Hairpin DNA as a Biobarcode Modified on Gold Nanoparticles for

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Hairpin DNA as a Novel Biobarcode Modified on Gold Nanoparticles for Electrochemical DNA Detection Hui-Fang Cui, Tai-Bin Xu, Yu-Long Sun, An-Wei Zhou, Yu-Han Cui, Wei Liu, and John H. T. Luong Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac504206n • Publication Date (Web): 20 Dec 2014 Downloaded from http://pubs.acs.org on December 21, 2014

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Analytical Chemistry Manuscript for Analytical Chemistry

Hairpin DNA as a Novel Biobarcode Modified on Gold Nanoparticles for Electrochemical DNA Detection

Hui-Fang Cui1,*, Tai-Bin Xu1, Yu-Long Sun1, An-Wei Zhou1, Yu-Han Cui1, Wei Liu1, John H.T. Luong2

1

School of Life Sciences, Zhengzhou University, 100# Science Avenue, Zhengzhou,

450001, PR China 2

Innovative Chromatography Group, Irish Separation Science Cluster (ISSC), Department

of Chemistry and Analytical, Biological Chemistry Research Facility (ABCRF), University College Cork, Cork, Ireland

∗

Corresponding to [email protected] (H. F. Cui), Tel: +86-371-67781325, Fax:

+86-371-67783235

1

ACS Paragon Plus Environment

Analytical Chemistry

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Manuscript for Analytical Chemistry

ABSTRACT: Hairpin DNA (hpDNA) as a novel biobarcode was conjugated with gold nanoparticles (AuNPs) and a reporter DNA (rpDNA) to form hpDNA/AuNP/rpDNA nanoparticles for the detection of an oligonucleotide sequence associated with H. pylori as a model target. The rpDNA is complementary to about a half portion of the target DNA sequence (tDNA). A capture DNA probe (cpDNA), complementary to the other half of the tDNA, was immobilized on the surface of a gold electrode. In the presence of tDNA, a sandwich structure of (hpDNA/AuNP/rpDNA)/tDNA/cpDNA was formed on the electrode surface. The differential pulse voltammetry (DPV) detection was based on [Ru(NH3)5(3-(2-phenanthren-9-yl-vinyl)-pyridine)]2+, an electroactive complex that binds to the sandwich structure by its intercalation with the hpDNA and the dsDNA of the sandwich structure. The several factors: high density of biobarcode hpDNA on the surface of AuNPs, multiple electroactive complex molecules intercalated with each hpDNA and dsDNA molecule, and the intercalation binding mode of the electroactive complex with the DNA sandwich structure, contribute to the DNA sensor with highly selective and sensitive sensing properties. The DNA sensor exhibited a detection limit of 1×10-15 M (i.e. 1 fM), the DNA levels in physiological samples, with linearity down to 2×10-15 M. It can differentiate even one single mismatched DNA from the complementary tDNA. This novel biobarcode based DNA sensing approach should provide a general platform for development of direct, simple, repetitive, sensitive and selective DNA sensors for various important applications in analytical, environmental and clinical chemistry. Sequence-specific DNA detection has been a topic of significant interest due to its widespread applications in clinical diagnostics, molecular biology, agriculture, forensic science, and pathogen detection. The DNA levels in physiological samples and other biological samples are often in femtomolar (fM) or even attomolar (aM), much lower than detection limits of various DNA sensing techniques.1 Therefore, it is a formidable task to develop an analytical tool or procedure for the detection of DNA with such sensitivity using very minute available DNA samples. The polymerase chain reaction (PCR) method has significantly advanced the analytical performance of sequence-specific DNA detection with remarkably high sensitivity by primer-mediated enzymatic amplification of DNA samples.2 However, the procedure is complex, expensive, time-consuming, labor-intensive and only provides a narrow target DNA quantification range after amplification.3 Among different strategies for signal amplification, the nanostructure-based labeling method in sandwich DNA sensing has emerged as an effective way to approach the PCR detection sensitivity.1d,4 Of notice is a PCR-less target DNA amplification method based 2


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on two-component single strand DNA (ssDNA) modified gold nanoparticles (AuNPs).1d One of the ssDNA components is complementary to a target sequence whereas the other is complementary to a barcode sequence, a unique identification tag for the target sequence. This amplification strategy detects a target DNA down to 0.5 aM, corresponding to 10 copies in the entire 30 μL sample. However, the barcode DNA must be released for the subsequent chip-based silver-enhanced detection, increasing the assay complexity. AuNPs have also been conjugated with an electrochemiluminescence-labeled oligonucleotide barcode for detection of a genetically modified microorganism.4a Again, the labeling step of the barcode adds complexity to the assay procedure. In some reports,4b,4f electrostatic interaction of cationic indicators with ssDNA are applied for signal indication and amplification. The electrostatic binding mode of indicators with the sandwich DNA sensing structure has an inevitable drawback of big background signals. Hairpin DNA (hpDNA) exhibits a stem-loop intramolecular base paring pattern that occurs when two regions of the same strand, complementary in nucleotide sequence when read in opposite directions, base-pair to form a double helix that ends in an unpaired loop. The double helix structure in hpDNA could intercalate with a double stranded DNA (dsDNA) intercalator. This property of hpDNA has been used to construct aptasensors5 for detecting a target protein5a or kanamycin5b, an antibiotic. In the aptasensor for protein, a hairpin DNA, a beacon type aptamer, intercalates with methylene blue, and changes its conformation upon binding to a target protein, releasing the intercalated dye.5a In contrast, upon addition of kanamycin, a kanamycin aptamer is induced into a specific conformation that contains a hairpin region, facilitating the intercalation of a luminescent intercalator.5b The combination of hpDNA with dsDNA intercalator has also been applied for detecting antitumor drug bleomycins6 that can selective cleave ssDNA, dsDNA and possibly also RNAs,7 and for carrying drugs in targeted cancer therapy.8 This paper reveals a direct, sensitive and selective electrochemical signal-on DNA biosensing approach using hpDNA as a novel biobarcode. The biobarcode, with 15 base pairs (bp), was modified on AuNPs with a high density together with a reporter DNA probe (referred to as rpDNA), resulting in hpDNA/AuNP/rpDNA nanoparticles. The rpDNA is complementary to about a half portion of the target DNA sequence (tDNA). A 3



