- Open Access
A cyclodextrin-based approach for selective detection of catecholamine hormone mixtures
© Yang et al; licensee Springer. 2014
- Received: 12 February 2014
- Accepted: 15 April 2014
- Published: 7 June 2014
This paper presents an electrochemical sensing approach that enables quantitative detection of three major catecholamine hormones from a mixture by specifically employing a chemically-modified microelectrode array with α-, β- and γ-cyclodextrin (CD) ‘catchers’ holding unique physical matching (size and shape) as well as chemical enticing (stereochemistry and surface charge) properties. The developed neurotransmitter sensor has selectively identified L-tyrosine, dihydroxyphenylalanine (L-DOPA) and dopamine in the absence of ascorbic acid. It exhibited the relatively linear sensitivities to each neurotransmitter with logarithmically increasing concentrations range of 5μM-10mM, while demonstrating stability up to 6 hours from the fabrication and the average accuracy of 91.2%.
- Catecholamine hormone
- Micro electrochemical sensor
Neurodegenerative diseases present an increasing burden for the world health care sector. Currently, 6.5 million Americans suffer from the neurodegenerative diseases such as Alzheimer’s disease (5.4 million), Parkinson’s disease (1 million), amyotrophic lateral sclerosis (30,000), and Huntington’s disease (15,000) [1–3]. Since the neurodegenerative diseases are primarily an age-based disease with the increasing incidence after age 65 years [4, 5] and the U.S. population continue to age , it is estimated that, by 2040, more than 13.1 million Americans will be diagnosed with neurodegenerative diseases. Such increase of the neurodegenerative disease population will cause the corresponding growth of social costs of treating neurodegenerative diseases; thus, developing effective treatments for the neurodegenerative diseases is an urgent and critical issue.
The detection of neurotransmitters has been performed largely in four ways: electrochemical detection (ECD) [10–17], fluorescence detection (FD) [18–21], chemiluminescence detection (CLD) [22–24], and mass spectrometric detection (MSD) . However, none of those approaches have been successful in selectively detecting the co-existing individual catecholamine neurotransmitters without using additional separation techniques such as liquid chromatography or capillary electrophoresis that require complex system, long analysis time, and high power consumption. Particularly, the ECD has been most frequently utilized for the neurotransmitter detection due to its advantages of high sensitivity, real time analysis, low cost, and easiness of system miniaturization [17, 26]; however, only the detection of a single catecholamine neurotransmitter has been successfully achieved because the conventional enzyme-based electrochemical sensors, based on the redox reaction of catechol groups with tyrosinase, was incapable of distinguishing the neurotransmitters because each DA and L-DOPA has identical catechol groups (two OH groups attached to one benzene ring) that similarly react with the enzyme attached to electrodes and generate redox reaction current.
Recently the use of cyclodextrins has been proven to detect dopamine (DA) despite the interference of ascorbic acid (AA) in a mixture [31–33]. Ascorbic acid has been known to interfere the detection of dopamine due to the overlap of the oxidation potentials and much higher (>100 times) concentrations. Based on these previous results, it was reasonably hypothesized that the proposed approach would properly function even in the presence of ascorbic acids. Thus, in this paper we report the simultaneous and quantitative detection approach to distinguish L-tyrosine, L-DOPA and Dopamine in the absence of ascorbic acids as the proof-of-concept. Particularly, we report the structure, fabrication and characterization of a micro neurotransmitter sensor as the proof-of-concept device, with particular foci on detecting DA as well as its closest pre-derivatives. The details will be reported of (1) microelectrode array fabrication, (2) electrode functionalization, (3) sensing sensitivity and selectivity and (4) simultaneous identification of the mixture ratios among L-tyrosine, L-DOPA and Dopamine.
Structure and operation principle
Note that in order to prove the selectivity of the method, one exemplary ascorbic acid (vitamin C) was mixed into a solution for in-vivo-like consideration while measuring the three neurotransmitters.
Micro electrode array fabrication
The microelectrode array was fabricated on a silicon substrate (Figure 3-left). First, a SiO2 layer (200 nm) was grown by wet oxidation, which served as an insulating layer. Second, a TiW/Au layer (30 nm/600 nm) was sputtered on top of the SiO2 layer, where the TiW layer enhanced the adhesion between the Au and SiO2. Third, the deposited metal layers were patterned using photolithography. KI and diluted H2O2 solution were used as metal etchant and Shipley 1813 positive photoresist as an etch mask. Finally, the patterned substrate was cleaned by Acetone, IPA, and DI water. The footprint of each electrode was 7.065 mm2.
