Anal Bioanal Chem DOI 10.1007/s00216-014-7882-9
RESEARCH PAPER
Zinc oxide nanoparticles based microfluidic immunosensor applied in congenital hypothyroidism screening Marco A. Seia & Sirley V. Pereira & Martin A. FernándezBaldo & Irma E. De Vito & Julio Raba & Germán A. Messina
Received: 14 March 2014 / Revised: 5 May 2014 / Accepted: 7 May 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract In this article, we present an innovative approach for congenital hypothyroidism (CHT) screening. This pathology is the most common preventable cause of mental retardation, affecting newborns around the world. Its consequences could be avoided with an early diagnosis through the thyrotropin (TSH) level measurement. To accomplish the determination of TSH, synthesized zinc oxide (ZnO) nanobeads (NBs) covered by chitosan (CH), ZnO-CH NBs, were covalently attached to the central channel of the designed microfluidic device. These beads were employed as platform for anti-TSH monoclonal antibody immobilization to specifically recognize and capture TSH in neonatal samples without any special pretreatment. Afterwards, the amount of this trapped hormone was quantified by horseradish peroxidase (HRP)conjugated anti-TSH antibody. HRP reacted with its enzymatic substrate in a redox process, which resulted in the appearance of a current whose magnitude was directly proportional to the level of TSH in the neonatal sample. The structure and morphology of synthesized ZnO-CH NBs were characterized by scanning electron microscopy (SEM) and X-ray diffraction (XRD). The calculated detection limits for electrochemical detection and the enzyme-linked immunosorbent assay procedure were 0.00087 μUI mL−1 and 0.015 μUI mL−1, respectively, and the within- and between-assay coefficients of variation were below 6.31 % for the proposed method. According to the cut-off value for TSH neonatal screening, a Electronic supplementary material The online version of this article (doi:10.1007/s00216-014-7882-9) contains supplementary material, which is available to authorized users. M. A. Seia : S. V. Pereira : M. A. Fernández-Baldo : I. E. De Vito : J. Raba : G. A. Messina (*) INQUISAL, Department of Chemistry, National University of San Luis, CONICET, Chacabuco 917. D5700BWS, San Luis, Argentina e-mail:
[email protected]
reasonably good limit of detection was achieved. These above-mentioned features make the system advantageous for routine clinical analysis adaptation. Keywords Hypothyroidism . Neonatal screening . Immunoassay . Electrochemical . Microfluidic system . Nanobeads
Introduction Congenital hypothyroidism (CHT) is an endocrine disorder with high incidence in newborns worldwide and the most common preventable cause of mental retardation [1]. Its main characteristic is a partial or total absence of thyroid hormone, which is especially necessary in the first 2–3 years of life. The prolonged deficiency of this hormone affects almost all life processes, but the most important for fetus and infant is the irreversible damage of the central nervous system. Children with CHT can present specific problems in the development, such as defective motor skills, learning difficulties, language, attention, memory, and behavioral concerns [2–4]. Systematic screening programs for thyroid function were introduced in many countries in the 1970s, allowing early detection and implementation of thyroid hormone therapy [5, 6]. The initial screening method was the T4 measurement in heel-prick blood samples. This has been superseded by thyrotropin (TSH) measurement in most programs around the world [7] because TSH screening is more cost effective in reducing false-positive and falsenegative cases for mass screening [8, 9]. In the newborn screening for CTH, a TSH cut-off point of 10 μU mL−1 allows children who would otherwise have escaped diagnosis to be identifed [10].
M.A. Seia et al.
