Surface-enhanced Raman scattering detection of wild-type and mutant p53 proteins at very low concentration in human serum Fabio Domenici, Anna Rita Bizzarri ⇑ , Salvatore Cannistraro Biophysics and Nanoscience Centre, CNISM, Facoltà di Scienze, Università della Tuscia, Largo dell’Università, 01100 Viterbo, Italy article info Article history: Received 15 April 2011 Received in revised form 20 September 2011 Accepted 3 October 2011 Available online 8 October 2011 Keywords: Raman-SERS p53 Ultrasensitive detection Atomic force microscopy abstract The development of ultrasensitive and rapid approaches to detect tumor markers at very low concentra- tions even in a physiological environment represents a challenge in nano-medicine. The p53 protein is at the center of the cellular network that protects organisms against the insurgence of tumors, most of which are related to alteration of p53 expression. Therefore p53 is regarded as a valuable prognostic mar- ker whose detection at high sensitivity may considerably contribute to early diagnosis of cancers. In this work we have applied an analytical method based on surface enhanced Raman spectroscopy with high sensitivity and rapidity to improve traditional bioaffinity techniques. The Raman reporter bifunctional linker 4-aminothiophenol (4-ATP) first assembled onto 50 nm gold nanoparticles (Nps) has then been azotated to bind low concentration wild-type and two mutated forms of p53 proteins. The Raman signal enhancement of the resulting p53-(4-ATP-Np) systems has been used to identify the p53 molecules cap- tured on a recognition substrate constituted by the azurin (Az) protein monolayer. Az has shown a strong association for both wild-type and mutated p53 proteins, allowing us to selectively detect these proteins at concentrations as low as 500 fM, in a human serum environment. Ó 2011 Elsevier Inc. All rights reserved. The p53 protein is directly involved in the chain of biochemical events that follow genotoxic damage, playing a pivotal role in tu- mor prevention [1–3]. Accordingly, it has great promise as a diag- nostic marker for monitoring the molecular machinery whose alteration results in early preneoplastic transformation. The p53 tumor suppression pathway is inactivated in almost all human cancers. In about 50% of them, this inactivation is a direct result of mutations in the p53 gene whereas the alteration of regulators of p53 occurs in many of the remainder [3–5]. An ever-growing number of publications have also reported an overexpressed level of alteration of the p53 protein, wild-type forms as well as structural mutants (non-null), i.e., the arginine 249 to serine (R249S) and cysteine 135 to valine (C135V) point mutations, as a consequence of neoplastic cell formation, tumor invasiveness, and genotoxic stresses [6–11]. These reports indicate that the level of p53 in human serum can be reasonably accurate in reflecting tissue alterations in p53 at the gene and/or protein level, thus leading to a potential convenient and noninvasive tumor screening approach. On such a basis, much attention has been devoted to developing procedures to detect the presence of p53 protein at very low con- centrations in a physiological environment. Among these proce- dures, the most common ones are traditional fluorescence-labeled immunological methods, such as the enzyme-linked immunosor- bent assay (ELISA) 1 [12,13], which require multiple steps, and rather long incubation periods. Beside these more traditional methods, novel label-free detec- tion techniques have paved the way for the realization of protein chip-based immunoassays with enhanced sensitivity and specific- ity [14–16]. In the last few years, applications involving nanoparti- cles (Nps) have received much attention in clinical diagnosis [15,17–19]. In particular, gold nanocolloids have been widely used to design immunoassay tests for tumor markers, based on their pe- culiar properties, such as a high surface-to-volume ratio, the possi- bility of suitable biomolecular conjugation, the rewarding chemical stability, and the collective electronic behavior at their surface. In- deed, their optical and electrochemical signal enhancement capa- bilities, combined to their ability to form hybrid assemblies with biomolecules [2,15], provide the basis for ultrasensitive and molec- ular specific detection. Within this context, the exploitation of the surface-enhanced Raman scattering (SERS) methodology [18–23] offers a great prom- ise for simplified, sensitive detection of biomolecular interactions and several advantages in early diagnostic over the previously 0003-2697/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2011.10.010 ⇑ Corresponding author. Fax: +39 0761357027. E-mail address: bizzarri@unitus.it (A.R. Bizzarri). 1 Abbreviations used: AFS, atomic force spectroscopy; 4-ATP, 4-aminothiophenol; Az, azurin; CT, charge transfer; DBD, DNA-binding domain; ELISA, enzyme-linked immunosorbent assay; EM, electromagnetic; Nps, nanoparticles; PBS, phosphate- buffered saline; SERS, surface-enhanced Raman scattering; SPR, surface plasmon resonance. Analytical Biochemistry 421 (2012) 9–15 Contents lists available at SciVerse ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio