Parallel Detection of Nucleic Acids Using an Electronic Chip Leyla Soleymani,1 Zhichao Fang,2 Shana O. Kelley2,3*, and Edward H. Sargent,1* 1 Department of Electrical and Computer Engineering, Faculty of Engineering, 2Division of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, and 3Department of Biochemistry, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada *e-mail:
[email protected];
[email protected] research laboratory and in clinical settings. Gene expression profiling based on electronic readout has long been cited as a promising approach that would enable a new family of chip-based devices with appropriate cost and sensitivity for direct medical testing. This would allow automated prostate cancer screening, and could improve the reliability of the diagnosis by increasing the number of samples analyzed. In this work, we designed and fabricated an electronic chip featuring an array of nano-structured microelectrodes. We then optimized an electrochemical strategy developed by our group [5] to readout the signals generated by the device in the presence of cancer related biomolecules.
Abstract Prostate cancer is the most commonly diagnosed cancer among North American men. The recent discovery of altered genes that play an underlying role in prostate cancer development has made the sequence-specific detection of nucleic acids especially important. Multiplexed detection using electronic readout would permit sensitive, reliable, fast and inexpensive identification of molecular biomarkers in clinical settings. This would improve early diagnosis, and would provide a route towards prognosis and new therapeutic possibilities. Here we report for the first time the development of a novel chip-based, addressable array of nano-textured microelectrodes (NMEs) that can be used in automated detection of a panel of prostate cancer-related gene fusions in clinical samples. The attomolar sensitivity along with the unique multiplexing capability of this system make it superior to the previously reported electrochemical nucleic acids sensors.
2. Device design and fabrication Electrochemical techniques have previously provided appropriate means for translating molecular recognition events into electrical signals.[6] These techniques are reported to have the sensitivity to detect as low as 80 copies of target nucleic acids molecules. [7] In addition, it has been cited that the use of threedimensional nanostructures can further improve the sensitivity of such techniques. [8] Although previous electrochemical techniques have shown the appropriate sensitivity for biomarker analysis, they are often serial techniques and rely on several labor intensive steps. Considering these findings along with the need to analyze several clinical samples in parallel, we developed an array based electronic chip featuring three-dimensional Nano-structured MicroElectrodes (NMEs). In our approach we used cost-effective conventional photolithography to position and address our electrodes; and then used electrodeposition to bring about, with a high degree of reproducibility, the nanostructuring of these microelectrodes. Figure 1 shows our 8-fold multiplexed passive chip. On a silicon substrate, a ~ 350 nm thick gold layer was patterned to connect eight 5-µm-wide Au wires to large
1. Introduction In recent years, there has been tremendous amount of research in identification of biomolecules specifically present in tumor tissue while absent in benign cases.[1] One of such biomolecules is the specific mRNA (messenger ribonucleic acid) responsible for the development of prostate cancer. This RNA molecule is formed in a majority of cases as a result of fusion of two previously separate genes. Since these gene fusions occur in early stages of prostate cancer they can be used in the development of an early diagnostics test. [2] Currently, Polymerase Chain Reaction (PCR) [3] and microarray techniques [4] are the main tools for gene expression profiling and have helped researchers in the discovery of a number of disease-related biomarkers. These tools lack the practicality and cost-effectiveness to be used outside of
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In order to use this chip-based platform for biosensing, we need to modify NMEs to selectively capture nucleic acids relevant to prostate cancer. This is performed by designing appropriate nucleic acids probes. These probes are made of Peptide Nucleic Acids (PNA). PNA is a man-made analog of DNA where the negative sugar phosphate backbone is replaced with a neutral peptide backbone. [9] The sequence of PNA is designed complementary to the naturally occurring prostate cancer related mRNA in cells. This PNA molecule is then modified with a sulfur terminating chemical group that specifically binds to noble metals. [10] Prior to every sensing experiment, solutions of probes are deposited on the electrodes for a period of 24 hours in order to cover electrodes with a monolayer of probe molecules.
metal pads for connection to off-chip instrumentation. A pinhole-free insulating SiO2 layer was deposited and patterned to create ~ 500 nm openings at the end of Au wires. This microfabricated chip formed the starting point for programmable bottom-up fabrication of NMEs. Electrodeposition was used to place individual Pd NMEs into half-micron openings of the insulating mask. This provided a wide range of morphologies and
Figure 3.Schematics showing NME modification using nucleic acids.