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capture DNA probe (cpDNA), complementary to the other half of the tDNA, was immobilized on the surface of a gold electrode. In the presence of tDNA, a sandwich structure of (hpDNA/AuNP/rpDNA)/tDNA/cpDNA forms on the electrode surface. An electrochemical

active

intercalator

of

dsDNA,

[Ru(NH3)5L]2+

where

L

is

3-(2-phenanthren-9-yl-vinyl)-pyridine,9 was bound to the sandwich structure by intercalation with the hpDNA and the dsDNA of the sandwich structure. About four bases of DNA bind to one [Ru(NH3)5L]2+ molecule.9a Electrochemical signal of the [Ru(NH3)5L]2+ intercalated DNA sandwich structure was detected directly at the electrode. As a model system, an oligonucleotide sequence associated with Helicobacter pylori （H. pylori）was selected. H. pylori is a crucial etiological agent in the pathogenesis of gastroduodenal diseases including peptic ulcer, mucosa-associated lymphoid tissue lymphoma and gastric adenocarcinoma.10 About 15 % of the world's human population has been infected with H. pylori.11 Therefore, sensitive and selective detection of the H. pylori DNA sequence is of great clinical importance for monitoring and management of gastroduodenal diseases.



EXPERIMENTAL SECTION

Materials and Chemicals. The chemicals, including hydrogen tetrachloroaurate (III) hydrate, 4-vinylpyridine, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), palladium triphenylphosphine, palladium acetate, 1-hexanethiol, hexaamineruthenium (Ⅲ) chloride, ammonium hexafluorophosphate, 9-bromophenantherene, silver trifluoroacetate, N,N, N',N'-tetramethylethylenediamine, and N,N-dimethylformamide (DMF) were purchased from Alfa Aesar (MA, USA). KNO3 (99.99% metals basis grade) was obtained from Aladdin Industrial (Shanghai, China). All other chemicals were of analytical grade and obtained from Sinopharm Chemical Reagent (Shanghai, China). The Tris-HCl buffer (10 mM, pH 7.0) was prepared from tris(hydroxymethyl) aminomethane and 0.1M HCl. The phosphate buffer solution (10 mM, PBS) contains 1.9 mM Na2HPO4 and 8.1 mM Na2HPO4. The TE buffer (pH 8.0) consists of 10 mM Tris-HCl buffer and 1 mM ethylene diamine tetraacetic acid (EDTA). The PBS/KNO3 buffer is the 4


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10 mM PBS with 100 mM KNO3. The immobilization buffer consists of 10 mM PBS, 1.0 mM EDTA and 0.6 M NaCl. The 6-mercapto-1-hexanol (MCH) aqueous solution (1 mM) was prepared by diluting the stock solution (100 mM in ethanol) with H2O. Deionized water obtained from a Millipore water system was used throughout the experiment. Oligonucleotide sequences were custom made by Invitrogen Biotech (Shanghai, China). The target sequence of a 43-mer oligonucleotide specific in H. pylori was selected by restriction endonuclease analysis on the UreB gene sequence of H. pylori. The oligonucleotide sequences used in this study are listed in Table 1. Cy5 and FAM are fluoresceins that were conjugated to the oligonucleotide sequences for facile characterization

of

hpDNA/AuNP/cpDNA.

ncDNA

and

mtDNA

denotes

for

non-complementary target and 1 nucleotide mismatched target (the mismatched nucleotide is italicized in bold ), respectively.

