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An immunoelectrochemical sensor for salivary cortisol measurement
Sensors and Actuators B 133 (2008) 533–537
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
An immunoelectrochemical sensor for salivary cortisol measurement Ke Sun, Niranjan Ramgir, Shekhar Bhansali ∗ Bio-MEMS and Microsystem Lab, Department of Electrical Engineering, University of South Florida, Tampa, FL 33620, United States
a r t i c l e
i n f o
Article history: Received 14 December 2007 Received in revised form 14 March 2008 Accepted 19 March 2008 Available online 28 March 2008 Keywords: Salivary cortisol Immunoelectrochemical sensor Cyclic voltammetry p-Nitrophenyle phosphate
a b s t r a c t An immunoelectrochemical sensor based on alkaline phosphatase (AP) enzyme for determination of salivary cortisol concentration is reported. Microfabricated Au electrodes encased in a microfluidic chamber were functionalized to immobilize the cortisol capture antibodies. The reaction product p-nitrophenol (pNP) generated by reacting the AP enzyme attached to the cortisol antigen via detector antibodies with the p-nitrophenyl phosphate (pNPP) solution. pNP was detected as an oxidative peak between 0.9 and 1.1 V (vs. Ag pseudo-reference electrode) using cyclic voltammetry (CV) at room temperature. The magnitude of the peak varies linearly with the cortisol concentration, and was used to quantify the concentration of cortisol in real saliva samples. This immunoelectrochemical detection method accurately measured cortisol in the collected saliva samples achieved to a concentration of 0.76 nmol/L with an incubation time of 10 min. We demonstrate successfully the approach for establishing diurnal cortisol concentration behavior for clinical purposes with numerous advantages: a much higher throughput capability, significantly lower amounts of the sample, sub-pmol/L range sensitivity, higher resolution at low mass ranges, and easy to use. © 2008 Elsevier B.V. All rights reserved.
1. Introduction As evaluating sweat or tear cortisol is only of theoretical importance and urinary cortisol of falling interest, salivary cortisol has become a valuable tool for both basic scientists and clinicians [1]. Numerous significant advantages over the blood cortisol evaluation have resulted in a steadily increasing interest in salivary cortisol measurement. It reflects the unbound fraction of circulating cortisol, and therefore it is not affected by alterations in cortisol-binding globulin [1]. Salivary cortisol concentration is not affected by saliva flow rate, making it a reliable end point. The advantages of collection at home at bedtime are obvious: the cost of an office or hospital visit is eliminated, and potential increases in cortisol levels because of anxiety or an unfamiliar environment is mitigated. The ease of sampling allows for sequential collection and hence the opportunity to examine cortisol levels as a function of time [2] and activity history [3]. Recently, researches on identification of abnormalities in salivary cortisol level indicating varieties of illnesses are executed. The salivary cortisol response to awakening in chronic fatigue syndrome (CFS) has been described as a non-invasive test of the capacity of the hypothalamic–pituitary–adrenal (HPA) axis to respond to stress [4]. Neuroendocrine profiles were obtained for subjects experiencing military survival training using saliva sam-
∗ Corresponding author. E-mail address: [email protected] (S. Bhansali). 0925-4005/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2008.03.018
ples collected at baseline and at four subsequent “stress” points [5]. Numerous other mind–body interrelationships have been explored using salivary hormone levels. The circadian rhythm of salivary cortisol levels has been studied in relationship to sleep activity in preterm infants [6]. Measurement of nighttime salivary cortisol levels provides excellent specificity and sensitivity to screen for Cushing’s syndrome [1]. The salivary cortisol test had better sensitivity and specificity for this syndrome than other detection methods, including a nighttime serum cortisol test and the dexamethasone suppression test. Abnormal circadian rhythms of salivary cortisol levels have also been observed in breast cancer patients [7]. Currently accepted methods for the clinical evaluation of stress biomarkers in saliva from patients include fluorescence enzymelinked immunosorbent assays (ELISA) or radioimmunoassay (RIA). However, these methods suffer from several disadvantages including interference from other molecules in the fluid environment, inefficient absorption, and lack of consistency in laboratory accuracy. Fluorescent polarization (FP) technology which has distinct advantages over previous methods was patented by Cullum et al. [8]. Similarly, UV detector is lack of sensitivity and this approach is not applicable for many antigens without strong UV absorbance. Laser-induced fluorescence (LIF) detection is a more general approach to improve sensitivity. However, it is difficult to use most laser instruments in samples that have not been purified or pretreated. Moreover, due to high cost, complex maintenance, low limit of detection (LOD), and because most biological fluids are strongly
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K. Sun et al. / Sensors and Actuators B 133 (2008) 533–537
luminescent when excited by the laser in the blue or green region of the spectrum, applications of LIF are limited. Electrochemical sensors with detection based on a change in an electrical quantity such as voltage, current, impedance or charge by a biochemical reaction are suitable for integration and microfabrication. In fact, miniaturized electrochemical sensors that combine the specificity and selectivity of biological components with the analytical advantages of electrochemical detection have been investigated [9,10]. Accompanied by the immunoassay methodology, label-free detection and quantification of the immune complex are enabled by modern technologies in immunoelectrochemical sensors. The fundamental of all immunoassay is the specificity of the molecular recognition of antigens by antibodies to form a stable complex. The use of high specific antibodies as bioaffinity interface for immunosensors greatly improved the selectivity, moreover, allows the detection with significantly low amounts of the sample and sub-pmol/L range sensitivity [11–14]. In the present work we report an immunoelectrochemical sensor based on alkaline phosphatase (AP) enzyme for determination of salivary cortisol concentration. Microfabricated Au electrodes encased in a microfluidic chamber were functionalized to immobilize the cortisol capture antibodies. AP enzyme attached to the cortisol antigen via detector antibodies generated p-nitrophenol (pNP) by reacting with the p-nitrophenyl phosphate (pNPP) solution. An oxidative peak of resulted pNP between 0.9 and 1.1 V (vs. Ag pseudo-reference electrode) was obtained using cyclic voltammetry (CV) at room temperature. The magnitude of the peak value varies linearly with the cortisol concentration, and was used to quantify the concentration of cortisol in real saliva samples. This immunoelectrochemical detection method accurately measured cortisol in the collected saliva samples achieved to a concentration of 0.76 nmol/L with incubation of 10 min. 2. Experimental 2.1. Chemicals and materials EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) and EZ-link sulfo-NHS–biotin were purchased from Pierce Biotechnology (part of ThermoFisher Scientific, Rockford, IL). Monoclonal Anti-Rabbit IgG (␥-chain specific)–Alkaline Phosphatase antibody produced in mouse, cortisol antibody (anti-cortisol), phosphate buffered saline (pH 7.4), hydrocortisone
Fig. 1. Schematic of the electrochemical cell.
