3-mercaptopropionic Acid Self-assembled Monolayer Research Articles

Fabrication of an electrochemical impedance sensor for ketamine hydrochloride and its application in the detection of doping substances in sports

This work reports an ultrasensitive impedimetric immunosensor for ketamine detection using self-assembled monolayers of 3-mercaptopropionic acid on gold electrodes. Monoclonal ketamine antibodies were covalently tethered via EDC/NHS crosslinking chemistry. Each fabrication step was validated by electrochemical and microscopic characterization. The optimized immunoelectrode demonstrated a broad dynamic range from 1 to 1000 ng/mL ketamine and record low detection limit of 0.5 ng/mL attributed to excellent antibody affinity. Analytical recovery of spiked serum and urine samples ranged 92–103% proving reliability despite biosample complexity. The simple, rapid and portable sensor surpasses existing chromatography and electrochemical techniques in terms of detection sensitivity as well as cost, instrumentation and training needs. The capabilities are attributed to rational antibody immobilization strategies enabling efficient binding events. The attractive detection limits coupled with label-free operation and microscale construct provide optimism regarding translation to viable clinical and on-site testing devices for toxicology, anti-doping and therapeutic applications.

Development of Graphene with Tungsten Disulfide Composite Layer Based SPR Biosensor for Biomedical Application

Surface Plasmon Resonance (SPR) biosensors have been widely used for biomedical applications due to their high sensitivity and label-free detection capabilities. However, their performance can be further enhanced by using advanced materials and signal-processing techniques. The objective of this study is to develop a composite layer-based SPR biosensor using Au, WS2, and Graphene layers and signal processing with MATLAB for enhanced sensitivity and the detection of DNA-DNA Hybridization. The composite layer-based SPR biosensor was fabricated by depositing a thin layer of Au on a glass substrate, followed by the deposition of WS2 and Graphene layers using a Chemical Vapor Deposition (CVD) technique. A self-assembled monolayer of 3-Mercaptopropionic Acid (MPA) was then attached to promote DNA immobilization. The performance of the biosensor was evaluated by detecting the hybridization of a single-stranded DNA (ssDNA) probe with a complementary ssDNA target. The sensor response was analyzed using MATLAB to enhance the sensitivity of the biosensor. The developed composite layer-based SPR biosensor exhibited a high sensitivity of 592 deg./RIU for the detection of DNA-DNA hybridization. 32.74% sensitivity has been increased. The signal processing with MATLAB significantly improved the signal-to-noise ratio and allowed for real-time monitoring of the biomolecular interactions. The composite layer-based SPR biosensor developed in this study demonstrated enhanced sensitivity for the detection of DNA-DNA hybridization in biomedical applications. The use of advanced materials such as Au, WS2, and Graphene layers, coupled with signal processing with MATLAB, can significantly improve the performance of SPR biosensors. This biosensor has great potential for use in various areas, including genetic testing, drug discovery, and disease diagnosis. Detected DNA-DNA hybridization is used in the biomedical field to identify and classify microorganisms by comparing the degree of genetic similarity between their DNA sequences.

Preparation and application of lysozyme molecularly imprinted surface plasmon resonance biosensors

The aim of this study is to fabricate surface plasmon resonance (SPR) biosensors for real-time detection of protein in complex media. Lysozyme (Lyz) was chosen as a model, and dopamine was used as functional monomer and cross-linker to prepare Lyz surface imprinted SPR biosensors (Lyz-MIPs). Non-covalent interaction was introduced into Lyz-MIPs via a self-assembled monolayer of 3-mercaptopropionic acid to improve the specificity, and protein-resistant poly(2-methyl-2-oxazoline-co-ethylene imine) was also introduced to resist nonspecific adsorption of polydopamine toward proteins in noncavity regions. The prepared Lyz-MIPs biosensor was characterized by X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, atomic force microscopy, variable angle spectroscopic ellipsometry, static water contact angle, and zeta potential measurements. SPR adsorption studies were carried out in aqueous Lyz solutions, and the limits of detection and quantification were obtained as 0.19 μg mL−1 and 0.58 μg mL−1, respectively. In addition, Lyz-MIPs biosensor exhibited high selectivity and good reusability. Finally, Lyz-MIPs biosensor was used to detect Lyz in complex media. The recovery of Lyz in simulated egg white was 95.3–105.0%, and 0.23 μg mL−1 Lyz was detected by using Lyz-MIPs biosensor in 20,000 times diluted hen egg white.

