A protein sensor based on nanostructured adaptive silver films (ASFs) is developed for soft protein adsorption and detection with surface-enhanced Raman scattering (SERS). Under protein deposition, the ASFs modify their local nanostructure so that the conformational state of proteins is preserved and SERS is optimized. The sensor is used to examine differences in Raman spectra of two insulin isomers, human insulin and its analogue insulin lispro. These two insulins differ only in the interchange of two neighboring amino acids; specifically, the propyl-lysyl sequence at the C-terminus of the B-chain in insulin lispro is inverted as compared to human insulin. This switch leads to conformational changes at the C-and N-termini and has an important clinical effect for diabetes treatment. The difference in SERS spectra for the two insulins was detected at a submonolayer density, 80 fmol/mm 2 , with only 25 amol in the probed area, and macroscopic enhancement factor 3 × 10 6 . The performed studies indicate that SERS detection using ASFs can lead to efficient proteomic sensing technology.

The interaction of biological molecules with a typical substrate for surface-enhanced Raman scattering (SERS) often leads to their structural and functional changes. In this work we describe SERS substrates, called adaptive silver films (ASFs), in which the biomaterial and the substrate act in concert to produce excellent Raman enhancement through local restructuring of the metal surface while at the same time preserving the properties (such as conformational state and binding activity) of the analyte. These adaptive substrates show great promise for SERS spectroscopy of many different types of biomolecules, and we provide several current examples of their use. IntroductionOne of main advantages of Raman scattering as a detection method for molecule sensing is well known. Raman spectra enable fingerprinting of molecules which is of particular interest for bio-applications. Surface enhanced Raman scattering (SERS) provides greater detection sensitivity than conventional Raman spectroscopy [1][2][3], and it is quickly gaining traction in the study of biological molecules adsorbed on a metal surface [4][5][6][7][8][9][10][11][12]. SERS spectroscopy allows for the detection and analysis of minute quantities of analytes because it is possible to obtain high-quality SERS spectra at submonolayer molecular coverage as a result of the large scattering enhancements. SERS has also been shown to be sensitive to molecular orientation and to the distance from the metal surface [13]. Thus, SERS is well-suited for biomolecule studies in which specificity and sensitivity to the conformational state and orientation of the molecule are very important.The SERS enhancement mechanism originates in part from the large local electromagnetic fields caused by resonant surface plasmons that can be optically excited at certain wavelengths for metal particles of different shapes or closely spaced groups of particles [14][15][16][17][18][19][20][21]. For aggregates of interacting particles, which are often structured as fractals, plasmon resonances can be excited in a very broad spectral range [22]. In addition to electromagnetic field enhancement, metal nanostructures and molecules can form charge-transfer complexes that provide further enhancement for SERS [23][24][25][26][27][28][29]. The resulting overall enhancement depends critically on the particle or aggregate nanostructure morphology [22,[30][31][32][33][34][35][36], and it can be as high as 10 5 to 10 8 for

A direct manifestation of electron energy quantization in metal nanoparticles is observed in two-photon excited luminescence. Experiments reveal the discrete spectra in broadband anti-Stokes photoluminescence from aggregates of silver colloid particles. A theory based on a spherical quantum-well model for metal nanoparticles is in good agreement with experimental observations.

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