DNA-based machine[37] or fluorescence polarization enhanced by gold nanoparticles.[38] Different metallic ions (such as Ca2+, Cu2+, Cd2+, Mg2+, 2+, Zn2+) have been used to investigate the selectivity of Ni the logic gate. The results in the Figure 5c show that these
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Figure 5. Highly reproducible SERS spectra generated by the metamaterials for the detection of mercury ions. a) SERS spectra of the GT-rich oligonucleotide under coordination of various concentrations of Hg2+ ranging from 0 to 4 × 106 ppb. The diagnostic Hoosteen band intensities at ≈1485 cm−1 show an inverse relationship with the Hg2+ concentrations. b) Statistic data represent the correlation of normalized SERS intensities at ≈1485 cm−1 as a function of Hg2+ concentrations for three parallel acquisitions. c) SERS spectra of the GT-rich oligonucleotide treated with various metallic ions at 1 mM concentration. d) The normalized SERS intensities at 1485 cm−1 indicate that the mercury ions were clearly differentiated from other cations at the same concentration.
metallic ions do not prevent the formation of Hoogsteen hydrogen bonding, their corresponding normalized Hoogsteen band intensities are much higher than that of the Hg2+treated sample (Figure 5d). Therefore, the SERS-based logic gate has not only ultrahigh sensitivity but also good selectivity for the detection of Hg2+. In summary, we have demonstrated for the first time the use of metamaterials as SERS-based logic gate operations stipulated by multiple inputs, serving as the intelligent sensing and diagnostics of mercury ions with ultrahigh sensitivity and selectivity. The logic gates’ operating and sensing principle are based on the metallophilic properties of a GT-rich oligonucleotide sequence to K+ or Hg2+ that can specifically trigger or interrupt the formation of Hoogsteen hydrogen bonding and in turn it could be monitored by Raman spectroscopy. The most notable attributes of the SERS-based logic gates developed in this effort are their label-free measurement, sensitivity, reversibility, reproducibility, and simplicity. In addition, as fabricated by electron beam lithography the SERS-generated metamaterials has not only good reproducibility and high degree of freedom in controlling the optical hot-spots, but is also compatible with prevailing microfluidic systems, the key element to interfacing optical or electrical circuits with biology. The concept of using lithographically fabricated metamaterials for SERS will open up a new approach for molecular logic gates that can be possibly operated down to single molecule level, and will be particularly beneficial for a variety of applications such as clinical diagnostics, environmental monitoring, food and environmental safety analysis, and biological computations.
Experimental Section Materials: The GT-rich oligonucleotide DNA (5′-GGT GGT GGT GGT TGT GGT GGT GGT GG-3′) was purchased from Integrated DNA Technologies, Singapore. HEPES buffer (50 mM HEPES buffer pH 7.4, 0.1% Triton X-100, 2% dimethyl sulfoxide), Hg(ClO4)2·H2O,
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KCl, MgCl2, Zn(NO3)2·6H2O, Cu(NO3)2·3H2O, CaCl2, Cd(NO3)2·3H2O, NiSO4, KI, and other essential chemicals were of analytical grade and obtained from Sigma-Aldrich, Singapore unless otherwise stated. All experiments were done using DNA-free water (1st Base, Singapore). Fabrication of metamaterial substrate: The U-shaped SRRs with line width of 45 nm were fabricated on 0.7 mm-thick ITO glass over an area of 40 µm × 40 µm by electron beam lithography as previously described elsewhere.