Investigating the substitution of intermolecular hydrogen bonds on the surface of self-assembled monolayer by scanning electrochemical microscopy

In this work, the substitution of intermolecular hydrogen bonds on the surface of 3-mercaptopropionic acid self-assembled monolayer (MPA SAM) was investigated using scanning electrochemical microscopy (SECM). • The substitution of intermolecular hydrogen bonds on the surface of MPA SAM was investigated by scanning electrochemical microscopy. • The formation of charge-assisted hydrogen bonds was more likely to induce intermolecular hydrogen bonds substitution. • All phenomena were properly characterized by the changes in current of substrate and tip electrodes. • The substitution of intermolecular hydrogen bonds is conductive to understanding the molecular selective recognition process on biofilm surface. Investigating the substitution of intermolecular hydrogen bonds on the simulated biofilm surface at molecular level is beneficial to understand the mechanism of molecular selective recognition occurring on biofilm surface. In this paper, the substitution of intermolecular hydrogen bonds on the surface of simulated biofilm, self-assembled monolayer of 3-mercaptopropionic acid (MPA SAM), is studied using scanning electrochemical microscopy (SECM). When acetic acid exists on MPA SAM surface, the intermolecular hydrogen bonds between acetic acid and MPA SAM are able to replace those between the redox mediator ferrocenemethanol (FcCH 2 OH) and MPA SAM, because the stronger charge-assisted hydrogen bonds (CAHB) formed between acetic acid and MPA SAM have the ability of substitution. The effect of the substitution of intermolecular hydrogen bonds is more significant with increasing the concentration of acetic acid within certain limits. This work may provide ideas for deeper understanding of molecular selective recognition on biofilm surface.

Carbon dots stabilized silver–lipid nano hybrids for sensitive label free DNA detection

Carbon dots have been extensively used for the development of fluorescent based molecular affinity sensors. However, label free DNA sensing by electrochemical method is not reported so far. Herein, we report carbon dots stabilized silver nanoparticles (CD-AgNPs) lipid nano hybrids as a sensitive and selective platform for label free electrochemical DNA sensing. The CD-AgNPs were synthesized by wet chemical method and then characterized by UV-visible, Fourier-transform Infra-red (FT-IR), dynamic light scattering (DLS) and high resolution transmission electron microscopy (HR-TEM) techniques. These CD-AgNPs were used for decorating the binary lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP) surface (named as lipid) and tethered on self-assembled monolayer of 3-mercaptopropionic acid (MPA) (MPA-lipid-CD-AgNPs). The formation of array of MPA-lipid-CD-AgNPs on Au electrode was confirmed by atomic force microscopy (AFM). Electrochemical behavior of MPA- lipid-CD-AgNPs was monitored in the presence of 1 mM potassium ferri/ferrocyanide (K3/K4 [Fe(CN)6]). The formation of layer-by-layer MPA-lipid-CD-AgNPs is indicated by increased anodic and cathodic peak (ΔEp) separation with decreased redox peak current of K3/K4 [Fe(CN)6]. Short chain DNA (30 mer oligonucleotide, representing the lung cancer) was used as a model system for label free DNA sensing. Un-hybridized (single stranded DNA), hybridized (complementary hybridized), single, double and triple base mismatched target DNA hybridized surfaces were efficiently discriminated at 1 µM target DNA concentration at the Au/MPA-lipid-CD-AgNPs electrode by change in the charge transfer resistance from impedance technique. Further, the modified electrode was successfully used to determine target DNA in a wide linear range from 10-16 to 10-11 M. The present work open doors for the utilization of CDs in molecular affinity based electrochemical sensor design and development.

Analysis of TiO2 Atomic Layer Deposition Surface Chemistry and Evidence of Propene Oligomerization Using Surface-Enhanced Raman Spectroscopy.

