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Sequence-specifi

School of Chemistry and Chemical Engine

Guangzhou, P. R. China. E-mail: cesllsh@m

Cite this: J. Anal. At. Spectrom., 2014,29, 1591

Received 2nd March 2014Accepted 22nd May 2014

DOI: 10.1039/c4ja00087k

www.rsc.org/jaas

This journal is © The Royal Society of C

c recognition of single-strandedDNA using atomic absorption spectrometry

Hong Zhang, Zhifang Zhu, Zunxiang Zeng and Liansheng Ling*

A method for the sequence-specific recognition of single-stranded DNA using atomic absorption

spectrometry has been developed based on the use of gold nanoparticle (AuNP) and silver-coated glass

modified with oligonucleotides. Silver-coated glass modified with the oligonucleotide 50-SH-T10-TCT

CTC CCA GGA CAG G-30 (Oligo 1) was used as the separation material and AuNP modified with

oligonucleotide 50-CAC AAA CAC GCA CCT C-T10-HS-30 (AuNP-Oligo 2) acted as the signal-reporting

component. When the target DNA (50-GAG GTG CGT GTT TGT GCC TGT CCT GGG AGA GA-30) was

added, it hybridized with Oligo 1 on the surface of the silver-coated glass and with Oligo 2 on the

surface of the AuNP. The concentration of the target DNA was represented by the number of AuNP

bound to the surface of the silver-coated glass and could therefore be determined using atomic

absorption spectrometry. The integrated absorbance was proportional to the concentration of the target

DNA over the range 10.0–200 nM with a limit of detection of 0.23 nM (3s/slope). The sensitivity of the

assay was further improved by using a layer-by-layer technique. All the processes were characterized

using scanning electron microscopy, the water contact angle and X-ray photoelectron spectroscopy.

Introduction

The sequence-specic recognition of DNA has attracted muchattention because of its potential applications in genetics,pathology, criminology, pharmacogenetics and food safety.1

Many methods have been developed for its recognition usingcolorimetry,2,3 chemiluminescence,4–6 uorescence,7,8 surface-enhanced Raman scattering spectroscopy,9,10 electrochem-istry,11,12 dynamic light scattering13,14 and mass spectrom-etry.15,16 However, to the best of our knowledge, atomicabsorption spectrometry (AAS) has not yet been applied toestablish a DNA sensor.

AAS is mainly used to detect metals and metalloids, forwhich it has both good sensitivity and selectivity. Nucleic acidsare composed of C, H, N, O, P and S, which are difficult to detectdirectly using AAS, it was therefore necessary to develop suitablelabels to solve this problem.17 Two main problems had to besolved to recognise DNA using AAS: how to tag the DNA with ametal element and how to separate the hybridized DNA fromthe free section of DNA.

Oligonucleotide-modied glass has been used to separatebound biomolecules from free molecules and several methodshave been reported for the functionalization of glass surfaces.DNA may be modied on a glass surface using physicalabsorption, chemisorption, covalent immobilization and othermethods.18–22 The most common approach is the covalent

ering, Sun Yat-Sen University, 510275,

ail.sysu.edu.cn; Tel: +86-20-84110156

hemistry 2014

binding of thiol-modied or amino-modied23–25 oligonucleo-tides on the surface of organosilane-coated glass. The prepa-ration of the organosilane-coated glass must be carefullycontrolled and a multi-step, complex and tedious method isrequired to prepare DNA-modied glass. Thiol-modied oligo-nucleotides have also been used to modify the surface of goldlms.26–28 Silver-coated glass, however, can easily be preparedusing the silver mirror reaction.29,30 In addition, thiol-func-tionalized oligonucleotides can self-assemble on the silversurface through Ag–S bonds.31,32 Thus, a thiol-functionalizedoligonucleotide can be immobilized on the surface of silver-coated glass, which might be an ideal separation material forthe proposed assay.

Gold nanoparticle (AuNP) have specic photonic propertiesand thiol-functionalized DNA can be assembled on theirsurfaces through Au–S bonds; many such DNA sensors havebeen reported.33–37 The concentration of gold in a solution ofAuNP can be determined by AAS, therefore AuNP were proposedas a label for this research. In the work reported here, thepossibility of the sequence-specic recognition of single-stranded DNA using silver-coated glass and AuNP followed byAAS was explored.

