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Localized Electrochemiluminescence from Nanoneedle Electrodes for Very-high-density Electrochemical Sensing Jingjing Zhang, Junyu Zhou, Chunxiu Tian, Shan Yang, Dechen Jiang, Xixiang Zhang, and Hong-Yuan Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02363 • Publication Date (Web): 28 Sep 2017 Downloaded from http://pubs.acs.org on September 29, 2017

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Analytical Chemistry

Localized Electrochemiluminescence from Nanoneedle Electrodes for Very-high-density Electrochemical Sensing Jingjing Zhang 1†, Junyu Zhou 1†, Chunxiu Tian2, Shan Yang2, Dechen Jiang*1, Xi-Xiang Zhang*2, Hong-Yuan Chen1 1

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical

Engineering Nanjing University, Nanjing, Jiangsu, 210093, China; 2

Physical Sciences and Engineering Division (PSE), King Abdullah University of Science and Technology,

Thuwal, 23955-6900, Kingdom of Saudi Arabia

Phone: 86-25-89684846 (D. J); 966-(0)12-808-2332 (X-X. Zhang); Fax: 86-25-89684846 (D. J); 966-2-8020221 (X-X. Zhang); E-mail: [email protected](D.J), [email protected]

†These authors contributed equally to this work.

ACS Paragon Plus Environment


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ABSTRACT In this paper, localized electrochemiluminescence (ECL) was visualized from nanoneedle electrodes that achieved very-high-density electrochemical sensing. The localized luminescence at the nanometer-sized tip observed was ascribed to enhanced mass transfer of the luminescence probe at the tip than on the planar surface surrounding the tip, which provided higher luminescence at the tip. The size of the luminescence spots was restricted in 15 µm permitting the electrochemical analysis with a density over 4 × 103 spots/mm2. The positive correlation between the luminescence intensity at the tips and the concentration of hydrogen peroxide supported the quantitative ECL analysis using nanoneedle electrodes. The further modification of glucose oxidase at the electrode surface conceptually demonstrated that the concentration of glucose ranged from 0.5 to 5 mM could be quantified using the luminescence at the tips, which could be further applied for the detection of multiple molecules in the complex bio-system. This successful localized ECL offers a specific strategy for the development of very-high-density electrochemical arrays without the complicate chip design.


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Introduction. A microarray is an array with probes spotted on a solid support that enables parallel processing and the detection of analytes in complicated samples.1-3 Owing to its high-throughput ability, the microarray has been most widely used for molecular biology and ‘-omics’ studies.4,5 The traditional microarray has a density of ~50 spots/mm2, where each spot is on the order of 100 µm in diameter. Recently, the rapid development of micro/nanofabrication and nanomaterials has increased the density of the array to more than 103 spots/mm2 (also referred to as a very-high-density sensing array) to fulfill a need in systems biology and genomics.6 As a highly sensitive, selective and unlabeled detection method, electrochemical analysis is one of the primary applications of the array.7,8 However, because contact pads and the connection lines in the array are required for the application of voltage and electrochemical signal recording, it is challenging to achieve very-highdensity electrochemical microarrays. Electrochemiluminescence (ECL) is the luminescence produced from electrochemically generated intermediates on an electrode, which can be imaged using a charge-coupled device (CCD).9-11

The

luminescence intensity in the image represents the spatial distribution of molecules that electrochemically react at the electrode. Assuming one pixel in the ECL image corresponds to one microelectrode, the whole electrode surface can be taken as the electrode assembly with a very high density. Only one pad is required for the application of the voltage on the electrode, avoiding the complexity in the fabrication of electrode array. As a result, the ECL based detection should provide a specific strategy to achieve very-high-density electrochemical analysis.

However, the relatively long lifetime (~millisecond) and large diffusion distance (~ a few