(␥-irradiated, cell culture tested), p-nitrophenyl phosphate (pNPP), Tris buffer (pH 9.2), Reagent grade 100% ethanol and gold were purchased from Sigma Aldrich (St. Louis, MO). dl-Thioctic acid (100%) was obtained from MP Biomedicals (Irvine, CA). XP SU-8 50 resist was purchased from Microchem Corp. (Newton, MA). 2.2. Electrochemical cell fabrication The immunoelectrochemical device was fabricated by photolithography, subsequent thermal deposition and lift-off tech˚ was used as nique on thermally oxidized silicon wafer. Cr (100 A) ˚ The gold electrodes were cleaned the adhesion layer for Au (1000 A). in piranha solution (3:1 mixture of sulphuric acid and hydrogen peroxide) to remove most organic matter and hydroxylate most surfaces (add OH groups), making them extremely hydrophilic. One of the Au electrodes was plated with Ag for use as a pseudo-reference electrode (pseudo-RE) using thermal evaporator. Microfabricated planar parallel Au fingers (100-m width and 10-m gap) as the interdigital working electrodes (WE-IDEs) are suitable for measuring small volume samples. IDE has been researched extensively, because it is one of the most effective structures for detecting electroactive species [15]. The repeated reduction and oxidation reactions on each electrode band give higher Faraday current. Such amplification of the limiting current by redox cycling has been effectively employed for improving the lower detection limit. A 50m thick SU-8 microfluidic chamber was finally fabricated to hold the reagents. The SU-8 chamber was then hard-baked at 180 ◦ C for 3 min to prevent any contamination (Fig. 1).
Fig. 2. Sandwich structure and electrochemical immunoassay protocol.
K. Sun et al. / Sensors and Actuators B 133 (2008) 533–537
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2.3. Saliva sample collection Subjects provide saliva more willingly than serum, and samples can be collected without medical help. Saliva samples about 1 mL were collected from volunteers from our laboratory and stored frozen in plastic sample vial. Separate saliva samples are collected at the following five time periods: 4 a.m. (during sleep), 8 a.m. (after wake), 12 p.m. (noon), 8 p.m. (before dinner) and 12 a.m. (midnight) on the same day. Samples in the morning were collected before brushing/flossing teeth, eating, drinking or applying makeup. For samples during other time, mouth was rinsed at least twice with cool water. Sample tubes were properly labeled and placed in the plastic zip-lock bag. Saliva samples were centrifuged after thawing to remove particulate matters and stored in freeze for later measurement [16]. 2.4. Immunoassay protocol The sandwich structure and conversion reaction of pNPP to pNP under the influence of AP enzyme and ultimately to hydroquinone upon electrooxiation is represented in Fig. 2. A multi-step process with the following steps: (a) activation of the electrode by overnight dipping 2% (w/v) thioctic acid in absolute ethanol; (b) application of 5 mmol/L EDC solution for 7 h and 1.8 mmol/L NHS–biotin solution for 3 h; (c) immobilization of 30 L cortisol antibody solution (anti-cortisol in 0.1 mol/L PBS) on electrodes followed by incubation at room temperature (RT) in humid condition for 30 min; (d) immobilization of 30 L cortisol antigen (diluted hydrocrotisone solution 1 mmol/L, 1 mol/L, 1 nmol/L, 1 pmol/L and 1 fmol/L were used for calibration purpose) by incubation at room temperature in humid condition for 5 min; (e) attachment of detector antibody with alkaline phosphatase (AP) by incubation at RT for 30 min; (f) 60 L of pNPP solution was added to the chamber to cover all the electrodes and the enzymatic reaction was allowed to proceed in dark at room temperature under humid condition; and (g) after 10 min, the enzymatic reaction was interrupted by adding 0.1 mol/L PBS (pH = 7.2) stopping solution. 2.5. Electrochemical measurement Cyclic voltammetry measurements were performed using an Autolab PGSTAT30 from Eco Chemic N.V. Modified Au WE and CE, and Ag deposited on the silicon chip served as the RE. The electrodes were connected through pogoTM pins to external electrochemical analyzer. All electrochemical measurements were performed in a standard three-electrode format at RT. pNPP was used as a substrate for electrochemical detection with AP enzyme, a useful enzyme for the heterogeneous electrochemical enzyme immunoassays [17]. One AP unit hydrolyzes 1 mol of pNPP per min at pH 9.8 and 37 ◦ C. The incubation time was varied and the optimum detection time was established at 10 min. 2.6. Optical and surface morphology studies Fourier transform infrared (PerkinElmer PE1600 FTIR) spectrometer in the range of 400–4000 cm−1 at a resolution of 1 cm−1 for the complimentary infrared absorption was used to confirm the effective chemical binding in the test protocol. 3. Results and discussions The effective binding in the test protocol was confirmed by FTIR studies on the samples in each stage of the functionalization process. The broad peak in the range of 925–1105 cm−1 is due to the asymmetric stretching of Si O Si (1014–1090 cm−1 ) bond. The
Fig. 3. FTIR spectra of gold electrodes before and after surface functionalization process.
