Dual Functional Graphene Derivative-Based Electrochemical

Article pubs.acs.org/ac

Dual Functional Graphene Derivative-Based Electrochemical Platforms for Detection of the TP53 Gene with Single Nucleotide Polymorphism Selectivity in Biological Samples Berta Esteban-Fernández de Á vila,† Elena Araque,† Susana Campuzano,*,† María Pedrero,† Berna Dalkiran,‡ Rodrigo Barderas,§ Reynaldo Villalonga,†,∥ Esma Kiliç,‡ and José M. Pingarrón*,†,∥ †

Departamento de Química Analítica, Facultad de CC. Químicas, Universidad Complutense de Madrid, E-28040 Madrid, Spain Faculty of Science, Department of Chemistry, Ankara University, 06100-Tandoğan, Ankara, Turkey § Departamento de Bioquímica y Biología Molecular, Facultad de CC. Químicas, Universidad Complutense de Madrid, E-28040 Madrid, Spain ∥ IMDEA Nanoscience, City University of Cantoblanco, 28049 Madrid, Spain ‡

S Supporting Information *

ABSTRACT: Novel disposable electrochemical DNA sensors were prepared for the detection of a target DNA sequence on the p53 tumor suppressor (TP53) gene. The electrochemical platform consisted of screen-printed carbon electrodes (SPCEs) functionalized with a water-soluble reduced graphene oxide− carboxymethylcellulose (rGO-CMC) hybrid nanomaterial. Two different configurations involving hairpin specific capture probes of different length covalently immobilized through carbodiimide chemistry on the surface of rGO-CMC-modified SPCEs were implemented and compared. Upon hybridization, a streptavidinperoxidase (Strep-HRP) conjugate was employed as an electrochemical indicator. Hybridization was monitored by recording the amperometric responses measured at −0.10 V (vs an Ag pseudo-reference electrode) upon the addition of 3,3′,5,5′tetramethylbenzidine (TMB) as a redox mediator and H2O2 as an enzyme substrate. The implemented DNA platforms allow single nucleotide polymorphism (SNP) discrimination in cDNAs from human breast cancer cell lines, which makes such platforms excellent as new diagnosis tools in clinical analysis.

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protein. Down-regulation of the TP53 gene, mutations, or altered p53 protein function (suggested by elevated expression) are the most frequently genetic events in human cancer, being present in more than 50% human tumors and cancer cell lines.1−4 Unlike the majority of tumor suppressor genes, which are usually inactivated during cancer progression by deletions or truncating mutations, the TP53 gene in human tumors is often found to undergo missense mutations, in which a single nucleotide is substituted by another. Approximately 90% of all mutations occur in exons 4−9, which encode the DNA binding domain of the protein and result in dysfunctional types of stable mutant p53 proteins whose accumulation is regarded as a hallmark of cancer cells. Of the mutations in this domain, ∼30% fall within six “hotspot” residues (residues R175, G245, R248, R249, R273, and R282) and are frequent in almost all types of cancer. Mutated TP53 genes typically occur with greater frequency in patients with late-stage cancer and at sites

ore than 11 million people are diagnosed with cancer every year. It is estimated that there will be 16 million new cases per year by 2020. Cancer is a cluster of diseases involving alterations in the status and expression of multiple genes that confer a survival advantage and undiminished proliferative potential to somatic or germinal cells. Alterations primarily in three main classes of genes (viz., (proto)oncogenes, tumor suppressor genes, and DNA repair genes) collectively contribute to the development of cancer.1 The p53 gene (TP53; 1182 bp) is one of the most important tumor suppressor genes, because of its dysfunction in the majority of human cancers. It normally acts as a mediator of several cellular functions, including growth arrest and apoptosis in response to DNA damage. It stops cell cycle in damaged cells until the alteration is properly repaired; otherwise, it initiates apoptosis cascade in damaged cells. If this guardian of the genome becomes inactivated upon mutation, it cannot execute its duty and more mutations will accumulate in the cell, eventually leading to cancer development. In addition to suppressing cancer development, normal TP53 gives sensitivity to chemotherapy and radiotherapy in tumor cells.2 The human TP53 gene is composed of 11 exons and codes a protein with 393 amino acids, the p53 © XXXX American Chemical Society

Received: October 29, 2014 Accepted: January 17, 2015

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DOI: 10.1021/ac504032d Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

