Thiol-SAM Concentration Effect on the Performance of Interdigitated Electrode-Based Redox-Free Biosensors.

Despite the direct, redox-free and simple detection non-faradaic impedimetric biosensors offer, considerable optimizations are required to enhance their performance for the detection of various biomarkers. Non-faradaic EIS sensors' performance depends on the interfacial capacitance between a polarized biosensor surface and the tested sample solution. Careful engineering and design of the interfacial capacitance is encouraged to magnify the redout signal upon bioreceptor-antigen interactions. One of the methods to achieve this goal is by optimizing the self-assembled monolayer concentration, which has not been reported for non-faradaic impedimetric sensors. Here, the impact of alkanethiolate (cysteamine) concentration on the performance of gold (Au) interdigitated electrode (Au-IDE) biosensors is reported. Six sets of biosensors were prepared, each with a different cysteamine concentration: 100 nM, 1 µM, 10 µM, 100 µM, 1 mM, and 10 mM. The biosensors were prepared for the direct detection of LDL cholesterol by attaching LDL antibodies on top of the cysteamine via a glutaraldehyde cross-linker. As the concentration of cysteamine increased from 100 nM to 100 µM, the sensitivity of the biosensor increased from 6.7 to 16.2 nF/ln (ng/mL). As the cysteamine concentration increased from 100 µM to 10 mM, the sensitivity deteriorated. The limit of detection (LoD) of the biosensor improved as the cysteamine increased from 100 nM to 100 µM (i.e., 400 ng/mL to 59 pg/mL). However, the LoD started to increase to 67 pg/mL and 16 ng/mL for 1 mM and 10 mM cysteamine concentrations, respectively. This shows that the cysteamine concentration has a detrimental effect on redox-free biosensors. The cysteamine layer has to be as thin as possible and uniformly cover the electrode surfaces to maximize positive readout signals and reduce negative signals, significantly improving both sensitivity and LoD.

Antibody functionalized capacitance sensor for label-free and real-time detection of bacteria and antibiotic susceptibility.

The effective management of infectious diseases and the growing concern of antibiotic resistance necessitates accurate and targeted therapies, highlighting the importance of antibiotic susceptibility testing. This study aimed to develop a real-time impedimetric biosensor for identifying and monitoring bacterial growth and antibiotic susceptibility. The biosensor employed a gold 8-channel disk-shaped microelectrode array with specific antibodies as bio-recognition elements. This setup was allowed for the analysis of bacterial samples, including Staphylococcus aureus, Bacillus cereus, and Micrococcus luteus. These microorganisms were successfully cultured and detected within 1 h of incubation even with a minimal bacterial concentration of 10 CFU/ml. Overall, the developed biosensor array exhibits promising capabilities for monitoring S. aureus, B. cereus and M. luteus, showcasing an excellent linear response ranging from 10 to 104 CFU/ml with a detection limit of 0.95, 1.22 and 1.04 CFU/mL respectively. Moreover, real-time monitoring of antibiotic susceptibility was facilitated by changes in capacitance, which dropped when bacteria were exposed to antibiotic doses higher than their minimum inhibitory concentration (MIC), indicating suppressed bacterial growth. The capacitance measurements enabled determination of half-maximal cytotoxic concentrations (CC50) values for each bacteria-antibiotic pair. As a proof-of-concept application, the developed sensor array was employed as a sensing platform for the real time detection of bacteria in milk samples, which ensured the reliability of the sensor for in-field detection of foodborne pathogens and rapid antimicrobial susceptibility tests (ASTs).

A new cysteamine-copper chemically modified screen-printed gold electrode for glyphosate determination.

A chemically modified screen-printed gold electrode has been prepared by covering the electrode surface with a cysteamine-copper self-assembled monolayer (SAM). The sensor was effective for the voltammetric sensing of glyphosate. The method exploits the interaction of glyphosate with copper ions complexed by cysteamine, which results in a decrease in the intensity of copper redox current. Cyclic voltammetry was employed as a measuring technique. When dealing with voltammograms with numerous peaks changing in shape and size, it is difficult to define which signal is the most significant for the analyte determination; in these cases, a helpful approach is chemometrics. In this work, PLS (Partial Least Square regression) has been applied to build models to correlate the signal with the glyphosate concentration in standard aqueous solutions and tap water samples (matrix-matched calibration). The method's figures of merits were evaluated, obtaining a limit of quantification of about 5 µM. The reliability of the proposed sensor was verified by analyzing tap water spiked with glyphosate; recoveries higher than 90 % were achieved.

