Microstructured Cystine Dendrites-Based Impedimetric Sensor for

ARTICLE pubs.acs.org/Biomac

Microstructured Cystine Dendrites-Based Impedimetric Sensor for Nucleic Acid Detection Chandra Mouli Pandey, Gajjala Sumana, and Bansi D. Malhotra* Department of Science & Technology Centre on Biomolecular Electronics, Biomedical Instrumentation Section, Materials Physics & Engineering Division, National Physical Laboratory (Council of Scientific & Industrial Research) Dr K. S. Krishnan Marg, New Delhi-110012, India ABSTRACT: We report results of the studies relating to the fabrication and characterization of novel biosensing electrode by covalent immobilization of DNA onto microstructural cystine (Cys) prepared by acoustic cavitation method. The TEM investigations of these structures reveal transformation of microstructured Cys from nanorods to dendritic structure under optimum conditions. The Cys dendrites (denCys) have been investigated by XRD, FT-IR, and SEM studies. These biosensing electrodes have been fabricated by immobilization of Escherichia coli (E. coli)-specific DNA probe onto the dendritic cystine. The results of the electrochemical impedance spectroscopy studies reveal that this nucleic acid sensor exhibits linear response to cDNA in the concentration range of 10
6 to 10
14 M with response time of 30 min. The biosensing characteristics show that the fabricated E. coli sensor can be reused about 4 times and is stable for ∼4 weeks. The studies on cross-reactivity of the sensor for other water-borne pathogens like Salmonella typhimurium, Neisseria meningitides, and Klebsiella pneumonia reveal specificity of the bioelectrode for E. coli detection.

’ INTRODUCTION The fabrication of nanostructured materials with controlled morphology has recently motivated many researchers to investigate their structure
property relationships. In this context, organic nanoparticles have attracted considerable interest in molecular biology, life sciences, and nanotechnology.1
3 These organic nanoparticles are employed as carriers for several classes of drugs including anticancer agents, antihypertensive agents, immunomodulators, harmones, therapeutics, and for effective drug delivery.4 The self-assembly of these low-dimensional organic nanostructures into 3D ordered superstructures such as multipods, snowflakes, dendritic, and other hierarchical structures5
7 has recently opened up new channels for designing novel materials with exciting applications. Efforts have recently been made to develop new synthetic methodologies for making novel nanostructures, achieving control over the size and morphology of the desired nanostructures and their further self-organization into multidimensional structures.8 Dendrimers, because of their conducive structure and ample functional groups on the surface, have been considered as an interesting matrix for fabrication of a biosensor as they provide enhanced surface coverage of the bioanalyte.9
12 These dendrimers have unique chemical and structural properties, such as structural homogeneity, integrity, controlled composition, and multiple homogeneous chain ends available for consecutive conjugation reaction, which make them as an ideal candidate for biosensing applications.13 It has recently been reported that organic dendrimers can be used to obtain increased concentration of hydrophobic molecules at the electrode
solution interface, with improved sensitivity and selectivity.14 Many conditions r 2011 American Chemical Society

are known to influence molecular parameters of the dendrimers such as size, shape, surface chemistry, flexibility, and topology.15,16 Because of their potential application in the field of biosensors and drug delivery, many researchers have reported on the synthesis of dendrites using polymers, metals, metal oxides, and semiconductors.17
21 The dendrites can be prepared using solvothermal method, hydrothermal method, solution-phase synthesis, and chemical vapor deposition method. Evenson et al. have shown that sodiumcarboxylate-terminated polyamidoamine (PAMAM) dendrimer self-assembles into the intertwined dendritic microfibrils using a solvent-casting method due to minimization of the surface tension during drying process of the dendrimer film on a silicone substrate.22 Martinovic et al. have developed a cobalt(II) salicylaldiimine metallo-dendrimer-based DNA biosensor and have reported high selectivity for the complementary oligonucleotide.23 Electrochemical DNA hybridization studies on G4 PAMAM dendrimer monolayer have been investigated by Zhu et al.24 Hong et al. have coupled amino-modified oligonucleotide with NH2-terminated dendrons, using di(N-succinimidyl) carbonate as a linker for application as a hybridization sensor.25 The remarkable properties of the dendritic materials present interesting opportunities for the design of highly sophisticated electroanalytical DNA biosensing devices. Because of their high surface area, nontoxicity, biocompatibility, and charge-sensitive conductance, they can be used as immobilization matrices for Received: April 11, 2011 Revised: May 19, 2011 Published: June 08, 2011 2925

