Journal of Electroanalytical Chemistry 751 (2015) 49–56

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Journal of Electroanalytical Chemistry

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

http://dx.doi.org/10.1016/j.jelechem.2015.05.0381572-6657/� 2015 Elsevier B.V. All rights reserved.

⇑ Corresponding authors.E-mail addresses: jaimee.ramirez@upr.edu (J.E. Ramirez-Vick), enrique.

melendez@upr.edu (E. Meléndez).

Wanda I. Pérez a, Yarelys Soto b, Jaime E. Ramirez-Vick c,⇑, Enrique Meléndez a,⇑a Department of Chemistry, University of Puerto Rico, PO Box 9019, Mayagüez, PR 00681, United Statesb Industrial Biotechnology Program, University of Puerto Rico, PO Box 9019, Mayagüez, PR 00681, United Statesc Engineering Science and Materials Department, University of Puerto Rico, PO Box 9019, Mayagüez, PR 00681, United States

a r t i c l e i n f o

Article history:Received 18 February 2015Received in revised form 7 May 2015Accepted 27 May 2015Available online 27 May 2015

Keywords:Gold nanostructured sensorBeta protein 1BiosensorDNA sensor

a b s t r a c t

Beta protein 1 (BP1) is a homeobox protein expressed in 80% of breast cancer cells in either estrogenreceptor (ER) positive or ER negative breast cancer. However, it is barely detectable in normal breast tis-sues. In this project we present an electrochemical DNA nanostructured gold biosensor for detection ofBP1. The gold sensor is first electrochemically nanostructured in 0.5 M sulfuric acid to reach superior con-ductivity, larger surface area, and higher stability. Nanostructured gold surface was characterized byatomic force microscopy (AFM) and scanning electron microscopy (SEM). The nanostructured gold sensoris then modified with double-stranded (ds) DNA mapping the genomic sequence that contains the bind-ing site for BP1. A redox-active probe (methylene blue) was intercalated in dsDNA to monitor the bindingevent 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 besensitive, selective, stable, and reusable allowing for its potential clinical use.

� 2015 Elsevier B.V. All rights reserved.

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 bycancer. The most common diagnosed cancer in woman is breastcancer. It is also the second leading cause of cancer death amongwomen around the world [1]. During 2014, around 29% of thenew cases of cancer in women were diagnosed to be breast cancer[2]. According to the American Cancer Society, over 231,840 newcases of invasive breast cancer are expected to be diagnosed inthe United States in 2015. Additionally, 60,290 new cases of carci-noma in situ breast cancer, which is the earliest form of breast can-cer and it is also non-invasive, will be diagnosed in 2015.Unfortunately, about 40,290 diagnosed women will die of thiscondition.

According to a statistical study, about 90% of the breast cancerpatients diagnosed with carcinoma in stage 0 will survive, empha-sizing the importance of early detection [3]. During the past decaderesearchers have driven their attention to search biomarkers forthe early detection of breast cancer. In 2002, Sidney et al. reportsa strong correlation between the expression of beta protein 1

(BP1) and ER in breast cancer cells [4]. BP1 is a member of thehomeobox gene superfamily of transcription factors (TFs), whichare expressed during embryonic development and are subse-quently re-expressed by several different types of cancers [5].This protein consists of a 60-amino acid helix-turn-helix structurewith three alpha helices connected by short loop regions in whichone of these interacts directly with DNA [6]. A remarkable featureof BP1 is that it is expressed in 100% of ER� tumors and in 73% ofER+ tumors [4–7], with the former being more invasive and with atypical poor prognosis when compared with the latter. Meanwhile,lack of BP1 expression was found in ER� normal breast cells andlow-level expression in ER+ normal breast cells. In addition, fre-quent expression of BP1 was also found in all tumor grades. Allthese properties make BP1 an ideal biomarker for early detectionof these types of breast cancer [4–7]. Recently researchers havedriven their attention in the development of biosensors for breastcancer detection using different biomarkers [8–10]. One of themost promising biomarkers for breast cancer detection is theabovementioned BP1 TF. The detection of breast cancer-relatedTFs, such as BP1, can be done using gel electrophoretic mobilityshift assays [11] and DNase footprinting assays [12], which arebased on migration retardation of protein–DNA complexes throughgel electrophoresis. However, these methods require long andlabor-intensive protocols and the use of an analytical laboratorywith specialized personnel, making them neither feasible for

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routine biomarker determination nor applicable for point-of-care(POC) testing. Thus, the challenge on the detection of TFs is thedevelopment of sensitive and simple methods which allow quan-tification of them [13].