The fabricated microelectrode array was functionalized by immobilizing CDs on its surface (Figure 3-right top). First, the fabricated device was soft-baked on a hot plate at 80°C for 10 min to remove residual water molecules on the electrode surface because water molecules can prevent uniform coating of CDs molecules. Second, it was treated in a 1 mM cysteamine for 12 hr, which was prepared in ethanol, for functionalizing amino-terminated monolayer. Third, the modified electrode array was immersed in a 30 mM of chlorobutyric acid (CA) for 2 hr, which was mixed with 0.1 M N-hydroxysuccinimide (NHS) and 0.4 M ethyl (dimethylaminopropyl) carbodiimide (EDC). This procedure enabled to form amide binding between the chlorobutyric acid and amino group, which changed the functionalized electrode to be hydrophilic. Finally, small droplets of the each mixture of 1 mM α-, β-, and γ-CDs and 20 ml of aqueous 4 M KOH were selectively deposited on top of each arrayed electrode at 65°C for 24 hr. The α-, β-, and γ-CDs and all the chemicals that used for CD immobilization such as Cysteamine (95%), CA, NHS, and EDC were purchased from Sigma-Aldrich Co., USA.
The functionalized microelectrode array was connected to an electrochemical measurement system using copper wire. The room temperature curable conductive paste (Silver conductive adhesive paste, Alfa Aesar) was used to bond the wire such that the immobilized CDs could avoid the thermal damage from conventional solder bonding. While curing the conductive paste, the microelectrode array was covered with a water-absorbed filter paper (1002–185, Whatman) to keep the CDs being sufficiently moisturized. After the conductive paste cured, an electrochemical chamber was built on top of the fabricated sensor chip by bonding a polycarbonate tube (inner diameter 10 mm, length 15 mm) with epoxy (Quick Set™, Loctite).
Analysis of the functionalized electrode
The functionalized microelectrode array was analyzed utilizing Fourier transform infrared spectrometry (FT-IR) and X-ray photoelectron spectroscopy (XPS) to confirm the immobilization of α-, β-, and γ-CDs. The FT-IR results identify existence of chemical bindings after each step of electrode functionalization over a wavelength range of 500-4000 nm (Perkin-Elmer Spectrum 100), while the XPS measurements identify the existing atoms on the electrode over electron beam power of 0-1000 eV (Kratos analytical Axis Ultra DLD).
The fabricated neurotransmitter sensor was connected to a potentiostat (Reference 600, Gamry Instruments) and test data was monitored using a labview-based data recording system (VFP600). The electrochemical cell was filled with target catecholamine hormone solution, and the fabricated sensor was tested in random mixtures of catecholamine hormone targets (L-tyrosine, L-DOPA, and DA), concentrations (5 μM-10 mM), and scan rate (1-100 mV/s). First, I-V curves of each electrode were measured in a specific neurotransmitter concentration to measure the individual responses of each CDs immobilized electrode. Next, by varying the neurotransmitter concentrations between 5 μM-10 mM at every decade, the response curves were constructed for each CD electrode and neurotransmitter. The slope of the curve provides the sensitivity of each electrode to each neurotransmitter. Then, from the collected sensitivity coefficients, the matrix of quantification was constructed to identify the individual concentrations from a mixture. Finally, the individual concentrations were extracted from random mixtures of L-tyrosine, L-DOPA and dopamine and compared to the original values, in order to validate the developed sensor and method. Measurement was repeated at least five times, and all the catecholamine hormones were purchased from Sigma-Aldrich Co., USA.
Results and discussion
XPS survey scan measurements, complementing the FT-IR spectra results, indicated that the key elements, such as nitrogen and chlorine, appeared and disappeared as the surface coating progresses. Figure 4-bottom displays the baseline binding energy of each element at the bare Au surface. In comparison, the peak of nitrogen (N 1 s) was observed at the binding energy of 397.1 eV after cysteamine treatment that creates amino termination. The peak for carbon (C 1 s) was also measured throughout the coating steps at the binding energy of 281.6 eV. Then, an additional peak of chlorine (Cl 2p) was measured at the binding energy at 192.6 eV, after chlorobutyric acid treatment that forms chlorine termination. Note that the oxygen (O 1 s) spectrum was observed as a distinctive peak at 529.4 eV, indicating the increased hydrophilicity on the functionalized Au surface. This enables the surface to easily interact with water molecules thus efficiently capture CDs. The peak of chlorine was not measured once the cholorobutyric acid replaced chlorine element with α-CDs. This confirms the existence of the α-CDs on the Au electrode.
