Development of Taste Sensor with Lipid/Polymer Membranes for Detection of Umami Substances Using Surface Modification.

A taste sensor employs various lipid/polymer membranes with specific physicochemical properties for taste classification and evaluation. However, phosphoric acid di(2-ethylhexyl) ester (PAEE), employed as one of the lipids for the taste sensors, exhibits insufficient selectivity for umami substances. The pH of sample solutions impacts the dissociation of lipids to influence the membrane potential, and the response to astringent substances makes accurate measurement of umami taste difficult. This study aims to develop a novel taste sensor for detecting umami substances like monosodium L-glutamate (MSG) through surface modification, i.e., a methodology previously applied to taste sensors for non-charged bitter substance measurement. Four kinds of modifiers were tested as membrane-modifying materials. By comparing the results obtained from these modifiers, the modifier structure suitable for measuring umami substances was identified. The findings revealed that the presence of carboxyl groups at para-position of the benzene ring, as well as intramolecular H-bonds between the carboxyl group and hydroxyl group, significantly affect the effectiveness of a modifier in the umami substance measurement. The taste sensor treated with this type of modifier showed excellent selectivity for umami substances.

Electrical Properties of Taste Sensors with Positively Charged Lipid Membranes Composed of Amines and Ammonium Salts.

Currently, taste sensors utilizing lipid polymer membranes are utilized to assess the taste of food products quantitatively. During this process, it is crucial to identify and quantify basic tastes, e.g., sourness and sweetness, while ensuring that there is no response to tasteless substances. For instance, suppression of responses to anions, like tasteless NO3- ions contained in vegetables, is essential. However, systematic electrochemical investigations have not been made to achieve this goal. In this study, we fabricated three positively charged lipid polymer membranes containing oleylamine (OAm), trioctylemethylammonium chloride (TOMACl), or tetradodecylammonium bromide (TDAB) as lipids, and sensors that consist of these membranes to investigate the potential change characteristics of these sensors in solutions containing different anions (F-, Cl-, Br-, NO3-, I-). The ability of each anion solution to reduce the positive charge on membranes and shift the membrane potential in the negative direction was in the following order: I- > NO3- > Br- > Cl- > F-. This order well reflected the order of size of the hydrated ions, related to their hydration energy. Additionally, the OAm sensor displayed low ion selectivity, whereas the TOMACl and TDAB sensors showed high ion selectivity related to the OAm sensor. Such features in ion selectivity are suggested to be due to the variation in positive charge with the pH of the environment and packing density of the OAm molecule in the case of the OAm sensor and due to the strong and constant positive charge created by complete ionization of lipids in the case of TOMACl and TDAB sensors. Furthermore, it was revealed that the ion selectivity varies by changing the lipid concentration in each membrane. These results contribute to developing sensor membranes that respond to different anion species selectively and creating taste sensors capable of suppressing responses to tasteless anions.

Research and development of taste sensors as a novel analytical tool.

Gustatory and olfactory receptors receive multiple chemical substances of different types simultaneously, but they can barely discriminate one chemical species from others. In this article, we describe a device used to measure taste, i.e., taste sensors. Toko and colleagues developed a taste sensor equipped with multiarray electrodes using a lipid/polymer membrane as the transducer in 1989. This sensor has a concept of global selectivity to decompose the characteristics of a chemical substance into taste qualities and to quantify them. The use of taste sensors has spread around the world. More than 600 examples of taste-sensing system have been used, while providing the first "taste scale" in the world. This article explains the principle of taste sensors and their application to foods and medicines, and also a novel type of taste sensor using allostery. Taste-sensor technology, the underlying principle of which is different from that of conventional analytical instruments, markedly affects many aspects including social economy as well as the food industry.

Prediction of dynamic allostery for the transmembrane domain of the sweet taste receptor subunit, TAS1R3.

The sweet taste receptor plays an essential role as an energy sensor by detecting carbohydrates. However, the dynamic mechanisms of receptor activation remain unclear. Here, we describe the interactions between the transmembrane domain of the G protein-coupled sweet receptor subunit, TAS1R3, and allosteric modulators. Molecular dynamics simulations reproduced species-specific sensitivity to ligands. We found that a human-specific sweetener, cyclamate, interacted with the mouse receptor as a negative allosteric modulator. Agonist-induced allostery during receptor activation was found to destabilize the intracellular part of the receptor, which potentially interfaces with the Gα subunit, through ionic lock opening. A common human variant (R757C) of the TAS1R3 exhibited a reduced response to sweet taste, in support of our predictions. Furthermore, histidine residues in the binding site acted as pH-sensitive microswitches to modulate the sensitivity to saccharin. This study provides important insights that may facilitate the prediction of dynamic activation mechanisms for other G protein-coupled receptors.