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of metastatic disease and in undifferentiated tumors.5 Some of these mutations are often associated with poor prognosis and are certain significant predictors of resistance to chemotherapy or radiotherapy.1,3 Although p53 is not a typical cancer-specific antigen, its central role in the control of cell growth and apoptosis and frequent mutations in tumors make that either mutation in the TP53 gene or overexpression of the p53 protein can be used to predict many aspects of the prognosis and outcome of patients with various types of cancer.1 Therefore, sensitive and rapid detection of the TP53 gene and its mutations are of great value, and a variety of methods for measuring the TP53 gene have been reported. These methods include reverse transcription polymerase chain reaction (RT-PCR),6 traditional nucleic acid probe,7 fluorescence,8 immunostaining,9,10 and immunohistochemistry.11,12 However, these methods are time-consuming and not sensitive.13 Recently, DNA biosensors for the detection of TP53 mutations have generated considerable interest for simple, rapid, and inexpensive testing of genetic and infectious diseases.14 All these systems are based on monitoring the hybridization reaction between a probe immobilized on the sensing surface and the complementary sequence or the mismatch one in solution with electrochemical,15−17 piezoelectric,18,19 fluorescence,20,21 surface plasmon resonance,22,23 and electrochemiluminescence13 transduction. Among them, electrochemical DNA biosensors allow the development of extremely sensitive and accurate, yet simple, inexpensive, real-time and robust gene-sensing platforms that meet the requirements of point-of-care (POC) clinical diagnostics.24−26 Although numerous electrochemical sensors have been developed for the detection of mutations in the TP53 gene,15,26−30 their selectivity for single base mismatch detection is limited and they have not been validated using complex biological or real patient samples. Therefore, increasing the probe DNA loading while keeping the accessibility for hybridization, minimizing background contributions even in the presence of complex sample matrices and getting full discrimination against single base mutations are still important challenges to face in work that involves electrochemical biosensors. Recent advances in nanomaterials science offer an unforeseeable opportunity of making new sensitive biosensors.31,32 No only do nanostructured electrochemical sensors exhibit significantly increased surface area for attaching probe molecules, but they also show improved electrochemical performance and beneficial orientation effects in probe immobilization. Earlier studies explored the use of carbon nanotubes (CNTs)33−35 and/or gold nanoparticles (AuNPs).36−38 Moreover, the use of DNA hairpin molecular beacons as capture probes exhibit thermodynamically preconditioned higher selectivity for single nucleotide polymorphism (SNP) than linear DNA,23,39 which is relevant for genetic biomarkers such as the TP53 gene, where the detection of SNPs is of great interest.15 This paper reports, for the first time, the development and analytical evaluation of nanostructured disposable integrated electrochemical DNA sensors for the detection of the TP53 gene corresponding oligonucleotide at the level of SNP, even in raw biological samples. Screen-printed carbon electrodes (SPCEs) modified with a functionalized hybrid nanomaterial, composed of reduced graphene oxide (rGO) and O-carboxymethylcellulose (CMC), were employed as platforms for the covalent immobilization of two different selective hairpin-forming capture probes. This functional hybrid nanomaterial, which has been demonstrated to provide excellent

transduction systems for the preparation of enzymatic biosensors,40 greatly improved the performance of the developed electrochemical DNA biosensor, offering important advantages over the use of the single nanomaterial (rGO) or other common nanomaterials such as multiwalled CNTs (MWCNTs). After a systematic optimization of the novel scaffolds, their performance toward the determination of the wild-type TP53 (wtTP53) gene and detection of a mutated mTP53 gene in complex biological samples such as undiluted human serum and saliva, and breast cancer cells cDNAs is evaluated.