propyl ammonium group, more pronounced with N N, N H, and N O functionalities were observed. A decrease in absorbance characterized by the formation of a polycarbon compound structure formed as a result of complex cortisol antigen–antibody binding was also visible. These results confirm the effective chemical binding involved in the testing protocol (Fig. 3). N-Hydroxysulfosuccinimide (Sulfo-NHS) esters of biotin are the most popular type of biotinylation reagent. NHS-activated biotins react efficiently with primary amine groups (–NH2 ) in pH 7–9 buffers to form stable amide bonds to capture cortisol antibody. The specific bonding between the antibody and antigen coupled with the enzymatic reaction between the AP enzyme attached to the cortisol antigen and the p-nitrophenyl phosphate (pNPP), helps to improve both the sensitivity and selectivity of the assay. pNPP is a widely used substrate for detecting alkaline phosphatase in ELISA applications [17]. When alkaline phosphatase dephosphorylates the pNPP substrate, a yellow water-soluble reaction product, p-nitrophenol (pNP), is formed. This reaction product absorbs light at 405 nm, which can also be detected by electrochemical reduction of its aromatic nitro group or by oxidation of its aromatic hydroxyl group. This enzymatic reaction product pNP is electroactive and underwent an irreversible oxidation between 0.9 and 1.1 V (vs. Ag pseudo-reference electrode) in 0.1 M Tris buffer [18]. pNP oxidation generally leads to the formation of intermediates, namely benzoquinone and hydroquinone, which are also known to be electroactive. The former is an oxidized compound that can be electroreduced, while the later one is a reduced compound that can be electrooxidized. Fig.4a shows the corresponding voltammograms recorded for the reaction product pNP collected with different concentrations of hydrocortisone (cortisol antigen) and detector antibody together with AP concentration of 5 mmol/L in Tris buffer solution with an incubation time of 10 min. All the tests were carried out with a test volume of 30 L at a scan rate of 50 mV/s and a potential scan from 0.4 to 1.1 V. The pNPP and AP reaction amplification extends the detection limit of the cortisol concentration. pNP, the reaction production was quantified by measurement of the height of its anodic peak as shown in Fig. 4a. The concentration of pNP is determined by the bonded detection antibody and AP group, which is linearly dependent on the cortisol concentration. Standard deviation of the measured peaks is 3.09 A and average peak value is 11.2 A. The magnitudes of these peaks drop dramatically with the decreasing hydrocortisone concentrations. Fig. 4b indicates the calibration
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K. Sun et al. / Sensors and Actuators B 133 (2008) 533–537
Fig. 4. (a) Voltammograms recorded for the reaction product pNP with different concentration of cortisol in Tris buffer solution with an incubation time of 10 min at a scan rate of 50 mV/s vs. Ag pseudo-reference electrode carried out on bare electrodes. Inset is a detailed view of the voltammograms of concentration below 1 nmol/L. (b) Calibration curve measured from hydrocortisone solution.