Development and Optimization of a Highly Sensitive Sensor to Quinine-Based Saltiness Enhancement Effect.

The saltiness enhancement effect can be produced by adding specific substances to dietary salt (sodium chloride). This effect has been used in salt-reduced food to help people forge healthy eating habits. Therefore, it is necessary to objectively evaluate the saltiness of food based on this effect. In a previous study, sensor electrodes based on lipid/polymer membrane with Na+ ionophore have been proposed to quantify the saltiness enhanced by branched-chain amino acids (BCAAs), citric acid, and tartaric acid. In this study, we developed a new saltiness sensor with the lipid/polymer membrane to quantify the saltiness enhancement effect of quinine by replacing a lipid that caused an unexpected initial drop in the previous study with another new lipid. As a result, the concentrations of lipid and ionophore were optimized to produce an expected response. Logarithmic responses have been found on both NaCl samples and quinine-added NaCl samples. The findings indicate the usage of lipid/polymer membranes on novel taste sensors to evaluate the saltiness enhancement effect accurately.

Identification of the Principle of Taste Sensors to Detect Non-Charged Bitter Substances by 1H-NMR Measurement.

A taste sensor with lipid/polymer membranes is attracting attention as a method to evaluate taste objectively. However, due to the characteristic of detecting taste by changes in membrane potential, taste sensors cannot measure non-charged bitter substances. Many foods and medicines contain non-charged bitter substances, and it is necessary to quantify these tastes with sensors. Therefore, we have been developing taste sensors to detect bitter tastes caused by non-charged substances such as caffeine. In previous studies, a sensor for detecting bitterness caused by caffeine and theobromine, theophylline, was developed, using a membrane modified with hydroxybenzoic acid (HBA) as the sensing part. The sensor was designed to form intramolecular hydrogen bonds (H-bonds) between the hydroxy group and carboxy group of HBA and to successively cause the intermolecular H-bonds between HBA and caffeine molecules to be measured. However, whether this sensing principle is correct or not cannot be confirmed from the results of taste sensor measurements. Therefore, in this study, we explored the interaction between HBA and caffeine by 1H-nuclear magnetic resonance spectroscopy (NMR). By the 1H NMR detection, we confirmed that both the substances interact with each other. Furthermore, the nuclear Overhauser effect (NOE) of intermolecular spatial conformation in solution was measured, by which 2,6-dihydroxybenzoic acid (2,6-DHBA) preferably interacted with caffeine via the H-bonding and stacking configuration between aromatic rings. Identifying the binding form of 2,6-DHBA to caffeine was estimated to predict how the two substances interact.

Effect of Hydroxybenzoic Acids on Caffeine Detection Using Taste Sensor with Lipid/Polymer Membranes.

A taste sensor with lipid/polymer membranes can objectively evaluate taste. As previously reported, caffeine can be detected electrically using lipid/polymer membranes modified with hydroxybenzoic acids (HBAs). However, a systematic understanding of how HBAs contribute to caffeine detection is still lacking. In this study, we used various HBAs such as 2,6-dihydroxybenzoic acid (2,6-DHBA) to modify lipid/polymer membranes, and we detected caffeine using a taste sensor with the modified membranes. The effect of the concentrations of the HBAs on caffeine detection was also discussed. The results of the caffeine detection indicated that the response to caffeine and the reference potential measured in a reference solution were affected by the log P and pKa of HBAs. Furthermore, the taste sensor displayed high sensitivity to caffeine when the reference potential was adjusted to an appropriate range by modification with 2,6-DHBA, where the slope of the change in reference potential with increasing 2,6-DHBA concentration was steep. This is helpful in order to improve the sensitivity of taste sensors to other taste substances, such as theophylline and theobromine, in the future.

Electrical Properties of Two Types of Membrane Component Used in Taste Sensors.