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Figure 1. Schematic showing the preparation and fabrication of microcystine and ssDNA/denCys/Au bioelectrode.

fabrication of nanoscale biosensing and bioelectronic devices. The dendritic-materials-based electrochemical DNA devices can be utilized to obtain improved sensitivity, selectivity, fast response time, and rapid recovery. Escherichia coli (E. coli) is a human intestinal pathogen that can result in life-threatening complications ranging from bloody diarrhea to renal failure. Conventional culture tests for determining coliforms and faecal coliforms require expertise, and are both expensive and time-consuming.26,27 There is thus an urgent need to fabricate a sensitive, specific, and rapid DNA sensor for detection of E. coli.28,29 We report results of the investigations conducted on cystine dendrite (denCys) prepared under desired physical conditions for biosensing application. These dendrites have multiple
COOH groups on its outer surface that are advantageous for covalent immobilization of DNA resulting in a stable genosensor for E. coli detection.

’ EXPERIMENTAL SECTION Materials. L-Cysteine (analytical reagent (AR), 98.5%), N-hydroxysuccinimide (NHS), N-ethyl-N-(3-dimethylaminopropyl carbodimide) (EDC), and all other reagents and solvents have been procured from SigmaAldrich (India). Deionized ultrapure water (Millipore, 18.0 MΩ cm
1) has been used for the preparation of aqueous solutions. E. coli specific probe (17 bases) identified from the 16s rRNA coding region of the E. coli genome, complementary, noncomplementary and one-base mismatch target sequence (probes I to IV) have been obtained from Sigma Aldrich, Milwaukee, WI 53209, USA. Clinical samples have been provided by the All India Institute of Medical Sciences (AIIMS), New Delhi, India. Probe I: DNA probe: amine (CH2)3-50 -GGT CCG CTT GCT CTC GC-30 (ssDNA) Probe II: complementary: 50 -GCG AGA GCA AGC GGA CC-30 (cDNA) Probe III: noncomplementary: 50 -CTA GTC GTA TAG TAG GC-30 (ncDNA) Probe IV: one-base mismatch: 50 -GCG AGA GAA AGC GGA CC-30 Synthesis of Microstructured Cystine. For the preparation of dendritic cystine microstructure, 10 mM L-cysteine aqueous solution is

prepared at pH 10.0 using HCl and sodium carbonate following sonication of the solution using ultrasonic bath for ∼15 min. The optimum time for growth of microstructured cystine dendrites is found to be 9 h at 25 °C.

Pretreatment and Fabrication of denCys Modified Gold Electrode. The gold (Au) electrode (0.5 cm2 diameter) is washed in

boiling 2.0 M KOH for ∼1 h, following ultrasonication in Piranha solution (3:1 H2SO4/H2O2) for 10 min, and the electrode is subsequently washed repeatedly with water. The electrode is voltammetrically cycled and characterized in 0.2 M H2SO4 from
0.5 to
1.4 V (vs Ag/AgCl) with a scan rate of 0.10 V/s until a stable cyclic voltammogram is obtained. To fabricate the denCys monolayer films, the Au electrode is dipped in the denCys solution overnight (10 h) at 27 °C, after which the modified electrode (denCys/Au electrode) is rinsed repeatedly with deionized water.