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EXPERIMENTAL SECTION Apparatus and Electrodes. Amperometric measurements were carried out with an ECO Chemie Autolab PGSTAT 101 potentiostat using the software package NOVA 1.7. A P-Selecta (Scharlab) ultrasonic bath was also employed. Screen-printed carbon electrodes (SPCEs, Model DRP-C110, purchased from Dropsens) consisting of a 4-mm smooth carbon working electrode, a carbon counter electrode, and a Ag pseudo-reference electrode, were used. A specific cable connector (ref DRP-CAC from DropSens, S.L.) acted as the interface between the SPCEs and the potentiostat. Homogenization of the solutions was facilitated with a Bunsen AGT-9 Vortex. A steam sterilizer (Raypa), a biological safety cabinet (Telstar Biostar), a temperature freezer (New Brunswick Scientific), a refrigerated centrifuge (Sigma, Model 1-15K), an infrared CO2 incubator (Forma Scientific), and an analogue block heater (Stuart, Model SBH130) were also used. Reagents and Solutions. 2-(N-morpholino)ethanesulfonic acid (MES), NaCl, KCl, sodium dihydrogen phosphate, and disodium hydrogen phosphate were purchased from Scharlab. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide (EDC), N-hydroxysulfosuccinimide (sulfo-NHS), casein, and a progesterone-deficient human serum (S7394) were purchased from Sigma− Aldrich. Saliva samples were freshly collected from volunteers in the laboratory, using the Salivette system. A water-soluble reduced graphene oxide−carboxymethylcellulose (rGO-CMC) hybrid nanomaterial was synthesized according to the protocol described by Araque et al.40 The characterization of this hybrid nanomaterial via atomic force microscopy (AFM) revealed the presence of platelets with an average thickness of 1.2−1.4 nm, fractal-like structures at the edges, and some pendant structures with a wire type shape and a monomolecular height of ∼0.5−0.7 nm. For comparison purposes, rGO without CMC attachedto it was also used. The DNA sequences used (see Table S1 in the Supporting Information) were those described by Farjami et al.15 and were synthesized by Sigma−Aldrich. These DNA oligonucleotides were dissolved in nuclease-free water to obtain a final concentration of 100 μM, aliquoted into smaller volumes and stored at −20 °C. A streptavidin peroxidase (Strep-HRP) conjugate from Roche (Reference No. 11 089 153 001, 500 U mL−1) and a 3,3′,5,5′tetramethylbenzidine/hydrogen peroxide (TMB/H2O2) K-Blue reagent solution from Neogen in a ready-to-use reagent format (K-Blue enhanced-activity substrate, also containing H2O2), were used for the detection step. All chemicals used were of analytical-reagent grade, and deionized water was obtained from a Millipore Milli-Q purification system (18.2 MΩ cm at 25 °C). Other solutions that were employed, and prepared in deionized water, included the following: 0.025 M MES buffer, pH 5.0; phosphate-buffered saline (PBS) consisting of 0.01 M phosphate buffer solution B



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Unless otherwise indicated, the reported data corresponded to the average of at least three replicates. A new SPCE was used for each measurement. Analysis of Spiked Biological Samples. The analysis of the samples (untreated commercial serum and fresh collected saliva) was accomplished using the protocol described for the preparation of the DNA sensor just substituting the hybridization buffer for the spiked biological sample under study (diluted or undiluted). Cell Lines, RNA Extraction, and cDNA Synthesis. MCF-7 and SK-BR-3 breast cancer cell lines were selected to check the genosensor performance, because these cell lines possessed wtTP53 or mTP53 mRNA, respectively. In addition, one nontumorigenic breast epithelial cell line (MCF-10A) with wtTP53 was used as the control. Cell lines were grown according to established protocols.41,42 Both cancer cell lines were grown in DMEM (Dulbecco’s modified Eagle’s medium), supplemented with 10% fetal bovine serum, penicillin and streptomycin, and 2.5 mM L-glutamine (GIBCO-Invitrogen, Carlsbad, CA, USA). MCF-10A cells were cultured in high-glucose DMEM/Ham’s Nutrient Mixture F12 (1:1) with 2.5 mM L-glutamine (Gibco), 5% (v/v) horse serum (Gibco), streptomycin, human insulin (Sigma), 0.5 mg mL−1 hydrocortisone (Sigma), 10 ng mL−1 epidermal growth factor (EGF), and 100 ng mL−1 cholera toxin (QuadraTech, Ltd.). RNA was extracted from cell lines using Tri Reagent (Molecular Research Center, Inc.) according to the manufacturer protocols.43 Total RNA was quantified with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc.). cDNA was synthesized using the Superscript III First Strand Synthesis kit (Invitrogen).42 cDNA quality was assessed by PCR using specific primers for human glyceraldehyde 3-phosphate dehydrogenase (GAPDH): sense, 5′-GGCTGAGAACGGGAAGCTTGT-3′, and antisense 5′-CGGCCATCACGCCACAGTTTC-3′.42 GAPDH PCR products were run in 1.5% agarose gels. We were able to amplify GAPDH in all cell lines, and then, we considered the quality of cDNA to be appropriate for the rest of the experiments. To perform the detection of the endogenous TP53 gene with the genosensor, the obtained cDNAs were appropriately diluted with hybridization buffer to provide a final concentration of 5 ng μL−1 and the same protocols for hybridization, labeling, and detection steps as described in previous sections were followed.

