Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2016 Aug 15.
Published in final edited form as: J Electroanal Chem (Lausanne). 2015 Aug 15;751:49–56. doi: 10.1016/j.jelechem.2015.05.038

Nanostructured gold dsDNA sensor for early detection of breast cancer by beta protein 1 (BP1)

Wanda I Pérez 1, Yarelys Soto 2, Jaime E Ramirez-Vick 3, Enrique Meléndez 1
PMCID: PMC4491849  NIHMSID: NIHMS697353  PMID: 26161048

Abstract

Beta protein 1 (BP1) is a homeobox protein expressed in 80% of breast cancer cells in either estrogen receptor (ER) positive or ER negative breast cancer. However, it is barely detectable in normal breast tissues. In this project we present an electrochemical DNA nanostructured gold biosensor for detection of BP1. The gold sensor is first electrochemically nanostructured in 0.5 M sulfuric acid to reach superior conductivity, larger surface area, and higher stability. Nanostructured gold surface was characterized by atomic force microscopy (AFM) and scanning electron microscopy (SEM). The nanostructured gold sensor is then modified with double-stranded (ds) DNA mapping the genomic sequence that contains the binding site for BP1. A redox-active probe (methylene blue) was intercalated in dsDNA to monitor the binding event of BP1. A linear correlation of the electrochemical response by concentration of BP1 was obtained (R2 = 0.998) with a limit of detection of 1.2 nM. This nanostructured gold dsDNA sensor is shown to be sensitive, selective, stable, and reusable allowing for its potential clinical use.

Keywords: Gold nanostructed sensor, beta protein 1, biosensor, DNA sensor

Graphical Abstract

graphic file with name nihms697353u1.jpg

1. Introduction

Cancer is one of the major causing deaths in the United States. Statistically, one of four deaths in the United States is caused by cancer. The most common diagnosed cancer in woman is breast cancer. It is also the second leading cause of cancer death among women around the world [1]. During 2014, around 29% of the new cases of cancer in women were diagnosed to be breast cancer [2]. According to the American Cancer Society, over 231,840 new cases of invasive breast cancer are expected to be diagnosed in the United States in 2015. Additionally, 60,290 new cases of carcinoma in situ breast cancer, which is the earliest form of breast cancer and it is also non-invasive, will be diagnosed in 2015. Unfortunately, about 40,290 diagnosed women will die of this condition.

According to a statistical study, about 90% of the breast cancer patients diagnosed with carcinoma in stage 0 will survive, emphasizing the importance of early detection [3]. During the past decade researchers have driven their attention to search biomarkers for the early detection of breast cancer. In 2002, Sidney et al. reports a strong correlation between the expression of beta protein 1 (BP1) and ER in breast cancer cells [4]. BP1 is a member of the homeobox gene superfamily of transcription factors (TFs), which are expressed during embryonic development and are subsequently re-expressed by several different types of cancers [5]. This protein consists of a 60-amino acid helix-turn-helix structure with three alpha helices connected by short loop regions in which one of these interacts directly with DNA [6]. A remarkable feature of BP1 is that it is expressed in 100% of ER− tumors and in 73% of ER+ tumors [4–7], with the former being more invasive and with a typical poor prognosis when compared with the latter. Meanwhile, lack of BP1 expression was found in ER− normal breast cells and low-level expression in ER+ normal breast cells. In addition, frequent expression of BP1 was also found in all tumor grades. All these properties make BP1 an ideal biomarker for early detection of these types of breast cancer [4–7]. Recently researchers have driven their attention in the development of biosensors for breast cancer detection using different biomarkers [8–10]. One of the most promising biomarkers for breast cancer detection is the abovementioned BP1 TF. The detection of breast cancer-related TFs, such as BP1, can be done using gel electrophoretic mobility shift assays [11] and DNase footprinting assays [12], which are based on migration retardation of protein-DNA complexes through gel electrophoresis. However, these methods require long and labor-intensive protocols and the use of an analytical laboratory with specialized personnel, making them neither feasible for routine biomarker determination nor applicable for point-of-care (POC) testing. Thus, the challenge on the detection of TFs is the development of sensitive and simple methods which allow quantification of them [13].

Electrochemical assays based on DNA-mediated charge transport at microelectrodes offer an alternative approach which can be sensitive, specific, fast, simple, and low-cost, making them suitable for POC diagnostics and multiplexed platforms [14]. This type of approach has been useful in assays to detect mutations and lesions as well as for the detection of protein binding at self-assembled DNA monolayers. However, its use for the detection of protein binding utilizing double-stranded (ds) DNA monolayers has been scarce and can be divided into electrodes modified with a hairpin oligonucleotide with conjugated redox-active probes [15–21], or with dsDNA that bends upon protein binding [22–24]. For the first type, TF binding stabilizes an oligonucleotide conformation which brings the redox-active prove close to the electrode leading to a large increase in Faradaic current signal. The second type capitalizes on the fact that upon binding some TFs bend the immobilized dsDNA probe restricting the flow of electrons. Both alternatives function as a switch, in which electrons are either transported or not to the electrode.

