Technical Note pubs.acs.org/ac
Flexible Microfabricated Film Sensors for the in Situ Quantum DotBased Voltammetric Detection of DNA Hybridization in Microwells Christos Kokkinos,*,† Anastasios Economou,‡ Thanasis Speliotis,⊥ Panagiota Petrou,§ and Sotirios Kakabakos§ †
Laboratory of Analytical Chemistry, Department of Chemistry, University of Ioannina, Ioannina, 45110, Greece Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Athens, 157 71, Greece ⊥ Institute of Materials Science, NCSR “Demokritos”, Aghia Paraskevi, Athens, 153 10, Greece § Immunoassay/Immunosensors Lab, Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety, NCSR “Demokritos”, Aghia Paraskevi, Athens, 153 10, Greece ‡
S Supporting Information *
ABSTRACT: A new flexible miniaturized integrated device was microfabricated for the in situ ultrasensitive voltammetric determination of DNA mutation in a microwell format, using quantum dots (QDs) labels. The integrated device consisted of thin Bi, Ag, and Pt films (serving as the working, reference, and counter electrode, respectively) deposited by sputtering on a flexible polyimide substrate. A DNA assay was employed in microwell format, where an immobilized complementary oligonucleotide probe hybridized with the biotinylated target oligonucleotide followed by reaction with streptavidin-conjugated PbS QDs. After the acidic dissolution of the QDs, the flexible sensor was rolled and inserted into the microwell and the Pb(II) released was determined in situ by anodic stripping voltammetry. Since the analysis took place directly in the microwell, the volume of the working solution was only 100 μL and the target DNA could be detected at a concentration down to 1.1 fmol L−1. The proposed flexible microdevice addresses the restrictions of conventional rigid electrodes while it provides a low cost integrated transducer for the ultrasensitive detection of important biomolecules.
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successful nontoxic electrodes so far,15,16 the use of electroplating is plagued by problems related to the formation, maintenance, and reproducibility of the Bi film.15,17 Moreover, existing ASV−QDs DNA assays involve the use of rigid “largesized” electrodes requiring the transfer of the dissolved QDs in a standard voltammetric cell and high dilution (in most cases about 50-fold) with supporting electrolyte.9−14 Due to the significant dilution of the samples, QD-loaded microbeads are used as labels in order to enhance the sensitivity and achieve limits of detection (LOD) at picomolar or femtomolar levels.11−13 In these labeling approaches, the surface of each nanosized microbead is loaded with a large number of QDs; hence, the quantity of QDs in every binding event is increased, and the signal is amplified. However, complex procedures are required for the preparation of these modified microbeads.11−13 In addition, the current ongoing transition from conventional rigid and brittle electrochemical transducers to foldable, rollable, thin, and stretchable sensors fabricated at a large scale has attracted considerable interest.18−21
ensitive, rapid, and cost-effective approaches for DNA detection have gained significance for the analysis of genetic and infectious diseases and for reliable forensic applications in molecular diagnostics.1 Although a number of DNA biosensing approaches have been reported, including surface plasmon resonance, radio frequency, and fluorescent assays,2−4 there has been a constant demand for small-sized, simple, and portable DNA detectors. Electrochemical sensors are readily integrated on a chip and can serve as transducers for the determination of nanoparticle tags, providing fast, simple, selective, and inexpensive labelbased bioassays.1,5−8 Especially, ultrasensitive DNA assays have been developed using quantum dots (QDs) as labels which are detected by anodic stripping voltammetry (ASV) after acidic dissolution. The high sensitivity of ASV−QDs detection is achieved not only by virtue of the voltammetric preconcentration step but also by the proper selection of transducer material and core type of QD nanoparticles.9−14 However, the major analytical applications based on the detection of DNA through ASV measurements of QD labels make use of bismuth- or toxic mercury-film electrodes (BiFEs and MFEs, respectively) prepared via electroplating protocols on various forms of rigid carbon substrates.10−14 Although BiFEs are the most © 2014 American Chemical Society
