Article pubs.acs.org/acssensors
One-Minute Fish Freshness Evaluation by Testing the Volatile Amine Gas with an Ultrasensitive Porous-Electrode-Capped Organic Gas Sensor System Liang-Yu Chang,†,# Ming-Yen Chuang,†,# Hsiao-Wen Zan,*,† Hsin-Fei Meng,*,‡ Chia-Jung Lu,§ Ping-Hung Yeh,∥ and Jian-Nan Chen⊥ †
Department of Photonics and Institute of Electro-Optical Engineering and ‡Institute of Physics, National Chiao Tung University, 1001, Ta Hsueh Rd., 300 Hsinchu, Taiwan § Department of Chemistry, National Taiwan Normal University, 162, Heping East Rd., Section 1, 106 Taipei, Taiwan ∥ Department of Physics, Tamkang University, 151, Yingzhuan Rd., Tamsui District, 25137 New Taipei City, Taiwan ⊥ Institute of Electronics Engineering, National Tsing Hua University, 101, Kuang-Fu Rd., Section 2, 300 Hsinchu, Taiwan S Supporting Information *
ABSTRACT: In this work, we successfully demonstrate a fast method to determine the fish freshness by using a sensing system containing an ultrasensitive amine gas sensor to detect the volatile amine gas from the raw fish meat. When traditional titration method takes 4 h and complicated steps to test the total volatile basic nitrogen (TVB-N) as a worldwide standard for fish freshness, our sensor takes 1 min to deliver an electrical sensing response that is highly correlated with the TVB-N value. When detecting a fresh fish with a TVB-N as 18 mg/100 g, the sensor delivers an effective ammonia concentration as 100 ppb. For TVB-N as 28−35 mg/100 g, a well-accepted freshness limit, the effective ammonia concentration is as 200−300 ppb. The ppb-regime sensitivity of the sensor and the humidity control in the sensing system are the keys to realizing fast and accurate detection. It is expected that the results in this report enable the development of on-site freshness detection and real-time monitoring in a fish factory. KEYWORDS: gas sensor, amine gas, fish freshness, volatile basic nitrogen, ammonia, dimethylamine, trimethylamine, P3HT he freshness of fish is a fundamental issue in the food industry. In addition to a bad smell, the spoiled fish causes a wide variety of health problems. For example, the spoiling bacteria produce histamine, which cannot be removed by cooking. Consumption of histamine may result in hypotension, edema, urticaria, diarrhea, and vomiting.1 The popularity of raw fish as food in some Asian nations makes the demands on fresh fish even tighter. In general, the price of fish highly depends on the warrant on its freshness. Conventionally the freshness of fish is evaluated by the concentration of the total volatile basic nitrogen (total VBN, TVB-N), usually in the units of mg/100 g.1 VBN includes mostly three kinds of gas molecules, ammonia (NH3), dimethylamine ((CH3)2NH, DMA), and trimethylamine (N(CH3)3, TMA). After the fish dies, the spoilage bacteria in the fish meat will convert the trimethylamine oxide (TMAO) in fresh meat into DMA and TMA. Simultaneously ammonia is produced by the bacteria due to the decomposition of urea and amino acids.2 Marine fishes usually contain more TMAO than freshwater fishes, and consequently more DMA and TMA, given the same decay time.3 Whereas the TVB-N value has been an international standard for fish freshness, its experimental determination is rather time-consuming. In practice, the volatile amines are released from the fish sample
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solution by adding potassium carbonate (K2CO3). The amine gas is then captured by boric acid through chemical reaction. The total amount of reaction is then determined by hydrogen chloride (HCl) titration. The whole process, including the preparation of fish sample solution, usually takes about 4 h. The slowness of the conventional titration method for TVB-N measurement makes it possible only for sampling of a small fraction of total fish products. So far, the overall quality control in the fish industry still mostly relies on human inspection of the color of the eyes or gills of the fish. Such control depends on the skill of the inspectors and inevitably has a large variation due to human factors. It will be of great importance for the fish industry if instant TVB-N data can be obtained at a low cost, such that generally accurate quality control can be performed at any point of the production chain. For example, the fish can be examined when it is purchased on the harbor, or checked on the fishery ship to prevent failure of the refrigeration system. Received: December 21, 2016 Accepted: April 11, 2017 Published: April 11, 2017 531
DOI: 10.1021/acssensors.6b00829 ACS Sens. 2017, 2, 531−539
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Figure 1. (a) Pictures of three kinds of fishes: Tilapia, Beltfish, and Mackerel. (b) Illustration of ammonia, DMA, and TMA production by the bacteria in fish. (c) Illustration of the P3HT gas sensor.