Most nano-gold (NG) sensors modified with single-stranded DNA are designed to characterize the hybridization between the two complimentary strands which allows electrochemical charge transport [14,20,24–27]. A series of conjugated charge donors or acceptors are employed as redox active probes interacting by π-stacked base pairs in dsDNA to monitor the electrochemical binding event [21,24,28–34]. In the case of TFs, the electrodes are modified with dsDNA containing the sequence which allows binding of a specific TF. Thus, this work proposes for the first time (1) the detection of a clinically-relevant transcription factor biomarker, (2) the electrochemical detection of a transcription factor using a double-stranded DNA probe on a nanostructured gold electrode, and (3) a limit of detection of 1.2 nM

2. Experimental section

2.1 Materials

Methylene Blue 82%, 6-mercapto-1-hexanol (MCH) 97%, and tris(hydroxymethyl)-aminomethane 99.9% were obtained from Sigma-Aldrich. The DNA sequences containing the BP1 binding motif ATATATATG were as follows: Forward 5′-TTAAAAAGATATATATATATGTTTTTCTAATGT-3′, (modified with 5′end thiol C6 linker) and reverse 5′-ACATTAGAAAAACATATATATATATCTTTT-TAA-3′ [6,7]. The following sequences without the BP1 binding sequence where used as the dsDNA negative control (Forward: 5′-TCTTAGAGGGAGGGCTGAGGGTTTGAAGTCCAA-CTCCTAAGCC-3′; Reverse: 5′-AGAATCTCCCTCCCGACTCCCAAACTTCAGGTTG-AGGATTCGG-3′) [6]. All DNA probes were obtained from Eurofins Genomics and the BP1 protein was obtained from Gen Way and used as received. Deionized water (18.2 MΩ) was used for all experiments.

2.2 Equipment

CP-II VEECO atomic force microscope (AFM) with scanning probe microscopy and lateral force microscopy and nanomanipulation capabilities with 100 _ 100 lm scanning area capacity is used to characterize the surface morphology. Scanning electron microscope (SEM) is used to characterize the surface morphology of the nanostructured electrode using a Field Emission Scanning Electron Microscope Supra 35 (Zeiss). The equipment allows analysis of features in the nanometer range using low accelerating voltage (3kV). Electrochemical experiments were carried on an Epsilon Potentiostat/Galvanostat (Bioanalytical Systems, Inc.) using a specially designed three-electrode electrochemical cell having an analytical gold sensor as working electrode (0.02 cm2), a platinum wire as counter (auxiliary) and Ag/AgCl paste covered wire as reference electrode. SRS Quartz crystal microbalance (QCM) Ti/Au, 5MHz electrode used for surface characterization was purchased from Stanford Research Systems.

2.3 Synthesis and characterization of gold nanoparticles

The gold electrode surface, before nanostructured, was polished prior to use with 0.5 μm alumina for 3 minutes and then cleaned in an ultrasonic bath with ethanol and deionized water for 5 minutes. The gold electrode is then electrochemically cleaned by cyclic voltammetry (CV) in 0.5 M sulfuric acid at 100 mVs−1 scan rate, between −0.35 to 1.5 V until no changes in voltamograms. After electrochemical cleaning of the gold electrode, the thick hydrous gold oxide layer accumulated on the gold electrode is removed in 0.5 M sulfuric acid by using repetitive square-wave voltammetry (SWV), with a potential range between −0.8 V and 2.5 V (versus silver/silver chloride electrode) at 2,000 Hz for 5 minutes. In the following step, the potential is held at −0.8 V until complete electro-reduction of the hydrous gold oxide layer is accomplished. The NG surface was characterized by cyclic voltametry in 0.5 H2SO4 between −0.35 to 1.5 V (versus silver/silver chloride electrode) at 100 mVs−1. After synthesis of gold nanoparticles, the gold reduction peak current at 0.942 V increases considerably. SEM was used to characterize the surface morphology of the nanostructured electrode. In addition, atomic force microscopy (AFM) was used to measure the surface topography.