containing 137 mM NaCl and 2.7 mM KCl, pH 7.2; and 0.1 M phosphate buffer, pH 7.0. Standard solutions of the oligonucleotides were prepared daily from the 100 μM stored aliquots (scpp53 and lcpp53 in 0.025 M MES buffer, pH 5.0 and TP53 and 1-m in 0.01 M PBS buffer, pH 7.2, supplemented with 0.5% of casein). A 0.5 mg mL−1 rGO-CMC hybrid nanomaterial dispersion prepared in deionized water was used to modify the SPCEs. Activation of the carboxylic groups was carried out using an EDC/sulfo-NHS mixture solution (50 mg mL−1 each prepared in MES buffer, pH 5.0). The blocking step was accomplished with a solution of skimmed UHT milk purchased in a local supermarket diluted 1:1 with 10 mM PBS buffer, pH 7.2. The 1:1000 Strep-HRP conjugate solution was prepared in 10 mM PBS buffer, pH 7.2, supplemented with 0.5% of casein. Preparation of the DNA Sensor and Assay Procedure. Modification of the SPCEs was accomplished by depositing a 10-μL aliquot of an aqueous dispersion of rGO-CMC (0.5 mg mL−1) on the working electrode surface and allowing it to dry at room temperature. Then, the −COOH groups of the rGO-CMC-modified SPCE surface were activated by depositing 10 μL of the EDC/sulfo-NHS mixture onto the working electrode. The reaction was allowed to proceed for 30 min at room temperature, and then the electrode was rinsed with MES buffer and dried under a nitrogen stream. The capture probe (cp) was covalently immobilized by dropping 10 μL of the corresponding capture probe solution (0.01 μM scpp53 or 10 μM lcpp53, prepared in MES buffer) on the activated electrodes and allowing incubation for 30 min. Subsequently, the scpp53- or lcpp53-modified SPCEs were washed with PBS to remove the unbound DNA and dried under a nitrogen stream. Thereafter, the unreacted activated groups on the rGO-CMC-SPCEs were blocked by dropping 10 μL of 1:1 PBS diluted skimmed milk solution on the working electrode and incubating for 30 min at 25 °C. After a washing step with PBS buffer (pH 7.2) and drying under a nitrogen stream, the hybridization process was accomplished by incubation of the cp-rGO-CMC-SPCEs with a 10-μL sample aliquot (synthetic target solution in PBS buffer containing 0.5% casein, spiked biological sample or diluted cDNA) during 30 or 15 min for the scpp53-modified SPCEs and lcpp53-modified SPCEs, respectively. After another washing step with PBS buffer (pH 7.2) and drying under a nitrogen stream, 10 μL of the Strep-HRP conjugate solution (1000 times diluted in PBS buffer containing 0.5% casein) was deposited on the modified working electrode surface for 30 min. Finally, the modified electrodes were thoroughly washed with 0.1 M sodium phosphate buffer solution (pH 7.0) and dried under a nitrogen stream before the electrochemical measurements. All the incubation steps were performed by placing the SPCE in a Petri dish in order to avoid evaporation of the drops deposited onto the working electrode surface. All procedures were carried out at room temperature. Amperometric Measurements. A 45-μL drop of 0.1 M sodium phosphate buffer solution (pH 7.0) was deposited onto the modified SPCE and the amperometric measurements were carried out at an applied potential of −0.10 V vs the Ag pseudo-reference electrode. After stabilization of the background current, 5 μL of the TMB-H2O2 solution was deposited on the working electrode surface and the resulting current was recorded. The analytical signal was the current measured 200 s after the addition of the TMB−H2O2 substrate.

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RESULTS AND DISCUSSION Two different DNA sensing platforms using SPCEs modified with rGO-CMC hybrid nanomaterial as scaffold for the immobilization of two amino-terminated DNA capture probes with hairpin structure and different length (33 vs 20 nts) were prepared and evaluated for the direct detection of the cancer biomarker TP53 gene sequence and discrimination between the healthy and SNP-containing DNA sequences in biological samples. The capture probes were DNA hairpin beacons complementary to the target sequence with dual modification: Biotin (Btn) at the 5′-end and amine (an amino-C7-modifier, AmC7) at the 3′-end through which they were immobilized on the rGO-CMC-SPCE scaffold (see Table S2 in the Supporting Information). The target sequence corresponded with a region of the wtTP53 gene and the 1-m sequence with a mutant TP53 genotype that contained the hot-spot mutation of G → A (SNP at codon 175, exon 5), leading to cancer-triggering deactivation of the tumor suppressor protein p53 responsible for cell cycle C



