Interfacial Molecular Recognition Research Articles

Improving Rare-Earth Mineral Separation with Insights from Molecular Recognition: Functionalized Hydroxamic Acid Adsorption onto Bastnäsite and Calcite.

Enhancing the separation of rare-earth elements (REEs) from gangue materials in mined ores requires an understanding of the fundamental interactions driving the adsorption of collector ligands onto mineral interfaces. In this work, we examine five functionalized hydroxamic acid ligands as potential collectors for the REE-containing bastnäsite mineral in froth flotation using density functional theory calculations and a suite of surface-sensitive analytical spectroscopies. These include vibrational sum frequency generation, attenuated total reflectance Fourier transform infrared, Raman, and X-ray photoelectron spectroscopies. Differences in the chemical makeup of these ligands on well-defined bastnäsite and calcite surfaces allow for a systematic relationship connecting the structure to adsorption activity to be framed in the context of interfacial molecular recognition. We show how the intramolecular hydrogen bonding of adsorbed ligands requires the inclusion of explicit water solvent molecules to correctly map energetic and structural trends measured by experiments. We anticipate that the results and insights from this work will motivate and inform the design of improved flotation collectors for REE ores.

Transfer of Thiolated DNA Staples from DNA Origami Nanostructures to Self-Assembled Monolayer-Passivated Gold Surfaces: Implications for Interfacial Molecular Recognition

We show that thiolated strands displayed on a DNA origami nanostructure can be transferred to a gold surface when the DNA origami tile is deposited onto a self-assembled monolayer (SAM) passivating the surface. Spatial statistical analysis revealed the ink molecule transfer yield to be 70%, comparable to the highest yield achieved with existing DNA-nanostructure-based nanoimprinting methods. The surface passivation reduces nonspecific adsorption and allows high-resolution, label-free characterization of the transferred spatial patterns. Therefore, our method offers a pathway toward forming complex single-molecule nanoarrays for elucidating the structure–function relationships of enzyme cascades and interfacial molecular recognition.

Single Molecule Profiling of Molecular Recognition at a Model Electrochemical Biosensor.

The spatial arrangement of target and probe molecules on the biosensor is a key aspect of the biointerface structure that ultimately determines the properties of interfacial molecular recognition and the performance of the biosensor. However, the spatial patterns of single molecules on practical biosensors have been unknown, making it difficult to rationally engineer biosensors. Here, we have used high-resolution atomic force microscopy to map closely spaced individual probes as well as discrete hybridization events on a functioning electrochemical DNA sensor surface. We also applied spatial statistical methods to characterize the spatial patterns at the single molecule level. We observed the emergence of heterogeneous spatiotemporal patterns of surface hybridization of hairpin probes. The clustering of target capture suggests that hybridization may be enhanced by proximity of probes and targets that are about 10 nm away. The unexpected enhancement was rationalized by the complex interplay between the nanoscale spatial organization of probe molecules, the conformational changes of the probe molecules, and target binding. Such molecular level knowledge may allow one to tailor the spatial patterns of the biosensor surfaces to improve the sensitivity and reproducibility.

Cucurbit[7]uril-Directed Assembly of Colloidal Membrane and Stimuli-Responsive Microcapsules at the liquid-liquid Interface.

Colloidal microcapsules based on supramolecular architectures feature attractive properties and offer new opportunities in diverse areas such as delivery, sensing, and catalysis. Herein, we report a new strategy to fabricate the colloidal membrane and stimuli-responsive microcapsules by utilizing cucurbit[7]uril-mediated interfacial host-guest molecular recognition. In contrast to the traditionally used cross-linking approach, this method exploits the engineered interaction between a nanoparticle ligand and cucurbit[7]uril to tune the interfacial energy and stabilize the colloidal assembly at the interface. These capsules provide a versatile platform for simultaneous encapsulation of dual cargos. Additionally, the dynamic nature of the supramolecular interactions allows triggered release of the encapsulated cargos through the orthogonal presentation of a high affinity guest molecule.

Designing Molecular Printboards: A Photolithographic Platform for Recodable Surfaces.

A light induced strategy for the design of β-cyclodextrin (CD) based supramolecular devices is introduced, presenting a novel tool to fabricate multifunctional biointerfaces. Precision photolithography of a modified β-CD was established on a light sensitive tetrazole surface immobilized on a bioinspired polydopamine (PDA) anchor layer via various shadow masks, as well as via direct laser writing (DLW), in order to craft any desired printboard design. Interfacial molecular recognition provided by light generated cavitate domains was demonstrated via spatially resolved encoding, erasing, and recoding of distinct supramolecular guest patterns. Thus, the light directed shaping of receptor monolayers introduces a powerful path to control supramolecular assemblies on various surfaces.