2.4 Immobilization of dsDNA onto gold nanostructured sensor

The BP1 binding sequence oligonucleotides were resuspended in an annealing buffer consisting of 10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM EDTA. Equimolar concentrations of the forward and reversed oligonucleotides were annealed at 94 °C for 4 minutes and then cooled at room temperature for 40 minutes. The resulting dsDNA was self-assembled into the NG surface for 18–24 h. After dsDNA addition to the NG surface, it was exposed to a solution of 1.0 mM mercaptohexanol (MCH) for 1 h (Scheme 1) to allow for surface passivation [35]. The co-adsorption of MCH onto DNA-modified gold surface removes the weakly, unspecifically adsorbed nucleic acids by forming a dense MCH sublayer. DNA sensor was then immersed in a solution on 20 μM MB for 20 minutes to intercalate it as redox-active probe [14,24]. Characterization of dsDNA and its binding to the NG surface were performed using CV to monitor changes in MB redox cycle. The CV experiments were carried out between −0.450 to 0 V at 100 mVs−1 in 20 mM Tris, 100 mM NaCl, pH 7.5, at room temperature under an inert atmosphere. A three-compartment electrochemical cell is used with a Pt wire auxiliary electrode, silver/silver chloride electrode as reference, and a 0.02 cm2 NG working electrode.

Scheme 1.

Scheme 1

Open in a new tab

Schematic representation of NG/dsDNA/MCH sensor construction.

2.5 Electrochemical characterization of DNA-binding event of BP1

The BP1 binding event was characterized by CV between −0.450 to 0 V (vs. Ag/AgCl) in 20 mM Tris, 100 mM NaCl, 20 μM MB, pH 7.5 at 100 mVs−1, using silver/silver chloride as reference, Pt wire as supporting electrode, NG/dsDNA/MCH as the working electrode and MB as redox-active probe. A stock solution of BP1 was prepared dissolving 0.01 mg of the protein in buffer. For the binding event characterization, the redox cycle current of MB was monitored with different dilutions of the BP1 stock solution (5.30 to 33.78 nM). The CVs were record after 2 minutes exposure time with the desired concentration. The NG/dsDNA/MCH was then incubated for two weeks and regeneration experiments were performed without loss of signal. Regeneration of the NG/dsDNA/MCH sensor was performed by a simple rinse in deionized water. Square wave voltametry was also performed after regeneration to confirm the BP1 binding event. This was carried out in 20 mM Tris, 100 mM NaCl, 20 μM MB, pH 7.5 from −0.6 to 0 V (vs. Ag/AgCl) at 600 Hz with an amplitude of 25 mV and a step size of 1 mV. It should be mentioned that both, CV and SWV can be used to monitor and quantify the BP1 binding. The experiments were run in triplicates.

3. Results and discussion

3.1 Characterization of gold nanostructures

The gold electrode surface was nanostructured with a modified procedure [36] using SWV in 0.5 M H2SO4 at 2,000 Hz for five minutes followed by electro-reduction at a fixed potential of −800 V. Figure 1 shows the cyclic voltammograms in 0.5 M H2SO4 at 100 mVs−1 of the gold electrode before electroreduction (black) and after electroreduction (red). The cyclic voltammogram for the clean gold electrode show two oxidation peaks related to the formation of gold oxides at 1.09 and 1.27 V and a reduction peak at 0.95 V [30]. At the end of the electroreduction process, the current of the reduction peak increases to 18 μA over the untreated gold electrode. The increase in reduction current of the gold reduction peak indicates the formation of gold nanostructures which increase both, the surface area and conductivity [30].

Figure 1.

Figure 1

Open in a new tab

Cyclic voltammograms of gold clean (black) and gold nanostructured sensor (red) performed in 0.5 M H2SO4 at a scan rate of 100 mVs−1. Methylene blue (MB) is the probe.

The NG surface topography was measured by AFM, and is shown in Figure 2. The gold surface topography before electroreduction (Figure 2A) shows a smooth surface topography with an average roughness of 2.089 nm while the NG electrode surface (Figure 2B) has a rough appearance with an average roughness of 7.202 nm. The increase in average roughness confirms the changes in surface area due to the formation of gold nanostructures. SEM of the clean gold electrode (Figure 2C) shows gold particles with a uniform size, while NG images (Figure 2D) shows small grains and nanoporous particles as reported in literature [30].

Figure 2.

Figure 2

Open in a new tab

A) AFM image of clean gold electrode, B) AFM image of gold nanostructed electrode, C) SEM image of clean gold electrode, and D) SEM image of NG electrode.

3.2 Immobilization of dsDNA onto gold nanostructured sensor

The complementary oligonucleotides with the BP1 binding sequence were first annealed by mixing them in equimolar concentrations on a thermostatic bath at 95°C for 4 min and cooled to room temperature for 40 min. Subsequently, the annealed dsDNA was exposed to the NG electrode, forming a self-assembled monolayer through the interaction between the thiol linker molecule and the NG surface [24,28]. The presence of the dsDNA attached to the gold electrode was confirmed by CV of the redox-active probe intercalated into the dsDNA (Figure 3). Figure 3A shows the CV of MB using the NG/MCH electrode (blue, without dsDNA). The MB has an anodic peak in −0.188 V. When it is intercalated onto the dsDNA attached to the NG/dsDNA/MCH electrode, the anodic peak potential is shifted to −0.253 V (Figure 3A, pink). The anodic peak potential shift of the MB is due to its intercalation into dsDNA. An increase in anodic peak current was also observed because the DNA is facilitating the charge transport.

