Facile Fabrication of a Sensor with a Bifunctional Interface for Logic

Nov 17, 2015 - Surface functionalization has attracted considerable interest from researchers because of its capability for facilitating the interface...
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Facile Fabrication of a Sensor with a Bifunctional Interface for Logic Analysis of the New Delhi Metallo-β-Lactamase (NDM)-Coding Gene Tsui-Ming Kuo,†,# Mo-Yuan Shen,‡,# Shih-Ying Huang,† Yaw-Kuen Li,*,‡ and Min-Chieh Chuang*,† †

Department of Chemistry, Tunghai University, Taichung 40704, Taiwan Department of Applied Chemistry, National Chiao Tung University, Hsinchu 30010, Taiwan

‡

S Supporting Information *

ABSTRACT: Surface functionalization has attracted considerable interest from researchers because of its capability for facilitating the interface communication between an energy transducer and a biological system. We report newly synthesized N-(4-aminobenzoyl)-N′-(4-maleimidobenzoyl)1,2-ethylenediamine (AME) to promote the modification of the surface with thiolated DNA and 3-((4-aminophenyl)dimethylammonio)propane-1-sulfonate (APSB) for the facile formation of a bifunctional interface forming an antifouling surface. Through the formation of diazonium ion, electrochemically reductive deposition of the two arylamines can fabricate simultaneously and effectively a bifunctional surface on a gold electrode and install a DNA probe to form a sensor; the sensor was applied to detect three genetic fragments of the New Delhi Metallo-β-Lactamase (NDM)-coding gene. Effects of the diazotization and the conditions of electrochemical deposition upon the sensing signals were investigated in connection with enumerating the accessible maleimide groups and assessing the diffusion resistance of the electroactive indicator. The detection limit given by the diazonium-constructed system was improved to attain the level of 54 pM, an advance over a conventional selfassembled monolayer. KEYWORDS: diazonium salts, genosensors, bifunctional layer, electrografting

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events in connection with tedious preparation frequently occur. For instance, we demonstrated an innovative scheme to identify three genetic fragments of the New Delhi Metallo-β-Lactamase (NDM)-coding gene for the simultaneous recognition of the NDM-specific and active site-characteristic genes.10 Such a mechanism relies on three hybridization events and two affinity conjugations (Supporting Information Figure S-1), which require sufficient space to accommodate the genetic fragments and the enzyme complexes for the purpose of minimizing steric hindrance. This objective can in principle be achieved on appropriately manipulating the coverage of a capture probe over the electrode surface. Consideration of the issues stated above has led us to explore the syntheses of two aniline derivatives to enable diazonium cations generated in situ in aqueous solution and their electrochemically reductive adsorption on a 16-well gold electrode array. With the added benefit that versatile substituent groups of arylamines including carboxyl,12,13 amine,14,15 biotin,16 boronic acid,17 maleimide,18 and azide or alkyne for click chemistry19 can be realized, we designed an aniline derivative bearing a maleimide moiety to realize a grafted layer with accessible sites of regulated number to facilitate binding to the thiolated DNA. Separately, an aniline derived from a zwitterionic molecule was synthesized20 and

unctionalization of the surface of a biosensor enables recognition of a selected target and resistance against nonspecific adsorption.1 Strategies to prepare a multifunctional interface in an efficient and facile manner are thus of vital significance in the design of a biosensor. Self-assembled monolayers (SAM) have been the means most widely utilized to achieve such an objective by virtue of their ease of preparation and the resulting well-defined monolayer,2−5 but the weak Au−S bond (enthalpy on the order of 170 kJ/mol) is likely to be subject to oxidation in ambient air and reagent media.6,7 Moreover, defects are likely to occur in layers of a short-chain alkanethiol, which yield a molecular assembly with decreased stability,8 thereby hampering the utilization of an alkanethiol SAM. Furthermore, the formation of a SAM on a gold electrode surface for multiple functionalities is a tedious and labor-intensive activity, as it is generally fabricated through incubating an electrode with varied thiolated substances for a few hours and in gradual steps. To facilitate the integrity against nonspecific adsorption of oligonucleotides and proteins, antifouling reagents such as dithiothreitol (DTT), 3-mercaptopropionic acid (3-MPA), polyethylene glycol (PEG), and bovine serum albumin (BSA) are typically applied throughout the protocol, which further prolongs the preparation. This issue arises particularly in a detection device intended to receive information from multiple targets as a basis for a decisive output.9−11 In such devices, complicated recognition © 2015 American Chemical Society

