Electrical Detection of C-Reactive Protein Using a Single Free-Standing, Thermally Controlled Piezoresistive Microcantilever for Highly Reproducible and Accurate Measurements
This study demonstrates a novel method for electrical detection of C-reactive protein (CRP) as a means of identifying an infection in the body, or as a cardiovascular disease risk assay. The method uses a single free-standing, thermally controlled piezoresistive microcantilever biosensor. In a commonly used sensing arrangement of conventional dual cantilevers in the Wheatstone bridge circuit, reference and gold-coated sensing cantilevers that inherently have heterogeneous surface materials and different multilayer structures may yield independent responses to the liquid environmental changes of chemical substances, flow field and temperature, leading to unwanted signal disturbance for biosensing targets. In this study, the single free-standing microcantilever for biosensing applications is employed to resolve the dual-beam problem of individual responses in chemical solutions and, in a thermally controlled system, to maintain its sensor performance due to the sensitive temperature effect. With this type of single temperature-controlled microcantilever sensor, the electrical detection of various CRP concentrations from 1 μg/mL to 200 μg/mL was performed, which covers the clinically relevant range. Induced surface stresses were measured at between 0.25 N/m and 3.4 N/m with high reproducibility. Moreover, the binding affinity (K D) of CRP and anti-CRP interaction was found to be 18.83 ± 2.99 μg/mL, which agreed with results in previous reported studies. This biosensing technique thus proves valuable in detecting inflammation, and in cardiovascular disease risk assays.
References
[1]
Lavrik, N.V.; Sepaniak, M.J.; Datskos, P.G. Cantilever transducers as a platform for chemical and biological sensors. Rev. Sci. Instrum. 2004, 75, 2229–2253.
[2]
Guanghua, W.; Ram, H.D.; Karolyn, M.H.; Thomas, T.; Richard, J.C.; Arun, M. Bioassay of prostate-specific antigen (PSA) using microcantilevers. Nat. Biotechnol. 2001, 19, 856–860.
[3]
Wee, K.W.; Kang, G.Y.; Park, J.; Kang, J.Y.; Yoon, D.S.; Park, J.H.; Kim, T.S. Novel electrical detection of label-free disease marker proteins using piezoresistive self-sensing micro-cantilevers. Biosens. Bioelectron. 2005, 20, 1932–1938.
[4]
Datskos, P.G.; Sauers, I. Detection of 2-mercaptoethanol using gold-coated micromachined cantilevers. Sens. Actuators B 1999, 61, 75–82.
[5]
Berger, R.; Delamarche, E.; Lang, H.P.; Gerber, C.; Gimzewski, J.K.; Meyer, E.; Guntherodt, H.-J. Surface stress in the self-assembly of alkanethiols on gold. Science 1997, 276, 2021–2024.
Mukhopadhyay, R.; Lorentzen, M.; Kjems, J.; Besenbacher, F. Nanomechanical sensing of DNA sequences using piezoresistive cantilevers. Langmuir 2005, 21, 8400–8408.
[8]
Zhang, Y.; Venkatachalan, S.P.; Xu, H.; Xu, X.; Joshi, P.; Ji, H.-F.; Schulte, M. Micromechanical measurement of membrane receptor binding for label-free drug discovery. Biosens. Bioelectron. 2004, 19, 1473–1478.
[9]
Lavrik, N.V.; Tipple, C.A.; Sepaniak, M.J.; Datskos, P.G. Enhanced chemi-mechanical transduction at nanostructured interfaces. Chem. Phys. Lett. 2001, 336, 371–376.
[10]
Datskos, P.G.; Lavrik, N.V.; Sepaniak, M.J. Detection of explosive compounds with the use of microcantilevers with nanoporous coatings. Sens. Lett. 2003, 1, 25–32.
[11]
Pei, J.; Tian, F.; Thundat, T. Glucose biosensor based on the microcantilever. Anal. Chem. 2003, 76, 292–297.
[12]
Ponchel, F.; Toomes, C.; Bransfield, K.; Leong, F.; Douglas, S.; Field, S.; Bell, S.; Combaret, V.; Puisieux, A.; Mighell, A.; et al. Real-time PCR based on SYBR-Green I fluorescence: An alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions. BMC Biotechnol. 2003, 3, doi:10.1186/1472-6750-3-18.
[13]
Lee, J.H.; Yoon, K.H.; Hwang, K.S.; Park, J.; Ahn, S.; Kim, T.S. Label free novel electrical detection using micromachined PZT monolithic thin film cantilever for the detection of C-reactive protein. Biosens. Bioelectron. 2004, 20, 269–275.
[14]
Lee, J.H.; Kim, T.S.; Yoon, K.H. Effect of mass and stress on resonant frequency shift of functionalized Pb(Zr0.52Ti0.48)O3 thin film microcantilever for the detection of C-reactive protein. Appl. Phys. Lett. 2004, 84, 3187–3189.
[15]
Liu, Y.; Li, X.; Zhang, Z.; Zuo, G.; Cheng, Z.; Yu, H. Nanogram per milliliter-level immunologic detection of alpha-fetoprotein with integrated rotating-resonance microcantilevers for early-stage diagnosis of heptocellular carcinoma. Biomed. Microdevices 2009, 11, 183–191.