To accomplish the determination of TSH, several immunoassay techniques have been used. Nowadays, great attention is paid to using electrochemical immunoassay (EI) [11–15], which has become one of the predominant analytical techniques due to its high sensitivity and good compatibility with advanced micromachining technologies [16–18]. Typical EI offers the possibility of being incorporated in a microfluidic system, which has been developed to perform a variety of biomedical and chemical analysis [19, 20]. Owing to the extremely small sample volume required in a microfluidic channel, a highly sensitive detection provided by the electrochemical techniques is of fundamental importance, either for the continuous monitoring of a reaction, as well as for the determination of analytes [19, 20]. The employment of EI and microfluidic technology allows the incorporation of different nanomaterials as platform of the specific immunoreactants, which, through an appropriate immobilization process, enable the fabrication of a biorecognition layer with desirable properties (i.e., large loading, wellpreserved bioactivity, and good reversibility) [21]. One of the solid supports employed are nanobeads (NBs). There are many benefits in the use of these NBs. The main advantage is the increase in the surface to volume ratio, whose direct consequence is the increment of the assay sensitivity because of the higher efficiency of interactions between samples and reagents [21]. The preference of zinc oxide has gained impetus during last few years because of the possibility of its relatively simple synthesis into nanoscale structures. Nanostructures like rods and particles have become the most promising research material because of their wide range of applications as biosensors, gas sensors, solar cells, ceramics, nanogenerators, photo detectors, catalysts, active fillers for rubber and plastic, UVabsorbers in cosmetics, and antivirus agent in coating, pigments, optical materials, cosmetics, photocatalytic, electrical and optoelectronic processes, systems, additives in many industrial products and in treatment of water and waste water also [22]. In the present work, ZnO NBs have been synthesized by solution-based approach and characterized by various techniques. Synthesized ZnO NBs were covalently incorporated in a designed microfluidic system and then functionalized with anti-TSH capture antibodies for neonatal TSH determination. Initially, the TSH protein in neonatal samples was captured by anti-TSH primary antibodies. Afterwards, the bounded hormone is recognized by specific secondary antibodies. Subsequently, horseradish peroxidase (HRP) enzyme-labeled secondary antibodies reacted with its enzymatic substrate, generating a product, which suffered an oxidation on the electrode surface. This redox process resulted in the appearance of a current whose magnitude was directly proportional to the level of TSH in the neonatal sample. To conclude, the
developed system represents an attractive and efficient analytical tool to be applied in the clinical diagnosis field.
Materials Reagents and solutions The following materials and chemicals were used as supplied. Sylgard 184 and AZ4330 photoresist (PR) were obtained from Dow Corning (Midland, MI, USA) and Clariant Corporation (Sommerville, NJ, USA), respectively. Glutaraldehyde (25 % aqueous solution) and hydrogen peroxide 30 % were purchased from Merck, Darmstadt. Chitosan (high purity; Mv, 140,000– 220,000), zinc nitrate tetrahydrate purum p.a. (crystallized, ≥ 99.0 % KT), hydrofluoric acid (HF), 3-aminopropyl triethoxysilane (APTES), and 4-tert-butylcatechol (4-TBC) were purchased from Sigma-Aldrich. The enzyme-linked immunosorbent assay (ELISA) test kit for the quantitative determination of TSH antibodies was purchased from RADIM S.p.A. Pomezia (Roma) Italia and was used in accordance with the manufacturer’s instructions. Mouse monoclonal antibody to TSH (2 mg mL−1) and HRP-conjugated anti-TSH antibody (0.9 mg mL−1) was purchased from Abcam (USA). All buffer solutions were prepared with Milli-Q water. Apparatus Amperometric measurements were performed using the BAS LC 4 C (Bioanalytical Systems, West Lafayette, IN, USA). The BAS 100 B electrochemical analyzer Bioanalytical Systems) was used for cyclic voltammetric analysis. The gold layer electrode was deposited at central channel (CC) by sputtering (SPI-Module Sputter Coater with Etch mode, Structure probe Inc., West Chester, PA, USA), and the gold thickness electrode was measured using a Quartz Crystal Thickness Monitor model 12161 (Structure Probe Inc., West Chester, PA, USA). The structure and morphology of prepared ZnO-CH NBs was characterized by a LEO 1450VP scanning electron microscope (SEM). A syringe pumps system (Baby Bee Syringe Pump, Bioanalytical Systems) was used for pumping, sample introduction and stopping flow. All solutions and reagent temperatures were conditioned before the experiment using a Vicking Masson II laboratory water bath (Vicking SRL, Buenos Aires, Argentina). Absorbance was detected by a Bio-Rad Benchmark microplate reader (Japan) and Beckman DU 520 general ultraviolet–visible spectrophotometer. All pH measurements were made with an Orion Expandable Ion Analyzer (Orion Research Inc., Cambridge, MA, USA) model EA 940 equipped with a glass combination electrode (Orion Research Inc.).