Table 1. Sequences of oligonucleotide used in this study Name

Sequence

tDNA

5’-TAATCGTGGATTACACCGGTATTTATAAAGCGGATATTGGTAT-3’

cpDNA

5’-HS-(CH2)6-ATACCAATATCCGCTTTAT-3’

rpDNA

5’-TACCGGTGTAATCCACGATTA(T)n-(CH2)3-HS-3’

rpDNA10T

5’-TACCGGTGTAATCCACGATTATTTTTTTTTT-(CH2)3-HS-3’

rpDNA15T

5’-TACCGGTGTAATCCACGATTATTTTTTTTTTTTTTT-(CH2)3-HS-3’

frpDNA

5’-Cy5-TACCGGTGTAATCCACGATTATTTTTTTTTTTTTTT-(CH2)3-HS-3’

hpDNA

5’-GCGCAACAAGAGTTCTTTTGAACTCTTGTTGCGCTTTTT-3’

HS-hpDNA

5’-GCGCAACAAGAGTTCTTTTGAACTCTTGTTGCGCTTTTT-(CH2)3-HS-3’

fhpDNA

5’-FAM-GCGCAACAAGAGTTCTTTTGAACTCTTGTTGCGCTTTTT-(CH2)3-HS-3’

ncDNA

5’-TGATAATGCTTAGGATCTACGTATATAGTCCATCAGGTTCGAT-3’

mtDNA

5’-TAATCGTGGATTACACCGGTATTTATAAAGCAGATATTGGTAT-3’

Instrumentation. Pristine and modified AuNPs were observed by transmission electron microscopy (TEM, FEI Tecnai G2 20, operated at 200kV). UV-vis spectroscopy 5



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was performed using a Shimadzu UV-2450 UV-Vis spectrophotometer (Shimadzu Scientific Instrument, Japan). Infrared spectra were recorded on a Nicolet 6700 FT-IR spectrometer (Thermo Scientific, USA). Fluorescence spectroscopy was performed using a spectrofluorometer F-4500FL (Hitachi, Japan). Mass spectroscopy was performed using a mass spectrometer LC-MSD-Trap-SL (Agilent Technologies, USA). Electrochemical measurement was carried out using an electrochemical analyzer [CHI 660C, Shanghai Chenhua Instrument (CHI), China] consisting of a three-electrode system: a working gold electrode, a platinum counter electrode and a 3 M KCl-Ag|AgCl reference electrode, at room temperature (~25°C). All potentials were quoted versus this reference electrode. The gold electrodes and the platinum electrodes are disk electrodes with a diameter of 2 mm (CHI).

Preparation of the [Ru(NH3)5L]2+ Complex. The [Ru(NH3)5L]2+ complex was prepared by mixing an equimolecular amount of the [Ru(NH3)5(H2O)]2+/DMF solution and the ligand L/water solution for 30 min at room temperature.9b The ligand L was synthesized as reported by Brown and Anson12 whereas [Ru(NH3)5(H2O)]2+ was prepared from [Ru(NH3)5Cl]2+ as described by Kuehn and Taube13 with some modification. In brief, silver trifluoroacetate was used directly, instead of being synthesized from Ag2O and trifluoroactic acid. The stock solutions (typically 2.0 mM) of L and [Ru(NH3)5(H2O)]2+ were prepared by dissolving the solids in DMF and water respectively, just prior to the preparation of the [Ru(NH3)5L]2+ complex.9b

Preparation

of hpDNA/AuNP/rpDNA Nanoparticles. Gold

nanoparticles

(AuNPs), 13 nm in diameter were prepared using the well-known Frens procedure.14 Briefly, an aqueous solution of HAuCl4 (0.294 mM, 100 mL) was brought to reflux with constant stirring, followed by the addition of 5 mL of a hot trisodium citrate solution (38.8 mM). Upon the reduction of HAuCl4, the solution color changed from pale yellow to deep red through light blue, blue and deep blue. The solution was refluxed for another 12 min, cooled to room temperature, and filtered through a 0.45 μm nylon filter (Micron Separations). The as-prepared AuNPs were characterized by UV-Vis spectroscopy, 6


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fluorescence spectroscopy and TEM. The concentration of the AuNPs solution was estimated based on its absorbance at 520 nm and the absorption coefficient of 13 nm AuNPs (ξ520=2.78).15 The concentration of the stock DNA solution (≈10-5 M, in TE buffer) was verified by UV absorbance at 260 nm, and the hairpin structure of hpDNA (including HS-hpDNA and frpDNA) was induced with MgCl2. The PBS buffer with 0.1 M MgCl2 was added to the 10-5 M HS-hpDNA solution (in TE buffer) to a final MgCl2 concentration of 0.01 M. The HS-hpDNA/MgCl2 solution was raised to 95 °C and kept at this temperature for 5 min to effect DNA denaturation. The hpDNA solution was then gradually cooled to room temperature to allow the formation of the hairpin structure, which was then evaluated by polyacrylamide gel electrophoresis (PAGE) (20% PAGE, 10 μL of 1 μM DNA per lane). A voltage of 100 V for 3 h was applied on the gel, and EB was used for staining. Before analysis, the HS-hpDNA sample was treated with 10 mM TCEP for 1 h at room temperature to reduce residual disulfide bonds. For the couplings of HS-hpDNA and rpDNA to AuNPs, 45 μL, 60 μL and 72 μL 1×10-5 M of the structure induced HS-hpDNA solution and 6 μL 1×10-5 M rpDNA solution (in TE buffer) (molar ratio of HS-hpDNA:rpDNA = 7.5:1, 10:1 and 12:1, respectively) was added into an aqueous solution of TCEP (6 μL, 50 mM). The DNA solution was adjusted to 80 μL with the addition of TE buffer and incubated at 30 °C for 1 h to reduce residual disulfide bonds. The DNA solution was then mixed with 500 μL of the AuNPs solution (1.8 nM, molar ratio of rpDNA:AuNP = 60:1), 10 μL of 0.1 M EDTA and 110 μL of 0.05 M PBS. The mixture was incubated at 30 °C for 5 h under gentle shaking, and protected from light. Then 2 M NaCl in the 10 mM PBS was gradually added into the reaction mixture with 30 min of time interval to a final NaCl concentration of 0.6 M within 4 h, followed by incubation for 16 h under the same reaction conditions. After centrifugation at 14,000 rpm for 20 min, the supernatant was collected for further analysis whereas AuNPs were resuspended in 1 mL of the immobilization buffer. The processes of centrifugation and resuspension were repeated twice. For the determination of the DNA ratio on AuNPs, frpDNA and fhpDNA were used instead of rpDNA and HS-hpDNA, respectively. The supernatant was subject to fluorescence analysis at the 7