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Scheme 1. Schematic Illustration of the Steps Involved in the Preparation of the Electrochemical DNA Biosensor for the Detection of the TP53 Gene Using rGO−CMC Hybrid Nanomaterial as a Scaffold ((a) the Long TP53 Capture Probe (lcpp53) or (b) the Short (spp53) TP53 Capture Probe) and Labeling with Strep-HRP (Relative Sizes of the Components Are Not Drawn on a Real Scale)

regulation.15 As illustrated in Scheme 1, both capture probes were covalently immobilized by carbodiimide chemistry in their folded states onto the −COOH rich rGO-CMC nanostructured platform through their amino moiety at the 3′-end. Moreover, labeling was performed through biotin at the 5′-end with Strep-HRP conjugate and amperometric detection of the hybridization extent was carried out using the TMB−H2O2 system. A typical “on−off” change of the electrochemical signal was observed (Figure 1) upon hybridization of the long capture probe to the target DNA, as a consequence of the remarkably increased distance between the attached Strep-HRP conjugate and the electrode surface, which hindered the electron transfer. Conversely, the use of the short capture probe produced a noticeable increase in the amperometric response after target hybridization (Figure 1), probably due to decreased hindering of the enzyme-catalyzed electron transfer to the electrode surface attributable to reduced steric effect as a consequence of the shorter duplex length. It is interesting to note that these findings are in agreement with the patterns observed by Farjami et al.,15 using thiolated capture probes labeled with Methylene Blue. In addition, it is important to highlight that no significant differences were observed for the amperometric measurements obtained in the absence or in the presence of 100 nM synthetic TP53 on un-

Figure 1. Amperometric responses obtained with the developed electrochemical platforms before hybridization (white bars) and after hybridization (gray bars) with 100 nM complementary target DNA using long TP53 capture probes (lcpp53) and short TP53 capture probes (scpp53). Other experimental conditions are described later in this paper in Table 1 (see the “selected values” column). Error bars are estimated as triple the standard deviation (n = 3).

modified SPCEs, unactivated rGO-CMC-SPCEs, or in the absence of capture probe (see Table S2 in the Supporting Information), thus demonstrating that neither the target nor the enzymatic D



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variables listed in Table 1 was the ratio between the current values measured at −0.10 V in the absence of 0.1 μM synthetic TP53 target (blank, i0) and in the presence of 0.1 μM synthetic TP53 target (signal, i0.1). For simplification, and taking into account the different signal outputs observed with both selected captures probes, the i0/i0.1 and i0.1/i0 ratio values were used to evaluate the results obtained with lcpp53 and scpp53, respectively. A 0.1 μM DNA target concentration was employed for optimization studies, since this concentration allowed one to obtain a current signal that differed significantly from the blank current with both DNA sensing platforms. As an example of these optimization studies, Figure 3 shows the results obtained

label were immobilized or nonspecifically adsorbed to a significant extent. Moreover, the influence of the nanostructuration employed to modify the SPCEs was compared using the functional hybrid nanomaterial (rGO-CMC), the single rGO or a widely used carbonaceous nanomaterial (MWCNTs) as electrode modifiers. Figure 2 shows the amperometric responses obtained

Figure 2. Comparative hybridization efficiency (gray bars) and background signal (white bars) for 100 nM complementary target DNA in the presence of scpp53 at rGO-CMC-SPCEs, rGO-SPCEs, and MWCNTs-SPCEs. Other experimental conditions are described later in this paper in Table 1 (see the “selected values” column). Error bars are estimated as triple the standard deviation (n = 3).

with scpp53 probes for 100 nM complementary target DNA, as well as the respective background signals. As is clearly seen, the performance of rGO-CMC-SPCEs is considerably better than that exhibited by rGO-SPCEs and MWCNTs-SPCEs, which reveals the unique advantages of rGO-CMC-SPCEs as a new platform for the preparation of electrochemical DNA biosensors. Similar results were obtained using the long lcpp53 capture probe. This improved electroanalytical behavior can be attributed to the fact that the functional rGO-CMC hybrid used as supporting scaffolds is able to dramatically increase the amount of immobilized probe (as a result of the high number of surface −COOH groups) with sufficient spacing for optimal hybridization.44 Optimization of Experimental Variables. The experimental variables involved in the preparation and functioning of both DNA biosensing platforms were optimized. The ranges tested for all the checked variables, and the corresponding selected values are collected in Table 1. Both the detection

Figure 3. Effect of the (a) lcpp53 and (b) scpp53 immobilization solution concentration on the amperometric responses obtained with the developed electrochemical platforms before and after hybridization in a 100 nM solution of the synthetic TP53 target. Other experimental conditions described in Table 1 (see the “selected values” column). Error bars are estimated as triple the standard deviation (n = 3).