Received: October 9, 2014 Accepted: December 16, 2014 Published: December 16, 2014 853
DOI: 10.1021/ac503791j Anal. Chem. 2015, 87, 853−857
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Here, we present a novel flexible microfabricated bismuthbased integrated device to perform QD-based electrochemical bioassays directly in microwells. This microdevice features a Bi working electrode (WE), a Ag reference electrode (RE), and a Pt counter electrode (CE), all deposited by sputtering on a flexible polyimide substrate. The pattern of the electrodes was defined via plastic masks, thus avoiding complex photolithographic or etching procedures.9,17,21 The bioanalytical capability of the proposed device was demonstrated for the detection of the C634R mutation of the RET gene (related to Multiple Endocrine Neoplasia Type 2 (MEN2)).22 The assay was designed as a representative case study of the detection of biotin-labeled DNA resulting from polymerase chain reaction (PCR) amplification of the target DNA fragment using specific biotinylated primers, an approach widely used in DNA analysis.23−25 The biotinylated target oligonucleotide was subjected to hybridization with the capture complementary probe, immobilized in a microwell, and further reacted with streptavidin-modified PbS quantum dots. After the acidic dissolution of PbS−QDs, the flexible device was rolled and inserted into the microwell for the square wave ASV (SWASV) detection of the Pb(II) released (Scheme 1). Thanks to the in
Technical Note
EXPERIMENTAL SECTION
Reagents. All stock solutions were prepared using distilled water. Sodium bis(2-ethylhexyl) sulfosuccinate (AOT), 2-(Nmorpholino) ethanesulfonic acid (MES), N-(3-dimethylamminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), and Nhydroxysulfosuccinimid sodium salt (NHS) were purchased from Sigma−Aldrich. Bovine serum albumin (BSA) and streptavidin (STV) were obtained from Pierce, and the metal targets were 99.9% purity and purchased from Williams Advanced Materials. DNA sequences were obtained from VCB Biotech. The capture complementary probe was 5′-amineCGA CGA GCT GTG CCG CAC GGT-3′, and the target oligonucleotide was 5′-biotin-ACC GTG CGG CAC AGC TCG TCG-3′. The single-base mismatch and noncomplementary oligonucleotides were 5′-biotin-ACC GTG CGG CGC AGC TCG TCG-3′ and 5′-biotin-TGG CGT ACT CCA CGA TGA GGA-3′, respectively. Polystyrene microwells were from Nalge Nunc International. All the other reagents were obtained from Sigma−Aldrich. Apparatus. Electrochemical measurements were performed with a PGSTAT101 potentiostat (Metrohm Autolab). The NOVA 1.8 software (Metrohm Autolab) was used for baseline correction of the voltammograms. The sputtering system was the CV401 (Cooke Vacuum Products). The surface structure of the Bi deposit was studied with an atomic force microscope (AFM) (SPM SMENA, NTMDT Co.) and a X-ray diffractometer (XRD) (Siemens D500, Bruker GmbH) using the Ni-filtered Cu Kα radiation. A field emission scanning electron microscope (FESEM) (JEOL JSM-7401f) was used for the imaging of STV-conjugated PbS QDs. Fabrication of the Devices. The microfabrication procedure and photographs of the devices are presented in Figure 1A,B. Polyimide (Kapton) film (DuPont) was used as flexible substrate, thanks to its physical and chemical advantages.26 Three polyester sheets (Mac Dermid), each one of which featured a slot (4 mm in width) for the electrode, were
Scheme 1. Illustration of the Hybridization Assay and Voltammetric Detection of DNA
situ electrochemical detection directly in the microwells, the proposed methodology minimizes the sample dilution leading to the reduction of the LOD at femtomolar levels without using any further signal amplifiers (i.e., QD-loaded microbeads). Moreover, the application of a thin-film microelectronic approach for the fabrication of the devices addresses the limitations of electroplating procedures for the fabrication of metal electrodes, such as the need of a plating solution and the strong influence of the electroplating variables (i.e., the pH of the solution and the concentration of the metal cation) over the structure of the generated metallic film.9,17 Finally, the use of flexible foil (instead of the expensive, rigid, and brittle silicon and carbon substrates), combined with sputter deposition of metal films, enables the cost-effective production of sensors on a large scale.