Immobilization of Probe DNA and Hybridization with Target DNA. The ssDNA/denCys/Au bioelectrodes for E. coli detection have been fabricated as shown in Figure 1. The formed denCys containing multiple thiol functional groups are self-assembled onto Au surface via formation of Au
S bond. Furthermore, this denCys/Au electrode having multiple
COOH group is activated by treating it with 2 mM EDC and 5 mM NHS (in 50 mM phosphate buffer solution, pH 5.5) for 1 h at 27 °C under dark conditions. Subsequently, 20 μL of 10
6 M amino-terminated probe DNA solution in PBS (pH. 7.0) is immobilized onto the modified electrode and dried at 30 °C for ∼12 h, followed by rinsing with tris-HCl buffer several times to remove any unbound probe. The hybridization studies of ssDNA/denCys/Au bioelectrode with complementary, noncomplementary, and one-base mismatch DNA sequences have been carried out in a humid chamber for ∼30 min at 25 °C. The care has been taken to wash these bioelectrodes with phosphate buffer to prevent any possible nonspecific binding. Pretreatment of Clinical Samples. DNA has been isolated from a panel of strains comprising of E. coli, Klebsiella pneumonia, Neisseria meningitides, and Salmonella typhimurium. For this purpose, a suspension of colonies is made by taking 100 μL of sterile Milli-Q water in a 1 mL Eppendorf and is then vortexed. The suspension is boiled for ∼10 min and is centrifuged at 8000 rpm for ∼5 min. To this, equal volume (100 μL) of 24:1 (v/v) chloroform/iso-amyl alcohol is added, followed 2926

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Figure 2. (A) TEM images of cystine aggregates synthesized under different concentrations and pH of L-cysteine solution: (i) 0.1 mM, pH 8.0; (ii) 10 mM, pH 8.0; (iii) 100 mM, pH 8.0; (iv) 0.1 mM, pH 10.0; (v) 10 mM, pH 10.0; and (vi) 10 mM, pH 10.0 (at higher magnification). (B) X-ray diffraction pattern of denCys.

by centrifuging at 12 000 rpm for 10 min. The aqueous layer containing DNA is carefully pipetted out. This extracted DNA is kept at 20 °C prior to being used. These clinical samples are prepared in Tris-HCl buffer and heated in a water bath (95 °C) for about 5 min followed by immediate chilling in ice bath to obtain denatured single-stranded DNA. The same aliquots of samples are subjected to sonication (15 min at 120 V) to break the long DNA strands into smaller fragments.30 Characterization. The structural and morphological investigations of denCys microstructures have been carried out using X-ray diffractometric (XRD, Cu KR radiation, Rigaku, miniflax 2) and transmission electron microscopic (TEM, Hitachi Model, H-800) studies. The scanning electron microscopic (SEM) images have been recorded using a JEOLJSM-6700F field-emitting scanning electron microscope (FESEM, 15 kV). For TEM analysis, a suitable amount of formed cystine solution

is dropped on Formvar-coated 200 mesh copper grids and is dried in the open atmosphere. Fourier transform infrared (FT-IR) spectroscopic measurements have been carried out using Perkin-Elmer spectrometer (model Spectrum BX) at 25 °C. The electrochemical studies have been conducted on an Autolab potentiostat/galvanostat (Eco Chemie, Utrecht, The Netherlands) using a three-electrode cell with Au as working electrode, platinum as an auxiliary electrode and Ag/AgCl as a reference electrode in phosphate buffer (PBS, 50 mM, pH 7.0, 0.9% NaCl) containing 1 mM [Fe(CN)6]3-/4-.

’ RESULTS AND DISCUSSION The results of TEM studies (Figure 2A) reveal that pH, concentration, and temperature play an important role in 2927

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Figure 3. (A) FT-IR spectrum of denCys. (B) SEM image of (i) denCys/Au electrode; at 2 KX (ii) ssDNA/denCys/Au bioelectrode; at 2 KX (iii) ssDNA/denCys/Au bioelectrode; at 5 KX.