curve that was plotted using the anodic peak voltage values for each curve against different cortisol concentrations with a variance (R) of 0.972. As a wide range of cortisol concentrations from 1 fmol/L to 1 mmol/L were measured, the x–y axes are exponentially described. Absence of cortisol concentration in the standard PBS (negative control) does not result in oxidation peak. Moreover, error bars were determined by performing six tests with the same concentration of cortisol in standard PBS solution. These cannot be identified when the concentration increases exponentially, as also revealed in Fig. 4b. The slight deviation from the linearity could be attributed to the error introduced by the sensor fabrication, washing efficiency and environmental effects which is about 15%. log10 (Current peak [A]) = 0.30845 × log10 (cortisol concentration [mol/L]) − 2.04134 A linear fit to the relationship between the hydrocortisone concentration and the current peak value (Fig. 4b) is described in the equation above, which was used to determine the salivary cortisol concentrations from the measured current peak values shown in Fig. 5a. In other words, the diurnal cortisol rhythm of
our subject is calculated and illustrated in Fig. 5b based on this equation. Salivary cortisol measurements are simple to obtain, easy to measure in most laboratories, and provide an indirect yet reliable and practical assessment of the serum free cortisol concentrations during critical illnesses [19]. Concentration of the cortisol in saliva normally ranges from sub-nmol/L to 100 nmol/L and also varies from person to person. A sharp rise to a morning challenge with a robust and short-term decline may indicate health in many populations, but a similar rise to morning challenge in a chronically stressed population may indicate a less functional cortisol response (e.g., the decline may be more gradual, indicating continued HPA over-stimulation). The “flatness” of a cortisol rhythm may be indicative of a long-term response to chronic stress (i.e., Kirschbaum’s burn-outs) or a variant within the normal range. In our presented results, a sharp increase after dinner and decrease during sleep were observed. Additionally, cortisol level was found dropping continuously during the daytime. Our findings are fairly consistent with the results from literatures [2]. However, as discussed above, cortisol levels are different from subjects to subjects and, importantly, are a function of stress and activities.
Fig. 5. (a) Voltammograms recorded for the reaction product pNP with saliva samples collected at (4:00 a.m., 8:00 a.m., 12:00 p.m. 8:00 p.m. and 12 a.m.) in Tris buffer solution with an incubation time of 10 min at a scan rate of 50 mV/s vs. Ag pseudo-reference electrode carried out on bare electrodes. (b) Calibration results on the saliva samples.
K. Sun et al. / Sensors and Actuators B 133 (2008) 533–537
4. Conclusions An immunoelectrochemical sensor based on alkaline phosphatase enzyme for determination of salivary cortisol concentration is reported. Cortisol levels to a concentration of 0.76 nmol/L were measured effectively in the collected saliva samples. We demonstrated successfully the approach for establishing diurnal cortisol concentration behavior for clinical purposes with numerous advantages like a much higher throughput capability, significantly lower amounts of the sample, sub-nmol/L range sensitivity, higher resolution at low mass ranges, and easy to use. Acknowledgements Authors would like to thank James and Esther King Biomedical Research Program administered by the Florida Department of Health and Office of Public Health Research, as well as Graduate Fellowships in the Functional Multiscale Materials by Design (FMMD) offered from Graduate School in University of South Florida.
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[11] N.S. Ramgir, et al., Voltammetric detection of cancer biomarkers exemplified by interleukin-10 and osteopontin with silica nanowires, J. Phys. Chem. C 111 (37) (2007) 13981–13987. [12] S.J. Patil, et al., Ultrasensitive electrochemical detection of cytokeratin-7, using Au nanowires based biosensor, Sens. Actuators B: Chem. 129 (2008) 859–865. [13] A. Kumar, et al., Ultrasensitive detection of cortisol with enzyme fragment complementation technology using functionalized nanowire, Biosens. Bioelectron. 22 (9-10) (2007) 2138–2144. [14] S. Aravamudhan, N.S. Ramgir, S. Bhansali, Electrochemical biosensor for targeted detection in blood using aligned Au nanowires, Sens. Actuators B: Chem. 127 (1) (2007) 29–35. [15] Y. Yi, J.-K. Park, Fabrication of electrochemical sensor with tunable electrode distance, J. Semiconduct. Technol. Sci. 5 (1) (2005) 30–37. [16] J.M. Dabbs, Salivary testosterone measurements: collecting, storing, and mailing saliva samples, Physiol. Behav. 49 (4) (1991) 815–817. [17] K. Jiao, W. Sun, H.Y. Wang, A new voltammetric enzyme immunoassay system for the detection of alkaline phosphatase, Chin. Chem. Lett. 13 (1) (2002) 69–70. ´ [18] P. Fanjul-Bolado, M. Gonzalez-Garc´ ıa, A. Costa-Garc´ıa, Anal. Bioanal. Chem. 385 (2006) 1202–1208. [19] B.M. Arafah, et al., Measurement of salivary cortisol concentration in the assessment of adrenal function in critically ill subjects: a surrogate marker of the circulating free cortisol, J. Clin. Endocrinol. Metab. 92 (8) (2007) 2965–2971.
Biographies References [1] D.A. Papanicolaou, et al., Nighttime salivary cortisol: a useful test for the diagnosis of Cushing’s syndrome, J. Clin. Endocrinol. Metab. 87 (10) (2002) 4515–4521. [2] B. Bandelow, et al., Diurnal variation of cortisol in panic disorder, Psychiatry Res. 95 (3) (2000) 245–250. [3] D.L. Rudolph, E. McAuley, Cortisol and affective responses to exercise, J. Sports Sci. 16 (2) (1998) 121–128. [4] A.D.L. Roberts, et al., Salivary cortisol response to awakening in chronic fatigue syndrome, Br. J. Psychiatry 184 (2) (2004) 136–141. [5] C.A. Morgan, et al., Hormone profiles in humans experiencing military survival training, Biol. Psychiatry 47 (10) (2000) 891–901. [6] S.R.R. Antonini, S.M. Jorge, A.C. Moreira, The emergence of salivary cortisol circadian rhythm and its relationship to sleep activity in preterm infants, Clin. Endocrinol. 52 (4) (2000) 423–426. [7] S.E. Sephton, et al., Diurnal cortisol rhythm as a predictor of breast cancer survival, J. Natl. Cancer Inst. 92 (12) (2000) 994–1000. [8] E. Cullum, M.C.A. Duplessis, L.J. Crepeau, Method for the detection of stress biomarkers including cortisol by fluorescence polarization, 2006, US. [9] M. Jia, Z. He, W. Jin, Capillary electrophoretic enzyme immunoassay with electrochemical detection for cortisol, J. Chromatogr. A 966 (1–2) (2002) 187–194. [10] M.M. Kushnir, et al., Cortisol and cortisone analysis in serum and plasma by atmospheric pressure photoionization tandem mass spectrometry, Clin. Biochem. 37 (5) (2004) 357–362.