The fish product can also be checked in the food process plant before packaging. To facilitate the fast detection of fish spoilage, several new methods have been proposed. Infrared spectroscopy has been used to study the decay of fish,4,5 but it is quite expensive and not so easy to identify the entire chemical structure of the molecules. Electrical characteristics of the whole fish were studied by an electrochemical method.6 The method detects the dissolved amine gas in the aqueous solution. Colorimetric sensors and the sensor array were utilized to detect the decay of meat, including fish.7−9 The monitoring of TMA, DMA, ammonia, and fish spoilage was also demonstrated by using metal oxide sensor array,10,11 operating at 250−300 °C. Here in our work, we utilize organic semiconductor gas sensors to realize the detection of fish freshness. Organic semiconductor chemical sensors have advantages such as low production cost, reusability, rapid reaction, and room temperature operation. 12,13 In our work, by measuring the concentration of the volatile base amines released naturally from the raw fish into the air, the detection takes only 1 min and correlates with the standard TVB-N results. The proposed method is in sharp contrast to the conventional titration method that measures the aqueous base amines. The gas sensor is an organic semiconductor diode with a porous top metal electrode. The current in the semiconductor will be confined in the electrode-overlapped regions and hence exhibits nanostructure. These nanostructures increase the surface to volume ratio in current distribution and thus lead to improved sensitivity. The current flows vertically to the semiconductor thin film, in contrast to the common structure with horizontal current flow. The vertical gas sensor was reported to have a very high sensitivity of about 100 ppb for ammonia in air.14 When the defrosted fish sample of about 1 g is placed inside a bag of dry air, a steady amine gas concentration will be reached quickly. The air in the bag is then pumped through a sensor chamber, and a NaOH dryer is used to desiccate the air before entering the chamber. The relative electrical current change, called response, can be registered in 60 s. The response is a weighted sum of ammonia, DMA, and TMA. The response data are collected for three kinds of fishes with various degrees of decay (depending on different storage conditions). At the same time the conventional titration VBN is also measured for the same storage conditions. We found that there exists a general relation between the slow conventional method and the rapid gas sensor method for all the data. The detecting range
corresponds to the TVB-N value from 10 to 70 mg/100 g, while the TVB-N smaller than 25 mg/100 g is usually required for a fresh fish. Furthermore, for freshwater fish, our gas sensor is more sensitive to the spoilage status than the conventional method. The gas sensor method is therefore a reliable substitution of the conventional VBN measurement. It is superior in applications as the data collection time is shortened from 4 h to only 60 s. It also has a low cost as the organic semiconductor is fabricated by a solution process. This new technology to detect total volatile base amine may have a significant impact on the fish industry by greatly simplifying the quality control approach.
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EXPERIMENTAL SECTION
Gas Sensing versus Conventional Method. The generation of the volatile base amines in spoiled fish is shown in Figure 1a. The pictures of three kinds of fishes studied in the work are shown, Tilapia (Oreochromis mossambicus), Beltfish (Trichiurus lepturus), and Mackerel (Scomber scombrus). The first is a freshwater fish and the other two are marine ones. All of them are common foods. After the fish dies, bacteria proliferates in its meat. One of the common kinds of bacteria which cause fish decay is Proteus mirabilis.15 As discussed above TMAO is present is many fishes, and the enzymes produced by the bacteria reduce TMAO into DMA and TMA. As for ammonia, it is mostly produced by the decomposition of urea and amino acids by bacteria activities. Note that even though all three molecules are gases at room temperature, their boiling points are very different: −33 °C for ammonia; 8 °C for DMA, and 5 °C for TMA. Ammonia is much more volatile than the other two amines. When the three amine gases are produced by bacteria the gas molecules will be released to the air as shown in Figure 1b. In a closed chamber the vapor pressure in the air is proportional to the concentration in the water part of the fish meat. The proportional coefficient should be however different for the three amine gases. Due to its lowest boiling point ammonia probably has the highest proportional coefficient. The air containing the amine gases is then pumped to a gas sensor based on an organic semiconductor. As shown in Figure 1c, the sensor has a sandwich structure. The red film is made of semiconducting conjugated polymer P3HT (poly(3-hexylthiophene)) deposited by a solution process like spin coating or blade coating, with a thickness of 40 nm. The chemical structure of P3HT is also shown in Figure 1c. The bottom electrode is indium−tin oxide (ITO), whereas the top electrode is a porous Al of 40 nm thickness. The top porous Al electrode contains a density of small round openings with diameters of 100 nm, which is made by colloid lithography. The detail of colloidal lithography process is given in the Supporting Information. The amine molecules can diffuse into P3HT through the gas pores as shown in Figure 1c. The reactions between P3HT and the amine gases are 532
DOI: 10.1021/acssensors.6b00829 ACS Sens. 2017, 2, 531−539