Figure 3.

Figure 3

Open in a new tab

Cyclic voltammograms of; A) NG/MCH sensor in 20 mM tris, 100 mM NaCl, 20 μM MB (blue) and NG/dsDNA/MCH with MB intercalated into dsDNA (pink). B) CV of NG/dsDNA/MCH in 20 mM tris, 100 mM NaCl, 20 μM MB (pink) and with 0.1M K2Fe(CN)6. MCH = 6-mercapto-1-hexanol.

Hybridization of the complementary strands to form the dsDNA was verified to detect any mismatch in the double strand formation. The mismatch can be detected through reduction of MB in presence of potassium ferricyanide, K3Fe(CN)6. The current flows through the π-stacked base pairs in the dsDNA transferring 1 electron to the intercalated MB. The MB is then reduced to produce leucomethylene blue (LB) which reduces ferricyanide to reoxidate MB [37,38]. The electro-reduction cycle resulted in amplification of the MB signal (Figure 3B). Meanwhile, in the case that the dsDNA contains any mismatched base pairs, the MB reduction to LB does not occur resulting in an oxidation current decrease of the MB [14,25].

3.3 Characterization of the binding event of beta protein 1 (BP1) to dsDNA

Characterization of the binding event of BP1 to the dsDNA was done by CV and SWV monitoring of the MB redox-active probe [29,31,39,40]. The MB was intercalated onto a dsDNA base pairs and the TF BP1 to the consensus binding sequence ATATATATG [6]. A negative control sequence, a dsDNA sequence without consensus motif, was also used to verify the selectivity. A schematic illustration of the binding event of BP1 to the dsDNA is shown in Scheme 1. After nanostructuring the gold electrode, the dsDNA is linked to the NG surface and passivated with MCH to prevent direct interactions of the protein with the gold surface. The MB is then intercalated at the top of the dsDNA by incubation of the NG/dsDNA/MCH sensor. The MB intercalated NG/dsDNA/MCH sensor is then monitored by CV as a function of BP1 concentration. When BP1 binds the dsDNA, the charge transport through the DNA to the MB is interrupted attenuating the DNA-mediated reduction of the redox-active probe [24].

Figure 4 shows the cyclic voltammograms of MB intercalated on the dsDNA bound to the NG surface as a function of BP1 concentration. The anodic peak current of MB decreases significantly after the addition of BP1. The anodic peak current of MB continues to gradually decrease with BP1 concentration. The percent response after the first addition of 5.30 nM BP1 was 16% reduction in the anodic current of MB. Binding of the BP1 TF to its target site, bends the DNA duplex and perturbs the base pair stack, attenuating the DNA-mediated reduction of the redox probe. The loss of signal becomes greater with concentration reaching a saturation point near 30 nM BP1.

Figure 4.

Figure 4

Open in a new tab

Cyclic voltammograms of 20 μM MB, in 20 mM tris, 100 mM NaCl at 100mVs−1 in presence of different concentrations of BP1 using the NG/dsDNA/MCH as working electrode and MB as redox-active probe.

3.4 Analytical performance of the biosensor

The analytical performance of the NG/dsDNA/MCH sensor was evaluated using three independent measurements using the same electrode. The NG/dsDNA/MCH sensor was incubated for 2 minutes on the desired concentration before the CV was recorded. A significant attenuation of the MB current response was observed while the BP1 concentration increases. The percent of current attenuation follows a good linear relation with BP1 concentrations (Figure 5), with a linear range from 5.3 to 23.6 nM with a correlation coefficient of 0.998 and a limit of detection of 1.2 nM (based on a linear regression). There is reproducibility higher than 90% on independent measurements. The NG/dsDNA/MCH biosensor also demonstrates to be stable and reusable with reproducible results. Comparing the NG/dsDNA/MCH with other biosensors (Table 1), our biosensor based on protein interaction is specific for breast cancer cells, identifying the BP1 protein, does not require pre-conditioning, can take instant measurements and is reusable.

Figure 5.

Figure 5

Open in a new tab

Calibration curve illustrating the percent of current change vs BP1 concentration (nM).

Table 1.

Comparison of analyte detection, linear range, advantages or disadvantages of electrochemical biosensors for breast cancer.