Atomic layer deposition (ALD) of TiO2 was performed in tandem with in situ surface-enhanced Raman spectroscopy (SERS) to monitor changes in the transient surface species across multiple ALD cycles. A self-assembled monolayer of 3-mercaptopropionic acid was used as a capture agent to ensure that nucleation of the titanium precursor (titanium tetraisopropoxide [TTIP]) occurs. Comparisons between the Raman spectra of the neat precursor and the SER spectra of the first ALD cycle of TiO2 reveal typical ligand exchange chemistry taking place, with self-limiting behavior and intact isopropoxide ligands. However, subsequent cycles show drastically different chemistry, with no isopropoxide ligands remaining at any point during the second and third cycles. Continuous exposure of either TTIP or isopropyl alcohol after the first cycle shows unlimited chemical vapor deposition (CVD)-type growth. Comparisons with alternative precursors (aluminum isopropoxide, titanium tert-butoxide, and titanium propoxide) and DFT calculations reveal that, for the TTIP precursor, isolated TiO2 sites play a role in the dehydration of off-gassing isopropyl alcohol. The resulting propene then undergoes oligomerization into six-carbon olefins before polymerizing into indistinguishable carbon products that accumulate on the surface. The emergence of the dehydration chemistry is expected to be exclusively the result of these isolated TiO2 sites and, as such, is expected to occur on other surfaces where TiO2 ALD is feasible. This work showcases how seemingly innocuous ALD can evolve into a CVD process when the products can participate in various side reactions with newly made surface sites.

Single-Use Disposable Electrochemical Label-Free Immunosensor for Detection of Glycated Hemoglobin (HbA1c) Using Differential Pulse Voltammetry (DPV).

A single-use disposable in vitro electrochemical immunosensor for the detection of HbA1c in undiluted human serum using differential pulse voltammetry (DPV) was developed. A three-electrode configuration electrochemical biosensor consisted of 10-nm-thin gold film working and counter electrodes and a thick-film printed Ag/AgCl reference electrode was fabricated on a polyethylene terephthalate (PET) substrate. Micro-fabrication techniques including sputtering vapor deposition and thick-film printing were used to fabricate the biosensor. This was a roll-to-roll cost-effective manufacturing process making the single-use disposable in vitro HbA1c biosensor a reality. Self-assembled monolayers of 3-Mercaptopropionic acid (MPA) were employed to covalently immobilize anti-HbA1c on the surface of gold electrodes. Electrochemical impedance spectroscopy (EIS) and X-ray photoelectron spectroscopy (XPS) confirmed the excellent coverage of MPA-SAM and the upward orientation of carboxylic groups. The hindering effect of HbA1c on the ferricyanide/ferrocyanide electron transfer reaction was exploited as the HbA1c detection mechanism. The biosensor showed a linear range of 7.5–20 µg/mL of HbA1c in 0.1 M PBS. Using undiluted human serum as the test medium, the biosensor presented an excellent linear behavior (R2 = 0.999) in the range of 0.1–0.25 mg/mL of HbA1c. The potential application of this biosensor for in vitro measurement of HbA1c for diabetic management was demonstrated.

Ligand-exchange assisted preparation of plasmonic Au/TiO2 nanotube arrays photoanodes for visible-light-driven photoelectrochemical water splitting

Visible-light-driven responsive Au/TiO2 nanotube arrays nanocomposites (LE-Au/TNTs) are prepared by depositing self-assembled monolayer of 3-mercaptopropionic acid (MPA) inside TNTs and then removing the ligands on the surface of Au through direct ligand-exchange method, which is beneficial for formation of intimate Au/TNTs Schottky contact after a mild annealing process. Under visible light illumination (λ > 400 nm), the photocurrent density of LE-Au/TNTs is 202 μA/cm2, which is the highest value ever reported in Au/TiO2 systems. Moreover, the incident photon to current conversion efficiency (IPCE) of LE-Au/TNTs at surface plasmonic resonance induced absorption peak (555 nm) is 7.4%. It is worth noted that the hydrogen evolution rates of the LE-Au/TNTs under simulated solar irradiation (AM1.5G, 100 mW/cm2) are 22.5 μmol/h, which is much higher than that of pristine TNTs (8.1 μmol/h). In addition, the LE-Au/TNTs show higher photoelectrochemical water splitting performance than the Au/TNTs prepared by direct impregnation-precipitation (IPAu/TNTs) strategy, which is ascribed to the close Schottky contact between Au and TNTs for better charge separation and transfer.

Morphological and Electrochemical Analysis of a Self-Assembled Monolayer of 3-Mercaptopropionic Acid on Gold Electrode

Morphological and Electrochemical Analysis of a Self-Assembled Monolayer of 3-Mercaptopropionic Acid on Gold Electrode

Determination of Subgroup J Avian Leukosis Virus by Surface Plasmon Resonance Immunosensor