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ACS Sensors explained by the redox reaction. The oxygen in air oxidizes P3HT and creates a certain level of hole charge carriers. When amine gases absorb on the P3HT chain, they react with sulfur atoms and form polarons to reduce the polymer chain. The hole concentration in P3HT therefore becomes lower and result in a decrease of conductance.16−18 Then, after removing amine molecules, the recovery of the conductance reveals that this reaction is more like a physical adsorption instead of formation of chemical bonds between amine molecules and P3HT. The redox mechanism also explains the results in our previous work in which the P3HT sensor exhibited higher sensitivity to strong polarity amine gases than to ethanol or acetone.14 The P3HT sensor can detect ammonia in dry air with concentration as low as 100 ppb. By using this gas sensor method in Figure 1 the TVB-N in mg/100 g trapped in fish can be inferred by the sensor response caused by the gas released into the air. The gas sensor method can take a response value once per 60 s. The detection of gas requires not only the sensor device but also the whole sensing system, which includes the pump, the gas tube, the desiccation cylinder, the sensor chamber, the electrical readout, and the computer to display the result. The flowchart of the sensing system is shown in Figure 2. It is compared with the conventional titration
water molecules the high water condensation on the sample should be avoided. Defrosting of 1−3 g fish meat can be done by hot air, hot water immersion, or electrical heating in tens of second as shown in Figure 3a. The fish sample is then placed inside a resealable food storage bag (Ziploc) of volume 1000 c.c. Dry air is delivered into the bag by a manual pump mounted with a cylinder tube filled with NaOH desiccant as shown in Figure 3b. The relative humidity of the air is about 10%. The full bag is then connected to the main system by the penetration of a sharp tube head into the bag as shown in Figure 3c. The tube is connected to a second NaOH dryer, then to the gas inlet of the sensor chamber. The gas outlet of the sensor chamber is finally connected to a pump, as shown in Figure 3c, which provides a steady flow rate of 500 c.c. per minute. The pumping starts before connection of the sample bag and the sensor current stays at a stable background value for a given gas flow rate. Once the gas bag is connected to the main system the air flowing through the sample is shifted from the dry air to the air in the bag containing the fish. As discussed already there is an equilibrium amine concentration in the bag determined by the aqueous VBN value in the fish meat. Due to the reduction effect of the amine gas there is a sharp drop of the electrical current of the sensor under a constant voltage bias normally at 2 V. The response is defined as the percentage of the current change at certain times after the sample bag connection. In most of the data below the time is 60 s after sample bag connection. In other words, the response can be obtained in 60 s. No chemical reaction is needed in such a method. For comparison, the conventional titration method for TVB-N is also shown in Figure 3d−f. The data in this work are taken at the Food Industry Research and Development Institute in Taiwan and the recipe below is their procedure. First, a boric acid absorbing liquid is prepared by dissolving 10 g boric acid in 200 mL ethanol (95%) and blending with 5 mL 0.03% bromocresol green (in 95% ethanol), 5 mL 0.06% methyl red (in 95% ethanol), and 790 mL distilled water. Then, a fish sample of 5 g and 45 mL of trichloroacetic acid (TCA) (2.2% in water solution) is mixed and homogenized. The acidity of TCA traps the VBN molecules as positive ions in water. The fluid is then filtered to obtain a clear sample liquid. The sample liquid of 1 mL is then placed in the outer chamber of the Conway dish shown in Figure 3e. The boric acid absorbing liquid is placed in the internal chamber of the Conway dish. In the outer chamber of the Conway dish the sample liquid is reacted with 1 mL of potassium carbonate (K2CO3) saturated solution, which is a strong base. The Conway then is covered with a
Figure 2. Flowchart of the sensing system. method in Figure 3. The fish meat of a few grams is first cut from the body. For frosted fish the sample needs to be defrosted first before measurement; otherwise, the low temperature of the sample would cause air moisture condensation. As the gas sensor also responds to
Figure 3. Comparison of the method used in this work and the conventional titration method. (a) Defrosting the fish sample by immersing it into the water. (b) Putting the fish sample in the Ziploc bag and filling with dry air. (c) Connecting the full bag with the sensing system by the penetration of a sharp tube head into the bag. The gas sensor interacts with the air drawn out from the bag and results in a change of current which reflects the amounts of the VBN. On the other hand, in the conventional titration method, the fish sample is first defrosted as in (a), followed by (d) mixing the fish sample with TCA and homogenizing. (e) Placing the extracted clear sample liquid and potassium carbonate saturated solution in the outer chamber of Conway dish while placing the prepared boric acid absorbing liquid in the internal chamber. Then, putting the Conway dish in the 37 °C oven for 1.5 h. (f) Titrating the boric acid absorbing liquid with HCl. 533
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Figure 4. Fabrication process of vertical P3HT gas sensor: (a) Sping-coating the P3HT on the ITO bottom electrode. (b) Blade-coating the PS nanospheres on the P3HT layer. (c) Thermal deposition of top Al electrode. (d) Removing the PS nanospheres to form a porous top electrode.