Ke Sun received his bachelor’s degree in electrical engineering from Beijing Institute of Technology (2003) Beijing China. He is currently pursuing his PhD in BioMEMS and Microsystem lab in the Department of Electrical Engineering at University of South Florida. His research interests include gold interactions with Si, gold affect on Si nanostructures, gold nanoparticles and their applications in physical, chemical, as well as chemical biosensors. Dr. Niranjan Ramgir completed his PhD (Nanoscience and Nanotechnology) in 2006, from the Physical and Materials Chemistry Division, National Chemical Laboratory, Pune India. He has a vast experience in synthesis (both chemical and physical methods) and characterization of semiconducting oxide and metallic nanostructures for specific applications towards gas sensing and field emission. Presently, he is working in the field of electrochemical biosensors. More specifically, on the detection of ovarian and lung cancer cells (biomarkers) using nano/bioMEMS, microfluidics and electrochemistry. Dr. Shekhar Bhansali is currently an associate professor in the Department of Electrical Engineering and Nanomaterials and Nanomanufacturing Research Center, University of South Florida. He received his PhD in Electrical Engineering (1997) from Royal Melbourne Institute of Technology, Melbourne, Australia. His current research interests are bioMEMS, nanostructures, micro-actuators and integrated systems.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
An immunoelectrochemical sensor for salivary cortisol measurement Ke Sun, Niranjan Ramgir, Shekhar Bhansali ∗ Bio-MEMS and Microsystem Lab, Department of Electrical Engineering, University of South Florida, Tampa, FL 33620, United States
a r t i c l e
i n f o
Article history: Received 14 December 2007 Received in revised form 14 March 2008 Accepted 19 March 2008 Available online 28 March 2008 Keywords: Salivary cortisol Immunoelectrochemical sensor Cyclic voltammetry p-Nitrophenyle phosphate
a b s t r a c t An immunoelectrochemical sensor based on alkaline phosphatase (AP) enzyme for determination of salivary cortisol concentration is reported. Microfabricated Au electrodes encased in a microfluidic chamber were functionalized to immobilize the cortisol capture antibodies. The reaction product p-nitrophenol (pNP) generated by reacting the AP enzyme attached to the cortisol antigen via detector antibodies with the p-nitrophenyl phosphate (pNPP) solution. pNP was detected as an oxidative peak between 0.9 and 1.1 V (vs. Ag pseudo-reference electrode) using cyclic voltammetry (CV) at room temperature. The magnitude of the peak varies linearly with the cortisol concentration, and was used to quantify the concentration of cortisol in real saliva samples. This immunoelectrochemical detection method accurately measured cortisol in the collected saliva samples achieved to a concentration of 0.76 nmol/L with an incubation time of 10 min. We demonstrate successfully the approach for establishing diurnal cortisol concentration behavior for clinical purposes with numerous advantages: a much higher throughput capability, significantly lower amounts of the sample, sub-pmol/L range sensitivity, higher resolution at low mass ranges, and easy to use. © 2008 Elsevier B.V. All rights reserved.