Modified electrodes Analyte Linear range Advantages/disadvantages References
anti-HER-3 HER-3 0.2 to 1.4 pg/mL HER-3 is present in a wide range to tumors cell lines
Incubation of 1 h with HER-3 sample
Single use electrode		 [41]
SAM-assisted SiNW Erα 1 pM to 1 fM Erα is present in breast cancer cell lines and non-tumorigenic breast cells
Incubation of 1 h with cells sample
Single use electrode		 [42]
Avastin-MGO-Au VEGF 31.25 – 2000 pg/mL VEGF is present in many human cancers
Incubation of 30 min with sample
Reusable		 [43]
VEGF-R1 10 – 70 pg/mL VEGF is secreted by tumor cells
Incubation of 1 h with VEGF sample
Single use electrode		 [44]
NG/dsDNA/MCH BP1 5.3 – 23.6 nM BP1 is expressed in Erα+ and Erα− breast cancer cells
Instant sample measurements
Reusable		 This work
Open in a new tab

HEGF-3 = Human epidermal grouth factor-3, SAM = self-assambled monolayer, SiNW = silicon nanowire, ERE = estrogen receptor element, Erα = Estrogen receptor alpha MGO = magnetic graphene oxide, VEGF-R1 = vascular endothelial growth factor receptor 1

3.5 Regeneration of the fabricated DNA sensor

The NG/dsDNA/MCH is a simple and sensitive biosensor for the early detection of breast cancer through the recognition of the BP1 TF. This TF biosensor is not only simple to use but it can be easily regenerated through a 30 s rinse with deionized water, removing all the protein bound to the dsDNA, allowing the reconstitution of the MB signal [21,24]. Additional measurements of BP1 binding were realized by SWV to prove the stability of the TF biosensor. Selection of the square wave frequency was performed to determine the MB redox signal at different frequencies after regeneration. The frequency which demonstrated the strongest MB signal was 600 Hz (Figure 6). Different SWV experiments were carried out as a function of BP1 concentration. A rapid response for the lowest concentration of BP1 was observed with a signal of about 16% less than the MB initial signal. Subsequent attenuation response with BP1 concentration is shown in Figure 7.

Figure 6.

Figure 6

Open in a new tab

Square wave voltametry of MB in NG/dsDNA/MCH sensor after regeneration at different frequencies. SWV was carried out in 20 mM tris, 100 mM NaCl from 60 Hz to 600 Hz with amplitude of 25 mV and a step size of 1 mV.

Figure 7.

Figure 7

Open in a new tab

Square wave voltametry of MB in NG/dsDNA/MCH sensor after regeneration at different concentrations of BP1. SWV was carried out in 20 mM tris, 100 mM NaCl at 600 Hz with amplitude of 25 mV and a step size of 1 mV.

3.6 Selectivity of NG/dsDNA/MCH biosensor

SWV was also used to assess the selectivity of the NG/dsDNA/MCH biosensor. A sequence of dsDNA without the BP1 binding motif was used as a negative control [6]. Figure 8 shows the SWV of MB in the TF biosensor modified using the negative control sequence. The MB intercalated onto the negative control sequence (black) shows a higher current reduction than the MB intercalated into the dsDNA BP1 binding motif (blue). However, upon addition of BP1 only a 3% of signal attenuation was observed with the negative control sequence (red) compared to a 16% with the genomic sequence containing the BP1 binding motif at the same TF concentration (cyan). No significant changes in attenuation response were observed at higher BP1 concentrations. These results suggest that the NG/dsDNA/MCH biosensor has a good selectivity due to the specific sequence for the BP1 binding site. In addition, the NG/dsDNA/MCH biosensor remained stable after two weeks of its preparation.

Figure 8.

Figure 8

Open in a new tab

Square wave voltametry of MB in NG/dsDNA/MCH sensor modified with the negative control DNA (black and red) and NG/dsDNA/MCH with the genomic sequence (blue and cyan). SWV was carried out in 20 mM tris, 100 mM NaCl at 600 Hz with amplitude of 25 mV and a step size of 1 mV.

4. Concluding remarks

A prototype NG/dsDNA/MCH biosensor using MB as redox-active probe was fabricated in a simple manner affording a sensitive BP1 biosensor. The NG/dsDNA/MCH sensor demonstrates to have rapid and sensitive response range from 5.30 to 33 nM of BP1 and a limit of detection of 1.2 nM. Women with breast cancer have BP1 levels higher than 39 ng/mL [45]. Thus, our biosensor has the potential to be a tool for breast cancer detection. Also, it has been demonstrated that NG/dsDNA/MCH biosensor is stable and can be regenerated. Regeneration of the sensor provides the advantage of a reusable sensor. Through the selective detection of BP1 at low concentrations, the NG/dsDNA/MCH biosensor is suitable for early detection of breast cancer. Although it has not been tested on real blood samples (complex matrix), the nanostructured gold dsDNA sensor presented here has shown to be sensitive, selective, stable, and reusable allowing its potential use for POS testing. This biosensor is a proof-of-concept that NG/dsDNA/MCH biosensor with the correct oligonucleotide sequence to recognize BP1 can be fabricated and has the potential to become a simple tool for breast cancer detection.