micrometer) of the electrochemically generated intermediates in the ECL process lead to the overlapping of the luminescence from the adjacent regions, which affects the accuracy of electrochemical quantification.12 Therefore, the generation of localized luminescence spots on the electrode to quantify the species is critical for achieving a very-high-density ECL sensing array. Currently, a bipolar electrode array has been developed based on the polarization of conducting wires in an electric field, where the luminescence was created at one end of the electrode to achieve localized luminescence.13,14 A nanoscale multichannel PET membrane with an ion-tracked intensity of 106/mm2 was reported as a bipolar electrode array for ECL sensing of hydrogen peroxide.15 Alternatively, a fiber optic bundle with a density of more than 104 fibers/mm2 was etched anisotropically and coated with gold.16 The incomplete insulation resulted in Au tips (1 µm in diameter) that generated localized luminescence. These two main breakthroughs demonstrate the feasibility of the application of localized ECL to generate a very-highdensity electrochemical sensing array. Further developments to fabricate electrodes into a chip and decrease the applied voltage avoiding a high current in solution should permit the very-high-density electrochemical sensing for the analysis of biological samples, such as cells and tissues. In this paper, the localized ECL was first visualized from nanoneedle electrode array and applied for veryhigh-density electrochemical sensing, as shown in Figure 1A. Only a low potential for the generation of ECL is applied on the electrodes that is suitable for the bioassay. The visualization of localized ECL depends on a



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well-known diffusion-based phenomenon: the mass transfer of aqueous species to the nanometer-sized tip is enhanced, as compared with that on the micrometer-sized planar electrode.17 Because the diffusion process dominates the electrochemical rate at the micro/nanoelectrode under over-potential, more luminescence probe should be oxidized at the nanoneedle tip than that on the surrounding micrometer-sized planar electrode, resulting in the visualization of luminescence spots at the tips.18, 19 The distance between the nanoneedles can be accurately controlled to a few micrometers via the microfabrication process, and therefore, the luminescence intensity at the tips could be used to quantify the target molecules for very-high-density electrochemical assay.

Experimental Section. Materials and Reagents. The photoresisit, AZ2020, and the developer were purchased from Microchem Corp (Newton, MA). The compound 8-amino-5-chloro-7-phenylpyrido [3, 4-d] pyridazine-1, 4(2H, 3H)-dione (L012) was obtained from Wako Chemical, Inc. (Richmond, VA). All other chemicals were from SigmaAldrich, unless indicated otherwise. Fabrication of nanoneedle electrode array. The fabrication of nanoneedle electrode array followed the protocol developed before20,21, as demonstrated in Figure S1 (supporting information). Briefly, 4-inch, ptype, silicon wafers with the resistivity in the range of 1-20 Ω•cm and a thickness of 500 µm were standard RCA cleaned and thermally oxidized to produce 200 nm SiO2 layer. The photoresist pattern was created by optical lithography and transferred to SiO2 pattern by reactive ion etching (RIE). After the RIE etching, the wafers were cleaned with acetone and isopropanol followed by N2 blow-drying to remove the photoresist residual. The wafers were then wet etched using a 40 wt% KOH aqueous solution at 80 ºC for 7 min. During the etching process, SiO2 acted as an etch mask. After wet etching, the wafers were rinsed in piranha solution (3:1 H2SO4:H2O2) and 0.49% HF solution to remove SiO2 and any other possible residuals acquired during previous processes to form nanoneedles. Finally, 10 nm Ti/100 nm Au layers were sputtered on the wafer to produce Au nanoneedle electrode array. CAUTION: Piranha solution is highly corrosive that reacts violently with most organics, and only small quantities should be prepared. Electrochemiluminescence imaging of a nanoneedle electrode array. The optical setup for the imaging was assembled with a 10X objective (Olympus, Japan), a tube and an electron-monitoring charge-coupled device (Evolve, Photometrics, Tucson, AZ). The nanoneedle electrode array was applied as the working electrodes, and an Ag/AgCl electrode and a Pt wire were connected as the reference and counter electrode, respectively. For the luminescence imaging of L012/hydrogen peroxide, a switching of the potential between 1 V (2 s) and 1 V (0.5 s) was continuously employed on the nanoneedle electrode array using a voltage generator (DG 1021, Rigol, China) to induce the luminescence. The focus at the tip of nanoneedles needed to be adjusted till bright and complete light-spots were observed under bright-field observation.


