Blog

Biosensors and Bioelectronics 25 (2010) 2296-2301 Contents lists available at ScienceDirect Biosensors and Bioelectronics journal homepage: www. elsevier. com/locate/bios Dithiobis(succinimidyl propionate) modified gold microarray electrode based electrochemical immunosensor for ultrasensitive detection of cortisol Sunil K. Arya , Ganna Chornokur a , Manju Venugopal b , Shekhar Bhansali a,’ Bio-MEMS and Micro Engineering, Universi EN3 118, ampa, FL 1 Swip nent page of Electrical E. Fowler Avenue, ded Therapeutics Inc. 5835 Peachtree Corners East, United States article info abstract suite D, Norcross, GA 30092, Gold microelectrode arrays functionalized with dithiobis(succinimidyl propionate) self-assembled monolayer (SAM) have been used to fabricate an ultrasensitive, disposable, electrochemical cortisol immunosensor. Cortisol specific monoclonal antibody (C-Mab) was covalently immobilized on the surface of gold microelectrode array and the sensors were exposed to solutions with different cortisol concentration.

After C- -lal Studia biosensor was successfully used for the measurement of cortisol in interstitial fluid in vitro. This research establishes the feasibility of using impedance based biosensor architecture for disposable, earable cortisol detector. 0 2010 Elsevier B. V. AII rights reserved. Article history: Received 22 December 2009 Received in revised form 9 March 2010 Accepted 10 March 2010 Available online 18 March 2010 Keywords: Cortisol Self-assembled monolayer Electrochemical impedance Immunosensor Dithiobis(succinimidyl propionate) Disposable biosensor 1.

Introduction Cortisol, a steroid hormone, is a biomarker for numerous diseases and is important for the regulation of blood pressure, glucose levels, and carbohydrate metabolism, within the physiologcal limit (Zhou et al. , 2004; Tai and welch, 2004; Stevens et al. , 2008). Abnormal increase in cortisol level inhibits inflammation, depresses immune system, increases fatty and amino acid levels in blood.

In addition, while excess cortisol levels contribute to the development of Cushings disease with the symptoms of obesity, fatigue and bone fragility, decreased cortisol levels lead to Addison’s disease which is manifested by weight loss, fatigue, and darkening of skin folds and scars (Zhou et al. , 2004; Stevens et ala, 2008). Cortisol in blood primarily exists in a bound state with corticosteroid-binding globulin (CBG). It has been reported that while nearly 90% of cortisol is bound, about 0% of it exists in a free biologically active form (Cook et al. , 1997) and can also be 21 it exists in a free biologically active form (Cook et al. 1997) and can also be found in bodily fluids like saliva, urine and interstitial fluids (ISF). Normal leve’ of cortisol in serum is generally in the range of 100-500 nM. There is a good correlation in the amount of free cortisol present in the saliva and the total cortisol present in the blood (Gozansky et al. , 2005), however, free cortisol levels in saliva Corresponding authors. Tel. : +1 8139747942. E-mail addresses: sunilarya333@gmail. com (S. K. Arya), bhansali@usf. edu (S. Bhansali). 956-5663/$ – see front matter C 201 0 Elsevier B. V. All rights reserved. 6 and urine are up to 100-fold lower than in serum (Morineau et al. 1997; Levine et ala, 2007). There has been growing interest in measurement of cortisol to establish whether cortisol variation can be used as a precursor to medically and psychologically relevant events, the most recent affliction being post-traumatic stress disorder (PTSD) (Hauer et al. , 2009; Lindley et al. , 2004). Measurement of cortisol requires reliable and accurate collection of any body fluid, e. g. , blood, saliva, or urine. Blood collection, however, requires trained medical personnel. Additionally, the trauma of venipuncture results in much reduced patient participation.

Hence, researchers have been exploring non- invasive/minimally invasive technlques that ensure: (a) h gh fidelity samples (cortisol is a small molecule that diffuses rapidly) and (b) minimal patient participation in the sample handling process molecule that diffuses rapidly) and (b) minimal patient participation in the sample handling process and trauma. These efforts have resulted in researchers exploring completely non- invasive sampling: (a) saliva or (b) urine for the free cortisol concentration estimation (Cook et al. , 1 997; Mitchell et al. 2009; Rovve et al. , 2007; Gatti et al. , 2005).

The need for the patient to collect samples at odd hours however adds to additional stress, thereby adding a bias constituent to the results. Collection of ISF or transdermal body fluid (TDF) IS a technique, which while minimally invasive, does not require the patient’s compliance as a simple harvesting device is attached to the patients. These harvesting devices can harvest ISF continually over 3-4 days without any additional compliance from the patient (Nindl et al. , 2006). The use of ISF collection approach necessitates development of a wearable biosensing technique that can ccurately measure cortisol S.