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emission wavelengths of 520 nm and 670 nm, for the determination of the concentration of fhpDNA and frpDNA, respectively. The average number of fhpDNA and frpDNA molecules coupled on each AuNP was then calculated by subtraction of the corresponding DNA concentration in a control experiment (without AuNPs), based on the DNA fluorescence-concentration calibration curves. The as-prepared hpDNA/AuNP/rpDNA nanoparticles were characterized by UV-Vis spectroscopy, FT-IR spectroscopy and TEM.

Binding Mode of the [Ru(NH3)5L]2+ Complex with hpDNA. Intercalation binding between [Ru(NH3)5L]2+ and dsDNA9a has an association constant (Ka) of  3.8×103 M-1. In this study, ethanol (to a final concentration of 10%, in volume ratio) was added into the PBS/KNO3 buffer together with the freshly prepared [Ru(NH3)5L]2+ solution, to completely dissolve [Ru(NH3)5L]2+. The binding mode of [Ru(NH3)5L]2+ in this solvent mixture with the hpDNA was investigated by using electrochemistry techniques and UV-Vis spectroscopy. The hairpin structure of hpDNA and HS-hpDNA was induced according to the method described earlier. For electrochemical investigation, HS-hpDNA (10-7 M HS-hpDNA in the immobilization buffer) was then reduced with TCEP and immobilized on the gold electrode by applying a constant voltage of +0.4 V for 500 s. Similarly, cpDNA was also immobilized onto the gold electrode by electrochemical deposition for comparison. The [Ru(NH3)5L]2+ complex was then immobilized onto the hpDNA/gold electrode from a 8 mM PBS buffer (pH 7.4) containing 80 mM KNO3, 50 μM [Ru(NH3)5L]2+ and 10% ethanol (volume ratio) by linear sweep voltammetry (LSV) with 200 cycles from -0.6 V to 0.1 V at the scan rate of 0.1 V s-1. The electrode was conditioned by soaking in the PBS/KNO3 buffer twice, and subject to differential pulse voltammetry (DPV) (initial voltage: -0.6 V, final voltage: +0.1 V, amplitude: 0.05 V, pulse width: 0.01 s, pulse period: 0.02 s) in the PBS/KNO3 buffer. The [Ru(NH3)5L]2+ bound hpDNA/gold electrode was submerged in the PBS/KNO3 buffer containing 10 mg mL-1 ethidium bromide (EB) overnight at 4 °C. For comparison, a control experiment with the [Ru(NH3)5L]2+ bound hpDNA/gold electrode submerged in the PBS/KNO3 buffer without EB was also performed. The electrodes were then washed with the PBS/KNO3 buffer twice, and subject to the DPV measurement. 8


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For UV-Vis spectroscopy, the hairpin structure induced hpDNA solution was mixed with the mixture of the freshly prepared [Ru(NH3)5L]2+, ethanol and the PBS/KNO3 solution, resulting in a 8 mM PBS containing 10% ethanol (volume ratio), 80 mM KNO3, 2×10-6 M hpDNA, with various concentrations of [Ru(NH3)5L]2+ (0, 5×10-6, 1×10-5, 2×10-5 and 5×10-5 M). The mixture was incubated at room temperature for 1 h under gentle shaking. The reaction mixture was subject to UV-Vis spectroscopy at 200–600 nm. The 8 mM PBS containing 10% ethanol and 80 mM KNO3 was used for blank subtraction.

Immobilization of Thiolated cpDNA on Gold Electrode. cpDNA with an alkylthiol moiety at its 5’ end was self-assembled on the gold electrode through the formation of the Au-S covalent bond. Prior to immobilization, 4 μL of the10 mM TCEP aqueous solution was added to 10 μL of the 10-5 M cpDNA solution in the TE buffer. The mixture was then allowed to react for 1 h at room temperature. The reduced 10-5 M cpDNA/TE solution was diluted to 100 nM with the immobilization buffer. For cpDNA immobilization, the gold electrode was immersed in 1 mL of the 100 nM cpDNA solution and a potential of +0.4 V was applied at the electrode for 500 s. After cleaning and soaking in the immobilization buffer twice, the electrode was passivated by submerging in aqueous 6-mercapto-1-hexanol (1 mM) at 30 ℃for 1 h. The passivated gold electrode was then washed and soaked in the immobilization buffer twice, ready for DNA hybridization.

