Noninvasive Label-Free Detection of Cortisol and Lactate Using Graphene Embedded Screen-Printed Electrode
Corresponding Author: Suresh Neethirajan
Nano-Micro Letters,
Vol. 10 No. 3 (2018), Article Number: 41
Abstract
A sensitive and specific immunosensor for the detection of the hormones cortisol and lactate in human or animal biological fluids, such as sweat and saliva, was devised using the label-free electrochemical chronoamperometric technique. By using these fluids instead of blood, the biosensor becomes noninvasive and is less stressful to the end user, who may be a small child or a farm animal. Electroreduced graphene oxide (e-RGO) was used as a synergistic platform for signal amplification and template for bioconjugation for the sensing mechanism on a screen-printed electrode. The cortisol and lactate antibodies were bioconjugated to the e-RGO using covalent carbodiimide chemistry. Label-free electrochemical chronoamperometric detection was used to analyze the response to the desired biomolecules over the wide detection range. A detection limit of 0.1 ng mL−1 for cortisol and 0.1 mM for lactate was established and a correlation between concentration and current was observed. A portable, handheld potentiostat assembled with Bluetooth communication and battery operation enables the developed system for point-of-care applications. A sandwich-like structure containing the sensing mechanisms as a prototype was designed to secure the biosensor to skin and use capillary action to draw sweat or other fluids toward the sensing mechanism. Overall, the immunosensor shows remarkable specificity, sensitivity as well as the noninvasive and point-of-care capabilities and allows the biosensor to be used as a versatile sensing platform in both developed and developing countries.
Highlights:
1 The as-electrodeposited 2D nanosheets exhibit enhanced electroconductive nature.
2 For the first time, a dual cortisol and lactate antibody-conjugated graphene biointerface has been developed. The developed assay is label free and applicable for the analysis of clinical samples.
3 The results are well correlated and validated with existing commercial ELISA kit. The time of detection is very low at ≤1 min.
Keywords
Download Citation
Endnote/Zotero/Mendeley (RIS)BibTeX
- A. Levine, O. Zagoory-Sharon, R. Feldman, J.G. Lewis, A. Weller, Measuring cortisol in human psychobiological studies. Physiol. Behav. 90(1), 43–53 (2007). https://doi.org/10.1016/j.physbeh.2006.08.025
- S. Choi, S. Kim, J.-S. Yang, J.-H. Lee, C. Joo, H.-I. Jung, Real-time measurement of human salivary cortisol for the assessment of psychological stress using a smartphone. Sens. Bio-Sensing Res. 2, 8–11 (2014). https://doi.org/10.1016/j.sbsr.2014.08.001
- S.E. Allen, J.L. Holm, Lactate: physiology and clinical utility. J. Vet. Emerg. Crit. Care 18(2), 123–132 (2008). https://doi.org/10.1111/j.1476-4431.2008.00286.x
- B. Phypers, J.T. Pierce, Lactate physiology in health and disease. Contin. Educ. Anaesth.: Crit. Care Pain 6(3), 128–132 (2006). https://doi.org/10.1093/bjaceaccp/mkl018
- J.B. Jeppesen, C. Mortensen, F. Bendtsen, S. Møller, Lactate metabolism in chronic liver disease. Scand. J. Clin. Lab. Invest. 73(4), 293–299 (2013). https://doi.org/10.3109/00365513.2013.773591