Electrochemical assays based on DNA-mediated charge transportat microelectrodes offer an alternative approach which can be sensi-tive, specific, fast, simple, and low-cost, making them suitable forPOC diagnostics and multiplexed platforms [14]. This type ofapproach has been useful in assays to detect mutations and lesionsas well as for the detection of protein binding at self-assembledDNA monolayers. However, its use for the detection of proteinbinding utilizing double-stranded (ds) DNA monolayers has beenscarce and can be divided into electrodes modified with a hairpinoligonucleotide with conjugated redox-active probes [15–21], orwith dsDNA that bends upon protein binding [22–24]. For the firsttype, TF binding stabilizes an oligonucleotide conformation whichbrings the redox-active prove close to the electrode leading to a largeincrease in Faradaic current signal. The second type capitalizes onthe fact that upon binding some TFs bend the immobilized dsDNAprobe restricting the flow of electrons. Both alternatives functionas a switch, in which electrons are either transported or not to theelectrode.

Most nano-gold (NG) sensors modified with single-strandedDNA are designed to characterize the hybridization between thetwo complimentary strands which allows electrochemical chargetransport [14,20,24–27]. A series of conjugated charge donors oracceptors are employed as redox active probes interacting by p-stacked base pairs in dsDNA to monitor the electrochemical bind-ing event [21,24,28–34]. In the case of TFs, the electrodes are mod-ified with dsDNA containing the sequence which allows binding ofa specific TF. Thus, this work proposes for the first time (1) thedetection of a clinically-relevant transcription factor biomarker,(2) the electrochemical detection of a transcription factor using adouble-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%, andtris(hydroxymethyl)-aminomethane 99.9% were obtained fromSigma–Aldrich. The DNA sequences containing the BP1 bindingmotif ATATATATG were as follows: Forward 50-TTAAAAAGATATA-TATATATGTTTTTCTAATGT-30, (modified with 50end thiol C6 linker)and reverse 50-ACATTAGAAAAACATATATATATATCTTTT-TAA-30

[6,7]. The following sequences without the BP1 binding sequencewhere used as the dsDNA negative control (Forward: 50-TCTTAG-AGGGAGGGCTGAGGGTTTGAAGTCCAA-CTCCTAAGCC-30; Reverse:50-AGAATCTCCCTCCCGACTCCCAAACTTCAGGTTG-AGGATTCGG-30)[6]. All DNA probes were obtained from Eurofins Genomics and theBP1 protein was obtained from Gen Way and used as received.Deionized water (18.2 MX) was used for all experiments.

2.2. Equipment

CP-II VEECO atomic force microscope (AFM) with scanningprobe microscopy and lateral force microscopy and nanomanipula-tion capabilities with 100_100 lm scanning area capacity is used tocharacterize the surface morphology. Scanning electron micro-scope (SEM) is used to characterize the surface morphology ofthe nanostructured electrode using a Field Emission ScanningElectron Microscope Supra 35 (Zeiss). The equipment allows anal-ysis of features in the nanometer range using low accelerating volt-age (3 kV). Electrochemical experiments were carried on an

Epsilon Potentiostat/Galvanostat (Bioanalytical Systems, Inc.)using a specially designed three-electrode electrochemical cellhaving an analytical gold sensor as working electrode (0.02 cm2),a platinum wire as counter (auxiliary) and Ag/AgCl paste coveredwire as reference electrode. SRS Quartz crystal microbalance(QCM) Ti/Au, 5 MHz electrode used for surface characterizationwas purchased from Stanford Research Systems.