Received: September 1, 2015 Accepted: November 17, 2015 Published: November 17, 2015 124

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peroxidase-conjugated FITC-antibody (α-FITC-HRP) (NOVUS Biologicals, Littleton, CO, USA), dithiothreitol (DTT) (Amresco, Solon, OH, USA) were obtained from the indicated sources. Buffers used in this work include (i) washing buffer (WB: phosphate 10 mM, NaCl 170 mM, MgCl2 1 mM, pH 7.6), (ii) immobilization buffer (IB: phosphate 10 mM, NaCl 1 M, MgCl21 mM, pH 7.5), (iii) hybridization buffer (HB: BSA 2.5% in IB), and (iv) binding buffer (BB: casein 0.5% in WB). The substrate solution for the GOx and HRP cascade reaction was composed of D-(+)-glucose (250 mM) and TMB (0.42 mM) in phosphate (0.2 M)/citrate buffer (0.1 M), pH 5.0. Synthesis of Arylamines. Synthesis of 3-((4-aminophenyl)dimethylammonio)propane-1-sulfonate (APSB)20 is illustrated in Supporting Information Scheme S-1. Detailed synthesis for N-(4aminobenzoyl)-N′-(4-maleimidobenzoyl)-1,2-ethylenediamine (AME) and chemicals used are illustrated in Supporting Information Scheme S-2. Electrode Functionalization. An acidic solution (HCl 0.5 M) containing AME or/and APSB at a desired concentration and sodium nitrite (1 equiv) was subjected to diazotization for 15 min with stirring in an ice bath. The mixture was immediately loaded over a gold working electrode (area 4.9 mm2) in the 16-sensor Au array chip (Genefluidics, Irwindale, CA USA) for grafting by electrochemical reduction (cyclic sweeping between 0 and −1 V at scan rate 100 mV/ s) of the diazonium generated in situ near 23 °C. After electrodeposition, the electrode was thoroughly rinsed with deionized H2O and dried with gaseous dinitrogen. In the electrode functionalization utilizing the consecutive (stepwise) electrografting, an electrografting process, stated as above, for AME was performed, followed by a repetitive procedure containing APSB. Detection of NDM-Encoding Gene. Capture DNA (C, 0.5 μM) was mixed with TCEP (100 μM) in IB and incubated for 60 min at 25 °C. Aliquant (5 μL) of this DNA/TCEP mixture was cast over each grafted working electrode, followed by incubation for 30 min at 25 °C in humidified surroundings. The C-assembled Au electrode was then rinsed with deionized H2O and dried with dinitrogen. Electrochemically quantitative estimation of C immobilized on the electrode surface was performed by following the procedures reported previously.31 In an investigation of electrografting of AME without blocking with APSB, DTT (100 μM in WB) was incubated on the electrode for 1 h to provide an antifouling ability. After being washed with cold deionized H2O and dried with dinitrogen, the modified Au electrodes were subsequently treated with MPA (1 mM in WB, 4 μL) for 1 h near 23 °C to avoid nonspecific binding of DNA. To form a bifunctional interface fabricated with AME and APSB, the blocking steps using both DTT and MPA were eliminated. Before challenging the system, targeted DNA (T) was mixed with reporter probes (5 μM, F and B in Supporting Information Table S-1) and incubated at 25 °C for 15 min. DNA hybrid (4 μL) was dropped onto the working electrode to hybridize with the assembled DNA probe at 25 °C for 30 min. After rinsing with ice-cold WB and drying with dinitrogen, the solution (2 μL) containing α-FITC-HRP (10 μg/ mL) and Av-GOx (50 μg/mL) in BB was cast over the electrode to incubate for 30 min at 25 °C. The sensors were subsequently washed and dried. To ensure reliable output signals throughout the investigations, the sensory chip was equipped with a 16-channel tailor-designed probing system connected to a multipotentiostat (CHI 1021C, CH Instruments, Austin, TX USA) to facilitate measurements. In the measurement, the substrate solution (35 μL) was loaded to cover the three electrodes of the sensors near 25 °C for 3 min to expedite the enzymatic reaction. Chronoamperometry at applied potential −0.15 V (vs pseudo-Au reference electrode located on the chip) was typically performed to sample the current of the system after 60 s. For the selectivity study, the three mutated targets (denoted as Q, N, and QN) with alternated nucleotides characteristic of mutations of asparagine and glutamine were performed to evaluate the selectivity of the sensing system. Antifouling. The gold electrode was grafted with APSB (concentration varied from 0.01 to 10 mM) with the same procedure illustrated above. The capture DNA (C), two reporter probes (F and B), and two enzyme complexes (α-FITC-HRP and Av-GOx) were