- K. Rathee, V. Dhull, R. Dhull, S. Singh, Biosensors based on electrochemical lactate detection: a comprehensive review. Biochem. Biophys. Rep. 5, 35–54 (2016). https://doi.org/10.1016/j.bbrep.2015.11.010
- Google Scholar
- B. Sun, Y. Gou, Y. Ma, X. Zheng, R. Bai, A.A. Ahmed Abdelmoaty, F. Hu, Investigate electrochemical immunosensor of cortisol based on gold nanops/magnetic functionalized reduced graphene oxide. Biosens. Bioelectron. 88, 55–62 (2017). https://doi.org/10.1016/j.bios.2016.07.047
- E.T.S.G. da Silva, D.E.P. Souto, J.T.C. Barragan, J. de F. Giarola, A.C.M. de Moraes, L.T. Kubota, Electrochemical biosensors in point-of-care devices: recent advances and future trends. ChemElectroChem 4, 778 (2017). https://doi.org/10.1002/celc.201600758
- S. Anastasova, B. Crewther, P. Bembnowicz, V. Curto, H.M. Ip, B. Rosa, G. Zhong-Yang, A wearable multisensing patch for continuous sweat monitoring. Biosens. Bioelectron. 93, 139–145 (2017). https://doi.org/10.1016/j.bios.2016.09.038
- R.D. Munje, S. Muthukumar, B. Jagannath, S. Prasad, A new paradigm in sweat based wearable diagnostics biosensors using room temperature ionic liquids (RTILs). Sci. Rep. 7, 1950 (2017). https://doi.org/10.1038/s41598-017-02133-0
- S. Ajami, F. Teimouri, Features and application of wearable biosensors in medical care. J. Res. Med. Sci. 20(12), 1208–1215 (2015). https://doi.org/10.4103/1735-1995.172991
- J. Wei, How wearables intersect with the cloud and the internet of things: considerations for the developers of wearables. IEEE Consum. Electron. Mag. 3(3), 53–56 (2014). https://doi.org/10.1109/MCE.2014.2317895
- G. Matzeu, L. Florea, D. Diamond, Advances in wearable chemical sensor design for monitoring biological fluids. Sens. Actuators B Chem. 211, 403–418 (2015). https://doi.org/10.1016/j.snb.2015.01.077
- S. Parvaneh, G.S. Grewal, E. Grewal, R.A. Menzies, T.K. Talal, D.G. Armstrong, E. Sternberg, B. Najafi, Stressing the dressing: assessing stress during wound care in real-time using wearable sensors. Wound Med. 4, 21–26 (2014). https://doi.org/10.1016/j.wndm.2014.01.003
- M.S. Khan, S.K. Misra, Z. Wang, E. Daza, A.S. Schwartz-Duval, J.M. Kus, D. Pan, D. Pan, Paper-based analytical biosensor chip designed from graphene-nanoplatelet-amphiphilic-di-block-co-polymer composite for cortisol detection in human saliva. Anal. Chem. 89(3), 2107–2115 (2017). https://doi.org/10.1021/acs.analchem.6b04769
- K.S. Kim, S.R. Lim, S.E. Kim, J.Y. Lee, C.H. Chung, W.S. Choe, P.J. Yoo, Highly sensitive and selective electrochemical cortisol sensor using bifunctional protein interlayer-modified graphene electrodes. Sens. Actuators B Chem. 242, 1121–1128 (2017). https://doi.org/10.1016/j.snb.2016.09.135
- S. Azzouzi, L. Rotariu, A.M. Benito, W.K. Maser, M. Ben Ali, C. Bala, A novel amperometric biosensor based on gold nanops anchored on reduced graphene oxide for sensitive detection of L-lactate tumor biomarker. Biosens. Bioelectron. 69(2), 280–286 (2015). https://doi.org/10.1016/j.bios.2015.03.012
- P. Labroo, Y. Cui, Graphene nano-ink biosensor arrays on a microfluidic paper for multiplexed detection of metabolites. Anal. Chim. Acta 813, 90–96 (2014). https://doi.org/10.1016/j.aca.2014.01.024
- A.K. Geim, K.S. Novoselov, The rise of graphene. Nat. Mater. 6, 183–191 (2007). https://doi.org/10.1038/nmat1849