Identification of each catecholamine neurotransmitter from a mixture
Experimental measurement-based quantification results of three neurotransmitter mixture cases with and without ascorbic acid (1 mM of vitamin C): (Case I) 0.035, 0.010 and 0.005 mM; (Case II) 0.39, 1.11, and 0.68 mM; and (Case III) 1.10, 3.24, and 2.37 mM of L-tyrosine, L-dopa and dopamine, respectively
Input (No ascorbic acid)
Accuracy output/input (error)
Estimated from measurement
Input (No ascorbic acid)
Accuracy output/input (error)
Estimated from measurement
Input (No ascorbic acid)
Accuracy output/input (error)
Estimated from measurement
A new neurotransmitter sensing technique has been examined to enables quantitative detection of three major catecholamine hormones from a mixture. Specifically, the sensing technique utilized three chemically-modified microelectrodes, respectively, with α-, β- and γ-cyclodextrin (CD) ‘catchers’. The sensing technique relied on different physical matching (size and shape) as well as chemical enticing (stereochemistry and surface charge) properties of α-, β-, and γ-CDs in order to produce statistically different affinities to each target catecholamine neurotransmitters of L-tyrosine, dihydroxyphenylalanine (L-DOPA) and dopamine. The developed neurotransmitter sensing technique has successfully identified three individual catecholamine hormones, without the existence of ascorbic acid, with respective CD ‘catchers’: α-CDs better responded to L-tyrosine and dopamine; β-CDs to L-DOPA and dopamine; and γ-CDs to L-tyrosine, respectively. It also demonstrated the linear sensitivities to each neurotransmitter with logarithmically increasing concentrations range of 5 μM-10 mM. The measurement accuracy changed over time, indicating the stability up to 6 hours from the fabrication. The demonstrated accuracy, over >5 tests near 1 mM of target concentrations, was within 91.2%, and the total device footprint was 13 × 21 mm2.
Jung Hoon Yang received the B.E. degree in chemical engineering from Kyounggi University, Suwon, Korea, in 2001, the M.S. degree in bio-microsystem technology from Korea University, Seoul, Korea, in 2003, and the Ph.D. degree in nano science and engineering from Waseda University, Tokyo, Japan, in 2007. From 2009 to 2010, Dr. Yang was a Postdoctoral Researcher in the department of electrical and computer engineering at the University of Utah, Salt Lake City, where he worked on electrochemical biosensors. He is currently a senior engineer at LG Chemical Development Corp., Daejeon, Korea. His research interests include biosensors, nano-particle functionalizations, MEMS process, microfabrication of carbon materials, enzymatic bio-fuel cell, electrochemistry, and surface chemistry.
Hyun Tae Kim received the B.E. degree in mechanical engineering from Korea University, Seoul, Korea, in 2006, and the M.S. degree in electrical engineering from the University of Utah, Salt Lake City, in 2012. He is currently working toward the Ph.D. degree in mechanical engineering at the University of Maryland, College Park. His research interests include micro sensors and actuators, bioMEMS, and medical robotics.
Hanseup Kim currently holds a position as an USTAR Assistant Professor of Electrical and Computer Engineering, of Mechanical Engineering, and of BioEngineering at the University of Utah in Salt Lake City, Utah since Fall 2009. He received his BS degree in Electrical Engineering from Seoul National University in 1997, and his MS and Ph.D. degrees in Electrical Engineering from the University of Michigan in 2002 and 2006, respectively. Between 2006 and 2009, he remained as a post-doctoral research fellow at the Center for Wireless Integrated MicroSystems (WIMS) in the University of Michigan. His research interests include: micro/nanofabrication technologies and structures, micro sensors and actuators, micropackaging, microfluidics, and bioMEMS.
Prof. Kim was awarded a National Science Foundation Faculty Early CAREER Award 2012 and a DARPA Young Faculty Award in 2011. He received the Best Paper Award with eight other co-authors from the International Conference on Commercialization of Micro and Nano Systems in 2008, the First Prize and the Best Paper Award with three other co-authors from the 38th International Design Automation Conference in 2001, and Rotary Club Ambassador Scholarship in 1999. He has been active in the field of solid state sensors, actuators and microsystems, and has been served as a Technical Program Committee member for MEMS 2013, PowerMEMS 2012, the Hilton Head Workshop 2012, and NanoUtah Conferences.
This work was supported by the Utah Science Technology and Research (USTAR) initiative program. Microfabrication was performed at the Utah NanoFabrication Cleanroom Facility. The authors thank Dr. Michael Free and Mr. Prashant Saraswat in Metallurgical Engineering at the University of Utah for their help on the electrochemical measurement utilizing a potentiostat.