Two novel Fourier transform-surface plasmon resonance immunosensors were fabricated for the determination of the subgroup J avian leukosis virus. The immunosensors were based on a functionalized gold substrate and immobilized gold–silver nanoparticles. For the first immunosensor, the gold substrate was functionalized with a dense self-assembled monolayer of 3-mercaptopropionic acid through gold–sulfur bonds, and then protein A which can combine subgroup J avian leucosis virus antibody on the antigen nonbinding site was modified on the sensor chip surface via covalent bonds formed between the amino groups and the activated carboxyl groups of 3-mercaptopropionic acid. After inactivation by ethanolamine, the sensor chip immobilized the antibody by the combination with protein A to produce a sensor. For the second immunosensor, 1,6- hexanedithiol formed a self-assembled monolayer on the sensor chip surface through gold–sulfur bonds. Then gold–silver nanoparticles were immobilized on the sensor chip by gold–sulfur and silver–sulfur bonds. The modification by various reagents and determination of the subgroup J avian leukosis virus antigen were the same as in the first immunosensor. The immunosensors determined the subgroup J avian leukosis virus antigen to analyte concentrations of 659 TCID 50 ·mL− 1 and 1054 TCID 50 ·mL− 1 , respectively. The result suggested that introducing gold–silver nanoparticles to the Fourier transform-surface plasmon resonance immunosensor is beneficial to effectively improving the sensitivity of the immunosensor. Moreover, the contrast experiment presented that the immunosensors showed high specificity for the subgroup J avian leucosis virus detection.

In situ synthesized gold nanoparticles for direct electrochemistry of horseradish peroxidase

In order to construct a three-dimensional electrode, in situ electrochemical deposition of gold nanoparticles onto a gold electrode coated with a self-assembled monolayer of 3-mercaptopropionic acid (Au NP/MPA/Au) was conducted. Horseradish peroxidase (HRP) was then immobilized into this three-dimensional electrode, leading to the realization of direct electron transfer of HRP. Scanning electron microscopy and electrochemical impedance spectroscopy were applied to characterize the electrode before and after HRP immobilization. The apparent Michaelis-Menten constant for immobilized HRP on a Au NP/MPA/Au electrode is 0.78mM, indicating high enzymatic activity of HRP. The HRP modified electrode was further utilized as a sensing platform for the electrocatalytic reduction and detection of hydrogen peroxide. For the detection of hydrogen peroxide on this electrode, the sensitivity is 311.72μAmM−1cm−2, the detection limit is 0.16μM, and the dynamic range is from 0.48μM to 1.2mM. Therefore this electrode is a promising device for sensitive and reproducible detection of H2O2.

Development of a label-free immunosensor system for detecting oocyte maturation-inducing hormone in fish

A label-free immunosensor for detecting the oocyte maturation-inducing hormone 17, 20β-dihydroxy-4-pregnen-3-one (DHP), was developed. A principle of the sensor system was based on differences in electrochemical activity changed by an immunoreaction in the absence and presence of DHP. For preparation of the immunosensor, anti-DHP IgG was immobilized on an Au working electrode modified with a self-assembled monolayer of 3-mercaptopropionic acid. The sensor was immersed into a sample solution containing DHP. DHP was determined by cyclic voltammetry. The immunosensor showed a specific response to DHP, and the oxidation peak current linearly decreased in the range of 7.8–500 pg ml−1 with a correlation coefficient of 0.996. The sensor system was applied to determine the DHP levels in plasma of goldfish and was compared with the DHP levels of the same samples determined using an ELISA as the conventional method. Good correlation was obtained between values determined using both methods in the range of 0.1–7.7 ng ml−1 (correlation coefficient 0.876). These findings suggest that the proposed label-free immunosensor can be used to analyze DHP levels in fish plasma samples.

A novel system combining biocatalytic dephosphorylation of l-ascorbic acid 2-phosphate and electrochemical oxidation of resulting ascorbic acid

An enzyme electrode was prepared with acid phosphatase (ACP) for development of a new electric power generation system using ascorbic acid 2-phosphate (AA2P) as a fuel. The properties of the electrode were investigated with respect to biocatalytic dephosphorylation of AA2P and electrochemical oxidation of resulting ascorbic acid (AA). The enzyme electrode was fabricated by immobilization of ACP through amide linkage onto a self-assembled monolayer of 3-mercaptopropionic acid on a gold electrode. AA2P was not oxidized on a bare gold electrode in the potential sweep range from −0.1 to +0.5 V vs. Ag/AgCl. However, the enzyme electrode gave an oxidation current in citric buffer solution of pH 5 containing 10 mM of AA2P. The oxidation current began to increase at +0.2 V, and reached to 5.0 μA cm −2 at +0.5 V. The potential +0.2 V corresponded to the onset of oxidation of ascorbic acid (AA). These results suggest that the oxidation current observed with the enzyme electrode is due to AA resulting from dephosphorylation of AA2P. The oxidation current increased with increasing concentration of AA2P and almost leveled off at around the concentration of 5 mM. Thus the enzyme electrode brought about biocatalytic conversion of AA2P to AA, followed by electrochemical oxidation of the AA. The oxidation current is likely to be controlled by the biocatalytic reaction.