Figure 1. (A) Steps for the fabrication of the Bi integrated sensor. (B) Photographs of the devices. (C) FESEM image of STV-conjugated PbS QDs. 854
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Analytical Chemistry
reproducible surface morphology, simplify the experimental procedure, and provide a wide scope for miniaturization and integration. These features were fully exploited in the present work in order to fabricate the flexible integrated electrochemical devices. The different fabrication parameters of the film sensors (the type of the flexible substrate material and the dimensions of the device as well as the size and thickness of the bismuth WE) were studied in connection to their intended use in microwells. In order to select the proper flexible substrate for the deposition of the metal electrodes, polyester and Kapton films were tested. The sputtered metals exhibited poor adhesion on the polyester sheets, irrespective of the addition of a 5 nm titanium buffer layer between the polyester substrate and the sputtered film. On the contrary, the metallic films adhered well to the Kapton substrates without the need of the titanium buffer layer. The size of the electrodes and of the entire device was selected considering the shape and the size of the microwells. The final shape of the device ensured that the electrodes did not overlap when the device was rolled and immersed into the microwell, while the device protruded sufficiently from the top of the microwell for easy connection of the electrodes to the potentiostat (Figure 1B). The thickness of the Bi WE was studied in the range of 200−800 nm (in 200 nm steps) by varying the sputter deposition time. Thin (200 nm) bismuth film resulted in lower reproducibility and sensor lifetime, while thick (600 and 800 nm) bismuth films led to wider Pb stripping peaks. The 400 nm thick bismuth WE exhibited the best analytical features. The surface characterization of the bismuth film WE was performed through AFM and XRD. The AFM image (Figure 2A) showed that the average surface roughness of the Bi layer was 133 nm. This rough structure is typical of low melting point metal films formed through sputtering, resulting in higher sensor active surface area.9 In the XRD spectrum of the Bi layer (Figure 2B), the numerous Bi-specific peaks suggested polycrystalline Bi deposition with rhombohedral orientation. No additional peaks were observed in the XRD spectrum, proving that no oxidation of Bi took place during the sputtering procedure. The polarization window of the flexible sputtered sputtered Bi WE was assessed through DC voltammetry in the range of −1.50 to 0.0 V (Figure 2C). The anodic potential limit was dictated by Bi oxidation which started at −0.27 V. On the other hand, the cathodic limit of the polarization range was set by the reduction of hydroxonium ions starting at −1.15 V. Therefore, the useful potential window ranged from −1.15 to −0.27 V. The flexible microfabricated Bi sensor was compared to the MFE and BiFE electroplated in situ on glassy carbon (Supporting Information) with regard to their respective SWASV response for Pb(II) (Figure 2D). The microfabricated Bi sensor exhibited an equally sharp and well-formed stripping peak for Pb with low background current contribution, while the response sensitivity toward Pb(II) at the microfabricated Bi electrode was comparable to that at the in situ electroplated BiFE and MFE. These results prove the suitability of the flexible sputtered Bi sensor to the stripping voltammetric determination of Pb(II). In order to increase the sensitivity of the ASV detection, the preconcentration time and the preconcentration potential were studied and selected using a solution of 5 pmol L−1 target oligonucleotide. The Pb stripping peak height showed a rectilinear dependence on the preconcentration time (Figure