1. Introduction As evaluating sweat or tear cortisol is only of theoretical importance and urinary cortisol of falling interest, salivary cortisol has become a valuable tool for both basic scientists and clinicians [1]. Numerous significant advantages over the blood cortisol evaluation have resulted in a steadily increasing interest in salivary cortisol measurement. It reflects the unbound fraction of circulating cortisol, and therefore it is not affected by alterations in cortisol-binding globulin [1]. Salivary cortisol concentration is not affected by saliva flow rate, making it a reliable end point. The advantages of collection at home at bedtime are obvious: the cost of an office or hospital visit is eliminated, and potential increases in cortisol levels because of anxiety or an unfamiliar environment is mitigated. The ease of sampling allows for sequential collection and hence the opportunity to examine cortisol levels as a function of time [2] and activity history [3]. Recently, researches on identification of abnormalities in salivary cortisol level indicating varieties of illnesses are executed. The salivary cortisol response to awakening in chronic fatigue syndrome (CFS) has been described as a non-invasive test of the capacity of the hypothalamic–pituitary–adrenal (HPA) axis to respond to stress [4]. Neuroendocrine profiles were obtained for subjects experiencing military survival training using saliva sam-
∗ Corresponding author. E-mail address: [email protected] (S. Bhansali). 0925-4005/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2008.03.018
ples collected at baseline and at four subsequent “stress” points [5]. Numerous other mind–body interrelationships have been explored using salivary hormone levels. The circadian rhythm of salivary cortisol levels has been studied in relationship to sleep activity in preterm infants [6]. Measurement of nighttime salivary cortisol levels provides excellent specificity and sensitivity to screen for Cushing’s syndrome [1]. The salivary cortisol test had better sensitivity and specificity for this syndrome than other detection methods, including a nighttime serum cortisol test and the dexamethasone suppression test. Abnormal circadian rhythms of salivary cortisol levels have also been observed in breast cancer patients [7]. Currently accepted methods for the clinical evaluation of stress biomarkers in saliva from patients include fluorescence enzymelinked immunosorbent assays (ELISA) or radioimmunoassay (RIA). However, these methods suffer from several disadvantages including interference from other molecules in the fluid environment, inefficient absorption, and lack of consistency in laboratory accuracy. Fluorescent polarization (FP) technology which has distinct advantages over previous methods was patented by Cullum et al. [8]. Similarly, UV detector is lack of sensitivity and this approach is not applicable for many antigens without strong UV absorbance. Laser-induced fluorescence (LIF) detection is a more general approach to improve sensitivity. However, it is difficult to use most laser instruments in samples that have not been purified or pretreated. Moreover, due to high cost, complex maintenance, low limit of detection (LOD), and because most biological fluids are strongly
534
K. Sun et al. / Sensors and Actuators B 133 (2008) 533–537
luminescent when excited by the laser in the blue or green region of the spectrum, applications of LIF are limited. Electrochemical sensors with detection based on a change in an electrical quantity such as voltage, current, impedance or charge by a biochemical reaction are suitable for integration and microfabrication. In fact, miniaturized electrochemical sensors that combine the specificity and selectivity of biological components with the analytical advantages of electrochemical detection have been investigated [9,10]. Accompanied by the immunoassay methodology, label-free detection and quantification of the immune complex are enabled by modern technologies in immunoelectrochemical sensors. The fundamental of all immunoassay is the specificity of the molecular recognition of antigens by antibodies to form a stable complex. The use of high specific antibodies as bioaffinity interface for immunosensors greatly improved the selectivity, moreover, allows the detection with significantly low amounts of the sample and sub-pmol/L range sensitivity [11–14]. In the present work we report an immunoelectrochemical sensor based on alkaline phosphatase (AP) enzyme for determination of salivary cortisol concentration. Microfabricated Au electrodes encased in a microfluidic chamber were functionalized to immobilize the cortisol capture antibodies. AP enzyme attached to the cortisol antigen via detector antibodies generated p-nitrophenol (pNP) by reacting with the p-nitrophenyl phosphate (pNPP) solution. An oxidative peak of resulted pNP between 0.9 and 1.1 V (vs. Ag pseudo-reference electrode) was obtained using cyclic voltammetry (CV) at room temperature. The magnitude of the peak value varies linearly with the cortisol concentration, and was used to quantify the concentration of cortisol in real saliva samples. This immunoelectrochemical detection method accurately measured cortisol in the collected saliva samples achieved to a concentration of 0.76 nmol/L with incubation of 10 min. 2. Experimental 2.1. Chemicals and materials EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) and EZ-link sulfo-NHS–biotin were purchased from Pierce Biotechnology (part of ThermoFisher Scientific, Rockford, IL). Monoclonal Anti-Rabbit IgG (␥-chain specific)–Alkaline Phosphatase antibody produced in mouse, cortisol antibody (anti-cortisol), phosphate buffered saline (pH 7.4), hydrocortisone
Fig. 1. Schematic of the electrochemical cell.
(␥-irradiated, cell culture tested), p-nitrophenyl phosphate (pNPP), Tris buffer (pH 9.2), Reagent grade 100% ethanol and gold were purchased from Sigma Aldrich (St. Louis, MO). dl-Thioctic acid (100%) was obtained from MP Biomedicals (Irvine, CA). XP SU-8 50 resist was purchased from Microchem Corp. (Newton, MA). 2.2. Electrochemical cell fabrication The immunoelectrochemical device was fabricated by photolithography, subsequent thermal deposition and lift-off tech˚ was used as nique on thermally oxidized silicon wafer. Cr (100 A) ˚ The gold electrodes were cleaned the adhesion layer for Au (1000 A). in piranha solution (3:1 mixture of sulphuric acid and hydrogen peroxide) to remove most organic matter and hydroxylate most surfaces (add OH groups), making them extremely hydrophilic. One of the Au electrodes was plated with Ag for use as a pseudo-reference electrode (pseudo-RE) using thermal evaporator. Microfabricated planar parallel Au fingers (100-m width and 10-m gap) as the interdigital working electrodes (WE-IDEs) are suitable for measuring small volume samples. IDE has been researched extensively, because it is one of the most effective structures for detecting electroactive species [15]. The repeated reduction and oxidation reactions on each electrode band give higher Faraday current. Such amplification of the limiting current by redox cycling has been effectively employed for improving the lower detection limit. A 50m thick SU-8 microfluidic chamber was finally fabricated to hold the reagents. The SU-8 chamber was then hard-baked at 180 ◦ C for 3 min to prevent any contamination (Fig. 1).
Fig. 2. Sandwich structure and electrochemical immunoassay protocol.