The lipid phosphoric acid di-n-decyl ester (PADE) has played an important role in the development of taste sensors. As previously reported, however, the concentration of PADE and pH of the solution affected the dissociation of H+, which made the measurement results less accurate and stable. In addition, PADE caused deterioration in the response to bitterness because PADE created the acidic environment in the membrane. To solve these problems, our past study tried to replace the PADE with a completely dissociated substance called tetrakis [3,5-bis (trifluoromethyl) phenyl] borate sodium salt dehydrate (TFPB) as lipid. To find out whether the two substances can be effectively replaced, it is necessary to perform an in-depth study on the properties of the two membranes themselves. In this study, we fabricated two types of membrane electrodes, based on PADE or TFPB, respectively, using 2-nitrophenyl octyl ether (NPOE) as a plasticizer. We measured the selectivity to cations such as Cs+, K+, Na+ and Li+, and also the membrane impedance of the membranes comprising PADE or TFPB of the different concentrations. As a result, we found that any concentration of PADE membranes always had low ion selectivity, while the ion selectivity of TFPB membranes was concentration-dependent, showing increasing ion selectivity with the TFPB concentrations. The ion selectivity order was Cs+>K+>Na+>Li+. The hydration of ions was considered to participate in this phenomenon. In addition, the membrane impedance decreased with increasing PADE and TFPB concentrations, while the magnitudes differed, implying that there is a difference in the dissociation of the two substances. The obtained results will contribute to the development of novel receptive membranes of taste sensors.

Bitterness compounds in coffee brew measured by analytical instruments and taste sensing system.

We investigated the bitter compounds in coffee brews using multivariate analysis of the data obtained from analytical instrument and electronic taste sensor experiments. Coffee brews were prepared from coffee beans roasted to four different degrees. Each brew was fractionated into four fractions by liquid-liquid extraction. The relative amounts of 30 compounds in each fraction were analyzed by analytical instruments, and the bitterness response value of each fraction was analyzed by a taste sensor. Candidate bitter compounds in the coffee brews were identified with reference to their variable importance in projection and by coefficient of projection to latent structure regression (PLS-R) analysis. PLS-R analysis suggested that nicotinic acid, l-lactic acid, and nicotinamide contributed to the bitterness of the coffee brews. In fact, the coffee brews with added nicotinic acid, l-lactic acid, and nicotinamide had an increased bitterness response value compared to those without.

Development of Taste Sensor to Detect Non-Charged Bitter Substances.

A taste sensor with lipid/polymer membranes is one of the devices that can evaluate taste objectively. However, the conventional taste sensor cannot measure non-charged bitter substances, such as caffeine contained in coffee, because the taste sensor uses the potentiometric measurement based mainly on change in surface electric charge density of the membrane. In this study, we aimed at the detection of typical non-charged bitter substances such as caffeine, theophylline and theobromine included in beverages and pharmaceutical products. The developed sensor is designed to detect the change in the membrane potential by using a kind of allosteric mechanism of breaking an intramolecular hydrogen bond between the carboxy group and hydroxy group of aromatic carboxylic acid (i.e., hydroxy-, dihydroxy-, and trihydroxybenzoic acids) when non-charged bitter substances are bound to the hydroxy group. As a result of surface modification by immersing the sensor electrode in a modification solution in which 2,6-dihydroxybenzoic acid was dissolved, it was confirmed that the sensor response increased with the concentration of caffeine as well as allied substances. The threshold and increase tendency were consistent with those of human senses. The detection mechanism is discussed by taking into account intramolecular and intermolecular hydrogen bonds, which cause allostery. These findings suggest that it is possible to evaluate bitterness caused by non-charged bitter substances objectively by using the taste sensor with allosteric mechanism.

Identification of characteristic compounds of moderate volatility in breast cancer cell lines.

In this study, we were challenging to identify characteristic compounds in breast cancer cell lines. GC analysis of extracts from the culture media of breast cancer cell lines (MCF-7, SK-BR-3, and YMB-1) using a solid-phase Porapak Q extraction revealed that two compounds of moderate volatility, 1-hexadecanol and 5-(Z)-dodecenoic acid, were detected with markedly higher amount than those in the medium of fibroblast cell line (KMST-6). Furthermore, LC-TOF/MS analysis of the extracts clarified that in addition to the above two fatty acids, the amounts of five unsaturated fatty acids [decenoic acid (C10:1), decadienoic acid (C10:2), 5-(Z)-dodecenoic acid (C12:1), 5-(Z)-tetradecenoic acid (C14:1), and tetradecadienoic acid (C14:2)] in MCF-7 medium were higher than those in medium of KMST-6. Interestingly, H2O2-oxidation of 5-(Z)-dodecenoic acid and 5-(Z)-tetradecenoic acid produced volatile aldehydes that were reported as specific volatiles in breath from various cancer patients, such as heptanal, octanal, nonanal, decanal, 2-(E)-nonenal, and 2-(E)-octenal. Thus, we concluded that these identified compounds over-produced in breast cancer cells in this study could serve as potential precursors producing reported cancer-specific volatiles.