4. Electronic readout
Figure 1. Schematics of the multiplexed chip. Top: overview of microfabricated chip, deposition using electrodeposition.
bottom:
NME
Electronic readout is performed by reporting the amount of electrostatic charge present on the electrodes. Each individual building block of a DNA molecule is called a nucleotide and has a negative charge associated with it. This means NMEs modified with double stranded DNA will contain more negative charge compared to single stranded uncharged PNA. This change in electrode’s overall charge is measured by an electrochemical technique called Cyclic Voltammetry. In this technique, a waveform shown in Figure 4a is applied while measuring the current passing through the electrode positioned in an electrochemical cell. This current is associated with the reduction of redox-active species. In case of DNA hybridization detection, redox species are carefully chosen to distinguish between single stranded and double stranded nucleic acids due to electrostatic interactions. Our approach relies on the electron acceptor Ru(NH3)63+, which is electrostatically attracted to electrode surfaces at levels correlated with the amount of bound nucleic acids. In order to amplify the electrochemical signal to make possible its detection using inexpensive instrumentation, Fe(CN)63, another redox compound is included during electrochemical readout. Role of Fe(CN)63- is to
growth rates depending on metal salt concentration, type of supporting electrolyte, electrodeposition potential, and plating duration. (Figure 2)
Figure 2. Scanning Electron Microscopy (SEM) images of different NMEs. a) zoomed out SEMs b) zoomed in SMEs of left: smooth hemispherical NME, middle: rough hemispherical NME, right: fractal NME.
3. Electrode modification using nucleic acids probes
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regenerate Ru(III) substrate for multiple redox cycles. [5] This reporter method is label-free and does not require the sample to be processed in any way – an important feature for a practical device. Figure 4b shows the cyclic voltammetry scans of a PNA modified electrode before and after hybridization with a 100 aM complementary target.
calculated for each concentration at each NME, and averaged between different trials. These are plotted in Figure 5 for three different NMEs. Detection limit for each structure is calculated by subtracting ∆I of a noncomplementary control at a high concentration (100fM) from all other ∆I’s. Detection limit is called as the first concentration at which the average ∆I is 3 times higher than the standard deviation at that concentration. This analysis shows the detection limit of fractal NME to be 10 aM and the lowest of all three structures. The rough hemispherical NME has a detection limit of 10 fM, and the smooth hemispherical NME has a detection limit of 100 fM. The role of nanostructuring is readily apparent in these experiments, which are the first to directly analyze the importance of this parameter in biosensing applications. We hypothesize that the more open structure of nanostructured sensing elements promotes better accessibility of the probe and hence more efficient complexation with the target sequence. The 10 aM sensitivity we observe here with our optimized NMEs and electrochemical reporter system is the lowest detection limit reported to date for a label- and PCR-free sensor; this corresponds to the detection of < 100 copies of the target sequence.
A
B
6. Dynamic range Although sensitivity of bioanalytical systems is extremely important in analyzing dilute clinical samples. In many applications, the quantity of biomarker present in cells sets the difference between healthy and diseased cells. [11] In order to address this issue we need a system with a large dynamic range. For this purpose, we take the lessons learnt from analyzing detection limits of different NMEs and make a complex multiplexed chip featuring NMEs with different degrees of nanostructuring. This is seen in the results of Figure 5. The signal changes of NMEs vary with concentrations of analytes in a log linear fashion. From this Figure, the fractal and rough hemispherical NMEs have a 2 decade log-linear range while the smooth hemispherical NME has 4 decades of log-linear dynamic range. Combining these structures on the same chip has improved the dynamic range of the whole device to 8 decades.