N-Heterocyclic carbene-based gold etchants.

N-Heterocyclic carbenes (NHCs) are an emerging alternative to thiols for the formation of stable self-assembled monolayers (SAMs) on gold. We examined several different species that have been used to produce NHC-based monolayers on gold, namely 1,3-diisopropyl-5-nitrobenzimidazolium iodide, 1,3-diisopropyl-5-nitrobenzimidazolium hydrogen carbonate, bis(1,3-diisopropyl-5-nitrobenzimidazolium)gold(I) iodide, and 1,3-diisopropyl-5-nitrobenzimidazole-2-ylidene. Contrary to expectation, solutions containing the first two species in tetrahydrofuran and dichloromethane caused visible loss of gold from thin-film-coated glass slides. The use of toluene solutions of all species resulted in no apparent dissolution of gold. We present scanning electron micrographs and elemental imaging analyses by energy dispersive X-ray spectroscopy to examine the effect of solutions of each species on the gold film. This work highlights the risk of unwanted etching during some routes to NHC-based surface functionalization but also the potential for deliberate etching, with the outcome determined by choice of chemically synthesized organic species and solvent.

Enhanced Triboelectric Charge Stability by Air-Stable Radicals.

This paper demonstrates that air-stable radicals enhance the stability of triboelectric charge on surfaces. While charge on surfaces is often undesirable (e.g., static discharge), improved charge retention can benefit specific applications such as air filtration. Here, it is shown that self-assembled monolayers (SAMs) containing air-stable radicals, 2,2,6,6-tetramethylpiperidin-1-yl)oxidanyl (TEMPO), hold the charge longer than those without TEMPO. Charging and retention are monitored by Kelvin Probe Force Microscopy (KPFM) as a function of time. Without the radicals on the surface, charge retention increases with the water contact angle (hydrophobicity), consistent with the understanding that surface water molecules can accelerate charge dissipation. Yet, the most prolonged charge retention is observed in surfaces treated with TEMPO, which are more hydrophilic than untreated control surfaces. The charge retention decreases with reducing radical density by etching the TEMPO-silane with tetrabutylammonium fluoride (TBAF) or scavenging the radicals with ascorbic acid. These results suggest a pathway toward increasing the lifetime of triboelectric charges, which may enhance air filtration, improve tribocharging for patterning charges on surfaces, or boost triboelectric energy harvesting.

Adsorption behavior of long-chain perfluoroalkyl substances on hydrophobic surface: A combined molecular characterization and simulation study.

Hydrophobic interaction is a prevalent sorption mechanism of poly- and perfluoroalkyl substances (PFAS) in natural and engineered environments. In this study, we combined quartz crystal microbalance with dissipation (QCM-D), atomic force microscope (AFM) with force mapping, and molecular dynamics (MD) simulation to probe the molecular behavior of PFAS at the hydrophobic interface. On a CH3-terminated self-assembled monolayer (SAM), perfluorononanoic acid (PFNA) showed ∼2-fold higher adsorption than perfluorooctane sulfonate (PFOS) that has the same fluorocarbon tail length but a different head group. Kinetic modeling using the linearized Avrami model suggests that the PFNA/PFOS-surface interaction mechanisms can evolve over time. This is confirmed by AFM force-distance measurements, which shows that while the adsorbed PFNA/PFOS molecules mostly lay flat, a portion of them formed aggregates/hierarchical structures of 1-10 nm in size after lateral diffusion on surface. PFOS showed a higher affinity to aggregate than PFNA. Association with air nanobubbles is observed for PFOS but not PFNA. MD simulations further showed that PFNA has a greater tendency than PFOS to have its tail inserted into the hydrophobic SAM, which can enhance adsorption but limit lateral diffusion, consistent with the relative behavior of PFNA/PFOS in QCM and AFM experiments. This integrative QCM-AFM-MD study reveals that the interfacial behavior of PFAS molecules can be heterogeneous even on a relatively homogeneous surface.

Survey of oligoethylene glycol-based self-assembled monolayers on electrochemical aptamer-based sensor in biological fluids.