Experimental sectionReagents

Quartz glass slides (10 mm � 10 mm � 1 mm) were purchasedfrom Guangliang High-tech Co. (Jiangsu, China). Silver nitrate,ammonia (25%) and glucose were purchased from Sinopharm

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Chemical Reagent Co. Ltd (Beijing, China). Tri-(2-carboxyethyl)phosphine (TCEP) and sodium citrate were purchased fromSigma-Aldrich Co. All oligonucleotides were synthesized bySangon Biotech Inc. (Shanghai, China). Phosphate bufferedsaline (PBS) buffer (pH 7.0, 10 mM) and acetate buffer (pH 5.0,10 mM) were used in our experiments. Deionized water wasused for all experiments. All the chemicals were of analyticalreagent grade.

Apparatus

The centrifugation experiments were carried out using a AnkeGL-20G-II centrifuge (Anting Scientic Instrument Factory,Shanghai, China). UV-visible absorption spectra were obtainedusing a TU-1901 double-beam spectrophotometer (Beijing Pur-kingje General Instrument Co. Ltd, China). The surfacemorphology and composition were investigated using an S-4800scanning electron microscope (SEM; Hitachi, Japan). Thesurface composition and chemical state of the samples wererecorded on an ESCALAB 250 X-ray photoelectron spectrometer(XPS; Thermo-VG Scientic, USA). Water contact angle (WCA)measurements were performed using a DSA 100 drop-shapeanalysis system (Kruss GmbH, Germany). Particle size wasrecorded with a JEM-2010HR transmission electronmicroscope(JEOL, Japan). All the samples for SEM, WCA and XPS werevacuum dried at 40 �C for 2 days prior to the measurements.

AAS measurements were made with a Z-2000 series polarizedZeeman atomic absorption spectrometer (Hitachi, Japan),equipped with a Hitachi 7J0-885 graphite tube. The measure-ments weremade using a gold hollow-cathode lamp with a lampcurrent of 10 mA, a wavelength of 242.8 nm and a slit width of1.3 nm. The injection volume was 20 mL and the time constantwas 0.1 s. The signal was measured by the integrated absor-bance (peak area), which had a better accuracy than the peakheight. NH4NO3 (20%) was used as the matrix modier. Thetemperature program for AAS is listed in Table 1.

Preparation of silver-coated glass and gold nanoparticles

Silver-coated glass was prepared using the traditional silvermirror reaction. The glass slide was thoroughly cleaned ultra-sonically with a cleanser, hot sodium hydroxide, nitric acid andethanol solution. It was then rinsed with a large amount ofultrapure water and dried in air. To obtain the Ag(NH3)2OHsolution, 1% aqueous ammonia solution was added dropwiseinto 5.0 mL of 3% silver nitrate solution with gentle stirringuntil most of the precipitate had dissolved. The glass slide was

Table 1 Graphite furnace temperature program for the assay

StepStart temp.(�C)

End temp.(�C)

Dry 80 140Ash 400 400Atomize 2400 2400Clean 2600 2600Cool 0 0

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then immersed in a mixture of 400 mL of Ag(NH3)2OH solutionand 800 mL of glucose solution (10%) at room temperature for5 min. A layer of shining silver mirror formed on the surface ofthe glass.

AuNP were prepared by reducing 1.0 mM of gold(III) chloridetrihydrate (HAuCl4$3H2O) (20 mL) with 38.8 mM sodium citrate(2.0 mL). The concentration of AuNP was about 11.5 nMaccording to UV-visible absorption spectrometry based on theextinction coefficient (2.6 � 108 M�1 cm�1). The average size ofthe AuNP was about 14 nm as determined from the measure-ments of 100 AuNP in a TEM image.

Preparation of silver-coated glass modied with Oligo 1

Oligo 1 (2.0 OD) was rst dissolved in 20 mL of 10 mM acetatebuffer solution. Then 8.8 mL of 20 mM TCEP was added and thesolution was kept at 25 �C to cleave the disulde bond at the 50

terminal of Oligo 1. Aer 2 h, the freshly unprotected Oligo 1was added to 5.0 mL of 10 mM PBS buffer (pH 7.0 with 0.1 MNaNO3). The glass was then immersed in PBS buffer for 12 h atroom temperature to obtain silver-coated glass functionalizedwith Oligo 1. Finally, the silver-coated glass functionalized withOligo 1 was rinsed with a large amount of PBS buffer.