obtaining the nanostructured morphology. At low pH (8.0) and 0.1 mM concentration, unaligned cystine rods are observed (Figure 2A (i)). However, when the concentration is increased to 10 mM, nanorods appear to align in a particular direction (Figure 2A (ii)). Upon further increasing the concentration to 100 mM, a number of nanospheres are formed that get arranged compactly into a spherical flowerlike microspheres with an average size of each sphere ∼350 nm (Figure 2A (iii)). Similarly, at pH 10.0 and 0.1 mM concentration, the enhanced density of aligned nanorods is observed (Figure 2A (iv)) with respect to the morphology at pH 8.0, which transforms to dendritic structure at 10 mM concentration (Figure 2A (v)). The TEM studies reveal that the growth of the dendrites is unidirectional and the size increases from several hundred nanometers to micrometer, whereas the particle size at the outer edges of the assemblies becomes larger than that at the center (Figure 2A (vi)). This can be attributed to acoustic cavitations, in which there is generation of H 3 and OH 3 radicals in water, which further react to produce H2O2 or HO2 (in the presence of O2).31,32 It appears that the formed product oxidizes the
SH group of the cysteine to form disulfide bonds resulting in the formation of Cys. The intermolecular interactions that are perhaps responsible for the formation of microstructured cystine aggregate are electrostatic interaction and hydrogen bonding, resulting because of changes in pH.33
36 With the variation of concentration and pH, the direction of intermolecular hydrogen bonding is perhaps affected, indicating that this dendrite aggregate orients in a particular direction. It appears that the growth process of Cys occurs in two steps, an initial nucleating stage and a subsequent crystal growth process. In the present study, we speculate that the formation of denCys is largely governed by the reactant concentration. At lower concentration, the crystalline phase of Cys is

critical for directing the intrinsic shapes of the crystals because of its characteristic symmetry and structure. When the reactant concentration is increased to 10 mM, it causes 3D growth and results in the formation of dendritic structure. Therefore, the post growth of the Cys is restrained to allow their growth to larger particle through oriented attachment and a larger size distribution. The formation of the denCys has been further confirmed using XRD studies (Figure 2B). The diffraction peaks show a pure monoclinic phase (space group: 12(5)) with lattice constants a = 18.27 Å, b = 5.24 Å, and c = 7.226 Å. These results are in agreement with the standard value obtained for the bulk monoclinic L-cystine (JCPDS 261776), indicating that the white sediment is Cys. However, the broad reflection planes arise because of the nanosize of the denCys. For FT-IR measurements, the formed denCys solution is kept at 25 °C for 24 h for the formation of sediments. With increasing time, the amount of sediment increases. These formed sediments of denCys have been used to record FT-IR spectra using KBr in the frequency region of 400
4000 cm
1 (Figure 3A). The characteristic bands seen at 3437 and 1490 cm
1 have been observed because of asymmetric stretching and deformation of N
H groups, respectively, revealing the presence of amino groups. The sharp absorption peaks, seen at 1590 and 1624 cm
1, are attributed to the asymmetric deformation of NH3+, and peaks at 1420 and 1490 cm
1 are due to the asymmetric stretching of COO
(electronegative atoms), confirming the zwitterionic nature of L-cystine in the solid state.37,38 The characteristic peaks of CH2
S asymmetric stretching found at 2919 cm
1, CH2
CO deformation (1420 cm
1), CH2 wagging and rocking vibrations at 1295 cm
1 and 782 cm
1, S
S stretching vibration at 448 cm
1, and C
S stretching vibration at 689 cm
1 reveal the formation of cystine. 2928

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electrode may be due to the well-oriented functional groups present in the denCys that perhaps help in covalent binding of the probe DNA. Figure 4A shows the cyclic voltammograms (CVs) obtained for bare Au electrode, denCys/Au electrode, and ssDNA/ denCys/Au bioelectrode in phosphate buffer (50 mM, pH 7.0, 0.9% NaCl) containing 1 mM [Fe(CN)6]3-/4-. The decrease in magnitude of the response current from 0.064 (Curve i) to 0.01 μA is observed in the case of denCys/Au electrode (Curve iii) with respect to that of the bare Au electrode in the potential range of
0.3 to 0.8 V at a scan rate of 50 mVs
1 . This is attributed to the stable self-assembled monolayer (SAM) of denCys, formed by S
Au bond on the surface of Au. Furthermore, increase in the peak current to 0.031 μA after immobilization of probe DNA on denCys/Au electrode (curve ii) reveals that denCys/Au SAM provides a favorable conformational environment for probe DNA loading resulting in improved electron transport between DNA and denCys/Au electrode. Figure 4B shows CV of ssDNA/ denCys/Au bioelectrode obtained as a function of scan rate (10
300 mV/s). The peak current exhibits a linear relationship with sweep rate (Figure 4B, inset (i)), suggesting that the electrochemical reaction is diffusion-controlled and follows eqs 1 and 2. The peak potential (E) increases as a function of scan rate (Figure 4B, inset (ii)), indicating facile charge transfer kinetics in the 10
300 mV/s range of scan rate, and follows eqs 3 and 4. I pa ðAÞ ½ssDNA=cystine=Au ¼ 0:014 + 1:240 μAðs=mVÞscan rate ðmV=sÞ with R ¼ 0:975 ð1Þ I pc ðAÞ ½ssDNA=cystine=Au ¼ 0:015 + 0:985 μAðs=mVÞscan rate ðmV=sÞ with R ¼ 0:936 ð2Þ Ep ðVÞ ½ssDNA=cystine=Au ¼ 0:155 ðVÞ 