[29,32] In brief, electron beam resist polymethyl methacrylate (950 PMMA A4, MicroChem, USA) was spin-coated at 4000 rpm for 1 min on the ITO glass, and baked at 180 °C for 20 min. The structures with designed geometrical parameters and periodicity were written using a JEOL 7001F SEM equipped with a nanometer pattern generation system (NPGS), and then developed in 1:3 methyl isobutyl ketone:isopropyl alcohol developer (MicroChem, USA) for 90 s. After the development, 30 nm Au film with a 2 nm Cr as an adhesive layer was deposited using thermal evaporation deposition (Elite Engineering, Singapore) at a base pressure of 3 × 10−7 Torr. Finally, the sample was immersed in acetone for at least 3 hr for lift-off, and washed thoroughly with isopropyl alcohol and water. Operation of SERS-based logic gates: The GT-rich DNA (10 µM) was first heated to 90 °C for 10 min and then immediately chilled in ice water for 2 hr. For the AND logic gate, the pretreated GT-rich DNA at a final effective concentration of 2 µM was subsequently introduced into HEPES buffer (input = 1,0) or HEPES buffer plus 20 mM K+ (input = 1,1). For the INHIBIT logic gate, final effective concentrations of the pretreated GT-rich DNA (2 µM) was then added into different solutions: HEPES buffer (input = 0,0), HEPES buffer plus 1 mM Hg2+ (input = 1,0), HEPES buffer plus 1 mM Hg2+ and 20 mM K+ (input = 1,1), HEPES buffer plus 20 mM K+ (input = 0,1). For an OR logic gate operation, an additional amount of 50 mM I− ion was subsequently introduced into the samples. The samples were then incubated for 2 hr at room temperature. Subsequently, an aliquot of the reacted solutions containing the GT-rich DNA and ions was dropped onto the U-shaped SRR substrate. Then, a glass coverslip (thickness no. 1) was placed on the SRR substrate and sealed with parafilm
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stripes to avoid evaporation. SERS measurement was performed on the SRR substrates using a micro-Raman spectrometer (Horiba-JY T64000) excited with a diode laser (λ = 785 nm) in the backscattering configuration. The back scattered signal was collected through a 50X objective lens, the laser power on the sample surface was measured about 2.5 mW, and acquisition time was 50 s. SERS-based logic gates for sensitive and selective detection of mercury ions: Volumes containing final effective concentrations of 2 µM preheated GT-rich DNA, HEPES buffer (50 mM HEPES buffer pH 7.4, 0.1% Triton X-100, 2% dimethyl sulfoxide), and each concentrations of Hg2+ ranging from 0 to 4×106 ppb were incubated for 2 hr at room temperature. For evaluating the selectivity, various metallic ions at 1 mM concentration (Ca2+, Cu2+, Cd2+, Mg2+, Ni2+, Zn2+) were used instead of Hg2+. The samples were also incubated for 2 hr at room temperature before the SERS analyses as described above.
Acknowledgements The author Q. X. thanks the strong support from Singapore National Research Foundation through a Fellowship grant (NRFRF2009–06), the support from Singapore Ministry of Education via two Tier 2 grants (MOE2011-T2–2–085 and MOE2011-T2–2–051), and a very strong support from Nanyang Technological University via start-up grant (M58110061).
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Intelligent and Ultrasensitive Analysis of Mercury Trace Contaminants via Plasmonic Metamaterial-Based Surface-Enhanced Raman Spectroscopy Cuong Cao, Jun Zhang, Shuzhou Li, and Qihua Xiong* Since the first introduction by Adleman in 1994,[1] the DNA logic gates have been considered as the future of computation technology where their small size is a distinct advantage over the conventional top-down semiconductor technology.[1–7] However, immediate interest has been particularly paid to diversifying applications of the logic circuits as smart sensing and diagnostic platforms owing to the unique properties of DNA such as self-assembly, specific recognition and conformation modulation upon exposing to external stimuli (i.e. metallic ions, proteins).[4,8–12] Along this line, DNA-based systems (e.g. DNAzymes,[4,8,9,13–16] molecular beacons,[3,17] Guanine-rich oligonucleotides (G-quadruplexes),[18,19] [20–22] have been devised on different nanometer aptamers scale carriers such as graphene,[23] graphene oxide,[20,23–25] solid-state nanochannels,[26] quantum dots,[27] and gold nanodisc arrays[28] for various logic gate operations and biosensing applications. Those DNA logic operations mostly rely on fluorescence and enzyme cascades to generate “ON” or “OFF” output signals which involve complex handling and analysis procedures, thus restricting the performance and applications of the sophisticated logic devices. In addition, it still remains very challenging to realize a label-free and switchable DNA logic gate-based biosensing platform that can selectively respond to extremely low concentration of the chemical and biological stimuli. To extend new dimensions towards this rapidly growing area, our work is motivated by the recent emergence of
