Herein we report an electrochemical DNA biosensor for the rapid detection of sequence (5’ AAT GGA TTT ATC TGC TCT TCG 3’) specific for the breast cancer 1 (BRCA1) gene. The proposed electrochemical genosensor is based on short oligonucleotide DNA probe immobilized onto zinc oxide nanowires (ZnONWs) chemically synthesized onto gold electrode via hydrothermal technique. The morphology studies of the ZnONWs, performed by field emission scanning electron microscopy (FESEM), showed that the ZnO nanowires are uniform, highly dense and oriented perpendicularly to the substrate. Recognition event between the DNA probe and the target was investigated by differential pulse voltammetry (DPV) in 0.1 M acetate buffer solution (ABS), pH 7.00; as a result of the hybridization, an oxidation signal was observed at +0.8 V. The influences of pH, target concentration, and non-complimentary DNA on biosensor performance were examined. The proposed DNA biosensor has the ability to detect the target sequence in the range of concentration between 10.0 and 100.0 μM with a detection limit of 3.32 μM. The experimental results demonstrated that the prepared ZnONWs/Au electrodes are suitable platform for the immobilization of DNA.
Breast cancer, that affects mainly inner lining of milk ducts, is one of the major causes of death of our times. Distant metastases are regarded as the major reason of death, so an early stage diagnosis, and subsequently treatment, of the cancer is highly required [
Ultimately site specific sensing platforms are strongly needed for early detection of breast cancer gene during biopsy. This is due to the fact that the existing nucleic acid (DNA) detection techniques, as northern blotting, ribonuclease protection assays, reverse transcription-polymerase chain reaction (RT-PCR) and DNA sequencing have several limitations, including low sensitivity, poor selectivity, expensive and nonlinearity to the target strength [
Biosensor is a compact analytical device having a biological recognition element intimately integrated with a physio-chemical transducer. The three main components of a biosensor are: the biological recognition elements such as enzyme, DNA, antibody, and nucleic acid, the transducer that converts the biological recognition event into a quantifiable signal and finally the signal processing system. The five principle transducer classes are optical, thermometric, electrochemical, piezoelectric, and magnetic. Electrochemical transduction due to its better sensitivity, selectivity, reproducibility, and easy maintenance as well as low cost, has gained a lot of popularity in DNA biosensors [
The basic principle of DNA biosensor usually relies on the immobilization of single stranded oligonucleotide (ssDNA) probe on the surface of an electrode providing; in this way the specificity towards a specific target DNA. Recognition event, based on DNA hybridization, is then converted into readable signal by transducer.
The performance of an electrochemical genosensor relies heavily on the properties of the supporting materials; these should provide a good environment for DNA immobilization, without compromising its biological activity, and providing good transduction abilities [
Recently, reports on electrochemical genosensors for the detection of breast cancer genes and cells have been reported [
Electrochemical measurements were performed at room temperature using AUTOLAB-PGSTAT 302N (Echo Chemie, The Netherlands). The three electrodes electrochemical systems consisted of: ZnONWs/Au as working electrode, a platinum wire as auxiliary electrode, and an Ag/AgCl as reference electrode. The DPV were performed in 15 mL of 0.1 M acetate buffer solution (ABS) (pH 7.00) from 0.4 V to 1.16 V with a modulation amplitude = 0.025 V, an interval time (t1) = 0.5 s, modulation time (t2) = 0.05 s, step potential = 0.005 V and a scan rate of 0.01 Vs−1. The acetate buffer solution was purged with nitrogen gas for 20 min prior to each experiment. The ZnONWs/Au electrode surface was characterized by field emission scanning electron microscope (FESEM).
Zinc nitrate hexahydrate, Zn (NO3)2·6H2O, hexamethylenetetramine, C6H12N4, and zinc acetate dehydrate, Zn (CH3COO)2∙2H2O were purchased from Sigma Aldrich, Germany. The 21-base pair single stranded DNAs were purchased from First BASE (IDT Inc, USA). The sequences of the different oligonucleotide are as follows (underlined are the mismatched bases):
Probe DNA: 5’ AAT GGA TTT ATC TGC TCT TCG 3’
Target DNA: 5’ CGA AGA GCA GAT AAA TCC ATT 3’
Three-base mismatch: 5’ CGA AGA GGA GAA AAA TCG ATT 3’; 5’ CAA AGA GCA GAT AGA TCC GTT 3’
The oligonucleotide stock solutions (100 µM) were prepared with deionized water and stored at 4˚C when not in use. Acetate buffer solution (ABS) of 0.1M concentration with different pH values were prepared by mixing different volumes of 0.1 M acetic acid and sodium acetate with deionized water.