utilized to functionalize a gold surface yielding resistance against nonspecific binding. To implement a multifunctional interface in a more efficient manner, concurrent electrografting of both aniline derivatives was performed (Scheme 1). Of Scheme 1. Schematic Diagram of the Interface Preparation to Detect NDM Genetic Fragmentsa

a

Step a: diazotization; step b: electrochemical reduction of diazonium cation; step c: immobilization of thiolated DNA; step d: formation of the biomolecular sensing system containing the targeted DNA, the reporter probes, and the enzyme complexes (see details in Supporting Information Figure S-1).

particular emphasis were the synthesis de novo of the binarycomponent arylamine layer (achieved via control of the ratio of maleimide to sulfobetaine) and the influence of the diazotization condition on the resulting signal output. Although binary-component films prepared through concurrent21−23 or consecutive24−29 assembly of two diazonium salts have been characterized and investigated in relation to the fabrication parameters, few authors30 have utilized grafting of diazonium salts to form mixed films for specific functionality. The novelty of this work beyond the preceding report that treats electron relay and antifouling in a bifunctional film resides in the design and synthesis of the maleimide-derived arylamine that embodies an increased molecular size, as opposed to the utilization of a sulfobetaine-modified aniline, aiming to minimize the steric hindrance during bioconjugation. The behavior imparted via the grafted aryl layer and its effect on the diffusion resistance of an electroactive indicator in the sensing scheme were also investigated in relation to the diazotization and electrochemical reductive conditions.

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EXPERIMENTAL SECTION

Reagents and Materials. Oligonucleotides were synthesized and purified using polyacrylamide gel electrophoresis (MDBio, Taipei, Taiwan). Oligonucleotides tagged with a thiol group, FITC, and biotin were synthesized (MWG Biotech, Ebersberg, Germany) and purified using high-performance liquid chromatography (HPLC). Sequences of all oligonucleotides are detailed in Supporting Information Table S-1. 3-Mercaptopropionic acid (MPA), magnesium chloride hexahydrate (MgCl2), 3,3′,5,5′-tetramethylbenzidine (TMB), D-(+)-glucose, dimethyl sulfoxide (DMSO), albumin from bovine serum (BSA), casein from bovine milk, citric acid monohydrate, sodium nitrite (NaNO2), Tris (2-carboxyethyl)-phosphine hydrochloride (TCEP), potassium hexacyanoferrate (III), potassium hexacyanoferrate (II) trihydrate, sodium chloride (NaCl), potassium chloride (KCl), and hexaammineruthenium(III) chloride (RuHex) were from SigmaAldrich (St. Louis, MO, USA). Potassium phosphate monobasic and dibasic (J. T. Baker, Phillipsburg, NJ, USA), avidin-conjugated glucose oxidase (Av-GOx) (Rockland, Gilbertsville, PA, USA), hydrogen 125

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currents in the first and subsequent sweeps were similar (Supporting Information Figure S-5). As there appeared to be no well-defined maximum for the reduction of APSB, voltammetric reduction was performed in the following experiments. Characterization of AME- and APSB-Grafted Surfaces. ATR infrared spectra were used to verify the grafting of the diazonium compounds and the retention of the functional groups involved. For AME (Figure 1C), the broad line at 3348 cm−1 is attributed to a NH-stretching vibrational mode. The sharp line at 1704 cm−1 is characteristic of the two carbonyl groups of maleimide.18 The line at 1513 cm−1 is associated with a benzene vibration. The C−N stretching vibrational mode appears at 1344 cm−1.18 This spectrum provides strong evidence that AME diazonium was grafted onto the gold electrode with the maleimide functional group. A grafting of phenyl sulfobetaine diazonium cation was confirmed also by the presence of infrared lines at 1210 and 1043 cm−1 (Figure 1D), corresponding to asymmetric and symmetric stretching vibrational modes of SO3−.34 The line at 927 cm−1 is associated with quaternary ammonium.35,36 The absorptions at 1643 and 3449 cm−1 are assigned to deformation and stretching vibrational modes of the OH group of water adsorbed on the grafted sulfobetaine.37,38 The film morphology was examined with an AFM (Supporting Information Figure S-6). Modification by the diazonium cations generated in situ decreased the roughness (Rmax) of the electrode surface from 7.5 nm (bare gold) to 3.6 nm (AME) and 3.3 nm (APSB). Reference to molecular dimensions of AME and APSB about 1.7 and 1.0 nm (predicted with ChemDraw), respectively, indicated that a multilayer was formed in the grafting after one voltammetric sweep of an aryl diazonium compound (10 mM). To acquire further insight into the molecular layer as a function of concentration of arylamine, we evaluated the AME-modified gold surface also with CV and EIS (Figure 2A). As expected, an increased AME concentration diminished the redox maximum current (inset) and increased the electron-transfer resistance of hexacyanoferrate(III)/ hexacyanoferrate(II). The declining magnitude obtained at 10 mM (curve f) is significantly greater than that at 1 mM (curve e), which is, however, differentiated with difficulty from those observed at 0.1 and 0.01 mM (data not shown). The barrier