K. Arya et al. / Biosensors and Bioelectronics 25 (2010) 2296 2301 2297 in ISF. This paper reports on the development of such a cortisol biosensor. The ISF is an extra cellular fluid that surrounds the cells in the human body and consists of small and moderate sized molecules, including glucose, ethanol, and cortisol. The homeostatic feedback loop in the body ensures that these molecules have a direct correlation to the concentration of molecules in blood (Stout et al. , 1999; Bantle and Thomas, 1997; Knoll et al. , 2002). ISF is especially attractive for cortisol mo 4 21

Bantle and Thomas, 1997; Knoll et al. , 2002). ISF is especially attractive for cortisol monitoring, as it can be drawn continuously from the dermis through an ablated stratum corneum by simply applying a small amount of vacuum. Currently, in clinical practice, total cortisol, which is the sum offree and protein bound fractions, is measured, however, free cortisol is the only biologically active fraction (Levine et al. , 2007; Stevens et al. , 2008). Hence, in order to diagnose and properly treat cortisol- related conditions, regular estimation of free cortisol IS required.

Current techniques for estimation of free cortisol, e. g. High Performance Liquid Chromatography (HPLC) (Oka et al. , 1987); fluorometric assay (Appel et al. , 2005); and reverse phase chromatography (Gatti et al. , 2005); require long analysis time, are expensive, and cannot be implemented at point of care. Alternate techniques of cortisol detection including radioimmunoassay (RIA), flow immunoassay and enzyme-linked immunosorbent assay (ELISA) (Cook et al. , 1997; Zhou et al. , 2004; Schmalzing et al. , 1995; Kaptein et al. , 1997; Koutny et al. , 1996; Sarkar et al. , 2007) are reliable and accurate.

However, these approaches are laborious, time-consuming, require large sample volume and nvolve the use of radioisotopes. Free cortisol can be measured experimentally by equilibrium dialysis and ultrafiltration techniques (Jerkunica et al. , 1980; Vogeser et al. , 2007), or it can be derived by calculation from Coolens equations (Dorin et al. , 2009). However, these methods a can be derived by calculation from Coolens equations (Dorin et al. , 2009). However, these methods are cumbersome as estimation requires assaying of both total cortisol and CBG on the same sample requiring 5 ml of blood per assay.

Recently, various biosensors have been reported for cortisol estimation (Stevens et al. 2008; Mitchell et al. , 2009; Kumar et al. , 2007; Sun et al. , 2008; Zhou et al. , 2004). However, all of these involve complicated system, tedious fabrication, indirect cortisol estimation, measurement at very high voltages or modification of analyte itself. Biosensor development using self-assembled monolayers (SAM) and electrochemical techniques have gained increased attention in recent years (Arya et al. , 2007a,b; Valera et al. , 2007; Geng et al. , 2008; Chen et al. , 2006; Maalouf et al. 2007; Khan and Dhayal, 2009; Loyprasert et al. , 2008, 2010; Kim et al. , 2010; Weng et al. , in press; Ng et al. 2010). SAM formation allows the binding of biomolecules in the closest vicinity of electrode surface, and electrochemlcal detection technique results in enhanced sensitivity, fast response, low cost and portability. The use of electrochemical impedance spectroscopy (EIS) coupled with SAMS is promising as they allow for the possibility of recording of direct signatures of bio-recognition events, occurring at the electrode surfaces through changes in capacitance and resistance (Azcon et ala, 2008).

In EIS, when a biological receptor binds to its counterpart, the measurement of changed impedance enables irect and label-fr receptor binds to its counterpart, the measurement of changed impedance enables direct and label-free measurement. The data points are generated using a small perturbation in signal, that reduces the matrix interference (Katz and Willner, 2003; Barsoukov and MacDonald, 2005). The main drawback of EIS, however, is decreased sensitivity and lower detection limits. This can be overcome through the use of microelectrodes.

Microelectrodes also lead to lower detection limits and minimization of an interference effect coming from nontarget analytes (Laschi and Mascini, 2006; Hagelsieb et al. , 2007; Thomas t al. , 2004; Radke and Alocilja, 2005; Valera et al. , 2007). n this work, we report on a successful fabrication of a label- free impedance based immunosensor for ultrasensitive cortisol testing. SAM formation utilized the dithiobis(succinimidyl propionate) (DTSP), which was used for an antibody immobilization onto Au microarray electrode.

The sensor was tested with ISF and the measurements were compared to ELISA. This biosensor enabled cortisol detection up to 1 PM within the 40 min analysis time 2. Materials and methods 2. 1. Chemicals and reagents Dithiobis(succinimidyl proplonate) (DTSP) and sodium orohydride (NaBH4 ) were purchased from ThermoFisher Scientific. Monoclonal cortisol antibody (anti-cortisol, C-Mab) 2330-4809 was procured from Abd serotec. Phosphate buffered saline and hydrocortisone (cortisol) were purchased from Sigma- Aldrich. SlJ8 resist was purchased from Microchem Corp.