Preparation of gold nanoparticle modied with Oligo 2

AuNP modied with Oligo 2 were prepared according to previ-ously published methods.38,39 Oligo 2 (2.0 OD) was rst dis-solved in 20 mL of 10 mM acetate buffer. Then 8.6 mL of 20 mMTCEP were added and the solution was kept at 25 �C for 2 h tocleave the disulde bond at the 30 terminal of Oligo 2. Subse-quently, 3.0 mL of 11.5 nM AuNP were added to the abovemixture and incubated for about 16 h at room temperature.Subsequently, 4.0 M NaNO3 was added stepwise to obtain thenal concentration of 0.1 M during a 44 hours of incubation.Excess reagents were removed from the AuNP-Oligo 2 bycentrifugation at 14000 rpm for 25 min. Aer removing thesuspension, the remaining red oily precipitate was washed withPBS buffer. This process was repeated three times. Finally, thered oily precipitate was redispersed in PBS buffer. AuNP modi-ed with the oligonucleotide 50-GAG GTG CGT GTT TGT G-T10-HS-30 (AuNP–Oligo 3) were prepared using the same procedureas for AuNP–Oligo 2.

Detection of target DNA

In the rst step, silver-coated glass functionalized with Oligo 1was immersed in 1.1 mL PBS buffer containing different

Ramp time (s) Hold time (s)Gas ow-rate(mL min�1)

40 0 20020 0 2000 5 300 4 2000 10 200

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concentrations of the target DNA and was allowed to hybridizewith the target DNA for 4 h at room temperature. The silver-coated glass functionalized with Oligo 1 was then washed threetimes with PBS buffer to remove the free target DNA. Next,3.7 nM AuNP–Oligo 2 solution was reacted with the target DNAbonded to the silver-coated glass for 4.5 h at room temperature.The silver-coated glass was then rinsed three times with PBSbuffer to remove free AuNP–Oligo 2. The double-stranded DNAwhich had formed and bonded on the surface of the silver-coated glass was dehybridized by the addition of 1.1 mL of50 mM NaOH solution for 1 h. Aqua regia was then added toneutralize any excess NaOH and to dissolve the AuNP. Finally,100 mL of 20% NH4NO3 were added to the sample to act as amatrix modier and the sample was diluted to 2.0 mL withwater for AAS analysis.

Fig. 2 Atomization curve of Au in (a) the absence and (b) the presence

Layer-by-layer amplication

Aer the rst layer of AuNP had bonded onto the silver-coatedglass, the silver-coated glass functionalized with Oligo 1 wasimmersed in a 2.0 nM solution of AuNP–Oligo 3 at roomtemperature for 45 min to form the second layer of AuNP. TheAuNP–Oligo 3 hybridized with the AuNP–Oligo 2, which wasalready linked to the surface of the silver-coated glass. Thesubstrate was rinsed three times with PBS buffer to remove freeAuNP–Oligo 3. It was then immersed in a 2.0 nM AuNP–Oligo 2solution for 45 min at room temperature to prepare the thirdlayer of AuNP. The substrate was rinsed three times with PBSbuffer to remove the free AuNP–Oligo 2. This hybridizationprocedure was repeated to generate multi-layers of AuNP on thesliver-coated glass by alternately immersing the silver-coatedglass into the solution of AuNP–Oligo 2 or AuNP–Oligo 3.

of the 273 nM target DNA.

Results and discussionScheme of the assay

A diagram of the assay is shown in Fig. 1. Silver-coated glass wasprepared using the silver mirror reaction and then Oligo 1 wasself-assembled on the surface of the silver-coated glass throughthe Ag–S bonds. Oligo 2 was modied on the surface of the

Fig. 1 Scheme for the sequence-specific recognition of single-stranded DNA with silver-coated glass and AuNP using AAS.

This journal is © The Royal Society of Chemistry 2014

AuNP (AuNP–Oligo 2). Oligonucleotide 50-GAG GTG CGT GTTTGT GCC TGT CCT GGG AGA GA-30 was designed as the targetDNA. It could hybridize with the Oligo 1 functionalized on thesilver-coated glass and with the AuNP–Oligo 2; the AuNP-Oligo 2was then immobilized on the surface of the silver-coated glassfunctionalized with Oligo 1 and the concentration of the AuNPcould be determined by AAS. Therefore the concentration of thetarget DNA could be determined from the absorbance ofelemental Au.