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Results and Discussion Localized electrochemiluminescence from nanoneedle electrode. As a conceptual presentation, the nanoneedle electrodes were generated from micropillars through wet etching following the literatures.20,21 The bright-field and scanning electron microscopy (SEM) images in Figures 1B-D indicated that the diameters of the tip and bottom in the nanoneedle were 750 nm and 3 µm, respectively, and the height from the top to the bottom was 6 µm. The distance between the centers of the adjacent nanoneedles was set to 25 µm, and therefore, the density of the electrodes in the array was 1.6 × 103 spots/mm2. Similarly, two additional nanoneedle electrodes with heights of 2 and 4 µm were prepared by decreasing the height of the micropillars during the initial fabrication process. To characterize the mass transfer of luminescence probe at the nanoneedle electrode, cyclic voltammetry was performed at the nanoneedle and planar electrodes in 10 mM phosphate saline buffer (PBS) containing 200 µM luminol (a classic luminescence probe), respectively. As shown in Figure S2 (supporting information), the peak oxidative current observed at the potential of ~ 0.6 V was attributed to electron transfer from luminol to the oxidized state (i.e.,3-aminophthalate).23 Compared with the peak oxidative current from planar electrode, ~ 78% more peak oxidative current was observed. Considering the presence of nanoneedles offered 8.3% more geometric surface area on the electrode, the oxidative-current-density at the nanoneedles was estimated to be ~ 9.4 fold higher than that from planar electrode suggesting enhanced mass transfer of the species at the nanoneedle. Because the luminescence intensity was controlled by the mass transfer of luminol to the electrode under the over-potential, a higher flow of luminol at the tip should generate enhanced luminescence. Furthermore, the diffusion of luminol to the nanoneedle was simulated using Comsol software, as modeled in Figure S3A (supporting information).22 To simplify the simulation, only electron transfer from luminol to the oxidized state (i.e.,3-aminophthalate) at a potential of 0.6 V was considered. As expected, the results in Figure S3B exhibited that the simulated oxidative-current-density of luminol at the nanometer-sized tip was 4 fold larger than that at the planar surface, confirming enhanced mass transfer of luminol at the tip. The discrepancy in the enhancement of the experimental and simulated currents might be caused by the relatively simple simulation mode and the possibly complex interaction among the nanoneedles at the electrode. The enhancement in the oxidative-current-density was weakened when the tip height was shortened from 6 to 2 µm. All these results supported that the radial diffusion of luminol was more pronounced on the longer nanoneedle facilitating the localized ECL. Luminol ECL from the nanoneedle electrode was performed in the presence of hydrogen peroxide to observe the luminescence on the nanoneedles. According to our previous potential mode for luminol ECL imaging,12 a switching of the potential between 1 V (2 s) and -1 V (0.5 s) was continuously employed on the electrode in 10 mM PBS with a physiological pH of 7.4. 200 µM L012 and 2 mM hydrogen peroxide were added into the buffer to induce the luminescence. L012, the luminol analog with higher luminescence intensity, was chosen to illustrate the luminol ECL.24 The luminescence image in Figure 2A displayed the bright luminescence spots on all of nanoneedle tips with a height of 6 µm demonstrating the visualization of



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the localized ECL. The luminescence at the tips was measured and shown in Figure 2B. The luminescence intensity at the tip was measured to be 1.75 fold (average) higher than that observed on the surrounding planar electrode. More discussion about the enhancement was described in Figure S4 (supporting information). This enhancement in the luminescence was smaller than that in the oxidative-current-density, which might be attributed to incomplete collection of luminescence experimentally. The size of the luminescence spot at the nanoneedle tip was measured to be 12.9 ± 1.4 µm, which was larger than the tip size. The enlargement of the size was due to the diffusion of the luminol intermediate and oxygen-containing species during the electrochemical reaction. More discussion to exclude the possibility of defocusing of the luminescence spots at nanoneedle tip was provided in Figure S5 (supporting information). The relative standard deviation of the luminescence intensity from 625 (25×25) nanoneedles was 2.65%, which confirmed the good reproducibility of the localized luminescence at the nanoneedles for the following electrochemical assay. A decrease in the height of the nanoneedle to 2 or 4 µm minimized the discrimination of the localized luminescence at the tip owing to the relatively slow diffusion of luminol on the shorter tips. On the contrary, the continuous increase in the height of the nanoneedle provided more tip-enhanced ECL, however, a higher nanoneedle required a micropillar with a larger diameter in the fabrication process resulting in a decrease in the nanoneedle density. Therefore, the nanoneedle with a height of 6 µm was set in the array. Similarly, Ru(bpy)32+/tripropylamine (TPA) as another classic luminescence probe was introduced on the nanoneedle electrode array, which emitted peak luminescence at the wavelength of ~ 610 nm under a positive potential.25 After continuously employing potentials at 1.3 V (2 s) and -1 V (0.5 s) on the electrode, the averaged 2.16 fold higher luminescence was observed at the nanoneedle tips in Figure 2C and D, which supported the generation of tip-enhanced ECL. The luminescence intensities at the 625 tips were nearly identical with a relative standard deviation of 2.7%. All of these results indicated that the localized ECL at the tip of nanoneedles could be achieved in multiple ECL systems. As a result, the visualization of localized luminescence provides the feasibility for very-high-density electrochemical assay. Very-high-density Electrochemical Sensing. In electrochemical sensing, the classic detection strategy involves the conversion of biomolecules into electrochemically active hydrogen peroxide using the corresponding oxidase, which is electrochemically oxidized on the electrode to provide electrons. Therefore, the visualization of hydrogen peroxide at all the nanoneedle electrodes is important for achieving very-high-density electrochemical arrays. The luminescence images of the nanoneedle array exposed to 10 mM PBS with 200 µM L012 and hydrogen peroxide were shown in Figure S6 (supporting information). Localized ECL spots were observed at all of the nanoneedle tips and the intensity was positively correlated with the hydrogen peroxide concentration from 0.5 to 5 mM, as shown in Figure 3A. The linear detection range was determined to be between 0.5 and 2 mM with a R-squared value of 0.95.