DNA Hybridization. For hybridization of tDNA with the cpDNA on the gold electrode, 100 μL of the tDNA solution (various concentrations in the immobilization buffer) was dropped onto the electrode surface. The electrode was inverted, covered with a pipette tip and sealed with parafilm. The electrode was kept at 40 ºC for 3 h under gentle shaking. After the electrode was washed with the immobilization buffer, 100 μL of the as-synthesized hpDNA/AuNP/rpDNA nanoparticle solution (1.8 nM, the concentration is based on AuNPs) was dropped onto the electrode surface to hybridize the labeled rpDNA with the immobilized tDNA. The electrode was sealed and kept at 47 ºC for 3 h under 9



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gentle shaking. The resulting electrode was washed and submerged in the immobilization buffer, ready for [Ru(NH3)5L]2+ intercalation and signal detection.

[Ru(NH3)5L]2+ Intercalation and Signal Detection. The [Ru(NH3)5L]2+ complex was bound to the sandwich structure of (hpDNA/AuNP/rpDNA)/tDNA/cpDNA on the gold electrode from the PBS buffer (8 mM, pH 7.4) containing 80 mM KNO3, 50 μM [Ru(NH3)5L]2+ and 10% ethanol (volume ratio) by LSV (200 cycles from -0.6 V to 0.1 V, scan rate: 0.1 V s-1). The electrode was cleaned by soaked in the blank PBS/KNO3 buffer twice, and subject to DPV (-0.6 V to +0.1 V, amplitude: 0.05 V, pulse width: 0.01 s, pulse period: 0.02 s) in the PBS/KNO3 buffer. All DPV curves are baseline-corrected using the software embedded in the CHI660C instrument. For the evaluation of the DNA sensing selectivity, control DNA sequences (100 fM), including the ncDNA and the mtDNA were introduced instead of the tDNA, using the same procedures as described for the tDNA. 

RESULTS AND DISCUSSION Binding Mode of the [Ru(NH3)5L]2+ Complex with hpDNA. The synthesized

[Ru(NH3)5L]2+ complex was characterized with the results shown in the Supporting Information (Figure S-1, S-2 and S-3). The DPV curve (Figure 1A) of the hpDNA/gold electrode (Curve a) bound with the [Ru(NH3)5L]2+ complex shows a very prominent oxidation peak at the peak potential (Ep) of  -0.32 V, compared to only a very small oxidation peak of the cpDNA/gold electrode (Curve c). After incubation with EB (Curve b), the oxidation peak at the hpDNA/gold electrode diminished 21.6%. Without EB (1.4%) (Figure 1A, Inset), the oxidation peak remained unchanged. Apparently, the [Ru(NH3)5L]2+ complex binds hpDNA mainly through intercalation under the electrochemical binding conditions of LSV. A very slight electrostatic interaction binding also exists, but only contributes a negligible DPV signal at the Ep of  -0.32 V. The high ion concentration in the binding solution (containing 80 mM KNO3 and 8 mM PBS) expels the [Ru(NH3)5L]2+ cation from accessing DNA molecules. The UV-Vis spectra of the hpDNA solution (with high ion concentration) in the presence of various concentrations of the [Ru(NH3)5L]2+ complex (Figure 1B) show that 10


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the DNA absorption value at 260 nm increases with increasing [Ru(NH3)5L]2+ complex concentration, confirming intercalation binding. The intercalation of the [Ru(NH3)5L]2+ complex with hpDNA leads to the elongation, unwinding and stiffening of dsDNA,16 exploding the N-containing bases and increasing their UV-Vis absorbance. The association constant (Ka) of the[Ru(NH3)5L]2+ complex with the hpDNA was determined from Eq. 1 according to Charak et al.17

A0 1  D  D   A  A0  DR  DR  K a CR

Eq. 1

where A0 and A represents the absorbance (at 260 nm) of the hpDNA solution and the hpDNA-[Ru(NH3)5L]2+ complex solution, respectively; εD and εDR represent the molar extinction coefficient of hpDNA and the hpDNA-[Ru(NH3)5L]2+ complex, respectively; and CR denotes the [Ru(NH3)5L]2+ complex concentration. From the 1/(A-A0) versus 1/CR plot (Figure 1B, Inset), the estimated Ka value was 3.3×104 M-1, significantly higher than the value of 3.8×103 M-1 as reported by Garcia et al.9a The higher affinity could be attributed to the presence of ethanol in the binding solution. Ethanol greatly improves the solubility of the [Ru(NH3)5L]2+ complex, enabling the availability of the intercalation molecules in the intercalation binding.

Figure 1. (A) The DPV curves of (a, b) hpDNA/gold and (c) cpDNA/gold electrodes bound with the [Ru(NH3)5L]2+ complex in the PBS/KNO3 solution, (a, c) before and (b) after incubation with EB. Inset: The percentage of diminishment of the DPV peak current (Ip) of the [Ru(NH3)5L]2+ bound hpDNA/gold after incubation (Ip1) compared to that before incubation (Ip0) with or without (WO) EB (n = 3). *** represents that the p value is less than 0.001 in the t-test. (B) UV-Vis spectra of 2×10-6 M hpDNA solution incubated 11



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with various concentrations of [Ru(NH3)5L]2+ (0, 5×10-6, 1×10-5, 2×10-5 and 5×10-5 M) in 8 mM PBS containing 80 mM KNO3 and 10% ethanol (volume ratio). Inset: The plot of 1/(A-A0) versus 1/CR has an interception value of 2.28 and a slope of 7×10-5.

