Letter pubs.acs.org/acssensors
Increasing the Detection Sensitivity for DNA-Morpholino Hybridization in Sub-Nanomolar Regime by Enhancing the Surface Ion Conductance of PEDOT:PSS Membrane in a Microchannel Xi Wei,†,‡ Prabodh Panindre,§ Qi Zhang,‡ and Yong-Ak Song*,†,‡ †
Division of Engineering, New York University Abu Dhabi, P.O. Box 129188, Abu Dhabi, United Arab Emirates Department of Chemical and Biomolecular Engineering and §Department of Mechanical Engineering, New York University Tandon School of Engineering, Brooklyn, 11201, United States
‡
S Supporting Information *
ABSTRACT: Electrokinetic concentration based on ion concentration polarization (ICP) offers a unique possibility to increase detection sensitivity and speed of surface-based biosensors for low-abundance biomolecules inside a microfluidic channel. To further improve the concentration performance, we investigated the effect of surface ion conductance of the ion-selective conductive polymer membrane, poly(3,4-ethylenedioxythiophene) polystyrenesulfonate (PEDOT:PSS), in a microfluidic channel. By increasing its thickness and surface charge, we could achieve a concentration increase of DNA by 6 orders of magnitude from an initial concentration of 100 fM within 10 min. As for the detection via surface hybridization on morpholino (MO) probes, DNA target concentration as low as 10 pM was detected within 15 min. This result means an improvement by 2 orders of magnitude in terms of the detection limit compared with our previous developed PEDOT:PSS membrane. These results demonstrate a potential application of the PEDOT:PSS membrane for the ICP-enhanced detection of DNA and other biomolecules in surface-based assays down to picomolar regimes. KEYWORDS: electrokinetic concentration, ion concentration polarization, PEDOT:PSS, surface ion conductance, microfluidics, DNA sensing
M
toward the ion depletion zone near the membrane and increases its concentration via electrokinetic trapping.2,20,21 The initiation of ICP is governed by the ratio of bulk conductance Gbulk to surface conductance Gσ,22 which is given by the inversed Dukhin number
ost clinically important biomarkers are present at subpicomolar regimes and pose a formidable challenge to any currently available biosensors in terms of speed and sensitivity.1−4 One of the promising avenues to detect those molecules with standard biosensors is to preconcentrate them at the site of detection by electrokinectic means inside a microchannel and increase their local concentration level above the limit of detection. Based on the nonlinear electrokinetic phenomenon of ion concentration polarization (ICP), several microfluidics devices have been developed to preconcentrate/ enrich biomolecular samples by using micro/nanochannel interfaces,2,5−8 utilizing perm selective membranes9 or embedding bipolar electrodes.10−12 These microfluidic preconcentration systems have been used to enhance both the reaction rate and the detection sensitivity of immunoassays,5,13−16 as well as of the standard enzymatic assays.17−19 Lee et al. reported concentration enhanced cell kinase assay in a micro/nanofluidic platform directly from cell lysates and demonstrated a 25-fold increase in reaction velocity and 65-fold enhancement in sensitivity.17 The formation of a highly concentrated biomolecular plug results from the ion selectivity of nanoporous membrane/channels in combination with an electroosmotic flow that transports biomolecules from the anodic reservoir © XXXX American Chemical Society
1/DU = G bulk /Gσ
(1)
The conductance ratio determines the amount of ionic current inside a nanoporous membrane/nanochannel. In the case of 1/DU ≪ 1, the counterions preferably go through the charged surface rather than the bulk solution, causing a propagation of ICP. Therefore, enhancing the surface conductance Gσ by increasing the thickness of the ion-selective membrane or its surface charge can be a viable option for further increasing the concentration performance by ICP.9 Our group has developed an electrokinetic concentrator using a cation-selective conductive polymer, poly(3,4-ethylenedioxythiophene) polystyrenesulfonate (PEDOT:PSS) that uniquely combines a cation selectivity based on the negatively Received: March 13, 2016 Accepted: July 5, 2016
A
DOI: 10.1021/acssensors.6b00169 ACS Sens. XXXX, XXX, XXX−XXX
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layer of ∼3 μm thick PEDOT:PSS membrane (denoted as “1X SH”) enhanced hybridization was used as the reference.26 As shown in Figure 2a−c, we could detect the DNA sample concentration as low as C0 = 1 pM with 2X SH membrane
charged sulfate group with a high electrical conductivity through the cationic PEDOT oligomers.23 Especially, the high electrical conductivity of PEDOT:PSS could possibly induce electrical field suppression in the vicinitysimilar to that occurs near a bipolar electrode perpendicular to the microchannelgenerates an electric field gradient for electrokinetic trapping.11,12,24,25 To further improve the concentration performance of the PEDOT:PSS membrane, we studied two alternative approaches to enhance surface ion conductance of the ion-selective membrane by increasing its thickness and surface charge. A single microfluidic channel in polydimethylsiloxane (PDMS) was fabricated using the standard soft lithography procedure.26 The microchannel was 17 μm high, 1 cm long, and 200 μm wide with one reservoir (4 mm in diameter) at each end. An ion-selective PEDOT:PSS membrane was printed on a glass substrate and was aligned to the circular pattern of the microchannel prior to reversible bonding, as shown in Figure 1a). Two types of commercially available PEDOT:PSS were
Figure 2. Characterization of ICP-induced electrokinetic concentration. Measurement of the fluorescence intensity of Cy5 tagged DNA with (a) 2X SH membrane, (b) 1X PH membrane, (c) 2X PH membrane, (d) 2X SH membrane spiked with lysed E. coli.