All other chemicals were of analytic Sigma-Aldrich. SI. J8 resist was purchased from Microchem Corp. All other chemicals were of analytical grade and were used without further purification. Working solutions of hydrocortisone were prepared by dilution in phosphate buffer saline (PBS) (10 mM, pH 7. 4). Commercially avallable Cortisol Luminescence Competitive Immunoassay manufactured by IBI_, Hamburg was used for the human ISF sample analysis which was performed by the Systems Laboratory (Dr. Clemens Kirschbaum) in Dresden, Germany. 2. 2.

Measurement and apparatus Electrochemical impedance (EIS) was utilized to characterize the EA/C-Mab/DTSP/ Au bio-electrodes and to estimate cortisol as a function of its concentration. EIS measurements were carried out at equilibrium potential called open circuit potential (OCP) without bias voltage in the frequency range of 0. 1—105 Hz with a 5 mv amplitude using Autolab Potentiostat/Galvanostat (Eco Chemie, Netherlands). Three-electrode cell configuration with AglAgCl as reference electrode and the gold electrode as a counter electrode was used for measurements.

EIS measurements were carried out using 30 1 of phosphate buffer saline (PBS) solution (1 0 mM, pH 7. 4) containing mixture of 5 mM (Ferrocyanide) and 6 5 mM of (Ferricyanide) i. e. 5 rnM F4CN)6 6 as a redox probe. Using the redox probe (5 mM change in charge transfer resistance at electrode/electrolyte interface has been investigated in electrochemical impedance. 2. 3. Fabrication of test chip he biosensor chips were fabricated on an oxidized 4 SI impedance. 2. . Fabrication of test chip The biosensor chips were fabricated on an oxidized 4 silicon wafer using standard photolithography techniques (Sun et ala, 2008).

Briefly, CM Au (200/2000 A) and crmg (200/3000 A) layers deposited using evaporation and were patterned, through liftoff. Gold microelectrode arrays with 5 m wide electrode fingers at a pitch of 15 m were used. As a final step, SU8 chamber patterned around the electrodes using SU8 50 to create a sample well around these electrodes (Fig. 1a). Further SlJ8 was hard baked at 200 • C to improve its resistance against hard solvents like acetone. 2. 4. SAM preparation and antibody immobilization The gold microelectrode array chips were pre-cleaned with acetone, isopropyl alcohol, and de-ionized water.

Next they were exposed to 2 mg ml—l solution of DTSP in acetone for 1 h for SAM formation. DTSP solution was first reduced using NaBH4 and then dispensed on the pre-cleaned chips at room temperature for SAM formation. The DTSP SAM modified electrodes were then rinsed with acetone to remove any unbound DTSP followed by 3-14- F4CN)6 2298 S. K. Arya et al. / Biosensors and Bioelectronics 25 (2010) 2296- Fig. 1 . Schematic for (a) gold microarray chip and (b) EA/C-Mab/ DTSP/Au bio-electrode fabrication. water rinsing and then utilized for antibody immobilization.

Cortisol antibodies were covalently attached to DTSP self- assembled monolayer by incubating the electrode in 30 1 ofl g ml-l antibody in phosphate buffer saline (PBS) solution (1 0 mM, in 30 1 ofl g ml-l antibody in phosphate buffer saline (PBS) solution (10 mM, pH 7. 4) for 1 h. Covalent binding (amide bond formation) results from the facile reaction between amino group of antibody and reactive succinimidyl group of the DTSP on the SAM surface. The sensor (C-Mab/DTSP/Au) was washed thoroughly with phosphate buffer (10 mM, pH 7. ) to remove any unbound biomolecules followed by a 10 min washing with 10% ethanolamine solution in PBS.

Ethanol amine was used to block unreacted succinimidyl group on DTSP SAM and to remove extra unbound antibodies onto the electrode surface. Fig. 1 schematically illustrates (a) gold microarray chip and (b) EA/C-Mab/DTSP/ALl bjo-electrode fabrication. The fabricated blo-electrodes were characterized using the electrochemical impedance technique. 3. Results and discussion 3. 1. Electrochemical impedance studies Electrochemical impedance spectroscopy (EIS) is a technique that utilizes an application of eriodic small amplitude AC signal over wide frequency range to get electrical response.

It is very sensitive and can be used as a characterization tool for studying the charge transfer processes occurring at the sensor-sample interface. Nyquist plots of impedance spectra In present studles have been exploited to study (i) charge transfer change at sensor-solution interfaces after DTSP SAM formation, C-Mab binding and ethanol amine blocking, (ii) the change of charge resistance with changing concentration of cortisol and to measure association constant for the cortisol-C- Mab interaction 0 DF 21