subsequently incubated with the grafted electrode consecutively with the same protocols illustrated. No T was added. The system was then measured with chronoamperometry (−0.15 V) in the presence of the substrate solution; the current sampled after 60 s is referred to as the signal given from nonspecific adsorption. Apparatus and Measurements. Both cyclic voltammetry (CV) and electrochemical impedance spectra (EIS) were recorded in a homemade three-electrode configuration equipped with the chip (working electrode), an additional Pt wire (counter electrode), and an Ag/AgCl electrode (CHI111 and CHI 660D, CH Instruments, Austin, TX USA). EIS experiments were conducted in a hexacyanoferrate(III)/hexacyanoferrate(II) solution (5 mM in KCl 0.1 M/WB) with a frequency scan from 100 kHz to 0.1 Hz at 0.24 V bias potential and 20 mV amplitude. Morphological measurements were conducted in air with an atomic force microscope (AFM, Agilent 5500, USA). The tapping mode was used to examine the surface profile to estimate the surface roughness. The absorbance of the reaction mixture after the sensing measurement was recorded at 650 nm with a spectrometer (UV/vis, Molecular Devices M2e, Sunnyvale, CA USA) adapted with a 24-well microplate. IR spectra were recorded in an attenuated-totalreflectance (ATR) mode (Fourier-transform spectrometer, PerkinElmer, Waltham, MA USA); the spectrum was corrected with a background spectrum from a bare gold electrode.

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RESULTS AND DISCUSSION Electrochemical Grafting. Electrochemical grafting of aryl diazonium generated in situ from AME and APSB was investigated with cyclic voltammetry (Figure 1A and B). For

Figure 1. Cyclic voltammograms (100 mV/s) of the gold electrode in the diazonium solution generated in situ containing (A) AME (10 mM) or (B) APSB (10 mM), and NaNO2 (10 mM) in aqueous HCl (0.5 M). The sweeping cycle number is printed as indicated on the figures. (C,D) ATR spectra obtained from a gold surface grafted with AME (C) and APSB (D). Spectra were corrected with the spectrum of a bare gold chip. The insets depict schematic grafting of AME (C) and APSB (D) on the gold electrode.

AME, the voltammogram presented an irreversible reduction wave near −0.8 V (vs Ag/AgCl) that declined with the increasing scans. For APSB, even though no cathodic signal was defined (Figure 1B), the cathodic current increased significantly at a potential more negative than −0.4 V. The current (−0.6 to −1.0 V) given in the first sweep was significantly greater than those in successive scans. This behavior has been demonstrated to result in an aryl film as either a monolayer or multilayer,32,33 and differed from the voltammogram recorded with the control solution (NaNO2 10 mM in aqueous HCl 0.5 M) of which the

Figure 2. (A) Nyquist plots of the Au electrode before (a) and after grafting in the diazonium cation solutions generated in situ containing 1 (b) and 10 (c) mM AME in hexacyanoferrate(III)/hexacyanoferrate(II) solution (5 mM, KCl 0.1 M; phosphate buffer 0.1 M, pH 7.2). Inset: cyclic voltammograms (curves d, e, f; 100 mV/s) corresponding to curves a, b, and c. One cycle of voltammetric sweep was performed for the reductive grafting. (B) Histograms representing the chronoamperometric current (at −0.15 V) of an APSB-modified gold electrode in the absence of a targeted DNA. Voltammetric reduction: 1 cycle. 126