The growth of ZnO nanowires was performed by hydrothermal method. Firstly, a gold electrode/substrate on silicon wafer purchased from Sigma Aldrich, Stockholm, Sweden was cleaned with isopropanol, washed with deionized water and finally dried at room temperature. As first step of the synthesis, a seed layer of zinc acetate dihydrate was prepared by spin coated for three times at 2500 r.p.m for 30 s onto the gold substrate. Following spin coating the substrate was annealed at 120˚C for 10 -20 mins. The seed particles containing electrodes were affixed onto a Teflon sample holder and then immersed in the growth solution of (0.075 M) zinc nitrate hexahydrate and (0.075 M) hexamethylenetetramine at 98˚C for 9 hours. After the growth of ZnO is completed, the grown nanostructures were washed with deionized water in order to remove eventual residual particles. Finally the ZnONWs/Au electrodes were left to dry at room temperature.
40 µL containing 80 µM of ssDNA molecules (probe) were drop casted onto the ZnONWs/Au electrode. The ssDNA molecules were left for immobilization onto ZnO NWs/Au electrode for 2 hours. Following the prepared electrodes were rinsed with ABS for 5 s to remove the unbound probes. The resulting electrode was labeled as ssDNA/ZnONWs/Au.
Hybridization assay was performed by spotting onto the sensor surface 40 µL of a solution containing the desired concentration, 80 µM of the complementary ssDNA, mismatches ssDNA and non-complementary ssDNA respectively. The hybridization was left to take place for 30 min.; following the electrode was then rinsed with ABS for 5 s to remove the non-hybridized targets ssDNA.
In
FESEM image of grown zinc oxide nanowires (ZnONWs) at the surface of gold electrode (A), and immobilized ssDNA onto the surface ZnONWs modified gold electrode (B)
The hybridization of the immobilized ssDNA probe with the complementary ssDNA (target) was studied by differential pulse voltammetry.
Differential pulse voltamograms (DPVs) that were obtained from immobilization of 80 µM ssDNA onto the surface of ZnONWs modified gold electrode after the probes were exposed to 100µM complimentary or target DNA (a), 3 mismatches or non-complimentary ssDNA (b) and (d), without ssDNA immobilization (c), for bare gold electrode (e) and ZnO NWs modified gold electrode-ssDNA (f). The DPVs were measured at a potential scanned between 0.4 to 1.16 V with a modulation amplitude = 0.025 V, interval time (t1) = 0.5 s, modulation time (t2) = 0.05 s and step potential = 0.005 V in 0.1 M acetate buffer solution at pH 7
The ZnONWs modified gold electrode revealed to be an excellent electrode substrate for the development of a sensitive DNA biosensor for the detection of breast cancer gene BRCA1. The FESEM image showed that nanostructure has been successfully grown at the surface of the gold electrode with an average diameter between 450 - 550 nm and with a good orientation. Furthermore we demonstrated that direct electrochemistry of DNA via the use of DPV measurement, was suitable for the selective detection of short DNA sequence associated with the BRCA1 gene, when DNA probes were chemisorbed onto the ZnONWs/Au surface. Presence of complementary DNA sequence resulted in a well-defined oxidation peak at ca. 0.8 V. The optimized pH condition for DNA biosensor operation was at pH 7. The response of the developed DNA biosensor was linear within complimentary target concentrations in between of 10 - 100 µM. In addition, the LOD was obtained on the li-
Current produced from DNA hybridization of 80 µM immobilized ssDNA on ZnONWs modified gold electrode to its target (BRCA1) in various concentrations (10 to 120 µM). The signal of DPV currents were measured at a potential scanned between 0.4 to 1.16 V with a modulation amplitude = 0.025 V, interval time (t1) = 0.5 s, modulation time (t2) = 0.05 s and step potential = 0.005 V in 0.1 M acetate buffer solutions (pH 7)
The pH effect on the biosensor response as indicated by the hybridization current with complementary DNA in 0.1 M ABS. The signal of DPV currents were measured at a potential scanned between 0.4 to 1.16 V with a modulation amplitude = 0.025 V, interval time (t1) = 0.5 s, modulation time (t2) = 0.05 s and step potential = 0.005 V in 0.1 M buffer solutions (pH 3-11)
Calibration plot presenting the changes of oxidation signals measured in the presence of DNA hybridization between 80 µM BRCA1 probe and various concentration levels of BRCA1 targets. The signal of DPV currents were measured at a potential scanned between 0.4 to 1.16 V with a modulation amplitude = 0.025 V, interval time (t1) = 0.5 s, modulation time (t2) = 0.05 s and step potential = 0.005 V in 0.1 M buffer solutions (pH 7)
near response of developed method, which was found to be 3.32 µM.
We would like to thank the Ministry of Higher Education Malaysia for the ERGS grant (600/RMI/st/ERGS/ 5/3/fst12/2011) and Universiti Teknologi MARA for financial support via postgraduate teaching assistant scheme (UPTA) to Nur Azimah Mansor for conducting this research.