Characterization of hpDNA/AuNP/rpDNA Nanoparticles. The two nucleotide

sequence regions of hpDNA, complementary in the opposite direction could form intra- as well as inter-molecular base pairs. The intra-molecular base pairing forms a stem-loop pattern, characteristics of hpDNA, while the inter-molecular hybridization forms a DNA dimer or even a tetramer. In the preparation of monodispersed hpDNA/AuNP/rpDNA nanoparticles, inter-molecular hybridization between two hpDNA single strand sequences must be avoided. Before modification on AuNPs, the stem-loop structure of HS-hpDNA was induced in the presence of Mg2+ and then verified by PAGE (Figure 2). The PAGE results confirmed that both the presence of Mg2+ and the denaturation/cooling steps contribute to the inhibition of the DNA dimer/tetramer formation, as well as the induction of the stem-loop structure formation. Therefore, the hpDNA (including HS-hpDNA and frpDNA) was induced by denaturation at 95 °C in 10 mM Na+ and 10 mM Mg2+ followed by cooling at room temperature (the condition for lane 6 in Figure 2).

Figure 2. The PAGE analysis result of the HS-hpDNA sequence after different treatments. From lane 1 to lane 7, the DNA sample treatment condition after melting was: (1) denaturation at 95 °C for 5 min followed by cooling at room temperature (RT); (2) keeping at RT in the presence of 10 mM Na+; (3) denaturation at 95 °C for 5 min (in 10 mM Na+) followed by cooling at RT; (4) denaturation at 95 °C for 5 min (in 10 mM Na+) followed by controlled slowly and gradually cooling; (5) keeping at RT in the presence of 10 mM Na+ and 10 mM Mg2+; (6) denaturation at 95 °C for 5 min (in 10 mM Na+ and 10 mM Mg2+) followed by 12


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cooling at RT; (7) denaturation at 95 °C for 5 min (in 10 mM Na+ and 10 mM Mg2+) followed by controlled slowly and gradually cooling. Lane 8: the standard DNA markers of 26, 34, 67, 89, 110, 147, 190, 242, 353, 404, 489 and 501 bp of DNA sequences.

The TEM micrograph of pristine AuNPs (Figures 3A) shows that AuNPs are monodispersed spherical particles with a narrow particle size distribution. Analysis using DigitalMicrograph software provided a mean diameter of 13±1 nm. The TEM micrographs of AuNPs after modification are illustrated in Figures 3B. No appreciable differences were observed for size, morphology, and monodispersity between AuNPs and hpDNA/AuNPs/rpDNA.

It

should

be

noted

that

monodispersity

of

hpDNA/gold-NPs/rpDNA is a prerequisite for the DNA sensor reported herein to achieve controllable and precise DNA sensing signal amplification.

Figure 3. TEM images of AuNPs (A) without and (B) with DNA (i.e. HS-hpDNA + rpDNA10T) modification.

The UV-Vis spectrum of AuNPs without modification displays an absorption peak at 520 nm (Curve a in Figure 4A), a characteristic surface plasmon resonance (SPR) band of 13～14 nm diameter gold particles.14b The size of AuNPs determined from the UV-Vis spectrum is consistent with the value observed from the TEM images. After modification, only a modest shift in the SPR peak from 520 to 528 nm was observed (Curve b in Figure 4A). This shift could be due to a slight decrease of the average inter-gold particle distance caused by a modest inter-molecular dimerization of HS-hpDNA. Alternatively, centrifugation of the DNA-modified particles during the course of preparation may also affect the particle size distribution, i.e., the position of the plasmon band.14b The 13



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absorption peak at 260 nm for the DNA modified AuNPs was more pronounced compared to the unmodified AuNPs, confirming the presence of DNA on the surface of AuNPs. For the FT-IR spectra (Figure 4B), an absorption peak at 1060 cm-1 appeared in the spectrum of HS-hpDNA modified AuNPs compared to that of the bare AuNPs. The spectrum of hpDNA presents an absorption peak at the same position, which is assigned to the symmetrical stretch vibration of PO2- group on the DNA backbone.18 The results suggest that hpDNA was conjugated to the surface of AuNPs.

Figure 4. (A) UV-Vis spectra of AuNPs (a) without and (b) with DNA (HS-hpDNA and rpDNA10T) modification. (B) FT-IR spectra of (a) HS-hpDNA, and (b, c) AuNPs, (b) with and (c) without HS-hpDNA modification. The ratio of hpDNA:rpDNA:AuNP was determined by fluorescence analysis of the concentration of fhpDNA and frpDNA in the supernatant of the synthesis system of fhpDNA/AuNP/frpDNA

nanoparticles.