A bioinspired peptide matrix for the detection of 2,4,6-trinitrotoluene (TNT).

A novel peptide-based three-dimensional probe called "peptide matrix," inspired by the antibody paratope region, was fabricated on a surface plasmon resonance (SPR) sensor chip to enhance the sensitivity of detecting the explosive 2,4,6-trinitrotoluene (TNT). Although peptide aptamer is an attractive candidate for a molecular recognition probe because of its ease of synthesis and chemical stability, it still has difficulty in applying to highly sensitive (i.e. parts-per-billion (ppb) or sub-ppb level) detections. Thus, we developed the concept of peptide matrix structure, which is constructed by consecutive disulfide bond formation between a large number of peptide fragments. This robust three-dimensional structure displays multiple binding sites which can efficiently associate with each TNT molecule. The peptide matrix lowered the dissociation constant (KD) by two orders of magnitude compared to the linear peptide aptamer, estimating KD as 10.1 nM, which is the lowest concentration reported by using peptide-based TNT probe. Furthermore, the concentration limit of detection of peptide matrix modified SPR sensor was 0.62 ppb, and hence comparable to single-chain variable fragment (scFv)-based TNT sensors. To our knowledge, this is the first report demonstrating peptide matrix fabrication and its application for small explosive molecule detection. This peptide matrix-based approach, which has the advantage of simple synthesis and high sensitivity, will be applicable to many other small-molecule label-free detections.

A Quantitative Method for Acesulfame K Using the Taste Sensor.

We have developed a method to quantify the sweetness of negatively charged high-potency sweeteners coexisting with other taste substances. This kind of sweetness sensor uses lipid polymer membranes as the taste-sensing part. Two types of outputs have been defined in the measurement of the taste sensor: one is the relative value and the other is the CPA (the change in membrane potential caused by adsorption) value. The CPA value shows a good selectivity for high-potency sweeteners. On the other hand, the relative value is several times higher than the CPA value, but the relative value is influenced by salty substances. In order to obtain both high sensitivity and selectivity, we established a model for predicting the concentration of sweeteners with a nonlinear regression analysis method using the relative values of both the sweetness sensor and the saltiness sensor. The analysis results showed good correlations with the estimated concentration of acesulfame potassium coexisting with salty substances, as represented by R2 = 0.99. This model can correspond well to the prediction of acesulfame K in a concentration of 0.2-0.7 mM, which is commonly used in food and beverages. The results obtained in this paper suggest that this method is useful for the evaluation of acesulfame K using the taste sensors.

Development of a Sensor with a Lipid/Polymer Membrane Comprising Na+ Ionophores to Evaluate the Saltiness Enhancement Effect.

The saltiness enhancement effect is the effect whereby saltiness is enhanced by adding specific substances to salt (sodium chloride). Since this effect can be used in the development of salt-reduced foods, a method to objectively evaluate the saltiness with this effect is required. A taste sensor with lipid/polymer membranes has been used to quantify the taste of food and beverages in recent years. The sensor electrodes of this taste sensor have the feature of selectively responding to each of the five basic tastes, which is realized by the lipid/polymer membranes. In this study, we developed a new saltiness sensor based on the lipid/polymer membrane with the aim of quantifying the saltiness enhancement effect. In addition to the conventional components of a lipid, plasticizer, and polymer supporting reagent, the membrane we developed comprises ionophores, which selectively capture sodium ions. As a result, the response of the sensor increased logarithmically with the activity of NaCl in measured samples, similarly to the taste response of humans. In addition, all of the sensor responses increased upon adding saltiness-enhancing substances, such as citric acid, tartaric acid and branched-chain amino acids (BCAAs), to NaCl samples. These findings suggest that it is possible to quantify the saltiness enhancement effect using a taste sensor with lipid/polymer membranes.