Table 1. Optimized Experimental Variables Affecting the Performance of the Developed Electrochemical DNA Platforms for the Determination of the Synthetic TP53 Target sccp53

in the optimization of the capture probes concentration used for their immobilization on the rCO-CMC-SPCEs. As it can be seen, the probe density strongly affected the target hybridization efficiency, reaching, in both cases, a maximum ratio value and showing a sharp decrease for large probe concentrations. These results are in agreement with earlier works reporting low hybridization efficiency for high-density probe films.32,46,47 The differences in the optimal capture probe concentration can be attributed to the different packing adopted on a surface, depending on the strand length. Probes shorter than 24 bases had a tendency to organize in end-tethered, highly extended configurations for which the long-term surface coverage is largely independent of oligonucleotide length. Conversely, longer probes (>24 bases) organize in less-ordered and more-flexible arrangements, presumably reflecting increasingly polymeric

lcpp53

variable

tested range

selected value

[rGO-CMC] [capture probe] tcapture probe thybridization

0−15 μg 0−0.5 μM 15−120 min 15−120 min

5.0 μg 0.01 μM 30 min 30 min

tested range

selected value

0−15 μg 0−25 μM 15−90 min 5−90 min

5.0 μg 10 μM 30 min 15 min

potential (−0.10 V vs Ag pseudo-reference electrode) and the volume of the TMB−H2O2 mixture (5 μL) were the same than those optimized previously for the electrochemical detection system used.45 In all cases, the criterion of selection for the E



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behavior in which coverage decreases notably with probe length.48 This behavior could explain why a much higher concentration of the lcpp53 (33 nts) was required to achieve an effective coverage than using the scpp53 (20 nts). It is worthy to note that an lcpp53 concentration of 0.01 μM did not show significant differences in the biosensor response in the absence or in the presence of 100 nM synthetic TP53. Analytical Characteristics. The analytical performance of the scpp53 and lcpp53-rGO-CMC-SPCEs sensing platforms were evaluated by constructing calibration curves for synthetic TP53 target under the optimized working conditions. The analytical characteristics obtained are summarized in Table 2.

responses toward 0 and 100 nM synthetic TP53 target. A control chart was constructed by setting the mean value from 10 i0/i0.1 or i0.1/i0 ratio measurements made the first day of the study as the central value, while the upper and lower limits of control were set at ±3SD of this initial value (results not shown). The ratio values for both DNA scaffolds (lcpp53- and scpp53-rGO-CMC-SPCEs) remained within the control limits for 15 days, indicating an acceptable storage stability of the prepared DNA sensors. Selectivity of the DNA Sensors. The selectivity of the new DNA sensors was investigated by performing control hybridization experiments for blank, target, noncomplementary, and single-base mismatched sequences. In this sense, it is important to mention that, since the TP53 gene shows little difference in expression between cancer and healthy tissues, it is much more relevant to implement a DNA sensing platform that is able to detect TP53 mutations than variations in gene expression.50 The results showed a signal not significantly different from that of the blank for the noncomplementary sequence, while a strong distinction was observed between perfectly matched and single-base mismatched DNA for lcpp53-rCO-CMC-SPCEs. As it can be deduced from Figure 4, only the perfectly matched

Table 2. Analytical Characteristics for the Determination of the Synthetic Tp53 Target with the Developed Electrochemical DNA Platforms sccp53 slope intercept Linear range, LR r limit of detection, LODa Limit of quantification, LOQb relative standard deviation, RSDn=10

−7

6.78 × 10 μA μM 2.96 × 10−8 A 0.01−0.1 μM 0.998 3.4 nM 11.2 nM 4.3%

lcpp53 −1

−5.20 × 10−7 μA μM−1 9.31 × 10−8 A 0.01−0.1 μM 0.996 2.9 nM 9.6 nM 6.1%

Calculated as 3 × sb/m, where sb was the standard deviation for 10 blank signal measurements and m is the slope value of the calibration plot. bCalculated as 10 × sb/m. a