2.3. Synthesis and characterization of gold nanoparticles

The gold electrode surface, before nanostructured, was polishedprior to use with 0.5 lm alumina for 3 min and then cleaned in anultrasonic bath with ethanol and deionized water for 5 min. Thegold electrode is then electrochemically cleaned by cyclic voltam-metry (CV) in 0.5 M sulfuric acid at 100 mV s�1 scan rate, between�0.35 and 1.5 V until no changes in voltammograms. After electro-chemical cleaning of the gold electrode, the thick hydrous goldoxide layer accumulated on the gold electrode is removed in0.5 M sulfuric acid by using repetitive square-wave voltammetry(SWV), with a potential range between�0.8 V and 2.5 V (versus sil-ver/silver chloride electrode) at 2000 Hz for 5 min. In the followingstep, the potential is held at�0.8 V until complete electro-reductionof the hydrous gold oxide layer is accomplished. The NG surfacewas characterized by cyclic voltammetry in 0.5 H2SO4 between�0.35 and 1.5 V (versus silver/silver chloride electrode) at100 mV s�1. After synthesis of gold nanoparticles, the gold reduc-tion peak current at 0.942 V increases considerably. SEM was usedto characterize the surface morphology of the nanostructuredelectrode. In addition, atomic force microscopy (AFM) was used tomeasure the surface topography.

2.4. Immobilization of dsDNA onto gold nanostructured sensor

The BP1 binding sequence oligonucleotides were resuspendedin an annealing buffer consisting of 10 mM Tris, pH 7.5, 50 mMNaCl, 1 mM EDTA. Equimolar concentrations of the forward andreversed oligonucleotides were annealed at 94 �C for 4 min andthen cooled at room temperature for 40 min. The resultingdsDNA was self-assembled into the NG surface for 18–24 h. AfterdsDNA addition to the NG surface, it was exposed to a solutionof 1.0 mM mercaptohexanol (MCH) for 1 h (Scheme 1) to allowfor surface passivation [35]. The co-adsorption of MCH ontoDNA-modified gold surface removes the weakly, unspecificallyadsorbed nucleic acids by forming a dense MCH sublayer. DNA sen-sor was then immersed in a solution on 20 lM MB for 20 min tointercalate it as redox-active probe [14,24]. Characterization ofdsDNA and its binding to the NG surface were performed usingCV to monitor changes in MB redox cycle. The CV experimentswere carried out between �0.450 and 0 V at 100 mV s�1 in20 mM Tris, 100 mM NaCl, pH 7.5, at room temperature under aninert atmosphere. A three-compartment electrochemical cell isused with a Pt wire auxiliary electrode, silver/silver chloride elec-trode as reference, and a 0.02 cm2 NG working electrode.

2.5. Electrochemical characterization of DNA-binding event of BP1

The BP1 binding event was characterized by CV between �0.450and 0 V (vs. Ag/AgCl) in 20 mM Tris, 100 mM NaCl, 20 lM MB, pH7.5 at 100 mV s�1, using silver/silver chloride as reference, Pt wireas supporting electrode, NG/dsDNA/MCH as the working electrodeand MB as redox-active probe. A stock solution of BP1 was prepareddissolving 0.01 mg of the protein in buffer. For the binding eventcharacterization, the redox cycle current of MB was monitored withdifferent dilutions of the BP1 stock solution (5.30–33.78 nM). TheCVs were record after 2 min exposure time with the desired concen-tration. The NG/dsDNA/MCH was then incubated for two weeks and

Scheme 1. Schematic representation of NG/dsDNA/MCH sensor construction.

W.I. Pérez et al. / Journal of Electroanalytical Chemistry 751 (2015) 49–56 51

regeneration experiments were performed without loss of signal.Regeneration of the NG/dsDNA/MCH sensor was performed by asimple rinse in deionized water. Square wavevoltammetry was alsoperformed after regeneration to confirm the BP1 binding event. Thiswas carried out in 20 mM Tris, 100 mM NaCl, 20 lM MB, pH 7.5from �0.6 to 0 V (vs. Ag/AgCl) at 600 Hz with an amplitude of25 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.

Fig. 1. Cyclic voltammograms of gold clean (black) and gold nanostructured sensor(red) performed in 0.5 M H2SO4 at a scan rate of 100 mV s�1. Methylene blue (MB) isthe probe. (For interpretation of the references to color in this figure legend, thereader is referred to the web version of this article.)