Biocatalyzed deposition amplification for detection of aflatoxin B 1 based on quartz crystal microbalance

An ultrasensitive piezoelectric method for the detection of the aflatoxin B 1 (AFB 1) based on the indirect competitive immunoassay and the biocatalyzed deposition amplification has been developed. In this method, the quartz crystal surface was coated with a self-assembled monolayer of 3-mercaptopropionic acid (MPA) for covalently immobilization of the BSA-AFB 1 conjugate, which could compete with the free AFB 1 for binding to the anti-AFB 1 antibody (MsIgG). After the competitive immunoreaction, the horseradish peroxidase (HRP) labeled goat anti-mouse IgG (G-Anti-MsIgG) was introduced into the detection cell to combine with the anti-AFB 1 antibody on the crystal surface. The enzyme labeled G-Anti-MsIgG as a biocatalyst could accelerate the oxidation of 4-chloro-1-naphthol by H 2O 2 to yield the insoluble product benzo-4-chlorohexadienone on the surface of quartz crystal microbalance (QCM), resulting in a mass increase that was reflected by a decrease in the resonance frequency of the QCM. The proposed approach could allow for the determination of AFB 1 in the concentration range of 0.01–10.0 ng mL −1. Furthermore, several artificially contaminated milk samples were analyzed with good recoveries obtained, which demonstrated the suitability of the proposed method for detecting AFB 1.

Counterion binding induces attractive interactions between negatively-charged self-assembled monolayer of 3-mercaptopropionic acid on Au(111) in reductive desorption

The electrochemical reductive desorption of the self-assembled monolayers of 3-mercaptopropionic acid in an aqueous alkaline solution gives a sharp peak with the full width at half maximum of about 20 mV irrespective of the type of cations in a linear scan voltammogram. This suggests that a strong attractive interaction exists between negatively charged carboxylate groups in the self-assembled monolayer surface due to the counterion binding, which not only simply stabilizes the adsorbed carboxylates but also makes the interaction between the adsorbed thiolates even attractive possibly by forming a two-dimensional ionic crystal. The effect of tetraalkylammonium ions on the shape of the voltammograms was also examined.

Voltammetric detection of cadmium ions at glutathione-modified gold electrodes

An electrochemical sensor for the detection of cadmium ions is described using immobilized glutathione as a selective ligand. First, a self-assembled monolayer of 3-mercaptopropionic acid (MPA) was formed on a gold electrode. The carboxyl terminus then allowed attachment of glutathione (GSH)via carbodiimide coupling to give the MPA-GSH modified electrode. A cadmium ion forms a complex with glutathione via the free sulfhydryl group and also to the carboxyl groups. The complexed ion is reduced by linear and Osteryoung square wave voltammetry with a detection limit of 5 nM. The effect of the kinetics of accumulation of cadmium on the measured current was investigated and modeled. Increasing the temperature of accumulation and electrochemical analysis caused an increase in the voltammetric peak of approximately 4% per degrees C around room temperature. The modified electrode could be regenerated, being stable for more than 16 repeated uses and more than two weeks if used once a day. Some interference from Pb(2+) and Cu(2+) was observed but the effects of Zn(2+), Ni(2+), Cr(3+) and Ba(2+) were insignificant.

A high sensitivity amperometric biosensor using a monomolecular layer of laccase as biorecognition element

Laccases from various sources were tested, and laccase from Rigidoporus lignosus was found to be the most active towards syringaldazine and ABTS, which are typical substrates of this class of enzymes, and towards the phenols found in olive oil mill wastewaters. This laccase was covalently immobilised by carbodiimide chemistry, on a self-assembled monolayer of 3-mercaptopropionic acid deposited on a gold surface. A flow biosensor, using the monolayer of laccase as bioelement and a glassy carbon electrode as amperometric transduction system, was developed. Although the amount of the immobilised enzyme (about 140 ng/cm 2 effective surface area) was tiny, the biosensor showed a sensitivity of 3 nA/μM when 1,4-hydroquinone was used as substrate, and a half-life of 35 days. The proposed device permits detection of phenols in aqueous solutions at concentrations in the low micromolar range, i.e. below European Community limits. The biosensor was successfully used to detect phenols in wastewaters from an olive oil mill after minimal sample preparation (incubation of the aqueous sample with sodium borohydride for a few minutes) to suppress the current due to oxidised compounds present in the wastewaters.

