Peptide Self-Assembled Monolayers for Label-Free and Unamplified Surface Plasmon Resonance Biosensing in Crude Cell Lysate Olivier R. Bolduc, Christopher M. Clouthier, Joelle N. Pelletier, and Jean-Franc ¸ ois Masson* De ´ partement de Chimie, Universite ´ de Montre ´ al, C. P. 6128 Succ. Centre-Ville, Montre ´al, Quebec, Canada, H3C 3J7 Short peptides, composed of polar or ionic amino acids, derived with a short organic thiol, significantly reduce nonspecific adsorption of proteins in complex biological matrices such as serum and crude cell lysate, which have nonspecific protein concentrations of 76 and 30-60 mg/ mL, respectively. Minimizing these nonspecific interac- tions has allowed rapid and direct quantification of -lactamase in a crude cell lysate using a surface plasmon resonance (SPR) biosensor. A library of short peptides with varying chain length and amino acid composition were synthesized using a solid-phase approach. A 3-mer- captopropionic acid (3-MPA) linker was covalently at- tached to the amino terminus of the peptides to subse- quently form a monolayer on gold in the form of 3-MPA- (AA) n -OH, where n is the length of the amino acid chain (n ) 2-5). Leu, Phe, Ser, Asp, and His were selected to investigate the effect on nonspecific adsorp- tion with different physicochemical properties of the sidechains; aliphatic, aromatic, polar, acid, and base. Advancing contact angles measured the hydrophobicity of each peptidic self-assembled monolayer (SAM) and showed that hydrophilicity of the gold surface improved as the chain length of the polar or ionic peptides increased, while aromatic and aliphatic peptides de- creased the hydrophilicity as the chain length in- creased. The nonspecific adsorption of undiluted bo- vine serum on SPR sensors prepared with the library of 3-MPA-(AA) n -OH showed that the lowest nonspe- cific adsorption occurred with polar or ionic amino acids with a chain length of n ) 5. We demonstrate that a monolayer composed of 3-MPA-(Ser) 5 -OH has significant advantages, including the following: (1) it minimizes nonspecific adsorption in undiluted bovine serum; (2) it provides a high surface concentration of immobilized antibodies; (3) it shows a great retention of activity for the antibodies; (4) it improves the response from -lactamase by ∼1 order of magnitude, compared to previous experiments; and (5) it allows direct quantification of submicromolar -lactamase concentration in a crude cell lysate with a nonspecific protein concentration of 30-60 mg/mL. The use of this peptide-based monolayer offers great advantages for quantitative SPR biosensing in complex biological media. Numerous biosensing techniques rely on the measurement of chemical or biological processes occurring on surfaces to detect molecules. In particular, affinity biosensors provide a measurable signal triggered by the binding of a molecule to a surface- immobilized receptor. Multiple physicochemical phenomena can be utilized to measure molecules with affinity biosensors, such as the change in mass, impedance, current, optical output or wavelength, or a change in refractive index, among others. 1 The latter is especially interesting in the case of measuring proteins, antibodies, or enzymes, because these molecules have a large molecular weight and high refractive index, resulting in a sensitive response using a refractive index sensor. To measure this response, surface plasmon resonance (SPR) is a label-free analyti- cal technique that allows real-time measurements of small changes of refractive index caused by the binding of a molecule with a molecular receptor such as DNA, enzymes, or antibodies. 2-4 The SPR effect occurs when a thin metallic film deposited on a dielectric material is excited in total internal reflection. SPR is sensitive to within 200-300 nm over the metallic surface, usually gold or silver. Thereby, any molecule migrating within this sensing volume with a refractive index different from the solution will cause a change in the SPR response. This results in SPR being sensitive to numerous categories of molecules, such as DNA or proteins. The broad sensitivity to many important classes of molecules makes SPR an interesting bioanalytical technology but, at the same time, greatly limits its application with real biological samples, because of nonspecific adsorption. Previous studies have demonstrated the ability of SPR affinity biosensors to efficiently detect or quantify specific biological markers in solutions such as buffers or strongly diluted biological matrices. 5 It is imperative to extend this technique to more-complex matrices such as cellular lysate, blood serum, urine, and blood. 6,7 However, for complex matrices containing high concentrations of proteins that have * To whom correspondence should be addressed. Tel.: 1-514-343-7342. Fax: 1-514-343-7586. E-mail: jf.masson@umontreal.ca. (1) Luppa, P. B.; Sokoll, L. J.; Chan, D. W. Clin. Chim. Acta 2001, 314, 1–26. (2) Homola, J. Anal. Bioanal. Chem. 2003, 377, 528–539. (3) Homola, J.; Yee, S. S.; Gauglitz, G. Sens. Actuators B 1999, 54, 3–15. (4) Phillips, K.; Cheng, Q. Anal. Bioanal. Chem. 2007, 387, 1831–1840. (5) Rich, R. L.; Myszka, D. G. Curr. Opin. Biotechnol. 2000, 11, 54–61. (6) Masson, J. F.; Battaglia, T. M.; Davidson, M. J.; Kim, Y. C.; Prakash, A. M. C.; Beaudoin, S.; Booksh, K. S. Talanta 2005, 67, 918–925. (7) Masson, J. F.; Battaglia, T. M.; Kim, Y. C.; Prakash, A.; Beaudoin, S.; Booksh, K. S. Talanta 2004, 64, 716–725. Anal. Chem. 2009, 81, 6779–6788 10.1021/ac900956y CCC: $40.75  2009 American Chemical Society 6779 Analytical Chemistry, Vol. 81, No. 16, August 15, 2009 Published on Web 07/16/2009 Downloaded by UNIV DE MONTREAL on August 14, 2009 Published on July 16, 2009 on http://pubs.acs.org | doi: 10.1021/ac900956y