The

fluorescence

intensity–concentration

calibration curves of fhpDNA and frpDNA were illustrated in Supplementary Information (Figure S-4). The fluorescence analysis data for the synthesis system of the feeding ratio of fhpDNA:frpDNA:AuNP = 600:60:1 (i.e. fhpDNA:frpDNA = 10:1) were listed in Table S-1 (Supporting Information). This feeding ratio resulted in an optimum tDNA sensing sensitivity for the as-constructed sandwich DNA sensor. The concentration of fhpDNA and frpDNA conjugated to AuNPs was 2.60×10-7 M and 6.30×10-8 M, respectively. Considering the estimated concentration of AuNPs of 1.80×10-9 M, the ratio of hpDNA:rpDNA:AuNP was calculated as 144:35:1. The larger size of HS-hpDNA exhibits a lower reaction rate constant and a lower conversion rate compared to rpDNA.

14


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Electrochemical DNA Sensing. Sensitive and selective detection of the H. pylori

DNA sequence is of great clinical importance for monitoring and assessment of gastroduodenal diseases. The fabrication procedures for the hpDNA based H. pylori DNA sensor are schematically shown in Scheme 1. The sandwich DNA structure of (hpDNA/AuNP/rpDNA)/tDNA/cpDNA was constructed on gold electrodes via thiolated cpDNA immobilization, electrode passivation, cpDNA/tDNA hybridization and tDNA/rpDNA hybridization. The gold electrode with the sandwich structure of hpDNA/AuNP/rpDNA)/tDNA/cpDNA on its surface was subject to repetitive linear stripping voltammetry (LSV) scanning in PBS containing [Ru(NH3)5L]2+, KNO3 and a very small amount of ethanol to allow [Ru(NH3)5L]2+ to intercalate into the dsDNA strands, including hpDNA, rpDNA/tDNA and tDNA/cpDNA, of the sandwich structure.

Scheme 1. The fabrication procedures for the hpDNA based sandwich DNA sensor and the signal detection strategy. The spacer length (i.e. the number of T at the 3’ end) of rpDNA may affect the availability of the rpDNA sequence for hybridization with tDNA, therefore the sensing sensitivity to tDNA, due to steric hindrance from hpDNA on the surface of AuNPs. The rpDNA15T fed with HS-hpDNA:rpDNA = 10:1 during the synthesis of the hpDNA/AuNP/rpDNA nanoparticles resulted in the sandwich DNA sensor with a significantly higher DPV peak current (Ip) (0.273 ± 0.036 μA, mean ± S.D.) in response 15



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to 10-13 M tDNA than that from the rpDNA10T (0.133 ± 0.012 μA, mean ± S.D.) (Figure 5A). Longer spacers could render more nucleotides of the rpDNA sequence exposed for hybridization with tDNA. The rpDNA15T was therefore used for all the following tDNA sensing experiments. The feeding ratio of HS-hpDNA to rpDNA15T was then varied to establish the optimum hpDNA/AuNP/rpDNA nanoparticles for the construction of the sandwich DNA sensor. The results (Figure 5B) show that the sensing signal of the DNA sensor from the feeding ratio of HS-hpDNA:rpDNA15T = 10:1 (0.273 ± 0.036 μA, mean ± S.D.) is significantly higher than those from the feeding ratio of HS-hpDNA:rpDNA15T = 7.5:1 (Ip = 0.163±0.022 μA, mean ± S.D.) and 12:1 (Ip = 0.116±0.006 μA, mean ± S.D.).

Figure 5. The DPV curves of the [Ru(NH3)5L]2+ intercalated (hpDNA/AuNP/rpDNA)/tDNA/cpDNA/gold electrode from the tDNA concentration of 10-13 M in the PBS/KNO3 solution. (A) The hpDNA/AuNP/rpDNA nanoparticles were synthesized from (a) rpDNA10T and (b) rpDNA15T at the HS-hpDNA:rpDNA feeding ration of 10:1. (B) The hpDNA/AuNP/rpDNA nanoparticle were synthesized from rpDNA15T at the HS-hpDNA:rpDNA15T feeding ratio of (a) 7.5:1, (b) 10:1 and (c) 12:1. Insets: The bar charts showing the corresponding DPV peak current (Ip) of the [Ru(NH3)5L]2+ intercalated DNA sandwich structure on the gold electrodes (n = 2-5). ** represents that the p value is less than 0.01 in the t-test. At the optimum rpDNA spacer length and the hpDNA:rpDNA ratio, DPV curves recorded from the [Ru(NH3)5L]2+ intercalated sandwich structure constructed from different concentrations of tDNA are shown in Figure 6A. The plot of Ip versus the logarithmic tDNA concentration (CDNA) is shown in Figure 6A (Inset). The lower 16


