New Insights into Surface-Enhanced Raman Spectroscopy Label-Free Detection of DNA on Ag°/TiO 2 Substrate Lijie He, †,‡ Michel Langlet, †,‡ Pierre Bouvier, †,‡ Christophe Calers, †,‡,§,∥ Claire-Marie Pradier, †,‡,§,∥ and Valerie Stambouli* ,†,‡ † Universite ́ Grenoble Alpes, LMGP, F-38000 Grenoble, France ‡ CNRS, LMGP, F-38000 Grenoble, France § Sorbonne Universite ́ s, UPMC Univ Paris 06, Laboratoire de Re ́ activite ́ de Surface, 4 place Jussieu, F-75005 Paris, France ∥ CNRS, UMR 7197, Laboratoire de Re ́ activite ́ de Surface, F-75005, Paris, France ABSTRACT: Four DNA polybases (polyA, polyC, polyG, and polyT), modified or not with an external NH 2 group, have been immobilized on an original, robust, and low-cost Ag°/TiO 2 surface-enhanced Raman spectroscopy (SERS) platform. The latter was elaborated through an optimized chemically assisted photocatalytic reduction process. The label-free SERS detection of these polybases has been performed, and the analysis of SERS spectra has been supported by X-ray photoelectron spectroscopy measurements. The high-resolution and signal/noise ratio of SERS spectra enabled us to clearly index the main and secondary Raman lines of all types of studied polybases and to propose some aspects of the polybase immobilization mechanism. Regardless of polybase type and presence or absence of NH 2 modification, an optimum detection is obtained for polybase concentrations lying in the 5−10 μM range, which gives rise to reproducible SERS spectra. This behavior has been interpreted in terms of distribution, orientation, and packing density of immobilized molecules. 1. INTRODUCTION The field of molecular in vitro diagnostics for point of care or environmental monitoring applications is in extensive develop- ment. The application requires the realization of portable, low- cost, and robust analytical platforms providing directly, rapidly, and simply the signal of the biological molecule or pathogen to be detected. 1 In this context, label-free DNA detection is under investigation notably for genome or disease diagnosis and forensic science. 2 The surface-enhanced Raman spectroscopy (SERS) technique perfectly matches these requirements as an ultrasensitive label-free method that produces molecule-specific vibrational spectra when a molecule is in close proximity with a roughened metal surface. Many recent studies report on label-free SERS detection of DNA strands before and/or after hybridization. 3−9 However, the comparison of the resulting spectra remains very challenging. Indeed, from one paper to another one, fluctuations in both band position and peak intensity are found, leading to difficulties in extracting sustainable data. Moreover, the important aspect related to data reproducibility and repeatability on sample surface is not often addressed. If the SERS technique is a powerful and ultrasensitive tool to detect an analyte, many experimental parameters may compromise the data reliability, for example, the Raman cross section of molecules to be detected, the molecule orientation relatively to both the laser beam polarization and the exalting surface, 10−13 the geometry of “hot spots”, 14 etc. In the specific case of DNA detection, two main parameters should be stressed. First, as for any other analyte, the characteristics of the SERS active substrate are crucial. Second, the other crucial parameter is related to the DNA molecule itself. This latter involves various levels of compositional, structural, and conformational organizations depending on environmental and experimental conditions. Both aspects are developed in the following. SERS active substrates proposed for the detection of DNA and its nucleobase derivatives can be classified into two main categories. The first category includes the SERS experiments performed in colloidal solutions. These latter involve either Ag° or Au° nanoparticles in various solutions. 3,4,6,12,15,16 In the second category, which is more relevant in view of portable biosensors, the dissolved molecules are brought and immobi- lized on a solid SERS platform. In this case, SERS measurements are done either in dried conditions or in liquid or humid conditions. These SERS solid platforms are elaborated according to two main strategies depending on the choice of “top-down” or “bottom-up” technology. The top- down strategy involves several routes of controlled etching of silver films. Historically, silver electrodes are electrochemically etched or roughened and SERS measurements are performed in liquid. 17−20 Alternatively, the bottom-up strategy is also under consideration because of its capacity to flexibly monitor the Received: July 25, 2014 Revised: October 3, 2014 Published: October 8, 2014 Article pubs.acs.org/JPCC © 2014 American Chemical Society 25658 dx.doi.org/10.1021/jp507462y | J. Phys. Chem. C 2014, 118, 25658−25670