3. Results and discussion

3.1. Characterization of gold nanostructures

The gold electrode surface was nanostructured with a modifiedprocedure [36] using SWV in 0.5 M H2SO4 at 2000 Hz for five min-utes followed by electro-reduction at a fixed potential of �800 V.Fig. 1 shows the cyclic voltammograms in 0.5 M H2SO4 at100 mV s�1 of the gold electrode before electroreduction (black)and after electroreduction (red). The cyclic voltammogram forthe clean gold electrode show two oxidation peaks related to theformation of gold oxides at 1.09 and 1.27 V and a reduction peakat 0.95 V [30]. At the end of the electroreduction process, the cur-rent of the reduction peak increases to 18 lA over the untreatedgold electrode. The increase in reduction current of the gold reduc-tion peak indicates the formation of gold nanostructures whichincrease both, the surface area and conductivity [30].

The NG surface topography was measured by AFM, and isshown in Fig. 2. The gold surface topography before electroreduc-tion (Fig. 2A) shows a smooth surface topography with an average

roughness of 2.089 nm while the NG electrode surface (Fig. 2B) hasa rough appearance with an average roughness of 7.202 nm. Theincrease in average roughness confirms the changes in surface areadue to the formation of gold nanostructures. SEM of the clean goldelectrode (Fig. 2C) shows gold particles with a uniform size, whileNG images (Fig. 2D) shows small grains and nanoporous particlesas reported in literature [30].

Fig. 2. (A) AFM image of clean gold electrode, (B) AFM image of gold nanostructured electrode, (C) SEM image of clean gold electrode, and (D) SEM image of NG electrode.

52 W.I. Pérez et al. / Journal of Electroanalytical Chemistry 751 (2015) 49–56

3.2. Immobilization of dsDNA onto gold nanostructured sensor

The complementary oligonucleotides with the BP1 bindingsequence were first annealed by mixing them in equimolar con-centrations on a thermostatic bath at 95 �C for 4 min and cooledto room temperature for 40 min. Subsequently, the annealeddsDNA was exposed to the NG electrode, forming a self-assembledmonolayer through the interaction between the thiol linker mole-cule and the NG surface [24,28]. The presence of the dsDNAattached to the gold electrode was confirmed by CV of the redox-active probe intercalated into the dsDNA (Fig. 3). Fig. 3 A showsthe CV of MB using the NG/MCH electrode (blue, withoutdsDNA). The MB has an anodic peak in �0.188 V. When it is inter-calated onto the dsDNA attached to the NG/dsDNA/MCH electrode,the anodic peak potential is shifted to�0.253 V (Fig. 3A, pink). Theanodic peak potential shift of the MB is due to its intercalation intodsDNA. An increase in anodic peak current was also observedbecause the DNA is facilitating the charge transport.

Hybridization of the complementary strands to form the dsDNAwas verified to detect any mismatch in the double strand forma-tion. The mismatch can be detected through reduction of MB inpresence of potassium ferricyanide, K3Fe(CN)6. The current flowsthrough the p-stacked base pairs in the dsDNA transferring 1 elec-tron to the intercalated MB. The MB is then reduced to produceleucomethylene blue (LB) which reduces ferricyanide to reoxidateMB [37,38]. The electro-reduction cycle resulted in amplification ofthe MB signal (Fig. 3B). Meanwhile, in the case that the dsDNA con-tains any mismatched base pairs, the MB reduction to LB does notoccur resulting in an oxidation current decrease of the MB [14,25].