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2.3. Saliva sample collection Subjects provide saliva more willingly than serum, and samples can be collected without medical help. Saliva samples about 1 mL were collected from volunteers from our laboratory and stored frozen in plastic sample vial. Separate saliva samples are collected at the following five time periods: 4 a.m. (during sleep), 8 a.m. (after wake), 12 p.m. (noon), 8 p.m. (before dinner) and 12 a.m. (midnight) on the same day. Samples in the morning were collected before brushing/flossing teeth, eating, drinking or applying makeup. For samples during other time, mouth was rinsed at least twice with cool water. Sample tubes were properly labeled and placed in the plastic zip-lock bag. Saliva samples were centrifuged after thawing to remove particulate matters and stored in freeze for later measurement [16]. 2.4. Immunoassay protocol The sandwich structure and conversion reaction of pNPP to pNP under the influence of AP enzyme and ultimately to hydroquinone upon electrooxiation is represented in Fig. 2. A multi-step process with the following steps: (a) activation of the electrode by overnight dipping 2% (w/v) thioctic acid in absolute ethanol; (b) application of 5 mmol/L EDC solution for 7 h and 1.8 mmol/L NHS–biotin solution for 3 h; (c) immobilization of 30 L cortisol antibody solution (anti-cortisol in 0.1 mol/L PBS) on electrodes followed by incubation at room temperature (RT) in humid condition for 30 min; (d) immobilization of 30 L cortisol antigen (diluted hydrocrotisone solution 1 mmol/L, 1 mol/L, 1 nmol/L, 1 pmol/L and 1 fmol/L were used for calibration purpose) by incubation at room temperature in humid condition for 5 min; (e) attachment of detector antibody with alkaline phosphatase (AP) by incubation at RT for 30 min; (f) 60 L of pNPP solution was added to the chamber to cover all the electrodes and the enzymatic reaction was allowed to proceed in dark at room temperature under humid condition; and (g) after 10 min, the enzymatic reaction was interrupted by adding 0.1 mol/L PBS (pH = 7.2) stopping solution. 2.5. Electrochemical measurement Cyclic voltammetry measurements were performed using an Autolab PGSTAT30 from Eco Chemic N.V. Modified Au WE and CE, and Ag deposited on the silicon chip served as the RE. The electrodes were connected through pogoTM pins to external electrochemical analyzer. All electrochemical measurements were performed in a standard three-electrode format at RT. pNPP was used as a substrate for electrochemical detection with AP enzyme, a useful enzyme for the heterogeneous electrochemical enzyme immunoassays [17]. One AP unit hydrolyzes 1 mol of pNPP per min at pH 9.8 and 37 ◦ C. The incubation time was varied and the optimum detection time was established at 10 min. 2.6. Optical and surface morphology studies Fourier transform infrared (PerkinElmer PE1600 FTIR) spectrometer in the range of 400–4000 cm−1 at a resolution of 1 cm−1 for the complimentary infrared absorption was used to confirm the effective chemical binding in the test protocol. 3. Results and discussions The effective binding in the test protocol was confirmed by FTIR studies on the samples in each stage of the functionalization process. The broad peak in the range of 925–1105 cm−1 is due to the asymmetric stretching of Si O Si (1014–1090 cm−1 ) bond. The
Fig. 3. FTIR spectra of gold electrodes before and after surface functionalization process.
propyl ammonium group, more pronounced with N N, N H, and N O functionalities were observed. A decrease in absorbance characterized by the formation of a polycarbon compound structure formed as a result of complex cortisol antigen–antibody binding was also visible. These results confirm the effective chemical binding involved in the testing protocol (Fig. 3). N-Hydroxysulfosuccinimide (Sulfo-NHS) esters of biotin are the most popular type of biotinylation reagent. NHS-activated biotins react efficiently with primary amine groups (–NH2 ) in pH 7–9 buffers to form stable amide bonds to capture cortisol antibody. The specific bonding between the antibody and antigen coupled with the enzymatic reaction between the AP enzyme attached to the cortisol antigen and the p-nitrophenyl phosphate (pNPP), helps to improve both the sensitivity and selectivity of the assay. pNPP is a widely used substrate for detecting alkaline phosphatase in ELISA applications [17]. When alkaline phosphatase dephosphorylates the pNPP substrate, a yellow water-soluble reaction product, p-nitrophenol (pNP), is formed. This reaction product absorbs light at 405 nm, which can also be detected by electrochemical reduction of its aromatic nitro group or by oxidation of its aromatic hydroxyl group. This enzymatic reaction product pNP is electroactive and underwent an irreversible oxidation between 0.9 and 1.1 V (vs. Ag pseudo-reference electrode) in 0.1 M Tris buffer [18]. pNP oxidation generally leads to the formation of intermediates, namely benzoquinone and hydroquinone, which are also known to be electroactive. The former is an oxidized compound that can be electroreduced, while the later one is a reduced compound that can be electrooxidized. Fig.4a shows the corresponding voltammograms recorded for the reaction product pNP collected with different concentrations of hydrocortisone (cortisol antigen) and detector antibody together with AP concentration of 5 mmol/L in Tris buffer solution with an incubation time of 10 min. All the tests were carried out with a test volume of 30 L at a scan rate of 50 mV/s and a potential scan from 0.4 to 1.1 V. The pNPP and AP reaction amplification extends the detection limit of the cortisol concentration. pNP, the reaction production was quantified by measurement of the height of its anodic peak as shown in Fig. 4a. The concentration of pNP is determined by the bonded detection antibody and AP group, which is linearly dependent on the cortisol concentration. Standard deviation of the measured peaks is 3.09 A and average peak value is 11.2 A. The magnitudes of these peaks drop dramatically with the decreasing hydrocortisone concentrations. Fig. 4b indicates the calibration
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Fig. 4. (a) Voltammograms recorded for the reaction product pNP with different concentration of cortisol in Tris buffer solution with an incubation time of 10 min at a scan rate of 50 mV/s vs. Ag pseudo-reference electrode carried out on bare electrodes. Inset is a detailed view of the voltammograms of concentration below 1 nmol/L. (b) Calibration curve measured from hydrocortisone solution.