A metal-ion-responsive adhesive material via switching of molecular recognition properties.

Common adhesives stick to a wide range of materials immediately after they are applied to the surfaces. To prevent indiscriminate sticking, smart adhesive materials that adhere to a specific target surface only under particular conditions are desired. Here we report a polymer hydrogel modified with both β-cyclodextrin (βCD) and 2,2′-bipyridyl (bpy) moieties (βCD–bpy gel) as a functional adhesive material responding to metal ions as chemical stimuli. The adhesive property of βCD–bpy gel based on interfacial molecular recognition is expressed by complexation of metal ions to bpy that controlled dissociation of supramolecular cross-linking of βCD–bpy. Moreover, adhesion of βCD–bpy gel exhibits selectivity on the kinds of metal ions, depending on the efficiency of metal–bpy complexes in cross-linking. Transduction of two independent chemical signals (metal ions and host–guest interactions) is achieved in this adhesion system, which leads to the development of highly orthogonal macroscopic joining of multiple objects.

Supramolecular chemistry at interfaces: host-guest interactions for fabricating multifunctional biointerfaces.

CONSPECTUS: Host-guest chemistry can greatly improve the selectivity of biomolecule-ligand binding on account of recognition-directed interactions. In addition, functional structures and the actuation of supramolecular assemblies in molecular systems can be controlled efficiently through various host-guest chemistry. Together, these highly selective, strong yet dynamic interactions can be exploited as an alternative methodology for applications in the field of programmable and controllable engineering of supramolecular soft materials through the reversible binding between complementary components. Many processes in living systems such as biotransformation, transportation of matter, and energy transduction begin with interfacial molecular recognition, which is greatly influenced by various external stimuli at biointerfaces. Detailed investigations about the molecular recognition at interfaces can result in a better understanding of life science, and further guide us in developing new biomaterials and medicines. In order to mimic complicated molecular-recognition systems observed in nature that adapt to changes in their environment, combining host-guest chemistry and surface science is critical for fabricating the next generation of multifunctional biointerfaces with efficient stimuli-responsiveness and good biocompatibility. In this Account, we will summarize some recent progress on multifunctional stimuli-responsive biointerfaces and biosurfaces fabricated by cyclodextrin- or cucurbituril-based host-guest chemistry and highlight their potential applications including drug delivery, bioelectrocatalysis, and reversible adsorption and resistance of peptides, proteins, and cells. In addition, these biointerfaces and biosurfaces demonstrate efficient response toward various external stimuli, such as UV light, pH, redox chemistry, and competitive guests. All of these external stimuli can aid in mimicking the biological stimuli evident in complex biological environments. We begin by reviewing the current state of stimuli-responsive supramolecular assemblies formed by host-guest interactions, discussing how to transfer host-guest chemistry from solution onto surfaces required for fabricating multifunctional biosurfaces and biointerfaces. Then, we present different stimuli-responsive biosurfaces and biointerfaces, which have been prepared through a combination of cyclodextrin- or cucurbituril-based host-guest chemistry and various surface technologies such as self-assembled monolayers or layer-by-layer assembly. Moreover, we discuss the applications of these biointerfaces and biosurfaces in the fields of drug release, reversible adsorption and release of some organic molecules, peptides, proteins, and cells, and photoswitchable bioelectrocatalysis. In addition, we summarize the merits and current limitations of these methods for fabricating multifunctional stimuli-responsive biointerfaces in a dynamic noncovalent manner. Finally, we present possible strategies for future designs of stimuli-responsive multifunctional biointerfaces and biosurfaces by combining host-guest chemistry with surface science, which will lead to further critical development of supramolecular chemistry at interfaces.