+ 0:051 ðsÞ Log½scan rateðmV=sÞ with R ¼ 0:981 ð3Þ Figure 4. (A) Cyclic voltammogram of (i) bare Au electrode, (ii) ssDNA/denCys/Au bioelectrode, and (iii) denCys/Au electrode. (B) Cyclic voltammogram of ssDNA/denCys/Au bioelectrode as a function of scan rate (10
300 mV/s), inset: (i) variation of current and scan rate and (ii) variation of potential with log of scan rate. (C) Nyquist diagram (Zim versus Zre) for the Faradic impedance measured in 50 mM PBS containing 1 mM [Fe(CN)6]3-/4- at pH 7.0 in the frequency for 105 to 1 Hz: (i) denCys/Au electrode, (ii) ssDNA/denCys/Au bioelectrode, (iii) bare Au electrode, and (iv) EDC/NHS activated denCys/Au electrode.

The SEM image of denCys/Au electrode (Figure 3B, image i) indicates well-isolated unidirectional aligned dendrites assembled at the Au substrate. The high magnified view is shown in the inset, revealing the width of the nanostructure from ∼100
200 nm. This structure probably assists immobilization of the probe DNA having optimum density due to the absence of the steric hindrance resulting in efficient hybridization of the target DNA. On immobilization of probe DNA, the modified surface containing globular DNA having shiny appearance is observed (image ii) that can be clearly visualized at higher magnification (image iii). The observed higher surface coverage of DNA onto denCys/Au

Ec ðVÞ ½ssDNA=cystine=Au ¼ 0:188 ðVÞ 


0:626 ðsÞ Log½scan rate ðmV=sÞ with R ¼ 0:994 ð4Þ The diffusion coefficient of the ssDNA/denCys/Au bioelectrode has been determined using the Randle Sevick equation I p ¼ ð2:69  105 Þn3=2 AD1=2 Cν1=2

ð5Þ

where Ip is the peak current (Ipa anodic and Ipc cathodic), n is the number of electrons, A is the area of electrode (0.5 cm2), D is the diffusion coefficient, C is the surface concentration, and ν is the scan rate (50 mV/s). The D value has been obtained as 1.8  10
6 cm2/s. The total surface concentration of probe DNA is calculated using Laviron’s theory39 and is found to be 2.67  10
10 mol cm
2, indicating high surface coverage of DNA onto denCys/Au electrode. The activity of the ssDNA/denCys/Au bioelectrode has been estimated as a function of pH varying from 6.0 to 8.0 at 25 °C. The high magnitude of response current obtained at pH 7.0 (data not shown) indicates that ssDNA/ denCys/Au bioelectrode is more active at pH 7.0, at which DNA molecules retain their natural structure and do not get denatured. Thus, all experiments have been conducted at pH 7.0 at 25 °C. 2929

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Figure 6. (A) Impedemetric response (Rct target/Rct ssDNA) of ssDNA/denCys/Au bioelectrode hybridized with clinical samples in 50 mM PBS containing 1 mM [Fe(CN)6]3-/4- at pH 7.0. (B) Comparison of the charge transfer resistance of ssDNA/denCys/Au bioelectrode with repeated denaturation and rehybridization process.