Figure 4. Cyclic voltammetry (CV) scans a. applied potential waveform b. CV scan of an NME before (dotted) and after (solid) hybridization with 100 aM of complementary target.
5. Detection limit In order to calculate the detection limit of each NME, we tested the electrodes using a series of complementary targets having different concentrations. These experiments are automated by software that calculates the difference between limiting reduction current before and after hybridization using the following equation.
∆I =
I ds − I ss I ss
Where Ids corresponds to the value of limiting current at -0.3 V after hybridization (solid curve in Figure 4b), and Iss corresponds to the current before hybridization (dotted line in Figure 4b). These changes of current are
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on the chip to thousands to increase the number of nucleic acids sequences detected in parallel.
9. References [1] J. A. Ludwig and J. N. Weinstein, "Biomarkers in Cancer Staging, Prognosis and Treatment Selection," Nat Rev Cancer, 2005, pp. 845-856. [2] S. A. Tomlins, D. R. Rhodes, S. Perner, S. M. Dhanasekaran, R. Mehra, X. Sun, S. Varambally, X. Cao, J. Tchinda, R. Kuefer, C. Lee, J. E. Montie, R. B. Shah, K. J. Pienta, M. A. Rubin and A. M. Chinnaiyan, "Recurrent Fusion of TMPRSS2 and ETS Transcription Factor Genes in Prostate Cancer," Science, 2005, pp. 644-648. [3] H.A. Erlich, Gelfand & R.K. Saiki, “Specific DNA amplification”, Nature ,1998, pp. 461 – 462. [4] R. Drmanac, S. Drmanac, Z. Strezoska, T. Paunesku, I. Labat, M Zeremski, J Snoddy, WK Funkhouser, B Koop, L Hood, “DNA sequence determination by hybridization: a strategy for efficient large-scale sequencing” Science, 1993, pp. 1649–1652, 1993 [5] M. A. Lapierre, M. O'Keefe, B. J. Taft and S. O. Kelley, "Electrocatalytic Detection of Pathogenic DNA Sequences and Antibiotic Resistance Markers," Anal. Chem., 2003, pp. 6327-6333. [6] E. Palecek, "Oscillographic Polarography of Highly Polymerized Deoxyribonucleic Acid," Nature, 1960, pp. 656657. [7] B. Munge, G. Liu, G. Collins and J. Wang, "Multiple Enzyme Layers on Carbon Nanotubes for Electrochemical Detection Down to 80 DNA Copies," Anal. Chem., 2005, pp. 4662-4666. [8] P. R. Nair and M. A. Alam, "Performance limits of nanobiosensors," Appl. Phys. Lett., 2006, pp. 233120-3. [9] M. Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M. Freier, D. A. Driver, R. H. Berg, S. K. Kim, B. Norden and P. E. Nielsen, "PNA hybridizes to complementary oligonucleotides obeying the Watson–Crick hydrogenbonding rules," Nature, 1993, pp. 566-568. [10] C. D. Bain and G. M. Whitesides, "Molecular-Level Control over Surface Order in Self-Assembled Monolayer Films of Thiols on Gold", Science,1988, pp. 62-63 [11] J. Lu, G. Getz, E. A. Miska, E. Alvarez-Saavedra, J. Lamb, D. Peck, A. Sweet-Cordero, B. L. Ebert, R. H. Mak, A. A. Ferrando, J. R. Downing, T. Jacks, H. R. Horvitz and T. R. Golub, "MicroRNA expression profiles classify human cancers," Nature, 2005, pp. 834-838.
Figure 5. Dynamic range and sensitivity of three different NMEs. Current changes resulting from hybridization with complementary targets are plotted at different concentrations. Each curve corresponds to a different NME. Scale bar is equivalent to 5 microns.
7. Conclusions In this paper, a new chip-based technique for parallel profiling of nucleic acids was developed. This chip can currently analyze 8 different nucleic acids sequences in parallel. It has a sensitivity of 10 aM, equivalent to