To investigate whether the assay was feasible, the atomiza-tion curve of Au was studied. As shown in Fig. 2, there wasalmost no absorption signal for Au in the absence of the targetDNA, whereas there was a strong signal of elemental Au in theatomization curve when 273 nM target DNA was added. These

Fig. 3 SEM images of silver-coated glass under different conditions.(a) Silver-coated glass without modification. (b) Silver-coated glassfunctionalized with Oligo 1 and AuNP–Oligo 2 in the absence of thetarget DNA. (c) Silver-coated glass functionalized with Oligo 1 andAuNP–Oligo 2 in the presence of 0.6 mM target DNA. (d) Silver-coatedglass functionalized with Oligo 1 and AuNP–Oligo 2 in the presence of0.6 mM target DNA and 50 mM NaOH solution. All free AuNP-Oligo 2had been washed away from the surface of the silver-coated glass.

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results showed that AAS could be used to develop the proposedDNA sensor.

Characterization of silver-coated glass using SEM

Silver-coated glass was prepared using the silver mirror reac-tion. The morphology of the silver-coated glass was observedusing SEM under different conditions (Fig. 3). In the absence ofthe target DNA, AuNP–Oligo 2 could not be adsorbed on thesurface of the silver-coated glass modied with Oligo 1; itappeared almost the same as the silver-coated glass withoutmodication. With the addition of 0.6 mM target DNA, manyAuNP were adsorbed onto the surface of the silver-coated glassmodied with Oligo 1, which indicated that hybridizationoccurred between the Oligo 1, Oligo 2 and the target DNA. Aerthe further addition of 50 mM NaOH solution and storage for 1h, most of the AuNP were removed from the silver-coated glassmodied with Oligo 1, showing that dehybridization hadoccurred.

Characterization of silver-coated glass using WCA

Nucleic acids are hydrophilic, whereas metallic silver and goldare relatively hydrophobic. Therefore, the surface wettabilityreects the surface structure and composition, which can bedetermined by measuring the WCA (Fig. 4).40 Silver-coated glassprepared using the silver mirror reaction had a WCA of 100.2�.Aer modication with Oligo 1, the WCA decreased sharply to36.9�, which indicated that hydrophilic DNA had beensuccessfully assembled on the surface of the silver-coated glass.The WCA further decreased to 33.9� with the addition of thetarget DNA, which might be because of the DNA hybridization.In the presence of AuNP–Oligo 2 and target DNA, a sandwichstructure formed. The AuNP–Oligo 2 were linked to the top ofthe surface and the linked AuNP changed the hydrophobicity;the WCA changed to 41.5�.41

Fig. 4 WCA of different surfaces. (a) Silver-coated glass, 100.2�; (b)silver-coated glass modified with Oligo 1, 36.9�; (c) silver-coated glassmodified with Oligo 1 and hybridized with 0.3 mM of target DNA, 33.9�;(d) sandwich system of silver-coated glass–double stranded DNA–AuNP, 41.5�.

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Characterization of silver-coated glass using XPS

XPS was used to investigate the surface composition of thesilver-coated glass. As shown in Fig. 5a and b, there was anobvious Ag 3d peak with the silver-coated glass; the high-reso-lution spectra showed two peaks at binding energies of 368.6and 374.6 eV, which could be ascribed to Ag 3d5/2 and Ag 3d3/2,respectively. These were in excellent agreement with the valuefor metallic silver42 and indicated that silver was successfullydeposited on the surface of the glass using the silver mirrorreaction. New peaks of N 1s, P 2p and S 2p are seen in Fig. 5cand d, showing that DNA had been successfully assembled onthe surface of the silver-coated glass. Aer hybridization withthe target DNA, there was no obvious change in the XPS image.Aer the further addition of AuNP–Oligo 2, new peaks appearedat 83.9 and 87.6 eV, which could be assigned to the peaks of Au4f7/2 and Au 4f5/2. These results showed that AuNP–Oligo 2 hadbeen linked to the silver-coated glass by hybridization with thetarget DNA.43,44

Calibration graph and detection limit for the determination oftarget DNA with AAS

The calibration graph for the assay is shown in Fig. 6. Theintegrated absorbance is proportional to the concentration oftarget DNA within the range 10.0–200 nM. The linear regres-sion equation is A¼ 0.0034C� 0.052 (C in nM, R¼ 0.9925); thedetection limit (DL) was 0.23 nM, which was obtained from theequation DL ¼ 3s/slope. To estimate the selectivity of theassay, the absorbance of an oligonucleotide mismatched byone base (50-GAG GTT CGT GTT TGT GCC TGT CCT GGG AGAGA-30) and an oligonucleotide mismatched by two bases (50-GAG GTT CGT GTA TGT GCC TGT CCT GGG AGA GA-30) werealso measured. The absorbance of the mismatched DNA waslower than that of the target DNA (Fig. 7); the integratedabsorbance of the DNA mismatched by one base and the DNAmismatched by two bases were 88% and 74% of the signal ofthe matched DNA, respectively, which revealed the sequenceselectivity of the assay.45,46