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maleimide groups of accessible number, are considered crucial factors to affect the sensing current. At 0.01 mM (a, b, c), the number of maleimide groups accessible to thiolated DNA increased with the sweeping cycle owing to an unsaturated coverage in forming a monolayer. As such, the number of maleimide groups dominated the resulting sensing current. When the concentration was 0.1 mM or greater, the density and thickness of the AME-modified layer prevailed wherein steric hindrance for the conjugation of enzyme complexes and the barrier effect against the TMB diffusion gradually arose. The increasing numbers of sweeps of voltammetric reduction (e.g., d → f or g → i) produced thicker grafting layers that promoted steric hindrance and the barrier effect. The absorbance measurement (Figure 3B) agrees with the maximum signal, at 0.1 mM. The sensing current in AME (10 mM) decreased from 55.5 through 39.5 to 19.0 nA (j, k, and l, respectively), but their characteristic absorbance was independent of the sweeping cycle (j′, k′, and l′). We construed this information to signify that only the maleimide group residing on the topmost layer is accessible to undertake the hybridization (DNA) and conjugation (enzyme complexes). The resulting thicker multilayer (e.g., l), nevertheless, exhibited a greater diffusion resistance (TMB) and thereby diminished the sensing current. A cyclic voltammogram (Figure 3C) recorded in the TMB/glucose substrate solution indicates a marked barrier effect at 10 mM, in terms of the significantly diminished redox features (a1, a2, c1, and c2 in green curve). These results indicate that, in the sensing scheme, not only the number of maleimide groups accessible to the thiolated DNA, but also the barrier effect, should be considered to dominate the ultimate sensing current. Influence of Electrografting Parameters on the Antifouling Ability. Although the APSB-modified electrode decreased the nonspecific binding of fetal bovine serum by 95%,20 its resistant capability against oligonucleotides is undocumented. We hence interrogated its utility against the oligonucleotides and the enzyme complexes in a consecutive incubation to mimic the extreme conditions toward the present sensor application. In the absence of T, the chronoamperometric currents (at 60 s) obtained from electrodes modified with APSB at varied concentration are represented in histogram fashion for comparison (Figure 2B). Relative to the signal recorded from the bare gold electrode, the grafting of APSB suppresses 56% (0.1 mM) and 80% (10 mM) of the background current. The residual signal obtained at the gold electrode modified with APSB (10 mM) was approximately 6.3 nA, which was comparable to the background current achieved on performing a conventional blocking approach (DTT and MPA), thereby verifying the antifouling utility. Modification of Binary Aryl Diazonium Cations Generated in Situ on Gold Electrodes. To form a bifunctional interface capable of bonding C and resulting in maximum resistance against a nonspecific adsorption of biomolecules, we performed electrografting of both AME and APSB either concurrently or consecutively. As shown in Figure 4A, the interface given with simultaneous electrografting exhibited sensing current 136 nA and background signal 7 nA. Both sensing and background currents were smaller than 227 and 28 nA of the stepwise electrografting. We ascribed the greater sensing current to an absence of competitive reaction of APSB. The increased background current was presumably due to the subsequent grafting of phenyl sulfobetaine diazonium cation onto the ortho position of the assembled AME, which

effect was ascribed to the large concentration of diazonium cation-induced thick, or a highly dense, molecular multilayer, of which the formation is a function of the amount of cation diffusing to the electrode surface and subsequent bonding between phenyl radicals and the assembled aryl molecules at their ortho position. Influence of Electrografting Parameters on the Sensing Signal. Relative to the results revealing the barrier effect of the electrochemically grafted layer, evaluation of the effect of diazotization conditions and parameters in the electrochemical reduction upon the resulting sensing current represented by the TMB redox reaction is beneficial to the interfacial design of sensors. Histograms representing the chronoamperometric current (at −0.15 V) are shown in Figure 3A. Spanning AME over 0.01−10 mM, there appears to be a maximum sensing current at 0.1 mM. The density and thickness of the modified aryl layer, as well as its bearing