Research Highlights.

  • Fabrication of re-usable nanostructured gold dsDNA sensor for early detection of breast cancer

  • Nanostructured gold dsDNA biosensor for breast cancer beta protein 1 (BP1)

  • NG-dsDNA sensor has a linear electrochemical response with a minimum detection of 5.30 nM

Acknowledgments

EM thanks the financial support of NIH-RISE 2 Best program (NIH-R25GM088023) for the research assistantships of Yarelys Soto-Echevarría (undergraduate student) and Wanda I. Pérez-Mercado (graduate student).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  • 1.DeSantis C, Ma J, Bryan L, Jemal A. Breast Cancer Statistics, 2013: Breast Cancer Statistics, 2013. CA Cancer J Clin. 2014;64:52–62. doi: 10.3322/caac.21203. [DOI] [PubMed] [Google Scholar]
  • 2.Siegel R, Ma J, Zou Z, Jemal A. Cancer Statistics, 2014: Cancer Statistics, 2014. CA Cancer J Clin. 2014;64(1):9–29. doi: 10.3322/caac.21208. [DOI] [PubMed] [Google Scholar]
  • 3.McDaniel RE, Maximov PY, Jordan VC. Vitamins & Hormones. Vol. 93. Elsevier; 2013. Estrogen-Mediated Mechanisms to Control the Growth and Apoptosis of Breast Cancer Cells; pp. 1–49. [DOI] [PubMed] [Google Scholar]
  • 4.Fu SW, Schwartz A, Stevenson H, Pinzone JJ, Davenport GJ, Orenstein JM, Gutierrez P, Simmens SJ, Abraham J, Poola I, et al. Correlation of Expression of BP1, a Homeobox Gene, with Estrogen Receptor Status in Breast Cancer. Breast Cancer Res BCR. 2003;5:R82–87. doi: 10.1186/bcr602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Haga SB, Fu S, Karp JE, Ross DD, Williams DM, Hankins WD, Behm F, Ruscetti FW, Chang M, Smith BD, et al. BP1, a New Homeobox Gene, Is Frequently Expressed in Acute Leukemias. Leukemia. 2000;14:1867–1875. doi: 10.1038/sj.leu.2401912. [DOI] [PubMed] [Google Scholar]
  • 6.Chase MB, Fu S, Haga SB, Davenport G, Stevenson H, Do K, Morgan D, Mah AL, Berg PE. BP1, a Homeodomain-Containing Isoform of DLX4, Represses the Beta-Globin Gene. Mol Cell Biol. 2002;22:2505–2514. doi: 10.1128/MCB.22.8.2505-2514.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Kluk BJ, Fu Y, Formolo TA, Zhang L, Hindle AK, Man Y, Siegel RS, Berg PE, Deng C, McCaffrey TA, et al. BP1, an Isoform of DLX4 Homeoprotein, Negatively Regulates BRCA1 in Sporadic Breast Cancer. Int J Biol Sci. 2010;6:513–524. doi: 10.7150/ijbs.6.513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Musayev J, Altiner C, Adiguzel Y, Kulah H, Eminoglu S, Akin T. Capturing and Detection of MCF-7 Breast Cancer Cells with a CMOS Image Sensor. Sens Actuators Phys. 2014;215:105–114. [Google Scholar]
  • 9.Ali MA, Mondal K, Singh C, Dhar Malhotra B, Sharma A. Anti-Epidermal Growth Factor Receptor Conjugated Mesoporous Zinc Oxide Nanofibers for Breast Cancer Diagnostics. Nanoscale. 2015;7(16):7234–7245. doi: 10.1039/c5nr00194c. [DOI] [PubMed] [Google Scholar]
  • 10.Mohanty P, Chen Y, Wang X, Hong MK, Rosenberg CL, Weaver DT, Erramilli S. Field Effect Transistor Nanosensor for Breast Cancer Diagnostics. 2014. ArXiv14011168 Q-Bio. [Google Scholar]
  • 11.Garner MM, Revzin A. A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 1981;9(13):3047–60. doi: 10.1093/nar/9.13.3047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Galas DJ, Schmitz A. DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res. 1978;5(9):3157–70. doi: 10.1093/nar/5.9.3157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Sanguinetti G, Lawrence ND, Rattray M. Probabilistic Inference of Transcription Factor Concentrations and Gene-Specific Regulatory Activities. Bioinformatics. 2006;22 (22):2775–2781. doi: 10.1093/bioinformatics/btl473. [DOI] [PubMed] [Google Scholar]
  • 14.Drummond TG, Hill MG, Barton JK. Electrochemical DNA Sensors. Nat Biotechnol. 2003;21:1192–1199. doi: 10.1038/nbt873. [DOI] [PubMed] [Google Scholar]