The relative standard deviation of the

luminescence intensity at all of the nanoneedle tips was calculated to be less than 3.2%, supporting the detection accuracy and reproducibility using our ECL-based nanoneedle array. The results in Figure 3B


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exhibited that the sizes of the luminescence spots using various concentrations of hydrogen peroxide were similar, which confirmed the presence of a diffusion layer of intermediates during the electrochemical reaction. All of the sizes were restricted to 15 µm, suggesting that the electrode density up to 4×103 spot/mm2 could be employed for bioanalysis. To demonstrate very-high-density electrochemical sensing, glucose as a classic biomolecule was analyzed using our nanoneedle electrode array. For the linkage of glucose oxidase onto the nanoneedle electrode array, the electrode was pretreated with Nafion, which introduced negative charges on the surface. Next, the array was exposed to glucose oxidase in the buffer (pH 4), resulting in electrostatic interactions between the positively charged glucose oxidase and negatively charged Nafion on the electrode surface.

The

luminescences at the nanoneedles before and after the modification of Nafion and glucose oxidase were shown in Figure S7 (supporting information). The introduction of Nafion at nanoneedle induced the increase in the luminescence. In the typical electrochemiluminescence measurement of L012, the oxidative product of L012 could be easily adsorbed at the electrode reducing the effective electrode surface so that less luminescence was collected. The presence of Nafion that could block the adsorption sites resulted in more luminescence from Nafion modified electrode than that from unmodified electrode during the exposure time.26 The further modification of glucose oxidase at the nanoneedle electrode exhibited a decrease in the luminescence, which supported the existence of an insulated layer of oxidase at the nanoneedle. After the chip was washed using PBS (pH 7.4), 10 mM PBS with 200 µM L012 and a glucose concentration of 0.5 to 5 mM was introduced, and the luminescence was recorded, as shown in Figures 4A-C. The luminescence spots that were observed at the nanoneedles were due to the hydrogen peroxide generated from the glucose and glucose oxidase.

Figures 4D and S8 (supporting information) showed the linear

correlation between the luminescence intensity and glucose concentration in the range of 0.5 and 2 mM with a R-squared value of 0.91, which supported that glucose could be detected using the nanoneedle electrode array. The relative standard deviation was less than 5.7%, which confirms the reproducibility of the detection. Since high salted buffer was reported to enhance the luminol luminescence,27 the concentration of PBS was regulated from 2 to 50 mM and the response of the nanoneedle electrode to 0.5 mM glucose was recorded. As shown in Figure S9 (supporting information), higher luminescence was observed in more salted buffer. However, for the future biological study, 10 mM PBS as the physiological buffer was still used in the whole experiments. The same strategy was applied to the detection of lactate and choline by modification of the electrode with lactate oxidase and choline oxidase, respectively. As shown in Figure S10 (supporting information), both of the molecules were successfully detected using our arrays. To achieve future high-throughput analysis of bio-samples with multiple analytes, the non-specific response of the nanoneedle electrode array to species other than the target must be minimized. Experimentally, the array that was modified with glucose oxidase was exposed to a solution containing lactate, urea, glycine or choline, and the luminescence intensities at the nanoneedle tips were measured. As shown in