As it can be seen, limit of detections (LODs) as low as 3.4 and 2.9 nM (34 and 29 fmol in 10 μL of sample), calculated according to the 3 × sb/m criterion, where sb was estimated as the standard deviation for 10 blank signal measurements and m is the slope value of the calibration plot, were obtained without any amplification step. Although these LOD values were comparatively higher than those reported with some other electrochemical sensors for the target wild-type TP53 gene (for instance, 10 aM32 or 800 pM49), most of them require multiple reagents and complex and time-consuming working protocols including amplification strategies.13,26,30,32,49 Moreover, no previous target labeling is required to perform the determination which constitutes also a very important practical advantage of the implemented methodologies. Nevertheless, a distinct advantage of our work relies on the high selectivity achieved as it is demonstrated below. In comparison with the DNA sensors reported by Fajarmi et al.,15 using thiolated capture probes, the scpp53 and lcpp53-rGO-CMC-SPCEs sensing platforms provided a similar sensitivity but much better selectivity in a considerably shorter assay time (2.5 h vs 28 h). The reproducibility of the amperometric responses for 100 nM synthetic TP53 target was evaluated with 10 different DNA sensors constructed using the same protocol. Relative standard deviation (RSD) values of 4.3% and 6.1% were obtained for scpp53- and lcpp53-rCO-CMC-SPCEs, respectively, which confirmed the reliability of the DNA sensor fabrication procedure including capture probe immobilization, target hybridization, and labeling steps, as well as the amperometric measurements. Moreover, the storage stability of the integrated DNA sensors was evaluated by preparing on the same day different cp-rGO-CMC-SPCEs which were stored at 4 °C. Their longterm stability was tested periodically by measuring the sensor

Figure 4. Selectivity tests using the lccp53- and scpp53-rCO-CMCSPCEs DNA sensors. Current values were measured in the absence (blank signal, white bars) or in the presence of 100 nM synthetic TP53 target (gray bars) and 1-m (lined bars) sequences. Experimental conditions described in Table 1 (see the “selected values” column). Error bars are estimated as triple the standard deviation (n = 3).

target DNA provided an amperometric signal significantly different from the blank. Although to a lower extent than that observed with lcpp53, a good discrimination was also observed for scpp53-rCO-CMC-SPCEs since the signal obtained for the single-base mismatched sequence was significantly higher (although lower than that obtained for the perfectly matched DNA) than that measured for the blank. Therefore, these results demonstrated that the lcpp53-based DNA sensor allowed SNP detection with complete discrimination between the fully matched and the G → A mutated TP53 gene sequence. That is a remarkable result, since, under the same conditions, the scpp53 could recognize sequence-specific TP53 sequences (wtTP53 and mTP53) with discrimination of up to 65.7%. Additional studies performed with the scpp53 sensor showed the discrimination could be improved by using shorter hybridization times (see Figure S1 in the Supporting Information), which is in agreement with data reported by Farjami et al.15 It should be highlighted that our DNA sensor exhibits an improved selectivity when it is compared with that reported for the thiolated capture probes immobilized onto gold electrodes and labeled with Methylene F



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Blue,15 as well as with that described using other linear capture probes.23,39 In addition, the implemented methodology did not involve a stringent wash process with precise temperature control and long analysis times. Electrochemical Detection of Synthetic Target DNA Hybridization in Spiked Complex Biological Samples. Taking into account the suitability of the lcpp53-based DNA biosensor to discriminate single nucleotide mismatches and, therefore, its higher relevance to accomplish the protocol strongly required in cancer diagnostic assays, only the lcpp53rGO-CMC-SPCEs platforms were used to test their applicability for the analysis of biological samples. Serum and saliva are complex biological samples with multiple components that can be nonspecifically adsorbed onto the sensing interface, interfering with the target DNA binding and/or increasing the background signal. Most importantly, the novel nanostructured interface allowed the direct detection of the target DNA with no significant matrix effect in raw human serum and saliva. Figure 5 shows the comparison of the amperometric responses

Figure 6. Detection of the endogenous TP53 gene status in 5 ng μL−1 cDNAs from different cell lines (MCF-10A, MCF-7, and SK-BR-3). For comparison purposes, the amperometric signals obtained for the blank (1), 100 nM synthetic TP53 target (2), and 1-m (3) are also shown. Experimental conditions described in Table 1 (see the “selected values” column). Error bars estimated as a triple of the standard deviation (n = 3).

175 (same G → A nucleotide change used to demonstrate the SNP selectivity of the TP53 developed sensor).52,53 The results displayed in Figure 6 show a clear agreement with these antecedents, since the sensor SNP-related response occurred only for SK-BR-3 cells. It is important to mention that, according to the literature, mutations in codons 175, 245, 248, 273, and 282 are the most common in both sporadic and familial tumors,54 which makes it the developed methodology an interesting tool for cancer-related research.