  • 15.Omidfar K, Khorsand F, Darziani Azizi M. New Analytical Applications of Gold Nanoparticles as Label in Antibody Based Sensors. Biosens Bioelectron. 2013;43:336–347. doi: 10.1016/j.bios.2012.12.045. [DOI] [PubMed] [Google Scholar]
  • 16.Ricci F, Bonham AJ, Mason AC, Reich NO, Plaxco KW. Reagentless, electrochemical approach for the specific detection of double- and single-stranded DNA binding proteins. Anal Chem. 2009;81(4):1608–14. doi: 10.1021/ac802365x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Lubin AA, Plaxco KW. Folding-based electrochemical biosensors: the case for responsive nucleic acid architectures. Acc Chem Res. 2010;43(4):496–505. doi: 10.1021/ar900165x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Vallée-Bélisle A, Bonham AJ, Reich NO, Ricci F, Plaxco KW. Transcription factor beacons for the quantitative detection of DNA binding activity. J Am Chem Soc. 2011;133(35):13836–9. doi: 10.1021/ja204775k. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Liang Z, Duanb Z, Li X, Liu F, Liu L, Wang K, Liu X. Determination of Transcription Nuclear Factor-Kappa B Using an Electrochemical, DNA-Based Nanoswitch. Anal Lett. 2014;47(16):2691–2698. [Google Scholar]
  • 20.Bonham AJ, Hsieh K, Ferguson BS, Vallée-Bélisle A, Ricci F, Soh HT, Plaxco KW. Quantification of Transcription Factor Binding in Cell Extracts Using an Electrochemical, Structure-Switching Biosensor. J Am Chem Soc. 2012;134:3346–3348. doi: 10.1021/ja2115663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Rowe AA, White RJ, Bonham AJ, Plaxco KW. Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules. J Vis Exp. 2011 doi: 10.3791/2922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Gorodetsky AA, Dietrich LE, Lee PE, Demple B, Newman DK, Barton JK. DNA binding shifts the redox potential of the transcription factor SoxR. Proc Natl Acad Sci U S A. 2008;105(10):3684–9. doi: 10.1073/pnas.0800093105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Williams K, Kim CS, Kim JR, Levicky R. Multimodal electrochemical sensing of transcription factor-operator complexes. Analyst. 2014;139(6):1463–71. doi: 10.1039/c3an02128a. [DOI] [PubMed] [Google Scholar]
  • 24.Gorodetsky AA, Ebrahim A, Barton JK. Electrical Detection of TATA Binding Protein at DNA-Modified Microelectrodes. J Am Chem Soc. 2008;130:2924–2925. doi: 10.1021/ja7106756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Gorodetsky AA, Buzzeo MC, Barton JK. DNA-Mediated Electrochemistry. Bioconjug Chem. 2008;19:2285–2296. doi: 10.1021/bc8003149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Genereux JC, Barton JK. Mechanisms for DNA Charge Transport. Chem Rev. 2010;110:1642–1662. doi: 10.1021/cr900228f. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Kelley SO, Jackson NM, Hill MG, Barton JK. Long-Range Electron Transfer through DNA Films. Angew Chem Int Ed. 1999;38:941–945. doi: 10.1002/(SICI)1521-3773(19990401)38:7<941::AID-ANIE941>3.0.CO;2-7. [DOI] [PubMed] [Google Scholar]
  • 28.Pheeney CG, Barton JK. DNA Electrochemistry with Tethered Methylene Blue. Langmuir. 2012;28:7063–7070. doi: 10.1021/la300566x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Pan D, Zuo X, Wan Y, Wang L, Zhang J, Song S, Fan C. Electrochemical Interrogation of Interactions between Surface-Confined DNA and Methylene Blue. Sensors. 2007;7:2671–2680. doi: 10.3390/s7112671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Zhong G, Liu A, Chen X, Wang K, Lian Z, Liu Q, Chen Y, Du M, Lin X. Electrochemical Biosensor Based on Nanoporous Gold Electrode for Detection of PML/RARα Fusion Gene. Biosens Bioelectron. 2011;26:3812–3817. doi: 10.1016/j.bios.2011.02.039. [DOI] [PubMed] [Google Scholar]