Supramolecular gel: From structure to function

With the development of molecular science, which focuses largely on the synthesis and functions of molecules linked together by covalent bonds, interest in supramolecular assemblies has increased. Supramolecular assemblies comprise ordered molecular systems that are held together by the cooperative or synergetic effects of non-covalent interactions such as hydrogen bonding, – stacking, electrostatic interactions, coordination, and hydrophobic interactions. Great effort has been devoted to designing and fabricating various supramolecular assemblies to produce functional soft materials that can be used in optoelectronic devices, smart responsive materials, high-performance catalytic systems, biomimetic and/or biocompatible materials and drug delivery systems. To date, many supramolecular functional systems have been constructed using “bottom-up” strategies, such as interfacial organization, surfactant-assisted assembly, molecular recognition, evaporation, electro-spinning and gelation techniques. Among the various assembly methods and molecular organized systems, gelation and gel systems have been intensely studied in recent years. Although polymeric gels have been investigated for a long time, the new low-molecular weight gels (organogels and hydrogels) have drawn renewed interest because of their facile fabrication and variety in design and functionalization. A variety of different gels and structurally organized nanomaterials can be obtained either through a simple heating and cooling process, or other stimulus of the gelator solutions. Because of the weak non-covalent interactions, the gels reversibly change between individual molecules and assemblies and are involved in assembly/disassembly processes. These fascinating characteristics of supramolecular gels allow them to be used in bionics, drug delivery systems, and smart materials. Furthermore, various well-defined nanostructures with controllable morphology, composition and even physicochemical properties can be easily synthesized in large quantities using supramolecular gelation processes. Thus, supramolecular gels are currently a hot topic and supramolecular science which involves collaborations between chemists, physicists, material scientists and engineers is evolving into an attractive interdisciplinary field. To promote the research and exchange ideas in the material science field in China, we organized a workshop at Shaanxi Normal University, Xi’an (8–10 November, 2011) under the support of the Chinese Chemical Society. In this forum, more than 50 scientists from Chinese universities and institutes discussed the history, current status, and the future trends in supramolecular gels and relevant functional materials. The forum is a great success and addresses many important issues. This special topic reports the results presented at the workshop and represents the recent progress made in the field of material sciences in China. We believe that these results will be of interest to the scientific and technological communities.

Characterization of surface modification on self-assembled monolayer-based piezoelectric crystal immunosensor for the quantification of serum α-fetoprotein

Self-assembled monolayers (SAMs) on coinage metallic material can provide versatile modeling systems for studies of interfacial electron transfer, biological interactions, molecular recognition and other interfacial phenomena. Recently, a bio-sensing system has been produced by analysis of the attachment of antibody using alkanethiols, to form SAMs on the face of Au-quartz crystal microbalance (QCM) surfaces. In this study, the attachment of anti-α-fetoprotein monoclonal antibody to a SAMs surface of 11-mercaptoundecanoic acid was achieved using water-soluble N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide as coupling agents. Surface analyses were utilized by X-ray photoelectron spectroscopy and atomic force microscopy. The quantization of immobilized antibody was characterized by the frequency shift of QCM and the radioactivity change of ¹²⁵I labeled antibody. The limit of detection and linear range of the calibration curve of the QCM method were 15 ng/ml and 15-850 ng/ml. The correlation coefficients of α-fetoprotein concentration between QCM and radioimmunoassay were 0.9903 and 0.9750 for the standards and serum samples, respectively. This report illustrates an investigation of SAMs for the preparation of covalently immobilized antibody biosensors.

Two-dimensional supramolecular assemblies involving neoglycoplipids: Self-organization and insertion properties into Langmuir monolayers

In nature, interfacial molecular recognition and chirality are of fundamental significance for the construction of biological assemblies. Lipid monolayers at liquid interface can be used as biomimetic models for studying molecular interactions in such assemblies. In this article, we will focus on the use of Langmuir monolayers for studying self-organization and insertion properties of several neoglycolipids. Two types of glycolipids have been considered, one in the context of the analysis of glycoconjugates of biological relevance, and one dealing with the ability of some glycoprobes to insert into a monolayer in relation with their efficiency for serving as membrane imaging systems.

In Situ Measurements of Aggregation and Disaggregation of Cu(II) Complex at Liquid/Liquid Interface

Aggregation of Cu(II)-5-(octadecyloxy)-2-(2-thiazolylazo)phenol (TARC18) complex at the heptane/water interface was measured directly by a centrifugal liquid membrane spectrometry and a two-phase microflow API-MS method. When the pH of an aqueous phase was increased from 4 to 6, the 1:1 complex of Cu(II)-TARC18, which was formed as a positively charged complex at the interface, formed further an aggregate, accompanied by the change of spectra suggesting its aggregation. The MS spectra of the interfacial species indicated the formation of 2:3 complex for Cu(II) and TARC18 under the conditions that the aggregate was formed. This observation allowed us to analyze the interfacial aggregation stoichiometrically: the aggregate of the 2:3 complex was formed from a 1:1 complex at the interface. The addition of purine base of adenine or guanine into the system resulted in the disruption of the aggregate by the formation of a new three-element complex of 1:1:1 for Cu(II), TARC18, and the base, showing a bathochromic shift in the spectra. Thus, the disaggregation experiment showed an interfacial molecular recognition ability of the Cu(II)-TARC18 aggregate for hydrophobic bases.