- Y. Yang, A.M. Asiri, Z. Tang, D. Du, Y. Lin, Graphene based materials for biomedical applications. Mater. Today 16(10), 365–373 (2013). https://doi.org/10.1016/j.mattod.2013.09.004
- S.K. Tuteja, M. Kukkar, C.R. Suri, A.K. Paul, A. Deep, One step in-situ synthesis of amine functionalized graphene for immunosensing of cardiac marker cTnI. Biosens. Bioelectron. 66, 129–135 (2015). https://doi.org/10.1016/j.bios.2014.10.072
- S. Li, A.N. Aphale, I.G. Macwan, P.K. Patra, W.G. Gonzalez, J. Miksovska, R.M. Leblanc, Graphene oxide as a quencher for fluorescent assay of amino acids, peptides, and proteins. ACS Appl. Mater. Interfaces 4(12), 7069–7075 (2012). https://doi.org/10.1021/am302704a
- S.K. Tuteja, P. Sabherwal, A. Deep, R. Rastogi, A.K. Paul, C.R. Suri, Biofunctionalized rebar graphene (f-RG) for label-free detection of cardiac marker troponin I. ACS Appl. Mater. Interfaces 6(17), 14767–14771 (2014). https://doi.org/10.1021/am503524e
- M. Pumera, Graphene in biosensing. Mater. Today 14(7–8), 308–315 (2011). https://doi.org/10.1016/S1369-7021(11)70160-2
- G. Wang, B. Wang, J. Park, Y. Wang, B. Sun, J. Yao, Highly efficient and large-scale synthesis of graphene by electrolytic exfoliation. Carbon 47(14), 3242–3246 (2009). https://doi.org/10.1016/j.carbon.2009.07.040
- A. Longo, R. Verucchi, L. Aversa, R. Tatti, A. Ambrosio, E. Orabona, U. Coscia, G. Carotenuto, P. Maddalena, Graphene oxide prepared by graphene nano-platelets and reduced by laser treatment. Nanotechnology 28(22), 224002 (2017). https://doi.org/10.1088/1361-6528/aa6c3c
- S.K. Tuteja, T. Duffield, S. Neethirajan, Graphene-based multiplexed disposable electrochemical biosensor for rapid on-farm monitoring of NEFA and βHBA dairy biomarkers. J. Mater. Chem. B 5(33), 6930–6940 (2017). https://doi.org/10.1039/C7TB01382E
- S.K. Tuteja, V. Priyanka, A. Bhalla, A.K. Deep, C.R.Suri Paul, Graphene-gated biochip for the detection of cardiac marker Troponin I. Anal. Chim. Acta 809, 148–154 (2014). https://doi.org/10.1016/j.aca.2013.11.047
- A.C. Ferrari, J. Robertson, Resonant Raman spectroscopy of disordered, amorphous, and diamond like carbon. Phys. Rev. B 64, 75414 (2001). https://doi.org/10.1103/PhysRevB.64.075414
- W.J. Basirun, M. Sookhakian, S. Baradaran, M.R. Mahmoudian, M. Ebadi, Solid-phase electrochemical reduction of graphene oxide films in alkaline solution. Nanoscale Res. Lett. 8, 397 (2013). https://doi.org/10.1186/1556-276X-8-397
- M. Koinuma, C. Ogata, Y. Kamei, K. Hatakeyama, H. Tateishi et al., Photochemical engineering of graphene oxide nanosheets. J. Phys. Chem. C 116(37), 19822–19827 (2012). https://doi.org/10.1021/jp305403r
- K. Krishnamoorthy, M. Veerapandian, K. Yun, S.J. Kim, The chemical and structural analysis of graphene oxide with different degrees of oxidation. Carbon 53(1), 38–49 (2013). https://doi.org/10.1016/j.carbon.2012.10.013
References
A. Levine, O. Zagoory-Sharon, R. Feldman, J.G. Lewis, A. Weller, Measuring cortisol in human psychobiological studies. Physiol. Behav. 90(1), 43–53 (2007). https://doi.org/10.1016/j.physbeh.2006.08.025
S. Choi, S. Kim, J.-S. Yang, J.-H. Lee, C. Joo, H.-I. Jung, Real-time measurement of human salivary cortisol for the assessment of psychological stress using a smartphone. Sens. Bio-Sensing Res. 2, 8–11 (2014). https://doi.org/10.1016/j.sbsr.2014.08.001