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CONCLUSIONS This work describes effective in practice disposable electrochemical DNA sensors using reduced graphene/CMC hybrid nanostructured scaffolds and DNA hairpin probes of different length for the detection of intact and SNP-containing TP53 gene sequences, one of the most popular genes in cancer research. The new bioplatforms, with a storage stability of 15 days, improved significantly the analytical performance when compared with other scaffolds prepared using only reduced graphene oxide or conventional MWCNTs. Limits of detection of ∼3 nM were obtained without any target or signal amplification. Moreover, the use of a 33 nts DNA hairpin beacon sequence provided increased selectivity for SNP detection, strongly required in cancer diagnostic assays, allowing complete discrimination between the fully matched and SNP-containing DNAs in only 30 min. Most interestingly, the sensor usefulness for the assessment of TP53 status in human cell lines has been demonstrated. It is important to point out that the detection of the TP53 gene status in any biological sample would require previous extraction of the total RNA followed by the synthesis of the total cDNA by RT-PCR. Therefore, it would be expected that the composition of the final RT-PCR mixture and the cDNA concentration would be similar for all the biological samples when using similar amounts of RNA. Accordingly, an analogous discrimination between a perfect match and a mutated sequence than that shown in bars “MCF-10A” or “MCF-7” and “SK-BR-3” in Figure 6 would be expectable for other samples. Such attractive features emphasize the crucial role of the rGO-CMC hybrid in minimizing background contributions and measuring low levels of the target DNA, thus offering great promise for the implementation of platforms that allow straightforward and fast mutation screening related to most human cancer types.

Figure 5. Comparison of the current values measured with lcpp53rCO-CMC-SPCEs for 0 nM (blank signal, white bars) and 100 nM synthetic TP53 target (gray bars) or 1-m (lined bars) sequences in hybridization buffer, undiluted human serum and saliva. Experimental conditions described in Table 1 (Selected values column). Error bars are estimated as triple the standard deviation (n = 3).

for 100 nM synthetic TP53 target concentration in either 100% human serum or saliva with those obtained working in the hybridization buffer solution. As it can be deduced, although with a lower sensitivity, it is possible to measure 100 nM of the synthetic TP53 target with single nucleotide mismatch discrimination both in untreated human serum and saliva samples. These results demonstrated that no significant adsorption of the different matrices components occurred on the lcpp53rGO-CMC-SPCEs. Application to the TP53 Gene Status Detection in cDNAs from Breast Cancer Cells. In order to demonstrate the real clinical applicability of the developed TP53 DNA sensor, the implemented methodology was applied to analyze the endogenous TP53 status in different human cell lines. The used methodology implies the synthesis of cDNA by RT-PCR from previously extracted total sample RNA, as described in the Experimental Section. Figure 6 shows the results obtained for three breast cell lines, MCF-10A and MCF-7 and SK-BR-3. The former is an epithelial nontumorigenic cell line, while the other two are cancer cell lines. Although it has been described that the wtTP53 gene is present in MCF-10A and MCF-7 cells,51 the cell line SK-BR-3 harbors a missense mutation in codon G



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ASSOCIATED CONTENT

S Supporting Information *

Tables showing the oligonucleotides used in this work (Table S1) and data on current values ratios measured at −0.10 V in the absence (blank, i0) and in the presence (signal, i0.1) of 100 nM synthetic TP53 target onto different biosensing scaffolds (Table S2), and a figure showing the effect of the hybridization time on the selectivity for the 1-m sequence using the scpp53-rCO-CMC-SPCEs (Figure S1) are provided as Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: +34 913944368. Fax: +34 913944329. E-mail: susanacr@ quim.ucm.es (S. Campuzano). *Tel.: +34 913944315. Fax: +34 913944329. E-mail: pingarro@ quim.ucm.es (J. M. Pingarrón). Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The financial support of the Spanish Ministerio de Economiá y Competitividad Research Projects (Nos. CTQ2011-24355, CTQ2012-34238, and S2013/MT-3029 (NANOAVANSENS Program from the Comunidad de Madrid)) are gratefully acknowledged. B. Esteban-Fernández de Á vila acknowledges a FPI fellowship from the Spanish Ministerio de Ciencia e Innovación. R. Villalonga acknowledges Ramón & Cajal contract from the Spanish Ministry of Science and Innovation. Rodrigo Barderas is supported by the Ramón y Cajal Programme of the MINECO.

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