Recognition and Dissociation Kinetics in the Interfacial Molecular Recognition of Barbituric Acid by Amphiphilic Melamine-Type Monolayers

Progress in the understanding of interfacial molecular recognition kinetics is obtained by use of the sweeping technique for experimental studies of the reaction kinetics between a host monolayer and a non-surface-active species dissolved in the aqueous subphase. The experimental results show that the interfacial recognition reaction between a 2C(11)H(23)-melamine (2,4-di(n-undecylamino)-6-amino-1,3,5-triazine) monolayer and dissolved barbituric acid is reversible when the 2C(11)H(23)-melamine/barbituric acid monolayer is transferred back onto a pure water subphase. The kinetics of the recognition and dissociation reaction is experimentally and theoretically investigated. The approximate additive theoretical model developed recently is extended to consider the dissociation kinetics of the interfacial supramolecular complex. The kinetic constants for the recognition and dissociation reactions in the mixed monolayer consisting of 2C(11)H(23)-melamine and 2C(11)H(23)-melamine/barbituric acid complex are determined. It is shown that the kinetic constant of the recognition reaction is nearly independent of temperature, whereas that of the dissociation reaction increases with increasing temperature.

MALDI-TOF MS sample preparation by using alkanethiolate self-assembled monolayers: A preliminary application for protein sample analysis

Samples originating from body fluids often contain a complex mixture of inorganic salts, buffers, chaotropic agents, surfactant/detergents, preservatives, and other solubilizing agents. The presence of those contaminants often precludes direct analysis by matrix-assisted laser-desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Self-assembled monolayers (SAMs) on coinage metal can provide versatile modeling systems for studies of interfacial electron transfer, biological interactions, molecular recognition and other interfacial phenomena. In this study, SAMs surface was used for MALDI-TOF MS sample cleanup application. Experimental results from MALDI-TOF MS have revealed the better S/N ratio and resolution of using functionalized SAMs surface for the demonstration of bovine serum albumin (BSA) in artificial human urine sample. This paper reports a surface modification and cleanup method that greatly simplifies this sample preparation process.

The Role of Nonsurface-Active Species at Interfacial Molecular Recognition by Melamine-Type Monolayers

The main characteristics of Langmuir monolayers are radically changed by molecular recognition of hydrogen bond nonsurface-active species. The change in the thermodynamic, phase, and structural features by molecular recognition of dissolved uracil or barbituric acid by 2,4-di(n-undecylamino)-6-amino-1,3,5-triazine (2C11H23-melamine) monolayers is characterized by combination of surface pressure studies with Brewster angle microscopy (BAM) imaging and Grazing incidence X-ray diffraction (GIXD) measurements. Phase behavior of the 2C11H23-melamine monolayer and morphology of the condensed phase domains are changed drastically, but in a specific way, by molecular recognition of uracil or barbituric acid. The main characteristics of the interfacial system can be essentially affected by the kinetics of the recognition process. Pure 2C11H23-melamine monolayers show only small compact, but nontextured domains. The monolayers of 2C11H23-melamine-uracil assemblies develop well-shaped circular condensed-phase domains having an inner texture with alkyl chains essentially oriented parallel to the periphery and having a striking tendency to two-dimensional (2D) Ostwald ripening. The 2C11H23-melamine-barbituric acid monolayers form large homogeneous areas of condensed phase that transfer at smaller areas per molecule to a homogeneous condensed monolayer. BAM imaging of corresponding assemblies with ((CH3(CH2)11O(CH2)3)2-melamine having modified alkyl chains demonstrates the specific effect of the monolayer component. GIXD results reveal that molecular recognition of pyrimidine derivatives gives rise only to quantitative changes in the two-dimensional lattice structure. The striking differences in the main characteristics between the supramolecular species are related to their different chemical structures. Quantum chemical calculations using the semiempirical PM3 method provide information about the different nature of the hydrogen-bonding-based supramolecular structures.