Figure 3. Histograms representing the chronoamperometric current (at −0.15 V) (A) and the corresponding absorbance (B) as functions of concentration of AME and cycle number of voltammetric reduction. (C) Cyclic voltammograms (20 mV/s) of the grafted gold chips (1 cycle voltammetric reduction) prepared with AME (0.01 (black), 0.1 (blue), 1 (red), and 10 mM (green)) recorded in the substrate solution [TMB 0.42 mM, glucose 250 mM, phosphate (0.2 M)/citrate (0.1 M) buffer, pH 5]. Legends a1 and a2 denote anodic redox peaks of TMB. Legends c1 and c2 denote cathodic redox peaks of TMB. 127

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Figure 4. (A) Chronoamperometric curves (at −0.15 V) derived from gold chips functionalized using concurrent (a, c) and consecutive (b, d) grafting in the presence (c, d) and absence (a, b) of the targeted DNA. Inset: Amperometric signals recorded at 60 s for each group. Blue and red bars are for background and sensing currents, respectively. The concentration for both AME and APSB in diazotization is 0.05 mM. (B) Chronoamperograms obtained for targeted DNA at varied concentrations (a → g: 0.01, 0.1, 0.5, 1, 10, 50, and 100 nM, respectively) using the mixed layer-functionalized electrodes. (C) Calibration curve corresponding to chronoamperometric current at t = 60 s. Inset: Magnified depiction for signals over 0−1 nM.

Table 1. Sensing and Background Currents of Gold Electrodes Functionalized with AME and APSB in Mixed Layers Prepared from Diazotization Solutions with Varied Molar Ratio [AME]/[APSB]a Sensing current (nA) Background current (nA) a

0.11

0.33

1

3

9

19

107.1 ± 4.2 8.4 ± 0.14

116.5 ± 13.4 10.1 ± 0.42

153.2 ± 9.8 7.7 ± 1.69

115.5 ± 12.8 12.2 ± 5.02

99.4 ± 12.7 7.5 ± 0.28

83.5 ± 10.6 8.7 ± 0.07

Concentration of total arylamine compounds: 0.1 mM.

led to a disordered molecular structure of phenyl sulfobetaine in a multilayer fashion related to the unsatisfactory antifouling capability. The concurrently derived gold electrode gave a greater ratio of sensing to background currents (S/N = 19 relative to 8 for stepwise grafting), favorable to the design of a multifunctional interface given with simplified and timeefficient procedures. The sensing and background currents acquired from the binary-component layers prepared with AME and APSB at varied molar ratios were also examined (Table 1). Under a total aniline derivative concentration of 0.1 mM, the sensing current increased and then decreased while the amount of AME in the diazotization solution increased. A similar trend was observed previously.39 The maximum sensing current was given at a unit ratio, the same as with the favorable equimolar binary components of aryl diazonium salts generated in situ reported previously.23 Although the increased sensing current given for ratios in the range 0.11−1 can be presumably ascribed to accessible maleimide group of increased number, the background current was minimally dependent on the ratio (8−12 nA), indicating that the grafting of APSB might be a prominent reaction in the electrochemically reductive adsorption, which was suggested by the less cathodic potential (−0.38 V) of grafting of APSB shown in Figure 1B. The coverage of capture DNA (C) immobilized on the electrografting layer was generally greater than the one prepared by SAM (Supporting Information Table S-2). In addition, the coverage was dependent on the concentration and the ratio of aniline derivatives, indicating that the effect of the ratio on the signal response of the present system was profound. Sensing Performance. The bifunctional gold electrode displayed a wide dynamic range for the detection of the New Delhi metallo-β-Lactamase (NDM)-coding gene. Figure 4B reveals chronoamperograms for levels of T increasing from 0.01 to 100 nM. Utilization of two different linear ranges (10 pM to 1 nM and 1 nM to 100 nM) enabled the assessment of