The ability to monitor levels of endogenous markers and clearance profiles of drugs and their metabolites can improve the quality of biomedical research and precision with which therapies are individualized. Towards this end, electrochemical aptamer-based (EAB) sensors have been developed that support the real-time monitoring of specific analytes in vivo with clinically relevant specificity and sensitivity. A challenge associated with the in vivo deployment of EAB sensors, however, is how to manage the signal drift which, although correctable, ultimately leads to unacceptably low signal-to-noise ratios, limiting the measurement duration. Motivated by the correction of signal drift, in this paper, we have explored the use of oligoethylene glycol (OEG), a widely employed antifouling coating, to reduce the signal drift in EAB sensors. Counter to expectations, however, when challenged in 37 °C whole blood in vitro, EAB sensors employing OEG-modified self-assembled monolayers exhibit both greater drift and reduced signal gain, compared with those employ a simple, hydroxyl-terminated monolayer. On the other hand, when EAB sensor was prepared with a mix monolayer using MCH and lipoamido OEG 2 alcohol, reduced signal noise was observed compared to the same sensor prepared with MCH presumably due to improved SAM construction. These results suggest broader exploration of antifouling materials will be required to improve the signal drift of EAB sensors.

Tailoring On-Surface Molecular Reactions and Assembly through Hydrogen-Modified Synthesis: From Triarylamine Monomer to 2D Covalent Organic Framework.

Relative to conventional wet-chemical synthesis techniques, on-surface synthesis of organic networks in ultrahigh vacuum has few control parameters. The molecular deposition rate and substrate temperature are typically the only synthesis variables to be adjusted dynamically. Here we demonstrate that reducing conditions in the vacuum environment can be created and controlled without dedicated sources─relying only on backfilled hydrogen gas and ion gauge filaments─and can dramatically influence the Ullmann-like on-surface reaction used for synthesizing two-dimensional covalent organic frameworks (2D COFs). Using tribromo dimethylmethylene-bridged triphenylamine ((Br3)DTPA) as monomer precursors, we find that atomic hydrogen (H•) blocks aryl-aryl bond formation to such an extent that we suspect this reaction may be a factor in limiting the ultimate size of 2D COFs created through on-surface synthesis. Conversely, we show that control of the relative monomer and hydrogen fluxes can be used to produce large self-assembled islands of monomers, dimers, or macrocycle hexamers, which are of interest in their own right. On-surface synthesis of oligomers, from a single precursor, circumvents potential challenges with their protracted wet-chemical synthesis and with multiple deposition sources. Using scanning tunneling microscopy and spectroscopy (STM/STS), we show that changes in the electronic states through this oligomer sequence provide an insightful view of the 2D COF (synthesized in the absence of atomic hydrogen) as the end point in an evolution of electronic structures from the monomer.

Patterning of Silicon Substrate with Self-Assembled Monolayers Using Vertically Aligned Carbon Nanotube Electron Sources.

We introduce a novel patterning technique based on e-beam lithography using vertically aligned carbon nanotube (VACNT) emitters with self-assembled monolayers (SAMs). A 20 µm line width of silicon wafer patterning was successfully demonstrated using octadecyl trichlorosilane (OTS) as a photoresist. To investigate surface modification by the irradiated electrons from the emitters, both contact angle measurement and energy dispersive X-ray (EDX) analysis were conducted. The patterning mechanism of the electron beam irradiated on OTS-coated substrate by our cold cathode electron beam (C-beam) was demonstrated by the analyzed results. The effect of current density and exposure time on the OTS patterning was studied and optimized for the Si wafer patterning in terms of the electronic properties of the VACNTs. The authors expect the new technique to contribute to the diverse applications to microelectromechanical (MEMS) technologies owing to the advantages of facile operation and precise dose control capability based on field electron emission current from the VACNT emitter arrays.

Nanometric Surface-Selective Regulation of Au/In2O3 Nanofibers as an Exhaled H2S Chemiresistor for Periodontitis Diagnosis.