S.E. Allen, J.L. Holm, Lactate: physiology and clinical utility. J. Vet. Emerg. Crit. Care 18(2), 123–132 (2008). https://doi.org/10.1111/j.1476-4431.2008.00286.x
B. Phypers, J.T. Pierce, Lactate physiology in health and disease. Contin. Educ. Anaesth.: Crit. Care Pain 6(3), 128–132 (2006). https://doi.org/10.1093/bjaceaccp/mkl018
J.B. Jeppesen, C. Mortensen, F. Bendtsen, S. Møller, Lactate metabolism in chronic liver disease. Scand. J. Clin. Lab. Invest. 73(4), 293–299 (2013). https://doi.org/10.3109/00365513.2013.773591
K. Rathee, V. Dhull, R. Dhull, S. Singh, Biosensors based on electrochemical lactate detection: a comprehensive review. Biochem. Biophys. Rep. 5, 35–54 (2016). https://doi.org/10.1016/j.bbrep.2015.11.010
Google Scholar
B. Sun, Y. Gou, Y. Ma, X. Zheng, R. Bai, A.A. Ahmed Abdelmoaty, F. Hu, Investigate electrochemical immunosensor of cortisol based on gold nanops/magnetic functionalized reduced graphene oxide. Biosens. Bioelectron. 88, 55–62 (2017). https://doi.org/10.1016/j.bios.2016.07.047
E.T.S.G. da Silva, D.E.P. Souto, J.T.C. Barragan, J. de F. Giarola, A.C.M. de Moraes, L.T. Kubota, Electrochemical biosensors in point-of-care devices: recent advances and future trends. ChemElectroChem 4, 778 (2017). https://doi.org/10.1002/celc.201600758
S. Anastasova, B. Crewther, P. Bembnowicz, V. Curto, H.M. Ip, B. Rosa, G. Zhong-Yang, A wearable multisensing patch for continuous sweat monitoring. Biosens. Bioelectron. 93, 139–145 (2017). https://doi.org/10.1016/j.bios.2016.09.038
R.D. Munje, S. Muthukumar, B. Jagannath, S. Prasad, A new paradigm in sweat based wearable diagnostics biosensors using room temperature ionic liquids (RTILs). Sci. Rep. 7, 1950 (2017). https://doi.org/10.1038/s41598-017-02133-0
S. Ajami, F. Teimouri, Features and application of wearable biosensors in medical care. J. Res. Med. Sci. 20(12), 1208–1215 (2015). https://doi.org/10.4103/1735-1995.172991
J. Wei, How wearables intersect with the cloud and the internet of things: considerations for the developers of wearables. IEEE Consum. Electron. Mag. 3(3), 53–56 (2014). https://doi.org/10.1109/MCE.2014.2317895
G. Matzeu, L. Florea, D. Diamond, Advances in wearable chemical sensor design for monitoring biological fluids. Sens. Actuators B Chem. 211, 403–418 (2015). https://doi.org/10.1016/j.snb.2015.01.077
S. Parvaneh, G.S. Grewal, E. Grewal, R.A. Menzies, T.K. Talal, D.G. Armstrong, E. Sternberg, B. Najafi, Stressing the dressing: assessing stress during wound care in real-time using wearable sensors. Wound Med. 4, 21–26 (2014). https://doi.org/10.1016/j.wndm.2014.01.003
M.S. Khan, S.K. Misra, Z. Wang, E. Daza, A.S. Schwartz-Duval, J.M. Kus, D. Pan, D. Pan, Paper-based analytical biosensor chip designed from graphene-nanoplatelet-amphiphilic-di-block-co-polymer composite for cortisol detection in human saliva. Anal. Chem. 89(3), 2107–2115 (2017). https://doi.org/10.1021/acs.analchem.6b04769
K.S. Kim, S.R. Lim, S.E. Kim, J.Y. Lee, C.H. Chung, W.S. Choe, P.J. Yoo, Highly sensitive and selective electrochemical cortisol sensor using bifunctional protein interlayer-modified graphene electrodes. Sens. Actuators B Chem. 242, 1121–1128 (2017). https://doi.org/10.1016/j.snb.2016.09.135