Figure 5. Real-time current sensing of the P3HT gas sensor to (a) ammonia, (b) DMA, and (c) TMA. (d) Sensing response as a function of concentration. The real-time current sensing of the P3HT gas sensor to (e) ammonia and DMA, and (f) ammonia and TMA. In (a), (b), (c), (e), and (f), colored areas represent the sensing duration to injected amine gases. The background is the dry air with a RH ∼ 10%.
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glass plate and put in the 37 °C oven. The OH− ions of the potassium carbonate will react with the positive amine ion like NH4+ to release the neutral NH3 molecules into the air. The released VBN molecules are then captured by the boric acid in the internal chamber of the Conway dish through chemical reaction. The color of the boric acid absorbing liquid changes as a result. The release of the amine molecules and the reaction with the boric acid is slow and takes about 1.5 h in oven. Finally HCl is added to convert the boric acid into its original form and color by titration as shown in Figure 3f. At the color turning point the total amount of HCl is equal to the TVB-N in the sample liquid. The conventional titration method takes about 4 h to attain the TVB-N value of the fish sample. Because of the sophisticated process for preparing the solutions and slow chemical reaction involving potassium carbonate and boric acid, the conventional method is time-consuming and has a large variation. Sensor Fabrication. The sensor fabrication process is shown in Figure 4. The indium tin oxide (ITO) serves as the bottom electrode of the gas sensor. A 40-nm-thick poly(3-hexylthiophene) (P3HT) (Rieke Metals, 2.5 wt % in chlorobenzene, molecular weight 50 000− 70 000) layer (the red layer in Figure 4a) is spin-coated on ITO, followed by 200 °C annealing for 10 min. By using the blade-coating method, the polystyrene (PS) nanospheres (Fluka, diameter 100 nm) is absorbed to the surface of P3HT, as shown in Figure 4b. In the method of colloid lithography, the PS nanospheres serve as the shadow mask during the thermal deposition of a 40-nm-thick aluminum top electrode (Figure 4c). After removing these nanospheres with the 3 M Scotch (810L-3PK) tape, a porous top electrode is formed (Figure 4d). The porous structure enables gas molecules to pass through the electrode and to diffuse into the active layer, the P3HT. The detail of colloidal lithography process is given in the Supporting Information.
RESULTS AND DISCUSSION Sensor Response to Calibrated Amine Gases. Before measuring the fish samples with unknown compositions, the vertical P3HT gas sensor is calibrated by various gas samples with known composition (ranged from 100 ppb to 1 ppm). In Figure 5a,b,c, we first measure the responses to the ammonia, DMA, and TMA individually. To prepare gases in ppb-regime concentration, a higher concentration (100 ppm) of these three gases is first obtained and extracted by syringes. 100 ppm ammonia is extracted from a gas cylinder (bought from Sinda Gases Co., Ltd.). 100 ppm DMA and TMA are obtained by evaporating the DMA and TMA water solution in a 1 L Tedlar bag (or a SamplePro FlexFilm bag, SKC Inc.) and diluting with pure nitrogen several times. During the measurement, there is a background flow of dry air. Then, by adjusting the injection rate of these gases with a syringe pump, these gases would be diluted and the concentrations would depend on the ratio between injection rate and background flow. Thus, we can obtain the concentration ranging from 100 ppb to 1 ppm. In Figure 5a,b,c, we measure the current of our sensor in every second (real-time measurement). In Figure 5a, for example, the color region indicates the period when we inject ammonia; otherwise, it is dry air with relative humidity of 10% (background). There is an overall background increase of the current in dry air. The drift of current may result from the gradual oxidation of the P3HT due to the presence of air and humidity; it also may result from the reduction of trap density in organic materials by applying continuous current flow during the real-time current sensing. However, the sensing response defined by the ratio between current variation and initial 534
DOI: 10.1021/acssensors.6b00829 ACS Sens. 2017, 2, 531−539
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Figure 6. (a) Effective ammonia concentration of the ventral, lateral, and dorsal parts of the fish as a function of storage time in 30 °C. (b) Effective ammonia concentration of the ventral part of the fish meat as a function of storage time in 22, 25, and 30 °C.