As one of the most prevalent diseases in the world, timely early intervention for periodontitis is a great challenge because the indicator is imperceptible. The exhaled H2S is considered to be a promising biomarker for fast and invasive periodontitis screening; however, the high-performance H2S gas sensor with excellent selectivity and sensitivity which is applicable to the oral cavity remains technically challenging. Herein, a self-assembled monolayer (SAM)-functionalized Au/In2O3 nanofiber (NF) sensor for H2S exhalation analysis was developed to flexibly and effectively modulate the selectivity of the sensor. Through optimizing the specific binding capacity to H2S by systematic adjustment with terminal groups and alkyl chains of SAMs, the sensing performance of the SAM-functionalized Au/In2O3 NF sensor is greatly enhanced. In the optimal (Au/In2O3-MPTES) sensor, the functionalization of the MPTES molecule could achieve significant response enhancement because of the stronger interaction between the sulfhydryl group at the end of the MPTES and H2S. Density functional theory simulation supports the proposed selective sensing mechanism via the analysis of adsorption energy and charge density distribution. The sensor exhibited a high response to H2S (1505.3-10 ppm) at an operating temperature of 100 °C with a low practical detection limit of 10 ppb and 13-145 fold enhanced selectivity. Furthermore, the Au/In2O3-MPTES sensor was successfully applied to distinguish the breath of healthy individuals and patients with severe periodontitis. This study provides novel design insights for the development of highly selective gas sensors for clinical aids in the diagnosis and detection of oral diseases such as periodontitis.

Determination of rSpike Protein by Specific Antibodies with Screen-Printed Carbon Electrode Modified by Electrodeposited Gold Nanostructures.

In this research, we assessed the applicability of electrochemical sensing techniques for detecting specific antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike proteins in the blood serum of patient samples following coronavirus disease 2019 (COVID-19). Herein, screen-printed carbon electrodes (SPCE) with electrodeposited gold nanostructures (AuNS) were modified with L-Cysteine for further covalent immobilization of recombinant SARS-CoV-2 spike proteins (rSpike). The affinity interactions of the rSpike protein with specific antibodies against this protein (anti-rSpike) were assessed using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) methods. It was revealed that the SPCE electroactive surface area increased from 1.49 ± 0.02 cm2 to 1.82 ± 0.01 cm2 when AuNS were electrodeposited, and the value of the heterogeneous electron transfer rate constant (k0) changed from 6.30 × 10-5 to 14.56 × 10-5. The performance of the developed electrochemical immunosensor was evaluated by calculating the limit of detection and limit of quantification, giving values of 0.27 nM and 0.81 nM for CV and 0.14 nM and 0.42 nM for DPV. Furthermore, a specificity test was performed with a solution of antibodies against bovine serum albumin as the control aliquot, which was used to assess nonspecific binding, and this evaluation revealed that the developed rSpike-based sensor exhibits low nonspecific binding towards anti-rSpike antibodies.

Electrochemical Determination of Interaction between SARS-CoV-2 Spike Protein and Specific Antibodies.

The serologic diagnosis of coronavirus disease 2019 (COVID-19) and the evaluation of vaccination effectiveness are identified by the presence of antibodies specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this paper, we present the electrochemical-based biosensing technique for the detection of antibodies specific to the SARS-CoV-2 proteins. Recombinant SARS-CoV-2 spike proteins (rSpike) were immobilised on the surface of a gold electrode modified by a self-assembled monolayer (SAM). This modified electrode was used as a sensitive element for the detection of polyclonal mouse antibodies against the rSpike (anti-rSpike). Electrochemical impedance spectroscopy (EIS) was used to observe the formation of immunocomplexes while cyclic voltammetry (CV) was used for additional analysis of the surface modifications. It was revealed that the impedimetric method and the elaborate experimental conditions are appropriate for the further development of electrochemical biosensors for the serological diagnosis of COVID-19 and/or the confirmation of successful vaccination against SARS-CoV-2.

Thermopower of Molecular Junction in Harsh Thermal Environments.

Molecular junctions can be miniaturized devices for heat-to-electricity conversion application, yet these operate only in mild thermal environments (less than 323 K) because thiol, the most widely used anchor moiety for chemisorption of active molecules onto surface of electrode, easily undergoes thermal degradation. N-Heterocyclic carbene (NHC) can be an alternative to traditional thiol anchor for producing ultrastable thermoelectric molecular junctions. Our experiments showed that the NHC-based molecular junctions withstood remarkably high temperatures up to 573 K, exhibiting consistent Seebeck effect and thermovoltage up to approximately |1900 µV|. Our work advances our understanding of molecule-electrode contact in the Seebeck effect, providing a roadmap for constructing robust and efficient organic thermoelectric devices.

Towards electrochemical surface plasmon resonance sensor based on the molecularly imprinted polypyrrole for glyphosate sensing.

