CONTENTS 1. Introduction 8471 2. Theoretical Background of Chiral SFG 8472 2.1. General Principles of SFG 8472 2.2. Effective Susceptibility of Chiral Surfaces 8473 2.3. Surface Susceptibility and Molecular Hyperpolarizability 8474 2.4. Chiral SFG Response: Bulk versus Interface 8476 3. Chiral SFG Experiments 8477 3.1. Polarization Settings for Chiral SFG Experiments 8477 3.2. Spectrometers for Chiral SFG Studies of Biomacromolecules 8478 3.3. Surface Platforms for Probing Biomacromolecules 8479 4. Structures of Biomacromolecules at Interfaces Probed by Chiral SFG 8480 4.1. Chiral Amide I Signals of Proteins or Peptides at Interfaces 8480 4.2. Chiral C−H Stretch Signals of DNA on Solid/ Water Interfaces 8480 4.3. Chiral N−H Stretch from Protein Backbone at Interfaces 8481 4.4. Chiral N−H Stretch and Amide I for Probing Secondary Structures at Interfaces 8482 4.5. Characterization of Various Vibrational Bands of Collagen 8484 4.6. Double Resonance for Detecting Chiral SFG Signal from Porphyrin J Aggregates 8484 5. Orientations of Biomacromolecules at Interfaces Probed by Chiral SFG 8485 5.1. Orientation of Antiparallel β-Sheet Structures at Interfaces 8485 5.2. Orientation of Parallel β-Sheet Structures at Interfaces 8486 6. Kinetics and Dynamics of Biomacromolecules at Interfaces Probed by Chiral SFG 8488 6.1. Kinetics of Protein Folding Probed by Chiral Amide I and N−H Stretch 8488 6.2. Kinetics of Proton Exchange in Protein Backbones Probed by Chiral N−H 8489 6.3. Kinetics of Protein Self-Assembly Probed by Chiral C−H Stretch of Protein Side Chains 8490 7. Calculations of Hyperpolarizability of Biomacromolecules 8491 7.1. Calculation of Hyperpolarizability of Biomacromolecules for Weak Vibrational Coupling 8491 7.2. Calculation of Hyperpolarizability of Biomacromolecules for Strong Vibrational Coupling 8492 7.3. Calculation of Hyperpolarizability by the ab Initio Quantum Chemistry Method 8492 7.4. Comparison of Calculation Methods for Hyperpolarizability of Biomacromolecules 8493 8. Summary and Outlook 8493 8.1. Summary 8493 8.2. Potential Applications 8493 8.3. Challenges and Outlooks 8494 Author Information 8495 Corresponding Author 8495 Notes 8495 Biographies 8495 References 8496 Note Added after ASAP Publication 8498

Surface-enhanced Raman spectroscopy (SERS) has emerged as a powerful tool to detect biomolecules in aqueous environments. However, it is challenging to identify protein structures at low concentrations, especially for the proteins existing in an equilibrium mixture of various conformations. Here, we develop an in situ optical tweezers-coupled Raman spectroscopy to visualize and control the hotspot between two Ag nanoparticle-coated silica beads, generating tunable and reproducible SERS enhancements with single-molecule level sensitivity. This dynamic SERS detection window is placed in a microfluidic flow chamber to detect the passing-by proteins, which precisely characterizes the structures of three globular proteins without perturbation to their native states. Moreover, it directly identifies the structural features of the transient species of alpha-synuclein among its predominant monomers at physiological concentration of 1 μM by reducing the ensemble averaging. Hence, this SERS platform holds the promise to resolve the structural details of dynamic, heterogeneous, and complex biological systems.

While surface enhanced Raman spectroscopy (SERS) based biosensing has demonstrated great potential for point-of-care diagnostics in the laboratory, its application in the field is limited by the short life time of commonly used silver based SERS active substrates. In this work, we report our attempt towards SERS based field biosensing, involving the development of a novel sustained and cost-effective substrate composed of silver nanoparticles protected by small nitrogen-doped Graphene Quantum Dots, i.e. Ag NP@N-GQD, and its systematic evaluation for glucose sensing. The new substrate demonstrated significantly stronger Raman enhancement compared to pure silver nanoparticles. More importantly, the new substrate preserved SERS performance in a normal indoor environment for at least 30 days in both the wet and dry states, in contrast to only 10 days for pure silver nanoparticles. The Ag NP@N-GQD thin film in the dry state was then successfully applied as a SERS substrate for glucose detection in mouse blood samples. The new substrate was synthesized under mild experimental conditions, and the cost increase due to N-GQD was negligible. These results suggest that the Ag NP@N-GQD is a cost-effective and sustained SERS substrate, the development of which represents an important step towards SERS based field biosensing.

Cell membranes are crucial to many biological processes. Because of their complexity, however, lipid bilayers are often used as model systems. Lipid structures influence the physical properties of bilayers, but their interplay, especially in multiple-component lipid bilayers, has not been fully explored. Here, we used the Langmuir-Blodgett method to make mono- and bilayers of 1,2-dihexadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), and 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phospho-L-serine (POPS) as well as their 1:1 binary mixtures. We studied the fluidity, stability, and rigidity of these structures using sum frequency generation (SFG) spectroscopy combined with analyses of surface pressure-area isotherms, compression modulus, and stability. Our results show that single-component bilayers, both saturated and unsaturated, may not be ideal membrane mimics because of their low fluidity and/or stability. However, the binary saturated and unsaturated DPPG/POPG and DPPG/POPS systems show not only high stability and fluidity but also high resistance to changes in surface pressure, especially in the range of 25-35 mN/m, the range typical of cell membranes. Because the ratio of saturated to unsaturated lipids is highly regulated in cells, our results underline the possibility of modulating biological properties using lipid compositions. Also, our use of flat optical windows as solid substrates in SFG experiments should make the SFG method more compatible with other techniques, enabling more comprehensive future surface characterizations of bilayers.