(Video S2), C0 = 10 pM with 1X PH membrane and C0 = 100 fM with 2X PH membrane (Video S3) compared to C0 = 100 pM, the current detection limit with 1X SH membrane. The concentrated DNA plug was stable with 15 min. With longer preconcentration time, however, the sample plug moved toward the cathodic reservoir due to the expansion of ion depletion zone. In terms of the concentration/enrichment factor, 2X SH membrane showed an enrichment factor (EF) between 104 and 105, while 1X PH membrane allowed a concentration increase by ∼104 compared with an enrichment factor of 102−103 of 1X SH membrane. 2X PH membrane reached the highest enrichment factor (EF) between 105−106 among all three testing membranes. Interestingly, for all three membranes, the EF achieved at the lowest detectable concentration C0 was always the highest compared with other initial DNA concentrations. In the case of 2X SH, the EF was 3 × 105 at C0 = 1 pM which was 3.5-, 18.2-, and 7.2-fold higher than the EF of C0 = 1 nM, 100 pM, and 10 pM, respectively. In the case of 1X PH, the EF for C0 = 10 pM was 3.2 × 104, which was 1.5and 4.2-fold higher than the EF for C0 = 1 nM and 100 pM, respectively. Similarly, in the case for 2X PH, the EF for C0 = 100 fM was 3.0 × 106, which was 259.4-, 83.1-, and 9.6-fold higher than the EF for C0 = 100 pM, 10 pM, and 1 pM, respectively. This phenomenon has also been reported by other research groups.2,11,28 A possible explanation for this difference in EF is that higher DNA concentration could increase the ionic strength at the depletion zone boundary, thus reducing the electric field gradient.2,11 Notably, the speed of DNA preconcentration induced by 6 μm PEDOT:PSS membrane (both 2X SH and 2X PH) was higher (an EF of 104−106 within 1 min) compared to the reported preconcentrating speed of ∼104 in 5 min with Nafion membrane.16 Our microfluidic concentrator also allowed to preconcentrate the same DNA target mixed with cell lysate from E. coli (see Video S4). Compared to the preconcentration without presence of cell lysate, however, it required 3 min to reach the concentration plateau and the limit of detection decreased to C0 = 10 pM with an EF of 103−104 (Figure 2d). Comparatively, when a noncomplementary DNA was added to the complementary
Figure 1. Schematic of a single microfluidic concentrator chip with its characterization. (a) Device design and its integration to an array of morpholino probes on a glass substrate. (b) Surface profiles of a ∼6μm-thick PEDOT:PSS (2X SH) membrane, a ∼3-μm-thick PH1000 PEDOT:PSS (1X PH) membrane, and a ∼6-μm-thick PEDOT:PSS (2X PH) membrane.
used in this study: (1) High conductivity grade PEDOT:PSS, 3−4% in H2O (Sigma-Aldrich, Cat. #655201, denoted as “SH”) with an electrical conductivity of σ ≈ 150 S/cm and a zeta potential of ζ = −66.7 ± 0.9 mV; (2) Clevios PH1000 (Heraeus, denoted as “PH”) with σ ≈ 833 S/cm and ζ = −85.9 + 1.3 mV after adding 10% ethylene glycol to improve its conductivity and adhesion (Table S1).27 In order to study the effect of membrane thickness and surface charge, we deposited a ∼6-μm-thick SH membrane by printing two layers on top of each other (denoted as “2X SH”) with a printing time of 30 s for each layer, a single layer of ∼3-μm-thick PH membrane (denoted as “1X PH”) and a ∼6-μm-thick PH membrane (denoted as “2X PH”) on a planar and a superaldehyde glass substrate (see Video S1). The estimated electrical conductance of all the PEDOT:PSS membranes including “1X SH” as a reference is listed in in Table S2. The surface profiles of the PEDOT:PSS membranes are shown in Figure 1b). To evaluate the enrichment efficiency of these two conductive polymer membranes, 50 μL of cyanine 5 (Cy5) labeled DNA target (5′ CAA CCG ATG CCA CAT CAT TAG CTA C-Cy5 3′) with an initial concentration (C0) ranging from 100 fM to 1 nM in 0.1× phosphate buffer saline (PBS) was loaded on the anodic reservoir. 100 μL of 0.1× PBS at pH 7.1 was loaded into the cathodic reservoir. A constant DC voltage of 50 V was applied across the channel to initiate ICP. Fluorescence signal intensity was measured to quantify the concentration increase by comparing it to the fluorescence signal intensities of reference DNA concentrations. A single B
DOI: 10.1021/acssensors.6b00169 ACS Sens. XXXX, XXX, XXX−XXX
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electrokinetic concentration performance compared with the reference PEDOT:PSS layer (1X SH). When an external DC voltage was applied across the channel, target DNA molecules were concentrated up to ∼106-fold within 10 min, enabling a detection limit at C0 = 100 fM. DNA hybridization experiment on MO probes confirmed a detection limit of 10 pM after 15 min of the target DNA concentration which is an improvement by 2 orders of magnitude compared with our previously published results. In addition, we demonstrated that the microfluidic concentrator can increase the DNA concentration even in the presence of cell lysate which opens up new exciting possibilities to apply this technique to complex biological samples directly. Following the two approaches outlined in this study, we can potentially further push the limit of detection for surface hybridization down to a few picomolar regimes in mass transport limited DNA-MO microarrays.