3.3. Characterization of the binding event of beta protein 1 (BP1) todsDNA

Characterization of the binding event of BP1 to the dsDNA wasdone by CV and SWV monitoring of the MB redox-active probe[29,31,39,40]. The MB was intercalated onto a dsDNA base pairsand the TF BP1 to the consensus binding sequence ATATATATG[6]. A negative control sequence, a dsDNA sequence without con-sensus motif, was also used to verify the selectivity. A schematicillustration of the binding event of BP1 to the dsDNA is shown inScheme 1. After nanostructuring the gold electrode, the dsDNA islinked to the NG surface and passivated with MCH to preventdirect interactions of the protein with the gold surface. The MB isthen intercalated at the top of the dsDNA by incubation of theNG/dsDNA/MCH sensor. The MB intercalated NG/dsDNA/MCH sen-sor is then monitored by CV as a function of BP1 concentration.When BP1 binds the dsDNA, the charge transport through theDNA to the MB is interrupted attenuating the DNA-mediatedreduction of the redox-active probe [24].

Fig. 4 shows the cyclic voltammograms of MB intercalated on thedsDNA bound to the NG surface as a function of BP1 concentration.The anodic peak current of MB decreases significantly after the addi-tion of BP1. The anodic peak current of MB continues to graduallydecrease with BP1 concentration. The percent response after thefirst addition of 5.30 nM BP1 was 16% reduction in the anodic cur-rent of MB. Binding of the BP1 TF to its target site, bends the DNAduplex and perturbs the base pair stack, attenuating the DNA-medi-ated reduction of the redox probe. The loss of signal becomes greaterwith concentration reaching a saturation point near 30 nM BP1.

Fig. 3. Cyclic voltammograms of; (A) NG/MCH sensor in 20 mM tris, 100 mM NaCl, 20 lM MB (blue) and NG/dsDNA/MCH with MB intercalated into dsDNA (pink). (B) CV ofNG/dsDNA/MCH in 20 mM tris, 100 mM NaCl, 20 lM MB (pink) and with 0.1 M K2Fe(CN)6. MCH = 6-mercapto-1-hexanol. (For interpretation of the references to color in thisfigure legend, the reader is referred to the web version of this article.)

Fig. 4. Cyclic voltammograms of 20 lM MB, in 20 mM tris, 100 mM NaCl at100 mV s�1 in presence of different concentrations of BP1 using the NG/dsDNA/MCHas working electrode and MB as redox-active probe. Fig. 5. Calibration curve illustrating the percent of current change vs. BP1

concentration (nM).

W.I. Pérez et al. / Journal of Electroanalytical Chemistry 751 (2015) 49–56 53

3.4. Analytical performance of the biosensor

The analytical performance of the NG/dsDNA/MCH sensor wasevaluated using three independent measurements using the sameelectrode. The NG/dsDNA/MCH sensor was incubated for 2 min onthe desired concentration before the CV was recorded. A significantattenuation of the MB current response was observed while theBP1 concentration increases. The percent of current attenuationfollows a good linear relation with BP1 concentrations (Fig. 5), witha linear range from 5.3 to 23.6 nM with a correlation coefficient of0.998 and a limit of detection of 1.2 nM (based on a linear regres-sion). There is reproducibility higher than 90% on independentmeasurements. The NG/dsDNA/MCH biosensor also demonstratesto be stable and reusable with reproducible results. Comparingthe NG/dsDNA/MCH with other biosensors (Table 1), our biosensorbased on protein interaction is specific for breast cancer cells, iden-tifying the BP1 protein, does not require pre-conditioning, can takeinstant measurements and is reusable.

3.5. Regeneration of the fabricated DNA sensor

The NG/dsDNA/MCH is a simple and sensitive biosensor for theearly detection of breast cancer through the recognition of the BP1

TF. This TF biosensor is not only simple to use but it can be easilyregenerated through a 30 s rinse with deionized water, removingall the protein bound to the dsDNA, allowing the reconstitutionof the MB signal [21,24]. Additional measurements of BP1 bindingwere realized by SWV to prove the stability of the TF biosensor.Selection of the square wave frequency was performed to deter-mine the MB redox signal at different frequencies after regenera-tion. The frequency which demonstrated the strongest MB signalwas 600 Hz (Fig. 6). Different SWV experiments were carried outas a function of BP1 concentration. A rapid response for the lowestconcentration of BP1 was observed with a signal of about 16% lessthan the MB initial signal. Subsequent attenuation response withBP1 concentration is shown in Fig. 7.