- Alzheimer’s Association (2012) Alz.org, Alzheimer’s fact sheet. : .Available from: , [http://www.alz.org/documents_custom/2012_facts_figures_fact_sheet.pdf]
- Parkinson’s Disease Foundation (2010) Parkinson’s fact sheet. : .Available from: , [http://www.pdf.org/pdf/fs_frequently_asked_questions_10.pdf]
- National Institute of Neurological Disorders and Stroke. (ᅟ) ALS(Amyotrophic Lateral Sclerosis) fact sheet, Huntington’s disease: hope through research. : .Available from: , [http://www.ninds.nih.gov/disorders/huntington/detail_huntington.htm]
- Van Den Eeden SK, Tanner CM, Bernstein AL, Fross RD, Leimpeter A, Bloch DA, Nelson LM: Incidence of Parkinson's disease: variation by age, gender, and race/ethnicity. Am J Epidemiol 2002, 157: 1015–1022. 10.1093/aje/kwg068View ArticleGoogle Scholar
- De Lau LML, Breteler MMB: Epidemiology of Parkinson's disease. Lancet Neurol 2006, 5: 525–535. 10.1016/S1474-4422(06)70471-9View ArticleGoogle Scholar
- Administration on Aging (ᅟ) Older population by age group: 1900–2050 with persons +65. : .Available from: , [http://www.aoa.gov/AoARoot/Aging_Statistics/future_growth/future_growth.aspx#age]
- Dauer W, Przedborski S: Parkinson's disease: mechanisms and models. Neuron 2003, 39: 889–909. 10.1016/S0896-6273(03)00568-3View ArticleGoogle Scholar
- Arias-Carrion O, Poppel E: Dopamine, learning, and reward-seeking behavior. Act Neurobiol Exp 2007, 67: 481–488.Google Scholar
- Shulman JM, De Jager PL, Feany MB: Parkinson's disease: genetics and pathogenesis. Annu Rev Pathol Mech Dis 2011, 6: 193–222. 10.1146/annurev-pathol-011110-130242View ArticleGoogle Scholar
- Zhao H, Zhang Y, Yuan Z: Study on the electrochemical behavior of dopamine with poly (sulfosalicylic acid) modified glassy carbon electrode. Anal Chim Acta 2001, 441: 117–122. 10.1016/S0003-2670(01)01086-8View ArticleGoogle Scholar
- Hjemdahl P: Catecholamine measurements by high-performance liquid chromatography. Am J Physiol 1984, 247: E13-E20.Google Scholar
- Bouchta D, Izaoumen N, Zejli H, Kaoutit ME, Temsamani KR: A novel electrochemical synthesis of poly-3-methylthiophene-γ-cyclodextrin film: Application for the analysis of chlorpromazine and some neurotransmitters. Biosen Bioelectron 2005, 20: 2228–2235. 10.1016/j.bios.2004.12.004View ArticleGoogle Scholar
- Majewska UE, Chmurski K, Biesiada K, Olszyna AR, Bilewicz R: Dopamine oxidation at per (6–‐deoxy–‐6–‐thio)–‐α–‐cyclodextrin monolayer modified gold electrodes. Electroanalysis 2006, 18: 1463–1470. 10.1002/elan.200603556View ArticleGoogle Scholar
- Alarcón-Angeles G, Pérez-López B, Palomar-Pardave M, Ramírez-Silva MT, Alegret S, Merkoçi A: Enhanced host–guest electrochemical recognition of dopamine using cyclodextrin in the presence of carbon nanotubes. Carbon 2008, 46: 898–906. 10.1016/j.carbon.2008.02.025View ArticleGoogle Scholar
- Yang J-H, Park JW, Kim H: Simultaneous detection of catecholamine neurotransmitters utilizing a cyclodextrin-based micro electrode array. MicroTAS 2010, 2010: 599–601.Google Scholar
- Yang J-H, Kim H-T, Park JW, Kim H: Cyclodextrin-based micro neurotransmitter sensor for selective catecholamine hormone detection. Transducers 2011, 2011: 2102–2105.Google Scholar
- Tang H, Lin P, Chan HLW, Yan F: Highly sensitive dopamine biosensors based on organic electrochemical transistors. Biosen Bioelectron 2011, 26: 4559–4563. 10.1016/j.bios.2011.05.025View ArticleGoogle Scholar
- Pagel P, Blome J, Wolf HU: High-performance liquid chromatographic separation and measurement of various biogenic compounds possibly involved in the pathomechanism of Parkinson’s disease. J Chromatogr B 2000, 746: 297–304. 10.1016/S0378-4347(00)00348-0View ArticleGoogle Scholar