concentration spanning over 4 orders of magnitude (Figure 4C). The sensitivity characteristic over 0.01−1 nM was 7.32 nA/nM (R2 = 0.996; see the inset). The limit of detection (LOD) (3σ) was estimated to be 54 pM (n = 3), which is less than that observed for the SAM electrode.10 The discriminating ability against the mutated targeted DNA was also interrogated, in which the substituted nucleotides would result in variations of corresponding amino acids, leading to a substantial loss of NDM catalytic activity. Mutations of asparagine and glutamine (i.e., N220 and Q123) which cause dramatic activity loss have been investigated.40−42 The wild-type target yields a greater outputted current (135.5 nA) which is discriminated from the other two single mutated targets (26.5 nA in Q123D and 29.3 nA in N220A), or the target with both mutation sites (11.8 nA) (Supporting Information Figure S-8). Even though the syntheses of AME and APSB facilitated formation of a bifunctional interface that is favorable for the detection of multiple fragments of the New Delhi metallo-betaLactamase (NDM)-coding gene, particular attention should be paid to the grafted constituents of the producing mixed layer23,43 that complicated the sensing and background currents (Table 1). The resulting mixed layer-functionalized electrodes exhibited a sensing current slightly less than an electrode fabricated with conventional SAM (Supporting Information Figure S-9), which is presumably ascribed to the greater diffusion resistance (for TMB) in the diazonium compoundgrafted interface. Although we did learn, from a preliminary trial (data not shown), that the stability of the sensing surfaces, fabricated on both self-assembly monolayer and electrografted diazonium film, is comparable within 5 days, systematic evaluation is required, yet is beyond the scope of the article. Prior reports have suggested that the electrografted diazonium film and the thiolated SAM hold a prevailing capability on the LOD and the sensitivity, respectively.44,45 While the carboxylterminated phenylamine layer was commonly formed to realize the coupling of amine-born biomolecules via the EDC/NHS 128

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ACS Sensors chemistry,30,46 a grafting of maleimide-substituent diazonium salt has enabled facile and spontaneous reaction binding to the thiolated single-stranded DNA.18 The synthesized AME herein prevails over such a prior maleimide-substituent compound18 that suffers from a small molecular size, and is thereby unable to provide sufficient space to facilitate properly the complex bioconjugations. The system remarkably and advantageously outperforms the SAM fabrication from the time- and laborintensive aspects, to substantially mitigate the duration of fabrication from 16 to 1 h.

CONCLUSIONS We synthesized two aniline derivatives to enable diazonium cations generated in situ and their electrochemically reductive adsorption on a 16-well gold chip to detect the multiple genetic fragments of the New Delhi metallo-β-lactamase (NDM)coding gene. The aniline derivative bearing a maleimide moiety (AME) was demonstrated to enable a grafted layer with a regulated number of sites accessible for facile binding to the thiolated DNA and to govern the resulting sensing signal. The other sulfobetaine-derived aniline was confirmed to offer antifouling capability efficiently against DNA and enzyme complexes. Through performing the concurrent grafting, the gradual and tedious preparation performed in SAM was improved, leading to a substantially decreased duration of fabrication from 16 to 1 h. The ultimate sensing current in the present sensing scheme was determined by not only the number of maleimide groups accessible to thiolated DNA, but also the barrier effect, which depended on the diazotation condition and the electrochemically reductive parameters. The greatest sensing signal of the system was obtained when the molar ratio of the two aniline derivatives was unity. The limit of detection was 54 pM with linear correlations over 0−1 nM and in a higher concentration range over 1−100 nM.

ACKNOWLEDGMENTS

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.5b00080. Schematic diagram of the logic sensing system; oligonucleotides used in this work; scheme for synthesis of APSB; Synthesis of AME; 1H NMR, 13C NMR, and ESI-MS of AME; Cyclic voltammograms of gold electrodes in solution containing NaNO2 and HCl; AFM images and surface profiles; chronocoulometric response curves; estimated surface coverage of DNA; amperometric signals given by targets with various nucleotide alterations; chronoamperograms of gold electrodes fabricated with SAM (PDF)

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Taiwan Ministry of Science and Technology supported this work under contracts 102-2113-M-029-003-MY2, 101-2113-M009-012-MY3, and 104-2113-M-029-003. We particularly appreciate Miss Ya-Chi Huang of Nano-Optoelectronics Laboratory (Tunghai University, Taiwan) and Dr. Sheng-Cih Huang (National Chiao Tung University, Taiwan) for assisting the AFM measurements and High Resolution Mass Spectrometry analysis, respectively. We also thank Professor Forest ShihSen Chien (Tunghai University, Taiwan) for valuable discussions and suggestions.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Fax: +886 36126976. Tel: +886 35731985. *E-mail: [email protected]. Fax: +886 4 2359 0426. Tel: +886 4 2359 0121 ext. 32218. Author Contributions #

Tsui-Ming Kuo and Mo-Yuan Shen contributed equally to this work. Notes

The authors declare no competing financial interest. 129

DOI: 10.1021/acssensors.5b00080 ACS Sens. 2016, 1, 124−130

Article

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DOI: 10.1021/acssensors.5b00080 ACS Sens. 2016, 1, 124−130