S. Azzouzi, L. Rotariu, A.M. Benito, W.K. Maser, M. Ben Ali, C. Bala, A novel amperometric biosensor based on gold nanops anchored on reduced graphene oxide for sensitive detection of L-lactate tumor biomarker. Biosens. Bioelectron. 69(2), 280–286 (2015). https://doi.org/10.1016/j.bios.2015.03.012
P. Labroo, Y. Cui, Graphene nano-ink biosensor arrays on a microfluidic paper for multiplexed detection of metabolites. Anal. Chim. Acta 813, 90–96 (2014). https://doi.org/10.1016/j.aca.2014.01.024
A.K. Geim, K.S. Novoselov, The rise of graphene. Nat. Mater. 6, 183–191 (2007). https://doi.org/10.1038/nmat1849
Y. Yang, A.M. Asiri, Z. Tang, D. Du, Y. Lin, Graphene based materials for biomedical applications. Mater. Today 16(10), 365–373 (2013). https://doi.org/10.1016/j.mattod.2013.09.004
S.K. Tuteja, M. Kukkar, C.R. Suri, A.K. Paul, A. Deep, One step in-situ synthesis of amine functionalized graphene for immunosensing of cardiac marker cTnI. Biosens. Bioelectron. 66, 129–135 (2015). https://doi.org/10.1016/j.bios.2014.10.072
S. Li, A.N. Aphale, I.G. Macwan, P.K. Patra, W.G. Gonzalez, J. Miksovska, R.M. Leblanc, Graphene oxide as a quencher for fluorescent assay of amino acids, peptides, and proteins. ACS Appl. Mater. Interfaces 4(12), 7069–7075 (2012). https://doi.org/10.1021/am302704a
S.K. Tuteja, P. Sabherwal, A. Deep, R. Rastogi, A.K. Paul, C.R. Suri, Biofunctionalized rebar graphene (f-RG) for label-free detection of cardiac marker troponin I. ACS Appl. Mater. Interfaces 6(17), 14767–14771 (2014). https://doi.org/10.1021/am503524e
M. Pumera, Graphene in biosensing. Mater. Today 14(7–8), 308–315 (2011). https://doi.org/10.1016/S1369-7021(11)70160-2
G. Wang, B. Wang, J. Park, Y. Wang, B. Sun, J. Yao, Highly efficient and large-scale synthesis of graphene by electrolytic exfoliation. Carbon 47(14), 3242–3246 (2009). https://doi.org/10.1016/j.carbon.2009.07.040
A. Longo, R. Verucchi, L. Aversa, R. Tatti, A. Ambrosio, E. Orabona, U. Coscia, G. Carotenuto, P. Maddalena, Graphene oxide prepared by graphene nano-platelets and reduced by laser treatment. Nanotechnology 28(22), 224002 (2017). https://doi.org/10.1088/1361-6528/aa6c3c
S.K. Tuteja, T. Duffield, S. Neethirajan, Graphene-based multiplexed disposable electrochemical biosensor for rapid on-farm monitoring of NEFA and βHBA dairy biomarkers. J. Mater. Chem. B 5(33), 6930–6940 (2017). https://doi.org/10.1039/C7TB01382E
S.K. Tuteja, V. Priyanka, A. Bhalla, A.K. Deep, C.R.Suri Paul, Graphene-gated biochip for the detection of cardiac marker Troponin I. Anal. Chim. Acta 809, 148–154 (2014). https://doi.org/10.1016/j.aca.2013.11.047
A.C. Ferrari, J. Robertson, Resonant Raman spectroscopy of disordered, amorphous, and diamond like carbon. Phys. Rev. B 64, 75414 (2001). https://doi.org/10.1103/PhysRevB.64.075414
W.J. Basirun, M. Sookhakian, S. Baradaran, M.R. Mahmoudian, M. Ebadi, Solid-phase electrochemical reduction of graphene oxide films in alkaline solution. Nanoscale Res. Lett. 8, 397 (2013). https://doi.org/10.1186/1556-276X-8-397
M. Koinuma, C. Ogata, Y. Kamei, K. Hatakeyama, H. Tateishi et al., Photochemical engineering of graphene oxide nanosheets. J. Phys. Chem. C 116(37), 19822–19827 (2012). https://doi.org/10.1021/jp305403r
K. Krishnamoorthy, M. Veerapandian, K. Yun, S.J. Kim, The chemical and structural analysis of graphene oxide with different degrees of oxidation. Carbon 53(1), 38–49 (2013). https://doi.org/10.1016/j.carbon.2012.10.013