An SPR Sensor Chip Based on Peptide-Modified Single-Walled Carbon Nanotubes with Enhanced Sensitivity and Selectivity in the Detection of 2,4,6-Trinitrotoluene Explosives.

In this study, we developed a surface plasmon resonance (SPR) sensor chip based on 2,4,6-trinitrotoluene (TNT) recognition peptide-modified single-walled carbon nanotubes (SWCNTs). The carboxylic acid-functionalized SWCNTs were immobilized on a 3-aminopropyltriethoxysilane (APTES)-modified SPR Au chip surface. Through π-stacking between the aromatic amino acids and SWCNTs, the TNT recognition peptide TNTHCDR3 was immobilized onto the surface of the SWCNTs. The peptide⁻SWCNTs-modified sensor surface was confirmed and evaluated by atomic force microscope (AFM) observation. The peptide⁻SWCNTs hybrid SPR sensor chip exhibited enhanced sensitivity with a limit of detection (LOD) of 772 ppb and highly selective detection compared with commercialized carboxymethylated dextran matrix sensor chips.

Effects of Manufacturing Processing Conditions on Retronasal-Aroma Odorants from a Milk Coffee Drink.

To develop a ready-to-drink (RTD) milk coffee that retains the original coffee flavor, the effects of manufacturing processing conditions on retronasal-aroma (RA) odorants were investigated by gas chromatography-olfactometry (CharmAnalysis™) using an RA simulator (RAS). Twenty-nine of 33 odorants detected in the RAS effluent (RAS odorants) were identified. The detected odorants were classified into 19 odor-description groups. The total odor intensity (charm value, CMV) of all coffee RAS odorants decreased approximately 68% following pH adjustment, whereas the total CMV increased 6% to 7% following ultra-high-temperature sterilization. The total CMV ratio (about 83%) of the milk coffee produced using a new blending-after-sterilization (BAS) process without pH adjustment of the coffee was greater than that (approximately 56%) prepared using a conventional blending-before-sterilization (BBS) process with pH adjustment. In BAS-processed milk coffees, the total CMV ratio (91%) with infusion (INF)-sterilized reconstituted milk (r-milk) was greater than that (83%) of plate (PLT)-sterilized r-milk. Principal component analysis of odor-description CMVs indicated that the effect of coffee pH adjustment on odor characteristics was greater than that of sterilization, that BAS and BBS samples differed, and that BAS milk coffee prepared using INF sterilization was more similar to homemade milk coffee (blending unsterilized coffee without pH adjustment with PLT-sterilized milk) than milk coffee prepared using PLT sterilization. In conclusion, the BAS process using INF sterilization is superior for manufacturing RTD milk coffee that retains odor characteristics similar to targeted homemade milk coffee. PRACTICAL APPLICATION: Ready-to-drink milk coffee beverages produced using conventional blending-before-sterilization methods do not retain their original coffee flavor following adjustment of the pH of the coffee during manufacturing. The use of newly developed blending-after-sterilization methods, by contrast, produces ready-to-drink milk coffee with an aroma more similar to that of homemade milk coffee, as demonstrated using an analytical system for characterizing food product aromas. The blending-after-sterilization process is now being used in Japan to produce ready-to-drink milk coffee beverages.

[Research of the Cancer-Odor].

Early detection and resection of cancer is the most effective in the treatment of solid cancer. Development of a new cancer detection method is expected to become a breakthrough to solve various problems for early detection. It has been reported that there is the specific odors of cancer by using bio olfaction such as dogs, and it has been recognized that there is the odors of cancer. Cancer cells acquire malignant traits as a result of metabolic changes originating from genetic mutation. The cancer specific odorous substances may be considered to be the end products of their metabolic changes. Omics researches such as genomics, proteomics, and metabolomics have been extensively performed to comprehensively analyze changes in DNA, RNA, protein, metabolism and its products specific to cancer for the purpose of developing a new cancer detection marker. It is thought that the research on the odor of cancer is also on the line of omics research. It is difficult to identify cancer-specific odorants buried in various environmental substances. However, it is expected that human will be able to acquire the technology, from the fact that they can be recognized by biological olfaction. We are continuing the research with the dream that identification of the odorous substances as a new cancer detection marker and sensor development for it will lead to the happiness of colleagues in the world.