used as plastic masks to define the area of the electrodes. The three electrodes were formed by successively sputtering each metal in the order of Ag (400 nm, constant current of 25 mA), Pt (400 nm, power of 30 W), and Bi (400 nm, constant current of 7 mA) on the Kapton film at the respective slots, each time using the appropriate polyester mask. The Kapton film was, then, cut into individual devices of 2 × 2 (length × width in cm) using a scissor. The electrodes were connected to the potentiostat with crocodile clips. Preparation of STV-Conjugated PbS QDs. The PbS QDs were synthesized and coupled with STV using a modified published procedure (Supporting Information).14 A FESEM image of the STV-conjugated PbS QDs is shown in Figure 1C. DNA Hybridization Assay. Microwells were coated with the capture probe by adding 100 μL of a 5 μg mL−1 solution of capture BSA−complementary oligonucleotide conjugate (Supporting Information) in phosphate buffered saline (PBS) (50 mmol L−1, pH 7.4) per microwell for 2 h at room temperature. After the immobilization of the capture probes, the microwells were washed twice with 300 μL of washing solution (PBS 50 mmol L−1, pH 7.4) to remove the excess of weakly absorbed probes, and the free microwell sites were blocked with BSA by adding 300 μL of a 10 mg mL−1 BSA solution in PBS per microwell for 1 h at room temperature. Then, the microwells were washed as described previously, and 100 μL of the target biotinylated oligonucleotide solution in 1× HEN (0.1 mol L−1 HEPES solution, 0.005 mol L−1 NaCl, 1 mmol L−1 EDTA, pH 8.0) was added per microwell and incubated for 30 min at room temperature under stirring. After the hybridization, the microwells were washed twice with 300 μL of 1× HEN buffer and incubated with 50 μL of a STV−PbS QDs solution for 30 min at room temperature under shaking. Finally, the wells were washed four times with 1× HEN buffer (Scheme 1). For the discrimination experiments between fully complementary and mismatching sequences, after hybridization, the microwells were sequentially rinsed with 300 μL of HEN buffers (from 1× HEN to 0.0625× HEN) for 1 min under shaking and finally with 300 μL of distilled water. As the forces between the DNA strands are electrostatic, rinsing with buffers of reduced ionic strength affects the hybridization of the mismatching sequences to a greater extent than that of the fully complementary ones. Detection by Anodic Stripping Voltammetry. Following the DNA assay, 50 μL of 0.01 mol L−1 HCl was added in the microwell and sonication was applied for 5 min to release Pb(II) from the PbS nanoparticles. Then, 50 μL of acetate buffer (0.1 mol L−1, pH 4.5) was added in the microwell in order to adjust the pH of the working solution to moderately acidic conditions (pH 4.5) to prevent hydrogen formation that can interfere with the accumulation procedure. The integrated sensor was rolled and inserted into the microwell, forming a mini cylindrical electrochemical cell, and Pb(II) was preconcentrated on the Bi electrode surface at −1.20 V for 240 s under shaking. After Pb preconcentration, the potential of the WE was scanned from −0.80 to −0.30 V in the SW mode (frequency: 50 Hz; pulse height: 40 mV; step increment: 4 mV). Finally, residual Pb was removed from the Bi WE at −0.30 V for 10 s.
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RESULTS AND DISCUSSION The rationale for applying the proposed thin-film microelectronic approach for the production of bismuth devices (as opposed to electroplating) is to ensure uniform and 855
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The analytical characteristics of the PbS−QDs DNA hybridization electrochemical assay were established by carrying out the assay with different concentrations of target DNA. The stripping response of Pb(II) was linearly related to the logarithm of the target DNA concentration in the range from 5 fmol L−1 to 5 nmol L−1 with a correlation coefficient of 0.994 (Figure 3A,B). The LOD, calculated as the target DNA
Figure 3. (A) SW voltammograms obtained with the flexible device in microwells for target DNA concentrations: 0, 5, and 50 fmol L−1; 0.5, 5, and 50 pmol L−1; 0.5 and 5 nmol L−1 from (a) to (h), respectively. (B) The corresponding calibration plot for the target DNA in the range from 5 fmol L−1 to 5 nmol L−1. Each point was the mean signal ± sd (n = 3) subtracting the mean value of the control (0 fmol L−1 target DNA). (C) SW voltammograms for: (a) blank solution with no target DNA; (b−d) 5 pmol L−1 of noncomplementary, single-base mismatch, and fully complementary sequences. Voltammetric parameters as noted in the text.