Dr. C. Cao, Dr. J. Zhang, Prof. Q.-H. Xiong Division of Physics and Applied Physics School of Physical and Mathematical Sciences Nanyang Technological University Singapore 637371 E-mail: [email protected] Prof. Q.-H. Xiong NOVITAS, Nanoelectronics Centre of Excellence School of Electrical and Electronic Engineering Nanyang Technological University Singapore 639798 Prof. S. Li Division of Materials Sciences School of Materials Sciences and Engineering Nanyang Technological University Singapore 639798 DOI: 10.1002/smll.201400165 small 2014, DOI: 10.1002/smll.201400165
plasmonic metamaterials capable of providing high electromagnetic enhancement (hot-spots) and high reproducibility for surface enhanced Raman scattering (SERS).[29–31] By engineering the geometry of plasmonic metamaterials which consist of densely packed periodic arrays of artificial split-ring resonators (SRRs) made of Au or Ag, the resonant excitation of plasmons can be manipulated and confined into the subwavelength hot-spots on the SRRs’ surface, and their electromagnetic responses can be tuned over a broad range of frequency from microwave to optical regimes.[29,32–35] The high electromagnetic enhancements allow significantly increased Raman scattering signals from molecules in close proximity to the SRRs’ surface. In this study, we describe AND, INHIBIT and OR logic gate operations based upon the metallophilic properties of a guanine- and thymine-rich oligonucleotide sequence to K+ or Hg2+ ions, which can specifically trigger or interrupt the formation of Hoogsteen hydrogen bonding that could be monitored by means of SERS with high sensitivity and selectivity. Significantly different from many other fluorescence-based or DNAzymebased logic gate operations which involve complex handling and analysis procedures, SERS obtained from the metamaterials is a direct measurement and can be implemented without the need of any labelling fluorescent dyes or enzymatic activities. Moreover, the molecular logic enables the ultrasensitive detection of mercury ions at a concentration as low as 2×10−4 ppb, which is at least 3 orders of magnitude improvement compared to the literature.[15,17,24,27,36–39] As shown in Figure 1a, the principle of the logic operations is based on the sequentially coordinating effects of Hg2+ and K+ on the conformational modulation of a short guanine (G)- and thymine (T)-rich oligonucleotide sequence ((GGT)4TG(TGG)4). In the presence of K+ cations, G-rich oligonucleotides are known to fold into a specific and stable three-dimensional shape, namely G-quadruplex where four G can self-assemble to form a distinct Hoogsteen hydrogenbonded square (i.e. G-tetrad or G-quartet) via C8=N7-H2.[40] The Hoogsteen hydrogen bonding results a sharp and strong peak centered at ≈1485 ± 3 cm−1 as measured by Raman scattering spectroscopy.[41,42] On the other hand, Hg2+ cations have been demonstrated to bridge specifically with two thymines by labile covalent bonds via N–Hg2+ to form a hairpin T–Hg2+–T complex with a binding constant of ≈8.9 × 1017 M−1,[17] which is much higher than that of K+ cations stacking with the quadruplex structure (≈5 × 106 M−1).[43] Therefore, it
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Figure 1. Combinational logic gate operations (AND, INH, and OR) using metamaterialsgenerated SERS. a) Schematic illustration of the Hoogsteen hydrogen bonding activation using the GT-rich oligonucleotide, Hg2+, K+, and I− ions as inputs. The output is translated into “ON” or “OFF” states of SERS signal at ≈1485 cm−1, which is a diagnostic marker of the C8=N7−H2 Hoogsteen hydrogen bonding of the folded G-quadruplex structure. b) Network map schematically represents the combination of AND, INHIBIT and OR gates for the switchable logic operations. c) SEM micrograph represents uniform and reproducible fabrication of U45 SRRs used as the SERS substrate. Inset is a magnified image, average width of the SRRs w = 45.2 ± 2.6 nm.