Figure 5. (A) Nyquist plots for the EIS measurements of (i) complementary ssDNA/denCys/Au, (ii) one base mismatch ssDNA/denCys/ Au, (iii) non complementary ssDNA/denCys/Au, and (iv) ssDNA/ denCys/Au bioelectrode in 50 mM PBS containing 1 mM [Fe(CN)6]3-/4at pH 7.0. (B) EIS response of ssDNA/denCys/Au bioelectrode as a function of cDNA concentration (10
6 to 10
14 M) in 50 mM PBS containing 1 mM [Fe(CN)6]3-/4- at pH 7.0. (C) Plot of the ratio of charge transfer resistance (Rct target/Rct ssDNA) versus the logarithm of the target DNA concentrations.

Electrochemical impedance spectroscopic (EIS) studies of the bare Au, denCys/Au electrode, and ssDNA/denCys/Au bioelectrode have been conducted in phosphate buffer (50 mM, pH 7.0, 0.9% NaCl) in the frequency range 0.01
105 Hz using [Fe(CN)6)]3-/4- as a redox marker. The equivalent circuit is outlined in Figure 4C (inset). The Rct corresponds to the interfacial electron-transfer resistance for redox marker, [Fe(CN)6)]3-/4-, Cdl is the double-layer capacitance, R is the ohmic resistance, and Zw is the Warburg impedance. According to the circuit, a typical

shape of a Faradic impedance spectrum (presented in the form of a Nyquist plot, Zim vs Zre) is a semicircle region lying on the Zre axis, followed by a straight line. The semicircle portion, observed at higher frequencies, corresponds to the electron-transfer-limited process, and Rct equals the respective semicircle diameter. The linear part is characteristic of the lower frequency range and represents the diffusion-limited electron-transfer process.40 We have utilized the semicircle diameter to observe the change of electronic transfer resistance (Rct). Figure 4C shows the Faradic impedance changes accompanying the stepwise electrode modification process. After modification of the Au surface with denCys monolayer, the interfacial electron-transfer resistance Rct corresponding to the respective semicircle diameter increases from 30 (curve (iii)) to 110 Ω (curve (i)). This increase in Rct value is due to the presence of increased negative charge from
COO
groups of denCys that perhaps perturb the interfacial electron-transfer rate between the electrode and the electrolyte solution. After activation with EDC/NHS, the negative charge of
COO
groups is sealed, and the available net positive charge attracts the negative redox marker, resulting in a decrease in Rct to 10 Ω (curve (iv)), even lower than that of bare Au electrode. After ssDNA is immobilized on the electrode surface, the electron-transfer probe by the negatively charged redox marker, [Fe(CN)6]3-/4-, is hindered again, resulting in increased Rct (45 Ω, curve ii). Figure 5A shows results of the selectivity studies conducted on the ssDNA/denCys/Au bioelectrode toward different target DNA sequences (complementary, noncomplementary, and one base mismatch) investigated using EIS. After incubation with the cDNA, the two-fold increase in the semicircle diameter 2930

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Table 1. Characteristics of ssDNA/denCys/Au Bioelectrode along with those reported in literature for other DNA sensor method of

validity with

SI. No.

immobilization matrix

immobilization

detection range

reusability

clinical samples

reference

1

cobalt(II) salicylaldiimine

covalent

0.34 pmol/L

no

23

metallodendrimer/Au 2

PAMAM/Au

covalent

1.1  10
10 to1.1  10
11 M

no

24

3

PAMAM/Au

covalent

10
11 to 10
8 M

no

25

4

denCys/Au

covalent

10
14 to 10
8 M

yes

present work

(curve (i)) with respect to ssDNA/denCys/Au bioelectrode (curve (iv)) is observed. This increase may be due to complete hybridization of the cDNA resulting in the double-stranded DNA (dsDNA) helix formation that increases the negative charge of the electrode surface and consequently the Rct. When the ssDNA/denCys/Au bioelectrode is treated with the ncDNA, the slight increase in the semicircle diameter (