Figure 2. (A) AFM image and (B) XRD spectrum of the microfabricated Bi sensor. (C) Polarization window of the Bi sensor assessed by DC voltammetry with scan rate of 50 mV s−1. (D) SW stripping peaks of 40 μg L−1 of Pb(II) on an in situ electroplated: (i) MFE; (ii) BiFE; and (iii) on a flexible microfabricated bismuth sensor. Preconcentration time: 120 s; other voltammetric parameters as noted in the text.
concentration corresponding to three times the standard deviation of the signal of the control (0 fmol L−1 target DNA), was 1.1 fmol L−1. Compared to other electrochemical assays without signal amplification27,28 and to those using QDs labels and ASV,9,10,14 the proposed method exhibited a significantly lower LOD. In addition, the LOD of the present method was comparable to, or lower than, that of stripping voltammetric assays employing QD-loaded microbeads with in situ electroplated MFEs and BiFEs (ranging from 0.22 fmol L−1 to 6.60 pmol L−1).11−13 The within-sensor reproducibility (expressed as the % RSD of eight repetitive measurements of 50 pmol L−1 target DNA at the same device) was 4.6% and the between-sensor reproducibility (in terms of % RSD at eight different devices) was lower than 11.4% over the whole calibration range, indicating satisfactory precision and fabrication reproducibility. Moreover, the electoanalytical response of the sensor remained stable for an average of 12 continuous ASV measurements (Figures S2 and S3 of the Supporting Information). The selectivity of the proposed DNA sensor was investigated against noncomplementary and single-base mismatch sequen-
S1A of the Supporting Information). At low preconcentration times, the Pb stripping current increased rapidly with the preconcentration time while at higher preconcentration times the rate of current increase dropped as saturation of the electrode was approached. On the basis of these results, a preconcentration time of 240 s was chosen combining good sensitivity and fast analysis. The Pb stripping current exhibited a sigmoidal shape dependence on the preconentration potential (Figure S1B of the Supporting Information). The Pb stripping current was low at more positive potentials since the potential was not sufficiently negative to initiate the reduction of Pb(II). The Pb stripping current increased as the preconcentration potential became more negative and leveled off at −1.20 V, since the reduction of Pb(II) was controlled by mass transfer. Thus, a preconcentration potential of −1.20 V was finally applied. 856
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(8) Wu, L.; Xiong, E.; Zhang, X.; Zhang, X.; Chen, J. Nano Today 2014, 9, 197−211. (9) Kokkinos, C.; Economou, A.; Petrou, P. S.; Kakabakos, S. E. Anal. Chem. 2013, 85, 10686−10691. (10) Huang, H.; Li, J.; Tan, Y.; Zhou, J.; Zhu, J. J. Analyst 2010, 135, 1773−1778. (11) Dong, H.; Yan, F.; Ji, H.; Wong, D. K.; Ju, H. Adv. Funct. Mater. 2010, 20, 1173−1179. (12) Xiang, Y.; Zhang, H.; Jiang, B.; Chai, Y.; Yuan, R. Anal. Chem. 2011, 83, 4302−4306. (13) Wang, J.; Liu, G. D.; Jan, M. R.; Zhu, Q. Electrochem. Commun. 2003, 5, 1000−1004. (14) Wang, J.; Liu, G.; Merkoci, A. J. Am. Chem. Soc. 2003, 125, 3214−3215. (15) Kokkinos, C.; Economou, A. Curr. Anal. Chem. 2008, 4, 183− 190. (16) Svancara, I.; Prior, C.; Hocevar, S. B.; Wang, J. Electroanalysis 2010, 22, 1405−1420. (17) Kokkinos, C.; Economou, A.; Raptis, I.; Speliotis, T. Electrochem. Commun. 