provides a rationale for a DNA-based detection of Hg2+ in which the formation of T–Hg2+–T complex in the presence of Hg2+ will inhibit the formation of G-quadruplex structure that in turn leads to the diminishment of the diagnostic Hoogsteen hydrogen bonding at ≈1485 cm−1. In addition, it has been previously reported that the binding constant of Hg2+ ions and iodide (I−) is as high as 5.6 × 1029,[44] therefore the introduction of I− could competitively disrupt the T–Hg2+–T bonding and lead to the reversible formation of the Hoogsteen band under the presence of K+. This will generate reversibly the combinational AND, INHIBIT and OR logic gates schematically represented in Figure 1b. This study illustrates the underlying metamaterial nanostructures of which we have systematically studied and engineered geometrical parameters of the SRRs.[29,32] Our previous results have shown that Au U-shaped SRRs with a line width of 45 nm (U45) give the strongest Raman signal for DNA samples as excited by a 785-nm diode laser with a parallel polarization to the gap of the SRRs.[32] Figure 1c shows representative SEM images of the U45 SRRs with uniformity of device width of 45.2 ± 2.6 nm across the arrays. Figure 2a and 2b depict the interaction of GT-rich DNA and K+ inputs for the development of a SERS-based AND
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logic gate operation using the U45 SRR as a substrate. The presence and absence of GT-rich DNA or K+ input are defined as 1 and 0, respectively. The intensity of Raman band at ≈1485 cm−1 normalized by its full width at half maximum (FWHM) is represented for the output 1 or 0. The combinations of four possible inputs are listed in the truth table (Figure 2c). The bare U45 SRRs (0,0) and the addition of K+ buffer (0,1) do not generate any noticeable SERS modes. When the GTrich DNA prepared in K+-free buffer is introduced (1,0), strong Raman bands at 860, 1005, 1128, 1274, 1237, 1374 cm−1 have been observed, and only weak hydrogen bonding of the Guanine N7 centered at 1492 cm−1 is present. However, in the presence of both GT-rich DNA and K+ buffer (1,1) the strong Hoogsteen band at ≈1485 cm−1 is generated, giving rise to the true value of output. This leads to the formation of AND logic operation as shown in the truth table (Figure 2c). The assignment of a few other strong peaks in the SERS spectra is discussed in detail in another work.[32] The INHIBIT molecular logic gate is presented in Figure 3a and 3b, where the DNA concentration is kept constantly, and the presence and absence of Hg2+ or K+ input are respectively defined as 1 and 0. The combinations of four possible inputs are listed in the truth table (Figure 3c). In the absence of inputs (0,0) or with the
Figure 2. a) SERS spectroscopy using U45 SRRs surface for the monitoring of Hoogsteen band formation at 1485 cm−1 of the GT-rich DNA (2 µM) under coordinating effects of 20 mM K+. b) and c) are normalized Raman intensity changes (ISERS/FWHM) at 1485 cm−1 and a truth table for the AND logic gate, respectively.
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Intelligent and Ultrasensitive Analysis of Mercury Trace Contaminants via Plasmonic Metamaterial-Based SERS
Figure 3. a) SERS spectroscopy using U45 SRRs surface for the monitoring of Hoogsteen band formation at 1485 cm−1 of the GT-rich DNA (2 µM) under coordinating effects of 1 mM Hg2+ and 20 mM K+. b) and c) are normalized Raman intensity changes (ISERS/FWHM) at 1485 cm−1 and a truth table for the INHIBIT logic gate, respectively.
Hg2+ alone (1,0), the GT-rich oligonucleotide is respectively in its unfolded state or in complexed form with T–Hg2+–T, and therefore no strong Hoogsteen band is observed. The intensity of the Hoogsteen hydrogen band is significantly increased when the K+ ions are introduced (0,1), giving rise to the output of 1. However, the band at ≈1485 cm−1 is completely diminished in the presence of both inputs (1,1), meaning that the coordination of Hg2+ in the T–Hg2+–T complex inhibits the formation of the Hoogsteen hydrogen bonding. As a means to evaluate the reversibility of the Hoogsteen hydrogen bonding, I− ions are subsequently introduced to the logic operation (Figure 4). In this case, the output
Figure 4. a) SERS spectra show that formation of the Hoogsteen band is switchable upon the introduction of 50 mM iodide anion (I−). The normalized Raman intensities at 1485 cm−1 are plotted in e), and f) is a truth table for the OR logic gate operation. small 2014, DOI: 10.1002/smll.201400165