[16]
Casas, J.P.; Shah, T.; Hingorani, A.D.; Danesh, J.; Pepys, M.B. C-reactive protein and coronary heart disease: A critical review. J. Int. Med. 2008, 264, 295–314.
[17]
Buckley, D.I.; Fu, R.; Freeman, M.; Rogers, K.; Helfand, M. C-reactive protein as a risk factor for coronary heart disease: A systematic review and meta-analyses for the US preventive services task force. Ann. Int. Med. 2009, 151, 483–495.
[18]
Chen, C.H.; Hwang, R.Z.; Huang, L.S.; Lin, S.M.; Chen, H.-C.; Yang, Y.C.; Lin, Y.T.; Yu, S.A.; Lin, Y.S.; Wang, Y.H.; et al. A wireless bio-MEMS sensor for C-reactive protein detection based on nanomechanics. IEEE Trans. Biomed. Eng. 2009, 56, 462–470.
[19]
Nordstr?m, M.; Keller, S.; Lillemose, M.; Johansson, A.; Dohn, S.; Haefliger, D.; Blagoi, G.; Havsteen-Jakobsen, M.; Boisen, A. SU-8 cantilevers for bio/chemical sensing; Fabrication, characterisation and development of novel read-out methods. Sensors 2008, 8, 1595–1612.
[20]
Boisen, A.; Thaysen, J.; Jensenius, H.; Hansen, O. Environmental sensors based on micromachined cantilevers with integrated read-out. Ultramicroscopy 2000, 82, 11–16.
[21]
Shekhawat, G.; Tark, S.-H.; Dravid, V.P. MOSFET-embedded microcantilevers for measuring deflection in biomolecular sensor. Science 2006, 311, 1592–1595.
[22]
Mostafa, S.; Lee, I.; Islam, S.K.; Eliza, S.A.; Shekhawat, G.; Dravid, V.P.; Tulip, F.S. Integrated MOSFET-embedded-cantilever-based biosensor characteristic for detection of anthrax simulant. IEEE Electron. Device Lett. 2011, 32, 408–410.
[23]
Watari, M.; Galbraith, J.; Lang, H.-P.; Sousa, M.; Hegner, M.; Gerber, C.; Horton, M.A.; McKendry, R.A. Investigating the molecular mechanisms of in-plane mechanochemistry on cantilever arrays. J. Am. Chem. Soc. 2006, 129, 601–609.
[24]
Ji, H.F.; Hansen, K.M.; Hu, Z.; Thundat, T. Detection of pH variation using modified microcantilever sensors. Sens. Actuators B 2001, 72, 233–238.
[25]
Choudhury, A.; Hesketh, P.; Thundat, T.; Hu, Z. A piezoresistive microcantilever array for surface stress measurement: Curvature model and fabrication. J. Micromech. Microeng. 2007, 17, 2065–2076.
[26]
Thaysen, J. Cantilever for Bio-Chemical Sensing Integrated in a Microliquid Handling System. Ph.D. Thesis, Technical University of Denmark, Lyngby, Denmark,, 2001.
[27]
Rasmussen, P.A.; Hansen, O.; Boisen, A. Cantilever surface stress sensors with single-crystalline silicon piezoresistors. Appl. Phys. Lett. 2005, 86, doi:10.1063/1.1900299.
[28]
Ji, H.; Dabestani, R.; Brown, G.; Britt, P. A novel self-assembled monolayer (SAM) coated microcantilever for low level caesium detection. Chem. Commun. 2000, 6, 457–458.
[29]
Rifai, N.; Ridker, P.M. High-sensitivity C-reactive protein: A novel and promising marker of coronary heart disease. Clin. Chem. 2001, 47, 403–411.
Wu, G.; Ji, H.; Hansen, K.; Thundat, T.; Datar, R.; Cote, R.; Hagan, M.F.; Chakraborty, A.K.; Majumdar, A. Origin of nanomechanical cantilever motion generated from biomolecular interactions. Proc. Natl. Acad. Sci. USA 2001, 98, 1560–1564.
[32]
Kuan, S.; Chi, S.-C.; Cheng, Y.-J.; Chia, T.-J.; Huang, L.-S. Binding kinetics of grouper nervous necrosis viruses with functionalized antimicrobial peptides by nanomechanical detection. Biosens. Bioelectron. 2012, 31, 116–123.
[33]
Gupta, A.K.; Nair, P.R.; Akin, D.; Ladisch, M.R.; Broyles, S.; Alam, M.A.; Bashir, R. Anomalous resonance in a nanomechanical biosensor. Proc. Natl. Acad. Sci. USA 2006, 103, 13362–13367.
[34]
R?cker, C.; Manolov, D.E.; Kuzmenkina, E.V.; Tron, K.; Slatosch, H.; Torzewski, J.; Nienhaus, G.U. Affinity of C-reactive protein toward FcγRI is strongly enhanced by the γ-chain. Am. J. Pathol. 2007, 170, 755–763.
[35]
Fan, Y.-J.; Sheen, H.-J.; Liu, Y.-H.; Tsai, J.-F.; Wu, T.-H.; Wu, K.-C.; Lin, S. Detection of C-reactive protein in evanescent wave field using microparticle-tracking velocimetry. Langmuir 2010, 26, 13751–13754.