Molecular Recognition of Dissolved Pyrimidine Derivatives by a Dialkyl Melamine‐Type Monolayer

Interfacial molecular recognition: Systems consisting of an amphiphilic melamine-type monolayer and a non-surface-active pyrimidine derivative (thymine, uracil) dissolved in the aqueous subphase are representative model arrangements for molecular recognition. The formation of interfacial supramolecular entities changes drastically—and specifically—the characteristic interfacial features of the amphiphilic monolayer (see picture).

Molecular Recognition Kinetics of Nonsurface Active Pyrimidine Derivatives Dissolved in the Aqueous Subphase by an Amphiphilic Melamine Type Monolayer: A Theoretical Approach

Experimental studies show a drastic change of the characteristic features (Pi-A isotherms, morphology of the condensed phase domains) of the 2C11H23-melamine monolayers by molecular recognition of the pyrimidine derivatives uracil and thymine, largely affected by the reaction kinetics of the recognition process. A new approximate additive theoretical model is introduced for describing the molecular recognition kinetics. The theoretical approach is based on two assumptions: (i) first-order reaction kinetics for the molecular recognition of the nonsurface active pyrimidine derivatives dissolved in the aqueous subphase by the melamine type monolayer and (ii) applicability of the Pi-A isotherm equations derived previously for the description of melamine type monolayers in the fluid (gaseous, LE) state and the phase transition region to the condensed state. The rate constants for the molecular recognition reaction between the melamine type monolayer and the dissolved thymine and uracil are estimated, which are roughly proportional to the bulk concentrations of the pyrimidine derivatives and depend only slightly on the temperature. The theoretical estimates agree satisfactorily with the experimental results obtained under various conditions (Pi = const and dA/dt = const). This fact supports the validity of the first model proposed for the interfacial molecular recognition kinetics with nonsurface active substrates dissolved in the aqueous subphase by complementary hydrogen bonding.

Interfacial Molecular Recognition of Dissolved Thymine by Medium Chain Dialkyl Melamine-Type Monolayers

Systems consisting of an amphiphilic melamine-type monolayer and a pyrimidine derivative dissolved in the aqueous subphase are good candidates for the formation of interfacial supramolecular assemblies by molecular recognition of hydrogen-bond nonsurface-active species. In the present work, the change in the thermodynamic, phase, and structural properties as a result of molecular recognition of dissolved thymine by 2,4-di(n-undecylamino)-6-amino-1,3,5-triazine (2 C11H23-melamine) monolayers is studied. The combination of surface pressure studies with Brewster angle microscopy (BAM) imaging and grazing incidence X-ray diffraction (GIXD) measurements is optimal for the characterization of the change in structure and phase behavior at the interfacial recognition process. The molecular recognition of the nonsurface-active thymine dissolved in aqueous subphase changes drastically the characteristic features (surface pressure-area isotherms, morphology of the condensed phase domains) of the 2 C11H23-melamine monolayer. It is demonstrated that the kinetics of the recognition process affect largely the main characteristics (phase behavior, morphology of the condensed phase domains) of the interfacial system. The monolayers of 2 C11H23-melamine-thymine assemblies form dumbbell-shaped condensed phase domains not yet observed in other Langmuir monolayers so far. GIXD results show that the molecular recognition of thymine causes only quantitative changes in the two-dimensional lattice structure. Complementary hydrogen bonding of two thymine molecules by one 2 C11H23-melamine molecule is concluded from the chemical structure of both components. Additional information about the nature of the hydrogen bonding on the basis of supramolecular assemblies is obtained by using the quantum chemical PM3 approximation. Energy and lengths of the hydrogen bonds of the optimized thymine-2 C11H23-melamine-thymine structure are calculated.

Proteomic Profiling of Erythrocyte Proteins by Proteolytic Digestion Chip and Identification Using Two-Dimensional Electrospray Ionization Tandem Mass Spectrometry