DNA for MO−DNA hybridization, a decrease of the detection limit was also observed in our previous study.26 Since DNA-MO surface hybridization is much slower (∼20to 40-fold) than in solution29,30 due to the diffusion-limited mass transport,3 the microfluidic electrokinetic concentrator can provide an effective means of accelerating the transport for faster detection.26 Using a MO-printed superaldehyde glass surface, the detection limit for the DNA surface hybridization was measured at C0 = 100 pM (see Video S5) for 2X SH, 500 pM for 1X PH, and 10 pM for 2X PH (see Video S6), respectively, compared to C0 = 1 nM with 1X SH (Figure 3a).
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.6b00169. Zeta potential of PEDOT:PSS; estimated electrical conductance (PDF) Layer by layer printing technique to deposit 2 layers of PEDOT:PSS membrane (AVI) DNA concentrating experiment at an initial concentration of 1 pM (MOV) DNA concentrating experiment at an initial concentration of 100 fM (MOV) DNA concentrating experiment at an initial concentration of 10 pM mixed with E. coli cell lysate (MOV) DNA-morpholino hybridization experiment at a DNA initial concentration of 100 pM (MOV) DNA-morpholino hybridization experiment at a DNA initial concentration of 10 pM (MOV)
Figure 3. ICP-enhanced morpholino-DNA hybridization. (a) Comparison of ICP-enhanced DNA hybridization results after 15 min of electrokinetic concentration from an initial DNA concentration of C0 = 10 pM to 10 nM with four types of PEDOT:PSS membranes: 1X SH (surface electrical conductance Cs = 45 × 10−3 S), 2X SH (2· Cs), 1X PH (5.6·Cs), 2X PH (11.2·Cs). (b) Enhancement of the DNA target from C0 = 100 pM in the microfluidic channel with 2X SH PEDOT:PSS membrane after 15 min and surface hybridization on an array of morpholino probes. 1 and 2: Fluorescence image of the concentrated DNA plug above printed morpholino probes and its 3D surface reconstruction with 15 min enrichment. 3 and 4: Fluorescence signal of the hybridized DNA after 15 min hybridization on MO probes and its 3D surface reconstruction. Scale bar: 40 μm.
The concentrated DNA from an initial concentration of C0 = 100 pM and its enhanced hybridization result on MO probes are illustrated shown in Figure 3b. At C0 = 1 nM, the hybridization signal using 2X SH was about 1.5-, 4.1-, and 57fold higher compared with the results obtained with 2X PH, 1X PH, and 1X SH membranes, respectively. 2X PH reached the best performance at C0 = 100 pM with 3-fold of signal increase compared to 2X SH membrane while hybridization signal was out of detection range with other membranes. In the control study without electrokinetic concentration, the detection limit for hybridization was at C0 = 10 nM after 2.5 h of incubation. At lower DNA concentrations, the fluorescence signal intensity for DNA-MO surface hybridization was below the limit of detection (LOD) even after prolonged incubation times (16 h for C0 = 1 nM, 36 h for C0 = 100 pM). This result validated the effectiveness of increasing the surface conductance of the PEDOT:PSS membrane by increasing its thickness (6 μm vs 3 μm) and surface charge (2X PH vs 2X SH), both leading to significantly faster DNA hybridization even in the subnanomolar target concentration regimes that usually require extensive incubation times or remain undetectable below the detection limit. Our electrokinetic concentrator can readily be integrated into a microfluidic channel and increase the local concentration of the analyte for enhanced detection with a surface-based assay. In this study, we explored two different avenues to increase the surface ionic conductance of the ion-selective membrane to achieve higher enhancement factor. Increasing the membrane thickness as well as its surface charge resulted in higher
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone: +971-2-628-4781. Fax: +971-2-659-0794. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully acknowledge the financial support from New York University Abu Dhabi (NYUAD) through the NYUAD Research Enhancement Fund 2013. The device fabrication was conducted in the microfabrication core facility of NYUAD.
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REFERENCES
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