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Figure 4E and Figure S11 (supporting information), no obvious increase in luminescence was observed at the nanoneedles guarantying the specificity of our array. Additionally, the accuracy of the response from our array was determined by comparison of the responses to only glucose and a mixture of glucose and lactate. An additional luminescence increase of only 7.5% was observed in the detection of the mixture in Figure 4E, which confirmed the accuracy of the detection method in this mixture. Recovery test was performed by adding 0.5 mM glucose into 10 mM PBS with 0.5 mM glucose, and the luminescence images were collected before and after the addition of glucose. According to the calibration curve in Figure S8, the difference in the luminescence from three independent measurements indicated 0.46 ~ 0.55 mM glucose added, which exhibited the recovery rate of 92~ 110%. The modification of different oxidases at the nanoneedles in special regions of the electrode could achieve the detection of multiple targets in the biological samples. To demonstrate this region-resolved sensing, a drop of solution with glucose oxidase was loaded at the nanoneedle electrode so that part of the nanoneedles at the electrode were modified with glucose oxidase and the other part of the nanoneedles was bare. After the exposure of the electrode to the buffer containing 0.5 mM glucose, the luminescence image was recorded and shown in Figure S12 (supporting information). The luminescence spots were observed only at the oxidase modified region, which suggested the feasibility of local detection of target molecules at specific region. Some bright luminescence observed at the edge of modification region, which might be ascribed to the local accumulation of oxidase at the edge of the droplet during drying. Therefore, to achieve the modification of specific oxidase at one or a few nanoneedles without the edge accumulation is critical for the future application in very-high-density electrochemical sensing.

Conclusion. In summary, nanoneedle electrodes with localized ECL were developed as novel very-high-density arrays for the electrochemical sensing of hydrogen peroxide, glucose, lactate and choline. The relative standard deviation of the luminescence intensity from the nanoneedles was less than 5.7%, demonstrating the good reproducibility of these electrodes in the array. In the conceptual presentation, the density of the electrodes was 1.6 × 103 electrodes/mm2, which can be up-regulated to a density of 4×103 electrodes/mm2 by decreasing the distance between the nanoneedles to ~15 µm. Multiple oxidases and receptors are being modified on the nanoneedle electrodes of one array, allowing a single array to be used for the very-high-density analysis of hundreds of molecules in a complex biosystem. Additionally, the achievement of localized ECL on the nanoneedle electrodes provides a specific platform for high throughput investigation of localized behavior of luminescence probes ECL to understand the ECL mechanism.

Acknowledgements This work was supported by National key research and development program (2016YFA0201203) and the National Natural Science Foundation of China (nos. 21327902 and 21575060). CXT, SY and XXZ would


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like to acknowledge the financial support from King Abdullah University of Science and Technology (KAUST).

Supporting Information Available: This material is available free of charge via the Internet at http://pubs.acs.org



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Figures and Captions. Figure 1: (A) Experimental setup for local tip-enhanced ECL from nanoneedle electrodes; (B) bright-field and (C) SEM image of nanoneedle electrodes; (D) SEM image of a single nanoneedle.

Figure 2: (A) Luminescence image of nanoneedle electrode in 10 mM PBS containing 200 µM L012 and 2 mM hydrogen peroxide; (B) luminescence intensity across the white line drawn in Figure 2A; (C) luminescence image of nanoneedle electrode in 10 mM PBS containing 10 mM Ru(bpy)32+ and 200 mM TPA; (D) luminescence intensity across the white line drawn in Figure 2C.

Figure 3: (A) Luminescence intensity at the nanoneedle tips in the presence of 200 µM L012 and 0.5, 1, 2 and 5 mM hydrogen peroxide; (B) size of the luminescence spots at 625 nanoneedle tips (the bars represent the standard deviation of each).

Figure 4: (A-C) Luminescence image of glucose oxidase-modified nanoneedle electrodes in the presence of 200 µM L012 and (A) 0.5, (B) 1 and (C) 5 mM glucose; (D) luminescence intensity at the nanoneedle tips with 0, 0.5, 1 and 5 mM glucose; (E) luminescence intensity collected with glucose oxidase in response to PBS, 5 mM lactate, 5 mM glucose and a mixture of 5 mM glucose and lactate.



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Figure 1.


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Figure 2.



Figure 3.

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Figure 4.



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Localized Electrochemiluminescence from Nanoneedle Electrodes for

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