Self-assembled monolayers (SAMs) on coinage metal provide versatile modeling systems for studies of interfacial electron transfer, biological interactions, molecular recognition, and other interfacial phenomena. The bonding of enzyme to SAMs of alkanethiols onto gold surfaces is exploited to produce an enzyme chip. In this work, the attachment of trypsin to a SAMs surface of 11-mercaptoundecanoic acid was achieved using water soluble N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide as coupling agent. A two-dimensional liquid-phase separation scheme coupled with mass spectrometry is presented for proteomic analysis of erythrocyte proteins. The application of proteomics, particularly with reference to analysis of proteins, will be described. Surface analyses have revealed that the X-ray Photoelectron Spectroscopy (XPS) C1s and N1s core levels illustrate the immobilization of trypsin. These data are also in good agreement with Fourier Transformed Infrared Reflection-Attenuated Total Reflection (FTIR-ATR) spectra for the peaks at Amide I and Amide II. Using two-dimensional nano-high performance liquid chromatography electrospray ionization tandem mass spectrometry (2D nano-HPLC-ESI-MS/MS) system observations, analytical results have demonstrated the erythrocyte proteins digestion of the immobilized trypsin on the functionalized SAMs surface. For such surfaces, it also shows the enzyme digestion ability of the immobilized trypsin. The experiment results revealed the identification of 272 proteins from erythrocyte protein sample. The terminal groups of the SAMs structure can be further functionalized with biomolecules or antibodies to develop surface-base diagnostics, biosensors, or biomaterials.

Supramolecular Assemblies and Molecular Recognition of Amphiphilic Schiff Bases with Barbituric Acid in Organized Molecular Films

A bolaform Schiff base, N,N'-bis(salicylidene)-1,10-decanediamine (BSC10), has been synthesized and its interfacial hydrogen bond formation or molecular recognition with barbituric acid was investigated in comparison with that of a single chain Schiff base, 2-hydroxybenzaldehyde-octadecylamine (HBOA). It has been found that while HBOA formed a monolayer at the air/water interface, the bolaform Schiff base formed a multilayer film with ordered layer structure on water surface. When the Schiff bases were spread on the subphase containing barbituric acid, both of the Schiff bases could form hydrogen bonds with barbituric acid in situ in the spreading films. As a result, an increase of the molecular areas in the isotherms was observed. The in situ H-bonded films could be transferred onto solid substrates, and the transferred multilayer films were characterized by various methods such as UV-vis and FT-IR spectrosopies. Spectral changes were observed for the films deposited from the barbituric acid subphase, which supported the hydrogen bond formation between the Schiff bases and barbituric acid. By measuring the MS-TOF of the deposited films dissolved in CHCl3 solution, it was concluded that a 2:1 complex of HBOA with barbituric acid and a 1:2 complex of BSC10 with barbituric acid were formed. On the other hand, when the multilayer films of both Schiff bases were immersed in an aqueous solution of barbituric acid, a similar molecular recognition through the hydrogen bond occurred. A clear conformational change of the alkyl spacer in the bolaform Schiff base was observed during the complex formation with the barbituric acid.

Characterization of trypsin immobilized on the functionable alkylthiolate self-assembled monolayers: A preliminary application for trypsin digestion chip on protein identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Self-assembled monolayers (SAMs) on coinage metal provide versatile modeling systems for studies of interfacial electron transfer, biological interactions, molecular recognition and other interfacial phenomena. Recently the bonding of enzyme to SAMs of alkanethiols onto Au electrode surfaces was exploited to produce a bio-sensing system. In this work, the attachment of trypsin to a SAMs surface of 11-mercaptoundecanoic acid was achieved using water soluble N-ethyl-N '-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide as coupling agent. The thickness of SAMs was determined by optical ellipsometer; contact angles of the modified Au surfaces were measured in air using a goniometer. The Second Harmony Generation data displays the last few percents of the alkylthiol molecules adsorbed and produced the complete monolayer by inducing the transition from a high number of gauche defects to an all-trans conformation. Using X-ray Photoelectron Spectroscopy (XPS) and Fourier-Transformed Infrared Reflection-Absorption and Attenuated Total Reflection Spectroscopes (FTIR-RAS and ATR), we examined the chemical structures of samples with different treatments. By matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), we demonstrated the digestion of bovine serum albumin (BSA) on the trypsin-immobilized SAMs surface. Experimental results have revealed that the XPS C1s core levels at 286.3 and 286.5 eV (Amine bond), 288.1 eV (Amide bond) and 289.3 eV (Carboxylic acid) illustrate the immobilization of trypsin. These data were also in good agreement with FTIR-ATR spectra for the peaks valued at 1659.4 cm(-1) (Amide I) and 1546.6 cm(-1) (Amide II). Using MALDI-TOF MS observations, analytical results have demonstrated the BSA digestion of the immobilized trypsin on the functionalized SAMs surface. For such surfaces, BSA was digested on the trypsin-immobilized SAMs surface, which shows the enzyme digestion ability of the immobilized trypsin. The terminal groups of the SAMs structure can be further functionalized with biomolecules or antibodies to develop surface-base diagnostics, biosensors, or biomaterials.