Microfluidic immunosensor for CHT screening
Fig. 1 Microfabrication process of glass microfluidoc chip with sealing based on the use of a thin layer of PDMS. Wet chemical etching of glass with 20 % HF for 4 min under continuous stirring: spin coating of a PDMS and photoresist layer over flat glass surface at 3,000 rpm during 15 s; plasma-oxidized treatment during 1 min
Methods Obtaining procedure for nanostructured particles: ZnO-CH NBs ZnO nanoparticles were synthesized by wet chemical method. Briefly, a 0.9 M NaOH aqueous solution was added under high-speed constant stirring and drop by drop to 0.5 M aqueous ethanol solution of zinc (Zn(NO3)2·4H2O). After addition of NaOH, the reaction was allowed to progress for 2 h. Then, it was kept standing overnight, and the supernatant was carefully separated. The remaining solution was centrifuged for 10 min, and the precipitate was removed. Finally, precipitated ZnO NBs were cleaned with deionized water and dried in air atmosphere at about 60 °C. During drying, Zn(OH)2 is converted into ZnO [23]. At the same time, a chitosan solution (0.5 %) was obtained through the dissolution of 0.025 g of CH in 5 mL of 0.05 mol L−1 acetate buffer solution. Then, 0.25 mg of ZnO NBs was dispersed in the CH solution. The mixture was stirred for 8 h at room temperature. Finally, a highly dispersed viscous preparation was achieved [24]. Microchip construction Figure 1 shows the developed microchip, whose design consisted of a T-type format with a CC (60 mm length and 100 μm diameter) and accessory channels (15 mm length and 70 μm diameter). The main body of the sensor was made of glass. The procedure for glass microchip fabrication (Fig. 1)
was carried out according to [25] with the following modifications: Firstly, the device layout was drawn using CorelDraw software version 11.0 (Corel Corporation) and printed on a high-resolution transparency film to be used as mask in the photolithographic step. The printed mask was placed on the top of a glass wafer previously coated with a 5-μm layer of AZ4330 PR. The substrate was exposed to UV radiation for 30 s and revealed in AZ 400K developer solution for 2 min. Glass channels were obtained employing an etching solution consisting of 20 % HF for 4 min under continuous stirring. The etching rate was 8±1 μm min−1. Following the etching step, substrates were rinsed with deionized water, and the photoresist layer was removed with acetone. To access the microfluidic network, holes were drilled on glass-etched channels with a Dremel tool (MultiPro 395JU model, USA) using 1-mm diamond drill bits. To achieve the final chip format, another glass plate was spin-coated with a thin polydimethylsiloxane (PDMS) layer at 3,000 rpm during 10 s. PDMS was prepared by a 10:1 mixture of Sylgard 184 elastomer and curing agent. The thickness of this layer was 50 μm. Before sealing, PDMS layer was cured at 100 °C during 5 min in a hot plate. Glass channels and PDMS-coated glass substrate were placed in an oxygen plasma cleaner (Plasma Technology PLAB SE80 plasma cleaner) and oxidized for 1 min. The two pieces were brought into contact immediately after the plasma treatment, obtaining a strong irreversible sealing. The final device format was achieved in