In this research the molecular imprinting technology was applied for the formation of glyphosate-sensitive layer. The glyphosate imprinted conducting polymer polypyrrole (MIPpy) was deposited on a gold chip/electrode and used as an electrochemical surface plasmon resonance (ESPR) sensor. The results described in this study disclose some restrictions and challenges, which arise during the development of glyphosate ESPR sensor based on the molecularly imprinted polymer development stage. It was demonstrated, that glyphosate could significantly affect the electrochemical deposition process of molecularly imprinted polymer on the electrode. The results of cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and surface plasmon resonance (SPR) have demonstrated that glyphosate molecules tend to interact with bare gold electrode and thus hinder the polypyrrole deposition. As a possible solution, the formation of a self-assembled monolayer (SAM) of 11-(1H-Pyrrol-1-yl)undecane-1-thiol (PUT) before electrochemical deposition of MIPpy and NIPpy was applied. Dissociation constant (KD) and free energy of Gibbs (ΔG0) values of glyphosate on MIPpy and Ppy without glyphosate imprints (NIPpy) were calculated. For the interaction of glyphosate with MIPpy the KD was determined as 38.18 ± 2.33⋅10-5 and ΔG0 as -19.51 ± 0.15 kJ/mol.

Electrochemical Biosensor for the Determination of Specific Antibodies against SARS-CoV-2 Spike Protein.

In this article, we report the development of an electrochemical biosensor for the determination of the SARS-CoV-2 spike protein (rS). A gold disc electrode was electrochemically modified to form the nanocrystalline gold structure on the surface. Then, it was further altered by a self-assembling monolayer based on a mixture of two alkane thiols: 11-mercaptoundecanoic acid (11-MUA) and 6-mercapto-1-hexanol (6-MCOH) (SAMmix). After activating carboxyl groups using a N-(3-dimethylaminopropyl)-N'-ethyl-carbodiimide hydrochloride and N-hydroxysuccinimide mixture, the rS protein was covalently immobilized on the top of the SAMmix. This electrode was used to design an electrochemical sensor suitable for determining antibodies against the SARS-CoV-2 rS protein (anti-rS). We assessed the association between the immobilized rS protein and the anti-rS antibody present in the blood serum of a SARS-CoV-2 infected person using three electrochemical methods: cyclic voltammetry, differential pulse voltammetry, and potential pulsed amperometry. The results demonstrated that differential pulse voltammetry and potential pulsed amperometry measurements displayed similar sensitivity. In contrast, the measurements performed by cyclic voltammetry suggest that this method is the most sensitive out of the three methods applied in this research.

Label-Free, Rapid and Facile Gold-Nanoparticles-Based Assay as a Potential Spectroscopic Tool for Trastuzumab Quantification.

Monoclonal antibody-based immunotherapy is one of the pillars of cancer treatment. However, for an efficient and personalized approach to the therapy, a quantitative evaluation of the right dose for each patient is required. In this study, we developed a simple, label-free, and rapid approach to quantify Trastuzumab, a humanized IgG1 monoclonal antibody used against human epidermal growth factor receptor 2 (HER2), overexpressed in breast cancer patients, based on localized surface plasmon resonance (LSPR). The central idea of this work was to use gold nanoparticles (AuNPs) as plasmonic scaffolds, decorated with HER2 binders mixed with oligo-ethylene glycol (OEG) molecules, to tune the surface density of the attached macromolecules and to minimize nonspecific binding events. Specifically, we characterized and optimized a self-assembled monolayer of mixed alkylthiols terminated with nitrilotriacetic acid (NTA), and OEG3 as a spacing ligand to achieve both excellent dispersibility and high reliability in protein immobilization. The successful immobilization of histidine-tagged HER2 (His-tagged HER2) on NTA via cobalt (II) chelates was demonstrated, confirming the fully functional attachment of the proteins to the AuNP surface. The proposed design demonstrates the capability of producing a clear readout that enables the transduction of a Trastuzumab/HER2 binding event into optical signals based on the wavelength shifts in LSPR, which allowed for detecting clinically relevant concentrations of Trastuzumab down to 300 ng/mL in the buffer and 2 µg/mL in the diluted serum. This strategy was found to be fast and highly specific to Trastuzumab. These findings make the present platform an auspicious tool for developing affordable bio-nanosensors.

Thermal and Thermoelectric Properties of SAM-Based Molecular Junctions.