curve that was plotted using the anodic peak voltage values for each curve against different cortisol concentrations with a variance (R) of 0.972. As a wide range of cortisol concentrations from 1 fmol/L to 1 mmol/L were measured, the x–y axes are exponentially described. Absence of cortisol concentration in the standard PBS (negative control) does not result in oxidation peak. Moreover, error bars were determined by performing six tests with the same concentration of cortisol in standard PBS solution. These cannot be identified when the concentration increases exponentially, as also revealed in Fig. 4b. The slight deviation from the linearity could be attributed to the error introduced by the sensor fabrication, washing efficiency and environmental effects which is about 15%. log10 (Current peak [A]) = 0.30845 × log10 (cortisol concentration [mol/L]) − 2.04134 A linear fit to the relationship between the hydrocortisone concentration and the current peak value (Fig. 4b) is described in the equation above, which was used to determine the salivary cortisol concentrations from the measured current peak values shown in Fig. 5a. In other words, the diurnal cortisol rhythm of
our subject is calculated and illustrated in Fig. 5b based on this equation. Salivary cortisol measurements are simple to obtain, easy to measure in most laboratories, and provide an indirect yet reliable and practical assessment of the serum free cortisol concentrations during critical illnesses [19]. Concentration of the cortisol in saliva normally ranges from sub-nmol/L to 100 nmol/L and also varies from person to person. A sharp rise to a morning challenge with a robust and short-term decline may indicate health in many populations, but a similar rise to morning challenge in a chronically stressed population may indicate a less functional cortisol response (e.g., the decline may be more gradual, indicating continued HPA over-stimulation). The “flatness” of a cortisol rhythm may be indicative of a long-term response to chronic stress (i.e., Kirschbaum’s burn-outs) or a variant within the normal range. In our presented results, a sharp increase after dinner and decrease during sleep were observed. Additionally, cortisol level was found dropping continuously during the daytime. Our findings are fairly consistent with the results from literatures [2]. However, as discussed above, cortisol levels are different from subjects to subjects and, importantly, are a function of stress and activities.
Fig. 5. (a) Voltammograms recorded for the reaction product pNP with saliva samples collected at (4:00 a.m., 8:00 a.m., 12:00 p.m. 8:00 p.m. and 12 a.m.) in Tris buffer solution with an incubation time of 10 min at a scan rate of 50 mV/s vs. Ag pseudo-reference electrode carried out on bare electrodes. (b) Calibration results on the saliva samples.
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4. Conclusions An immunoelectrochemical sensor based on alkaline phosphatase enzyme for determination of salivary cortisol concentration is reported. Cortisol levels to a concentration of 0.76 nmol/L were measured effectively in the collected saliva samples. We demonstrated successfully the approach for establishing diurnal cortisol concentration behavior for clinical purposes with numerous advantages like a much higher throughput capability, significantly lower amounts of the sample, sub-nmol/L range sensitivity, higher resolution at low mass ranges, and easy to use. Acknowledgements Authors would like to thank James and Esther King Biomedical Research Program administered by the Florida Department of Health and Office of Public Health Research, as well as Graduate Fellowships in the Functional Multiscale Materials by Design (FMMD) offered from Graduate School in University of South Florida.
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Ke Sun received his bachelor’s degree in electrical engineering from Beijing Institute of Technology (2003) Beijing China. He is currently pursuing his PhD in BioMEMS and Microsystem lab in the Department of Electrical Engineering at University of South Florida. His research interests include gold interactions with Si, gold affect on Si nanostructures, gold nanoparticles and their applications in physical, chemical, as well as chemical biosensors. Dr. Niranjan Ramgir completed his PhD (Nanoscience and Nanotechnology) in 2006, from the Physical and Materials Chemistry Division, National Chemical Laboratory, Pune India. He has a vast experience in synthesis (both chemical and physical methods) and characterization of semiconducting oxide and metallic nanostructures for specific applications towards gas sensing and field emission. Presently, he is working in the field of electrochemical biosensors. More specifically, on the detection of ovarian and lung cancer cells (biomarkers) using nano/bioMEMS, microfluidics and electrochemistry. Dr. Shekhar Bhansali is currently an associate professor in the Department of Electrical Engineering and Nanomaterials and Nanomanufacturing Research Center, University of South Florida. He received his PhD in Electrical Engineering (1997) from Royal Melbourne Institute of Technology, Melbourne, Australia. His current research interests are bioMEMS, nanostructures, micro-actuators and integrated systems.