Effects of Processing Conditions During Manufacture on Retronasal-Aroma Compounds from a Milk Coffee Drink.

To develop a ready-to-drink (RTD) milk coffee retaining the original coffee flavor, the effects of processing conditions during manufacture on retronasal-arma (RA) compounds from the milk coffee were investigated by gas chromatography-mass spectrometry using an RA simulator (RAS). Thirteen of 46 detected compounds in the RAS effluent (RAS compounds) decreased significantly following pH adjustment of coffee (from pH 5.1 to 6.8) and 5 compounds increased. RAS compounds from coffee tended to decrease through the pH adjustment and subsequent sterilization. Significantly higher amounts of 13 RAS compounds were released from the milk coffee produced using a blending-after-sterilization (BAS) process without the pH adjustment than from that using a blending-before-sterilization (BBS) process with the pH adjustment. In BAS-processed milk coffee, significantly lower amounts of 8 high-volatility compounds and 1H-pyrrole were released from coffee containing infusion-sterilized (INF) milk than from coffee containing plate-sterilized (PLT) milk, whereas 3 low-volatility compounds were released significantly more from coffee using PLT milk. Principal component analysis revealed that the effect of the manufacturing process (BAS, BBS, or homemade (blending unsterilized coffee without pH adjustment with sterilized milk)) on milk coffee volatiles was larger than that of the sterilization method (INF or PLT) for milk, and that the sterilization method could result in different RAS volatile characteristics in BAS and homemade processes. In conclusion, a BAS process was found to be superior to a BBS process for the manufacture of an RTD milk coffee that retains volatile characteristics similar to that of a homemade milk coffee. PRACTICAL APPLICATION: Ready-to-drink (RTD) milk coffee manufactured using the conventional blending-before-sterilization process does not retain its original coffee flavor due to pH adjustment of the coffee during the process. The new blending-after-sterilization (BAS) process enabled the production of RTD milk coffee whose volatiles are closer to that of homemade milk coffee, as demonstrated by the results of RAS-GC-MS analysis. The BAS process has already been applied to the manufacture of RTD milk coffees in Japan.

Improved Durability and Sensitivity of Bitterness-Sensing Membrane for Medicines.

This paper reports the improvement of a bitterness sensor based on a lipid polymer membrane consisting of phosphoric acid di-n-decyl ester (PADE) as a lipid and bis(1-butylpentyl) adipate (BBPA) and tributyl o-acetylcitrate (TBAC) as plasticizers. Although the commercialized bitterness sensor (BT0) has high sensitivity and selectivity to the bitterness of medicines, the sensor response gradually decreases to almost zero after two years at room temperature and humidity in a laboratory. To reveal the reason for the deterioration of the response, we investigated sensor membranes by measuring the membrane potential, contact angle, and adsorption amount, as well as by performing gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS). We found that the change in the surface charge density caused by the hydrolysis of TBAC led to the deterioration of the response. The acidic environment generated by PADE promoted TBAC hydrolysis. Finally, we succeeded in fabricating a new membrane for sensing the bitterness of medicines with higher durability and sensitivity by adjusting the proportions of the lipid and plasticizers.

Rational Design of Peptide-Functionalized Surface Plasmon Resonance Sensor for Specific Detection of TNT Explosive.

In this study, a rationally-designed 2,4,6-trinitrotoluene (TNT) binding peptide derived from an amino acid sequence of the complementarity-determining region (CDR) of an anti-TNT monoclonal antibody was used for TNT detection based on a maleimide-functionalized surface plasmon resonance (SPR) sensor. By antigen-docking simulation and screening, the TNT binding candidate peptides were obtained as TNTHCDR1 derived from the heavy chain of CDR1, TNTHCDR2 derived from CDR2, and TNTHCDR3 from CDR3 of an anti-TNT antibody. The binding events between candidate peptides and TNT were evaluated using the SPR sensor by direct determination based on the 3-aminopropyltriethoxysilane (APTES) surface. The TNT binding peptide was directly immobilized on the maleimide-functionalized sensor chip surface from N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS). The results demonstrated that peptide TNTHCDR3 was identified and selected as a TNT binding peptide among the other two candidate peptides. Five kinds of TNT analogues were also investigated to testify the selectivity of TNT binding peptide TNTHCDR3. Furthermore, the results indicated that the APTES-GMBS-based SPR sensor chip procedure featured a great potential application for the direct detection of TNT.