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detection limit (signal to noise ratio: 2) of the DNA sensor is 1×10-15 M (i.e. 1 fM), reaching the DNA levels in physiological samples. The DNA sensor exhibits good linearity for the semi logarithmic curve of Ip versus log(CDNA) with the concentrations above 2×10-15 M (regression coefficient of 0.973). The high detection sensitivity is contributed mainly by the magnifying effect from the multiple hpDNA and [Ru(NH3)5L]2+ molecules. As characterized, 144 hpDNA molecules were immobilized on each AuNP, and one hpDNA could intercalate theoretically with about seven [Ru(NH3)5L]2+ molecules, calculated based on the report that about four bases of DNA bind to one [Ru(NH3)5L]2+ molecule.9a The 35 rpDNA molecules on each AuNP would favor the efficient hybridization of rpDNA with tDNA. In order to investigate the tDNA sensing selectivity of the hpDNA based sandwich DNA sensor, control DNA sequences for tDNA, including10-13 M ncDNA and 10-13 M mtDNA were detected at the above-mentioned optimal conditions. The DPV curves are illustrated in Figure 6B. The Ip value for the ncDNA is very small (7.39 nA), the same as that for the blank sample. For the mtDNA, the detection signal is 0.065 μA, about one fourth of the sensing signal for the complementary tDNA (0.273 μA), and 8 times of that for the ncDNA. This result indicates that the DNA sensor exhibits very good sensing selectivity to tDNA, and can differentiate even one single mismatched DNA from the complementary tDNA. The [Ru(NH3)5L]2+ molecule binds to dsDNA and hpDNA in an intercalation binding mode. The high concentration (i.e. 80 mM) of the electrolyte KNO3 in the [Ru(NH3)5L]2+ solution would facilitate the formation of a K+-hydration shell around the DNA molecules on the gold electrode to prevent electrostatic interaction between DNA and [Ru(NH3)5L]2+ molecules which in turn minimizes the response signal to ncDNA and the blank sample. The sensing properties of the hpDNA-based DNA sensor in comparisons with those of some reported nanostructure-based sandwich DNA sensors are listed in Table 2. In addition to the advantages of simple procedure and small blank signal, the DNA sensor reported in this paper exhibits similar sensitivity to most of the other reported results. It should be noted that the assay in this paper uses pristine gold electrode, and only needs 17



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one signal amplification step. Modification of the electrode with some hot nanomaterials should further increase the detection sensitivity.

Figure 6. DPV curves recorded from the [Ru(NH3)5L]2+ intercalated sandwich DNA structure on gold electrodes, at the HS-hpDNA:rpDNA15T feeding ratio of 10:1. (A) The sandwich DNA structures were constructed from difference concentrations of tDNA. Inset: The working curve of Ip versus the logarithmic function of tDNA concentration (CDNA). (B) The sandwich DNA structures were constructed from (a) 10-13 M ncDNA, (b) 10-13 M mtDNA and (c) 10-13 M tDNA.

Table 2. The sensing properties of some nanostructure-based sandwich DNA sensors

Labeling Method

Solid support for cpDNA

Lower detection limit 5×10-19 M

Advantages

Reference

ssDNA as biobarcode on AuNPs. The barcode are then released from the AuNPs for the second signal amplification

Magnetic microparticles

Extremely high sensitivity

1d

Electrochemiluminescen ce labeled ssDNA as biobarcode on AuNPs

Magnetic beads

1×10-15 M

High sensitivity

4a

ssDNA as biobarcode on AuNPs. Cationic indicators

Dendrimers modified gold electrode

1.4×10-14 M

High sensitivity

4b

18


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electrostatically bind to the barcode ssDNA as biobarcode on AuNPs. Cationic indicators electrostatically bind to the barcode

Gold nanorods decorated reduced graphene oxide (rGO) sheets on glassy carbon electrode

3.5×10-17 M

High sensitivity

4f

Chemiluminescence functionalized AuNP chains

AuNPs modified gold electrode

3.3×10-16 M

High sensitivity

4d

1×10-15 M

High sensitivity, simple procedure, and small blank signal

This paper

Pristine gold Hairpin DNA as electrode biobarcode on AuNPs. Indicators intercalate into the barcode



CONCLUSION

A highly sensitive and selective electrochemical DNA sensor, for an oligonucleotide sequence associated with H. pylori as a model target, was developed based on hpDNA as a novel biobarcode modified on AuNPs. Direct intercalative binding of the [Ru(NH3)5L]2+ complex with hpDNA/dsDNA obviates complicated covalent modification procedures and releasing of biobarcode from the sensing structure, and minimizes background and interferences. High ratios of hpDNA to rpDNA on the AuNPs, and multiple [Ru(NH3)5L]2+ complex molecules intercalated with one hpDNA/dsDNA molecule, are able to extensively amplify the electrochemical sensing signal. Modification of the gold electrode with nanostructures should further enhance the DNA detection sensitivity. This novel biobarcode based DNA sensing approach should provide a general platform for development of direct, simple, repetitive, sensitive and selective DNA sensors for various important applications in analytical, environmental and clinical chemistry. 

ASSOCIATE CONTENT 19



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Supporting Information Available

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. 

AUTHOR INFORMATION Corresponding author

* E-mail: [email protected]. Phone: +86-371-67781325. Fax: +86-371-67783235. 

ACKNOWLEDGMENTS

This work was financially supported by the National Natural Science Foundation of China (NSFC 21345007), the National Recruitment Program of High-End Foreign Experts (China, GDW20124100167), and the Henan Open-up and Collaboration Program of Science and Technology (132106000070).



REFERENCES

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Hairpin DNA as a Biobarcode Modified on Gold Nanoparticles for

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