  • 31.Tuite E, Kelly JM. The Interaction of Methylene Blue, Azure B, and Thionine with DNA: Formation of Complexes with Polynucleotides and Mononucleotides as Model Systems. Biopolymers. 1995;35:419–433. [Google Scholar]
  • 32.Zhao GC, Zhu JJ, Zhang JJ, Chen HY. Voltammetric Studies of the Interaction of Methylene Blue with DNA by Means of B-Cyclodextrin. Anal Chim Acta. 1999;394:337–344. [Google Scholar]
  • 33.Cañete SJP, Yang W, Lai RY. Folding-Based Electrochemical DNA Sensor Fabricated by “click” Chemistry. Chem Commun. 2009:4835–4837. doi: 10.1039/b911273a. [DOI] [PubMed] [Google Scholar]
  • 34.Rohs R, Sklenar H. Methylene Blue Binding to DNA with Alternating AT Base Sequence: Minor Groove Binding Is Favored over Intercalation. J Biomol Struct Dyn. 2004;21:699–711. doi: 10.1080/07391102.2004.10506960. [DOI] [PubMed] [Google Scholar]
  • 35.Arinaga K, Rant U, Tornow M, Fujita S, Abstreiter G, Yokoyama N. The Role of Surface Charging during the Coadsorption of Mercaptohexanol to DNA Layers on Gold: Direct Observation of Desorption and Layer Reorientation. Langmuir. 2006;22:5560–5562. doi: 10.1021/la060324m. [DOI] [PubMed] [Google Scholar]
  • 36.Zhong G. Detection of Femtomolar Level Osteosarcoma-Related Gene via a Chronocoulometric DNA Biosensor Based on Nanostructure Gold Electrode. Int J Nanomedicine. 2012:527. doi: 10.2147/IJN.S27794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Kelley S. Single-Base Mismatch Detection Based on Charge Transduction through DNA. Nucleic Acids Res. 1999;27:4830–4837. doi: 10.1093/nar/27.24.4830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Boon EM, Ceres DM, Drummond TG, Hill MG, Barton JK. Mutation Detection by Electrocatalysis at DNA-Modified Electrodes. Nat Biotechnol. 2000;18:1096–1100. doi: 10.1038/80301. [DOI] [PubMed] [Google Scholar]
  • 39.Bradley DF, Stellwagen NC, O’konski CT, Paulson CM. Electric Birefringence and Dichroism of Acridine Orange and Methylene Blue Complexes with Polynucleotides. Biopolymers. 1972;11:645–652. doi: 10.1002/bip.1972.360110309. [DOI] [PubMed] [Google Scholar]
  • 40.Tuite E, Norden B. Sequence-Specific Interactions of Methylene Blue with Polynucleotides and DNA: A Spectroscopic Study. J Am Chem Soc. 1994;116:7548–7556. [Google Scholar]
  • 41.Canbaz MÇ, Şimşek ÇS, Sezgintürk MK. Electrochemical Biosensor Based on Self-Assembled Monolayers Modified with Gold Nanoparticles for Detection of HER-3. Anal Chim Acta. 2014;814:31–38. doi: 10.1016/j.aca.2014.01.041. [DOI] [PubMed] [Google Scholar]
  • 42.Zhang GJ, Huang MJ, Ang JJ, Liu ET, Desai KV. Self-Assembled Monolayer-Assisted Silicon Nanowire Biosensor for Detection of protein–DNA Interactions in Nuclear Extracts from Breast Cancer Cell. Biosens Bioelectron. 2011;26:3233–3239. doi: 10.1016/j.bios.2010.12.032. [DOI] [PubMed] [Google Scholar]
  • 43.Lin CW, Wei KC, Liao S, Huang CY, Sun CL, Wu PJ, Lu YJ, Yang HW, Ma CCM. A Reusable Magnetic Graphene Oxide-Modified Biosensor for Vascular Endothelial Growth Factor Detection in Cancer Diagnosis. Biosens Bioelectron. 2015;67:431–437. doi: 10.1016/j.bios.2014.08.080. [DOI] [PubMed] [Google Scholar]
  • 44.Sezgintürk MK. A New Impedimetric Biosensor Utilizing VEGF Receptor-1 (Flt-1): Early Diagnosis of Vascular Endothelial Growth Factor in Breast Cancer. Biosens Bioelectron. 2011;26:4032–4039. doi: 10.1016/j.bios.2011.03.025. [DOI] [PubMed] [Google Scholar]
  • 45.Ng EH, Ji CY, Tan PH, Lin V, Soo KC, Lee KO. Altered serum levels of insulin-like growth-factor binding proteins in breast cancer patients. Ann Surg Oncol. 1998;5:194–201. doi: 10.1007/BF02303854. [DOI] [PubMed] [Google Scholar]

RESOURCES