of the INHIBIT logic gate (output 2) is used as one of the inputs. Figure 4a and 4b show the SERS spectra and the normalized Raman intensities at ≈1485 cm−1 for monitoring the reformation of the Hoogsteen band. It should be noted that K+ has been already introduced in the buffer solution, thus G-quadruplex formation will be generated as long as the free GT-rich DNA is present. I− ions strongly bind with Hg2+ to break the bridge between thymine and Hg2+ and to liberate the GT-rich oligonucleotide in such a way that the G-quadruplex could be reformed by stacking with K+. Therefore, in the absence of inputs (0,0) the T–Hg2+–T complex could not be disrupted, resulting in the output 3 of 0. However, the output 3 is true if either the output 2 or I− ion is true ((0,1), (1,0), or (1,1)), leading to an OR logic operation as shown in the truth table (Figure 4c). Bivalent mercury ions Hg2+ are the most stable inorganic forms of mercury contaminant in environment and food products and are responsible for a number of life-long fatal effects in human health such as kidney damage, brain damage, and other chronic diseases.[45–47] According to the United States Environmental Protection Agency (EPA), the maximum amount of mercury should be lower than ppb and ppm levels for drinking water and food products, respectively.[45,46] However, because mercury has a strong bioaccumulation effect through the food chain,[47] therefore there exists a great demand and also a significant challenge for development of a method that allows facilely monitoring the concentration of mercury below the defined exposure limit level. The principle of molecular logic gates discussed in Figure 1 presents a rationale for ultrahigh sensitive detection of Hg2+ ions: the trace amount of Hg2+ ions bind to the GTrich oligonucleotides to form hairpin structures thus strongly inhibiting the formation of the quadruplex structures. As a result, the 1485 cm−1 Raman fingerprint of the Hoogsteen hydrogen bonding diminishes. This suggests that lower Hg2+ ion concentration actually leads to a stronger the Raman fingerprint band, which is reminiscent of the recent “inverse sensitivity” mechanism discussed in Au nanostructures.[48] Figure 5a shows the representative SERS spectra of the GT-rich oligonucleotide under coordination of various concentrations of Hg2+ ranging from 0 to 4 × 106 ppb, where the Hoogsteen bands at ≈1485 cm−1 are inversely proportional to the Hg2+ concentrations. The intensities were normalized and plotted statistically as shown in Figure 5b, where a threshold level defined as three times of standard deviation from the blank sample is used to identify detection limit of the assay (L.O.D). The bar graph indicates that concentrations of Hg2+ ranging from 2 × 10−4 to 4 × 106 ppb could be detected, and the lowest detectable concentration (2 × 10−4 ppb) is four orders of magnitude lower than the exposure limit allowed by EPA. Strikingly, the detection limit of the assay far exceeds all reported sensitivity of Hg2+ detections, including the L.O.D of 20 ppb[36] or 2 ppb[37] for colorimetric detections using DNA-functionalized gold nanoparticles, 0.2 ppb for DNA-based machine[37] or fluorescence polarization enhanced by gold nanoparticles.[38] Different metallic ions (such as Ca2+, Cu2+, Cd2+, Mg2+, 2+, Zn2+) have been used to investigate the selectivity of Ni the logic gate. The results in the Figure 5c show that these
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Figure 5. Highly reproducible SERS spectra generated by the metamaterials for the detection of mercury ions. a) SERS spectra of the GT-rich oligonucleotide under coordination of various concentrations of Hg2+ ranging from 0 to 4 × 106 ppb. The diagnostic Hoosteen band intensities at ≈1485 cm−1 show an inverse relationship with the Hg2+ concentrations. b) Statistic data represent the correlation of normalized SERS intensities at ≈1485 cm−1 as a function of Hg2+ concentrations for three parallel acquisitions. c) SERS spectra of the GT-rich oligonucleotide treated with various metallic ions at 1 mM concentration. d) The normalized SERS intensities at 1485 cm−1 indicate that the mercury ions were clearly differentiated from other cations at the same concentration.
metallic ions do not prevent the formation of Hoogsteen hydrogen bonding, their corresponding normalized Hoogsteen band intensities are much higher than that of the Hg2+treated sample (Figure 5d). Therefore, the SERS-based logic gate has not only ultrahigh sensitivity but also good selectivity for the detection of Hg2+. In summary, we have demonstrated for the first time the use of metamaterials as SERS-based logic gate operations stipulated by multiple inputs, serving as the intelligent sensing and diagnostics of mercury ions with ultrahigh sensitivity and selectivity. The logic gates’ operating and sensing principle are based on the metallophilic properties of a GT-rich oligonucleotide sequence to K+ or Hg2+ that can specifically trigger or interrupt the formation of Hoogsteen hydrogen bonding and in turn it could be monitored by Raman spectroscopy. The most notable attributes of the SERS-based logic gates developed in this effort are their label-free measurement, sensitivity, reversibility, reproducibility, and simplicity. In addition, as fabricated by electron beam lithography the SERS-generated metamaterials has not only good reproducibility and high degree of freedom in controlling the optical hot-spots, but is also compatible with prevailing microfluidic systems, the key element to interfacing optical or electrical circuits with biology. The concept of using lithographically fabricated metamaterials for SERS will open up a new approach for molecular logic gates that can be possibly operated down to single molecule level, and will be particularly beneficial for a variety of applications such as clinical diagnostics, environmental monitoring, food and environmental safety analysis, and biological computations.