Improvement of the sensitivity of the assay using a layer-by-layer technique

To further improve the sensitivity of the assay, the signal wasamplied by a layer-by-layer technique. As shown in Fig. 8, therst layer of AuNP was formed by hybridizing the target DNAwith AuNP–Oligo 2 and Oligo 1 on the silver-coated glass.AuNP–Oligo 3 was hybridized with the AuNP–Oligo 2 on thesurface of silver-coated glass and formed a second layer ofAuNP. Multi-layers of AuNP could be formed by alternatelyadding AuNP–Oligo 2 and AuNP–Oligo 3 to the surface ofsilver-coated glass modied with Oligo 1; this was conrmedby SEM.

As shown in Fig. 9, in the absence of the target DNA, therewas little change in the SEM image with increasing layers ofAuNP. However, when the 5.0 nM target DNA was added, theSEM image changed dramatically with increasing numbers oflayers of AuNP. There were only small amounts of AuNP on the

This journal is © The Royal Society of Chemistry 2014

Fig. 5 XPS spectra of silver-coated glass under different conditions. (a and b) Silver-coated glass; (c and d) silver-coated glass modified withOligo 1; (e and f) silver-coated glass modified with Oligo 1 hybridized with 0.6 mM of target DNA; (g and h) sandwich system of silver-coatedglass–double stranded DNA–AuNP. Parts (b), (d), (f), and (h) are the high-resolution spectra with a step-size of 0.05 eV.

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surface of the silver-coated glass for one-layer samples, but thenumber of AuNP obviously increased for the ve-layer and ten-layer samples. This indicates that the layer-by-layer techniquemay increase the sensitivity of the assay.

The results for absorbance coincided with the SEM result.As shown in Fig. 10, for ve layers of AuNP, the absorbance of ablank sample was almost equal to that of 40 pM of target DNA,which indicated that 40 pM of target DNA was not detectable.

This journal is © The Royal Society of Chemistry 2014

For ten layers of AuNP, the blank signal increased to0.1654, whereas that of the 40 pM of target DNA was 0.2562and the standard deviation of the blank sample was 0.0023(n > 6), indicating that the 40 pM of target DNA wasdetectable. These results show that the sensitivity of the assaycould be increased using a layer-by-layer technique; thesensitivity of the assay was comparable with that of the uo-rescent method.7

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Fig. 10 Absorbance of Au in the presence of 0 pM, 40 pM, 200 pM and1.0 nM of target DNA for five layer and ten layer AuNP.

Fig. 8 Scheme for the construction of the AuNP multi-layers.

Fig. 6 Calibration graph for the assay.

Fig. 7 Sequence selectivity of the assay. The concentration of thetarget DNA, the DNA mismatched by one base and the DNA mis-matched by two bases was 150 nM.

Fig. 9 SEM images of silver-coated glass modified with Oligo 1 in thepresence and absence of 5.0 nM of target DNA under differentconditions. One layer of AuNP on the surface of silver-coated glass in(a) the absence and (b) the presence of 5.0 nM of target DNA; fivelayers of AuNP on the surface of silver-coated glass in (c) the absenceand (d) the presence of 5.0 nM of target DNA; ten layers of AuNP onthe surface of silver-coated glass in (e) the absence and (f) the pres-ence of 5.0 nM of target DNA.

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Conclusions

AAS was successfully applied to the sequence-specic recogni-tion of single-stranded DNA. AuNP–Oligo 2 acted as the signal-reporting component and silver-coated glass modied withOligo 1 was used as the separation material; this was able toseparate free AuNP–Oligo 2 from those that were hybridized.

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The concentration of the target DNA was related to the signal ofthe AuNP and the absorbance was proportional to the concen-tration of the target DNA over the range 10.0–200.0 nM, with aDL of 0.23 nM. The sensitivity of the assay could be furtherimproved using a layer-by-layer technique. This assay opens upa new direction for the application of AAS, enabling it to be usedin other DNA-related projects in the future.

Acknowledgements

This work was supported by National Natural Science Founda-tion of China (no. 21375153), the Fundamental Research Fundsfor the Central Universities (no. 13lgzd05) and the open projectof Beijing National Laboratory for Molecular Sciences.

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