- Wood AT, Hall MR: Reversed-phase high-performance liquid chromatography of catecholamines and indoleamines using a simple gradient solvent system and native fluorescence detection. J Chromatogr B 2000, 744: 221–225. 10.1016/S0378-4347(00)00249-8View ArticleGoogle Scholar
- Wang HY, Sun Y, Tang B: Study on fluorescence property of dopamine and determination of dopamine by fluorimetry. Talanta 2002, 57: 899–907. 10.1016/S0039-9140(02)00123-6View ArticleGoogle Scholar
- Balaji J, Reddy CS, Kaushalya SK, Maiti S: Microfluorometric detection of catecholamines with multiphoton-excited fluorescence. Appl Optics 2004, 43: 2412–2417. 10.1364/AO.43.002412View ArticleGoogle Scholar
- Tsunoda M, Takezawa K, Santa T, Imai K: Simultaneous automatic determination of catecholamines and their 3-o-methyl metabolites in rat plasma by high-performance liquid chromatography using peroxyoxalate chemiluminescence reaction. Anal Biochem 1999, 269: 386–392. 10.1006/abio.1999.4043View ArticleGoogle Scholar
- Ragab GH, Nohta H, Zaitsu K: Chemiluminescence determination of catecholamines in human blood plasma using 1,2-bis(3-chlorophenyl)ethylenediamine as pre-column derivatizing reagent for liquid chromatography. Anal Chim Acta 2000, 403: 155–160. 10.1016/S0003-2670(99)00637-6View ArticleGoogle Scholar
- Nalewajko E, Wiszowata A, Kojli A: Determination of catecholamines by flow-injection analysis and high-performance liquid chromatography with chemiluminescence detection. J Pharm Biomed Anal 2007, 43: 1673–1681. 10.1016/j.jpba.2006.12.021View ArticleGoogle Scholar
- Hows MEP, Lacroix L, Heidbreder C, Organ AJ, Shah AJ: High-performance liquid chromatography/tandem mass spectrometric assay for the simultaneous measurement of dopamine, norepinephrine, 5-hydroxytryptamine and cocaine in biological samples. J Neurosci Methods 2004, 138: 123–132. 10.1016/j.jneumeth.2004.03.021View ArticleGoogle Scholar
- Tsunoda M: Recent advances in methods for the analysis of catecholamines and their metabolites. Anal Bioanal Chem 2006, 386: 506–514. 10.1007/s00216-006-0675-zView ArticleGoogle Scholar
- Ferancova A, Korgova E, Labuda J, Zima J, Barek J: Cyclodextrin modified carbon paste based electrodes as sensors for the determination of carcinogenic polycyclic aromatic amines. Electroanalysis 2002, 23: 1668–1673. 10.1002/elan.200290009View ArticleGoogle Scholar
- Bouzitoun M, Mlika R, Gam H, Ouada HB, Majdoub M, Sfihi H: A non-water-soluble modified β-cyclodextrin for sensitive electrode. Mater Sci Eng C 2006, 26: 481–485. 10.1016/j.msec.2005.10.065View ArticleGoogle Scholar
- Li W, Jin G, Chen H, Kong J: Highly sensitive and reproducible cyclodextrin-modified gold electrodes for probing trace lead in blood. Talanta 2009, 78: 717–722. 10.1016/j.talanta.2008.12.030View ArticleGoogle Scholar
- Tredici I, Merli D, Zavarise F, Profumo A: α-Cyclodextrins chemically modified gold electrode for the determination of nitroaromatic compounds. J Electroanal Chem 2010, 645: 22–27. 10.1016/j.jelechem.2010.03.036View ArticleGoogle Scholar
- Fukuda T, Maeda Y, Kitano H: Stereoselective inclusion of DOPA derivatives by a self-assembled monolayer of thiolated cyclodextrin on a gold electrode. Langmuir 1999, 15: 1887–1890. 10.1021/la981269nView ArticleGoogle Scholar
- Fragoso A, Almirall E, Cao R, Echegoyen L, González-Jonte R: A supramolecular approach to the selective detection of dopamine in the presence of ascorbate. Chem Commun 2004, 19: 2230–2231. 10.1039/b407792jView ArticleGoogle Scholar
- Palomar-Pardavé M, Alarcón-Ángeles G, Ramírez-Silva MT: Electrochemical and spectrophotometric determination of the formation constants of the ascorbic acid-β-cyclodextrin and dopamine-β-cyclodextrin inclusion complexes. J Incl Phenom Macrocycl Chem 2011, 69: 91–99. 10.1007/s10847-010-9818-0View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.