2007, 9, 2795−2800. (18) Windmiller, J. R.; Wang, J. Electroanalysis 2013, 25, 29−46. (19) Benight, S. J.; Wang, C.; Tok, J. B.H.; Bao, Z. Prog. Polym. Sci. 2013, 38, 1961−1977. (20) Pang, C.; Lee, C.; Suh, K. Y. J. Appl. Polym. Sci. 2013, 130, 1429−1441. (21) Kim, D. H.; Lu, N.; Huang, Y.; Rogers, J. A. Mater. Res. Soc. 2012, 37, 226−235. (22) Geoffrey, W.; Krampitz, M. D.; Jeffrey, A.; Norton, M. D. Cancer 2014, 120, 1920−1931. (23) Liao, K. T.; Cheng, J. T.; Li, C. L.; Liu, R. T.; Huang, H. J. Biosens. Bioelectron. 2009, 24, 1899−1904. (24) Campuzano, S.; Pedrero, M.; Garcia, J. L.; Garcia, E.; Garcia, P.; Pingarron, J. M. Anal. Bioanal. Chem. 2011, 399, 2413−2420. (25) Kalogianni, D. P.; Boutsika, L. M.; Kouremenou, P. G.; Christopoulos, T. K.; Ioannou, P. C. Anal. Bioanal. Chem. 2011, 400, 1145−1152. (26) Monereo, O.; Claramunt, S.; Marigorta, M. M.; Boix, M.; Leghrib, R.; Prades, J. D.; Cornet, A.; Merino, P.; Merino, C.; Cirera, A. Talanta 2013, 107, 239−247. (27) Jin, Y.; Yao, X.; Liu, Q.; Li, J. Biosens. Bioelectron. 2007, 22, 1126−1130. (28) Moreno-Hagelsieb, L.; Foultier, B.; Laurent, G.; Pampin, R.; Remacle, J.; Raskin, J. P.; Flandre, D. Biosens. Bioelectron. 2007, 22, 2199−2207. (29) Xue, Q.; Lv, Y.; Zhang, Y.; Xu, S.; Li, R.; Yue, Q.; Li, H.; Wang, L.; Gu, X.; Zhang, S.; Liu, J. Biosens. Bioelectron. 2014, 61, 351−356. (30) Liu, S.; Cheng, C.; Liu, T.; Wang, L.; Gong, H.; Li, F. Biosens. Bioelectron. 2015, 63, 99−104.
ces, respectively (Figure 3C). The fully complementary target sequence showed a response 5.7 (±0.5) (n = 3) times higher than that of the single-base mismatch sequence, presenting selectivity comparable to other electrochemical and fluorescent assays.9−11,29,30 The stripping peak current of the noncomplementary sequence was 5% of that of the fully matching sequence and comparable with that of the control signal (0 fmol L−1 target DNA). These results indicate that the proposed voltammetric DNA sensing platform can determine a single nucleotide polymorphism with high sensitivity ensuring satisfactory discrimination between single-base mismatching and fully matching sequences.
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CONCLUSIONS AND OUTLOOK In conclusion, we have demonstrated the utility of a new type of flexible miniaturized integrated Bi-based device featuring sputtered metal film electrodes for the ultrasensitive stripping voltammetric analysis of DNA mutation using QDs as labels. The flexibility of the proposed device allows the detection directly in microwells, reducing the volume of the working solution at the microliter scale and lowering the LOD at femtomolar levels, which is comparable to, or lower than, methods employing QD-loaded microbeads as labels. These disposable flexible bismuth sensors are environmentally friendly, inexpensive, easily mass produced and overcome the disadvantages of existing rigid voltammetric sensors. Finally, the sensors exhibit a multielement capability of simultaneously detecting Pb(II), Cd(II), and Zn(II) by ASV (Supporting Information). Therefore, the use of PbS, CdS, and ZnS QDs, as voltammetric labels, is expected to allow multiplexed detection of important biomolecules in a single assay.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone:+306972937675. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The research project is implemented within the framework of the Action “Supporting Postdoctoral Researchers” of the Operational Program “Education and Lifelong Learning” and is co-financed by the European Social Fund (ESF) and the Greek State.
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