In molecular thermoelectrics, the thermopower of molecular junctions is closely interlinked with their thermal properties; however, the detailed relationship between them remains uncertain. This study systematically investigates the thermal properties of self-assembled monolayer (SAM)-based molecular junctions and relates them to the thermoelectric performance of the junctions. The electrode temperatures for the bare AuTS, AuTS/EGaIn, and AuTS/TPT SAM//Ga2O3/EGaIn samples placed on a hot chuck were measured under different conditions, such as air vs vacuum and the presence and absence of thermal grease, which generates a heat conduction channel from a hot chuck to gold. It was revealed that the SAM was the most efficient thermal resistor, which was responsible for the creation of a temperature differential (ΔT) across the junction; ΔT in an air atmosphere is overestimated to some extent, and air mainly contributes to large dispersions of thermovoltage (ΔV) data. While junction measurements in air were possible at low ΔT (up to 13 K), the new optimal condition, under a vacuum and with thermal grease, allowed us to examine a wide temperature range up to ΔT = 40 K and obtain a more reliable Seebeck coefficient (S, µV/K). The value of S under the new condition was ∼1.4 times higher than that measured in air without thermal grease. Our study shows the potential of liquid-metal-based junctions to reliably investigate heat conduction across nanometer-thick organic films and elaborates on how the thermal properties of molecular junctions affect their thermoelectric performance.

Streptavidin Coverage on Biotinylated Surfaces.

Biosensors and other biological platform technologies require the functionalization of their surface with receptors to enhance affinity and selectivity. Control over the functionalization density is required to tune the platform's properties. Streptavidin (SAv) monolayers are widely used to immobilize biotinylated proteins, receptors, and DNA. The SAv density on a surface can be varied easily, but the predictability is dependent on the method by which the SAv is immobilized. In this study we show a method to quantitatively predict the SAv coverage on biotinylated surfaces. The method is validated by measuring the SAv coverage on supported lipid bilayers with a range of biotin contents and two different main phase lipids and by using quartz crystal microbalance and localized surface plasmon resonance. We explore a predictive model of the biotin-dependent SAv coverage without any fit parameters. Model and data allow to predict the SAv coverage based on the biotin coverage, in both the low- and high-density regimes. This is of special importance in applications with multivalent binding where control over surface receptor density is required, but a direct measurement is not possible.

SARS-CoV-2 spike protein detection through a plasmonic D-shaped plastic optical fiber aptasensor.

A specific aptameric sequence has been immobilized on short polyethyleneglycol (PEG) interface on gold nano-film deposited on a D-shaped plastic optical fiber (POFs) probe, and the protein binding has been monitored exploiting the very sensitive surface plasmon resonance (SPR) phenomenon. The receptor-binding domain (RBD) of the SARS-CoV-2 spike glycoprotein has been specifically used to develop an aptasensor. Surface analysis techniques coupled to fluorescence microscopy and plasmonic analysis have been utilized to characterize the biointerface. Spanning a wide protein range (25 ÷ 1000 nM), the SARS-Cov-2 spike protein was detected with a Limit of Detection (LoD) of about 37 nM. Different interferents (BSA, AH1N1 hemagglutinin protein and MERS spike protein) have been tested confirming the specificity of our aptasensor. Finally, a preliminary test in diluted human serum encouraged its application in a point-of-care device, since POF-based aptasensor represent a potentially low-cost compact biosensor, characterized by a rapid response, a small size and could be an ideal laboratory portable diagnostic tool.

Bioactive Coatings on Titanium: A Review on Hydroxylation, Self-Assembled Monolayers (SAMs) and Surface Modification Strategies.

Titanium (Ti) and its alloys have been demonstrated over the last decades to play an important role as inert materials in the field of orthopedic and dental implants. Nevertheless, with the widespread use of Ti, implant-associated rejection issues have arisen. To overcome these problems, antibacterial properties, fast and adequate osseointegration and long-term stability are essential features. Indeed, surface modification is currently presented as a versatile strategy for developing Ti coatings with all these challenging requirements and achieve a successful performance of the implant. Numerous approaches have been investigated to obtain stable and well-organized Ti coatings that promote the tailoring of surface chemical functionalization regardless of the geometry and shape of the implant. However, among all the approaches available in the literature to functionalize the Ti surface, a promising strategy is the combination of surface pre-activation treatments typically followed by the development of intermediate anchoring layers (self-assembled monolayers, SAMs) that serve as the supporting linkage of a final active layer. Therefore, this paper aims to review the latest approaches in the biomedical area to obtain bioactive coatings onto Ti surfaces with a special focus on (i) the most employed methods for Ti surface hydroxylation, (ii) SAMs-mediated active coatings development, and (iii) the latest advances in active agent immobilization and polymeric coatings for controlled release on Ti surfaces.