Table 1Comparison 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 0.2–1.4 pg/mL HER-3 is present in a wide range to tumors cell lines [41]Incubation of 1 h with HER-3 sampleSingle use electrode

SAM-assisted SiNW Era 1 pM to 1 fM Era is present in breast cancer cell lines and non-tumorigenic breast cells [42]Incubation of 1 h with cells sampleSingle use electrode

Avastin-MGO-Au VEGF 31.25–2000 pg/mL VEGF is present in many human cancers [43]Incubation of 30 min with sampleReusable

VEGF-R1 10–70 pg/mL VEGF is secreted by tumor cells [44]Incubation of 1 h with VEGF sampleSingle use electrode

NG/dsDNA/MCH BP1 5.3–23.6 nM BP1 is expressed in Era+ and Era� breast cancer cells This workInstant sample measurementsReusable

HEGF-3 = Human epidermal growth factor-3, SAM = self-assembled monolayer, SiNW = silicon nanowire, ERE = estrogen receptor element, Era = estrogen receptor alphaMGO = magnetic graphene oxide, VEGF-R1 = vascular endothelial growth factor receptor 1.

Fig. 7. Square wave voltammetry of MB in NG/dsDNA/MCH sensor after regener-ation 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.

Fig. 6. Square wave voltammetry of MB in NG/dsDNA/MCH sensor after regener-ation at different frequencies. SWV was carried out in 20 mM tris, 100 mM NaClfrom 60 Hz to 600 Hz with amplitude of 25 mV and a step size of 1 mV.

54 W.I. Pérez et al. / Journal of Electroanalytical Chemistry 751 (2015) 49–56

3.6. Selectivity of NG/dsDNA/MCH biosensor

SWV was also used to assess the selectivity of theNG/dsDNA/MCH biosensor. A sequence of dsDNA without the BP1

binding motif was used as a negative control [6]. Fig. 8 showsthe SWV of MB in the TF biosensor modified using the negativecontrol sequence. The MB intercalated onto the negative controlsequence (black) shows a higher current reduction than the MBintercalated into the dsDNA BP1 binding motif (blue). However,upon addition of BP1 only a 3% of signal attenuation was observedwith the negative control sequence (red) compared to a 16% withthe genomic sequence containing the BP1 binding motif at thesame TF concentration (cyan). No significant changes in attenua-tion response were observed at higher BP1 concentrations. Theseresults suggest that the NG/dsDNA/MCH biosensor has a goodselectivity due to the specific sequence for the BP1 binding site.In addition, the NG/dsDNA/MCH biosensor remained stable aftertwo weeks of its preparation.

4. Concluding remarks

A prototype NG/dsDNA/MCH biosensor using MB as redox-ac-tive probe was fabricated in a simple manner affording a sensitiveBP1 biosensor. The NG/dsDNA/MCH sensor demonstrates to haverapid 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 haveBP1 levels higher than 39 ng/mL [45]. Thus, our biosensor has thepotential to be a tool for breast cancer detection. Also, it has beendemonstrated that NG/dsDNA/MCH biosensor is stable and can beregenerated. Regeneration of the sensor provides the advantage ofa reusable sensor. Through the selective detection of BP1 at lowconcentrations, the NG/dsDNA/MCH biosensor is suitable for earlydetection of breast cancer. Although it has not been tested on realblood samples (complex matrix), the nanostructured gold dsDNAsensor presented here has shown to be sensitive, selective, stable,and reusable allowing its potential use for POS testing. This

Fig. 8. Square wave voltammetry of MB in NG/dsDNA/MCH sensor modified withthe negative control DNA (black and red) and NG/dsDNA/MCH with the genomicsequence (blue and cyan). SWV was carried out in 20 mM tris, 100 mM NaCl at600 Hz with amplitude of 25 mV and a step size of 1 mV. (For interpretation of thereferences to color in this figure legend, the reader is referred to the web version ofthis article.)

W.I. Pérez et al. / Journal of Electroanalytical Chemistry 751 (2015) 49–56 55

biosensor is a proof-of-concept that NG/dsDNA/MCH biosensor withthe correct oligonucleotide sequence to recognize BP1 can be fabri-cated and has the potential to become a simple tool for breast can-cer detection.

Acknowledgements

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

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