Experimental Section Materials: The GT-rich oligonucleotide DNA (5′-GGT GGT GGT GGT TGT GGT GGT GGT GG-3′) was purchased from Integrated DNA Technologies, Singapore. HEPES buffer (50 mM HEPES buffer pH 7.4, 0.1% Triton X-100, 2% dimethyl sulfoxide), Hg(ClO4)2·H2O,
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KCl, MgCl2, Zn(NO3)2·6H2O, Cu(NO3)2·3H2O, CaCl2, Cd(NO3)2·3H2O, NiSO4, KI, and other essential chemicals were of analytical grade and obtained from Sigma-Aldrich, Singapore unless otherwise stated. All experiments were done using DNA-free water (1st Base, Singapore). Fabrication of metamaterial substrate: The U-shaped SRRs with line width of 45 nm were fabricated on 0.7 mm-thick ITO glass over an area of 40 µm × 40 µm by electron beam lithography as previously described elsewhere.[29,32] In brief, electron beam resist polymethyl methacrylate (950 PMMA A4, MicroChem, USA) was spin-coated at 4000 rpm for 1 min on the ITO glass, and baked at 180 °C for 20 min. The structures with designed geometrical parameters and periodicity were written using a JEOL 7001F SEM equipped with a nanometer pattern generation system (NPGS), and then developed in 1:3 methyl isobutyl ketone:isopropyl alcohol developer (MicroChem, USA) for 90 s. After the development, 30 nm Au film with a 2 nm Cr as an adhesive layer was deposited using thermal evaporation deposition (Elite Engineering, Singapore) at a base pressure of 3 × 10−7 Torr. Finally, the sample was immersed in acetone for at least 3 hr for lift-off, and washed thoroughly with isopropyl alcohol and water. Operation of SERS-based logic gates: The GT-rich DNA (10 µM) was first heated to 90 °C for 10 min and then immediately chilled in ice water for 2 hr. For the AND logic gate, the pretreated GT-rich DNA at a final effective concentration of 2 µM was subsequently introduced into HEPES buffer (input = 1,0) or HEPES buffer plus 20 mM K+ (input = 1,1). For the INHIBIT logic gate, final effective concentrations of the pretreated GT-rich DNA (2 µM) was then added into different solutions: HEPES buffer (input = 0,0), HEPES buffer plus 1 mM Hg2+ (input = 1,0), HEPES buffer plus 1 mM Hg2+ and 20 mM K+ (input = 1,1), HEPES buffer plus 20 mM K+ (input = 0,1). For an OR logic gate operation, an additional amount of 50 mM I− ion was subsequently introduced into the samples. The samples were then incubated for 2 hr at room temperature. Subsequently, an aliquot of the reacted solutions containing the GT-rich DNA and ions was dropped onto the U-shaped SRR substrate. Then, a glass coverslip (thickness no. 1) was placed on the SRR substrate and sealed with parafilm
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stripes to avoid evaporation. SERS measurement was performed on the SRR substrates using a micro-Raman spectrometer (Horiba-JY T64000) excited with a diode laser (λ = 785 nm) in the backscattering configuration. The back scattered signal was collected through a 50X objective lens, the laser power on the sample surface was measured about 2.5 mW, and acquisition time was 50 s. SERS-based logic gates for sensitive and selective detection of mercury ions: Volumes containing final effective concentrations of 2 µM preheated GT-rich DNA, HEPES buffer (50 mM HEPES buffer pH 7.4, 0.1% Triton X-100, 2% dimethyl sulfoxide), and each concentrations of Hg2+ ranging from 0 to 4×106 ppb were incubated for 2 hr at room temperature. For evaluating the selectivity, various metallic ions at 1 mM concentration (Ca2+, Cu2+, Cd2+, Mg2+, Ni2+, Zn2+) were used instead of Hg2+. The samples were also incubated for 2 hr at room temperature before the SERS analyses as described above.
Acknowledgements The author Q. X. thanks the strong support from Singapore National Research Foundation through a Fellowship grant (NRFRF2009–06), the support from Singapore Ministry of Education via two Tier 2 grants (MOE2011-T2–2–085 and MOE2011-T2–2–051), and a very strong support from Nanyang Technological University via start-up grant (M58110061).
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