In situ scanning tunneling microscopy observation of self-assembled monolayers of 3-mercaptopropionic acid on Au(111) in perchloric acid solution

In situ scanning tunneling microscopy (STM) observation was performed to study the self-assembled monolayers of 3-mercaptopropionic acid (MPA) on Au(111) in aqueous perchloric acid solution. It was seen that the MPA molecules formed a monolayer composed of molecularly ordered domains. High-resolution STM imaging revealed that each individual MPA molecule appeared as two spots, corresponding to the carboxylic functional group of the MPA molecule, and the ordered MPA monolayer was explained by a rhombic (3×3) structure. The packing arrangement of the MPA monolayer is close to that of the (√3×√3) R30° structure, but possesses slightly different positions of molecules, indicating that three adjacent MPA molecules in triangular positions are snuggled up due to intermolecular hydrogen bonding. The reductive desorption reaction in alkaline solution was used to estimate the surface concentration of MPA molecules on Au(111), which is consistent with the (3×3) structure obtained from the STM observation. The in situ STM imaging successfully visualized the interfacial structure of MPA monolayers self-assembled on Au(111) at a molecular scale.

Ordered structures of self-assembled monolayers of 3-mercaptopropionic acid on Au(111): in situ scanning tunneling microscopy study

In situ scanning tunneling microscopy (STM) and cyclic voltammetry were employed to investigate self-assembled monolayers of 3-mercaptopropionic acid (MPA) on Au(111) in perchloric acid solutions. In situ STM revealed that the MPA monolayer was composed of translationally and rotationally ordered MPA domains, and some disordered portions also coexisted on the surface, particularly near the etch pits. The ordered monolayer exhibited a molecularly ordered (3 × 3) structure, where three MPA molecules in triangular positions stand fairly close together due to intermolecular hydrogen bonding. Two distinctly different phase boundaries were found between the ordered (3 × 3) domains, and high-resolution STM images disclosed the molecular arrangement of the MPA monolayers. The MPA monolayer desorbed at negative potentials, causing transformation of the surface into the Au(111)-(√3 × 23) reconstruction. The in situ STM successfully elucidated the interfacial structure and phase boundary of the MPA monolayers on Au(111) under electrochemical conditions.

In situ STM imaging of individual molecules in two-component self-assembled monolayers of 3-mercaptopropionic acid and 1-decanethiol on Au(111)

Two-component self-assembled monolayers composed of 3-mercaptopropionic acid (MPA) and 1-decanethiol (CH3(HC2)9SH:C10SH) on Au(111) were investigated with in situ scanning tunneling microscopy (STM) and cyclic voltammetry, where the monolayers I and II were prepared in 0.1 mM ethanol+water solutions with the ratios of MPA:C10SH=95:5 and 90:10, respectively. In situ STM images revealed that both monolayer I and II consisted of phase-separated domains with molecularly ordered structures, each of which was predominantly formed by one of the constituent molecules. Based on the STM images, the surface fractions of constituent molecules were evaluated as MPA:C10SH=0.61:0.28 for monolayer I, and MPA:C10SH=0.34:0.57 for monolayer II. Cyclic voltammograms of the reductive desorption of the thiols provided the surface fractions of MPA:C10SH=0.69:0.31 for monolayer I, and MPA:C10SH=0.41 and 0.59 for monolayer II, which were in good agreement with the results obtained from STM images. Molecular resolution imaging allowed us to visualize the individual MPA and C10SH molecules and to determine the interfacial structures in the molecularly ordered domains of the phase-separated monolayer. The C10SH domains in the monolayer exhibited ordered phases with densely packed (√3×√3)R30° and p(3×2√3R−30°) structures, which are well-characterized structures for alkanethiol monolayers. On the other hand, a completely different molecular arrangement of MPA defined as a (3×3) structure was consistently observed in the MPA domains, where the molecular arrangement is almost the same as that of (√3×√3)R30° but intermolecular hydrogen bonding is thought to exist in the three neighboring MPA molecules located around the corner of the (3×3) unit cell. It was demonstrated that individual molecules of the monolayer constituents were successfully imaged in the two-component, phase-separated monolayer in solution.