current will not be affected by the baseline drift.14,16 The response is the relative current decrease in the color region of 60 s and is represented in a percentage in the figure. The response to 100, 300, and 500 ppb and 1 ppm is 8%, 25%, 34%, and 48.5%, respectively. We also measure the real-time current sensing of DMA and TMA, as shown in Figure 5b,c. The results of sensing responses in Figure 5a,b,c are summarized in Figure 5d as a function of gas concentration. As shown in Figure 5d there is a strong positive correlation between the ammonia concentration and the response. The error bars represent the standard deviation obtained from 6 individual devices. The DMA and TMA detection results using 2 individual devices are also plotted. No significant deviation can be observed. For a given concentration, the order of sensitivity is ammonia > DMA > TMA as shown in Figure 5d. We then try to mix these amine gases to see what will happen to the sensing response as they exist at the same time. In Figure 5e,f, we mix ammonia with DMA and with TMA, respectively. The ammonia concentration is marked in red in the figures and the concentration of DMA and TMA is marked in black in the Figure 5e,f, respectively. It is shown that when the gas contains two amine gases the responses can be roughly added. For example, 1 ppm of TMA alone gives 21% in Figure 5c, and 300 ppb of ammonia alone gives 25% in Figure 5a. The addition is 46% as shown in Figure 5f, while the response of the mixed gas is 37.5% which is slightly lower than the added value. Similarly, the response for 1 ppm of DMA alone is 38% in Figure 5b, and for 300 ppb of ammonia alone is 25% in Figure 5a. The addition is 63%, while the mixed gas gives 56% in Figure 5e which is close to the added value. For unknown gas samples from the fish below, the response is regarded as a weighted sum of the three VBN gases with the highest weight from ammonia as shown in Figure 5d. In addition to amine gas, the spoilage process also generates sulfur compounds, aromatics, N-cyclic compounds, and some acids.19,20 The concentration of all these volatile gases increases with spoilage time. Here in this work, we use the proposed sensor to detect the total volatile gas. The sensor is sensitive to amine gas, and may also detect other compounds together. The question is whether the standard TVB-N by conventional method and the new gas sensor method follow the same mathematical relation for various kinds of fish types and storage conditions. If the answer is positive, the simple gas sensor method can be used to replace the complicated titration method for TVB-N. Fish Testing Using Gas Sensor. In a previous section, we already demonstrated that the proposed gas sensor is sensitive to ammonia, TMA, and DMA in the ppb regime. About 10%
current variation can be obtained when using the proposed sensor to detect 100 ppb ammonia, 500 ppb TMA, and 300 ppb DMA in a dry air background (RH = 10%) (Figure 5d). Now we start to use the proposed sensor to test the volatile gas collected from fish. As described in the Experimental Section, the volatile gas is collected from a bag with a fish sample. Because amine molecules are easily dissolved in water, it is necessary to avoid the water accumulation in the sample bag. Hence, the fish sample taken from a frosted fresh fish has to be defrosted first, and the bag has to be filled with dry air (RH = 10%). After preparing the dry background, in the following sections, fish samples with different conditions are then used to study the influences of the storage time, the fish parts, the storage temperature, and the fish species. In the following section, we used a total of five sensor devices to measure the fish freshness. To minimize the influence of device-to-device variation, for every sensor device, we established its individual calibration curve by plotting its response (current variation ratio) as a function of standard ammonia concentration. When using the sensor to detect fish freshness, the sensor response (i.e., the current variation ratio) is then plotted on each calibration curve to find out the corresponding ef fective ammonia concentration without the need to consider the device-to-device variation. First, we use Mackerel (Scomber scombrus) to do the testing since Mackerel is known to spoil easily. As shown in Figure 6a, the sensor response (i.e., the current variation ratio) is plotted as a function of the storage time when the 1 g samples of fish meat are collected from ventral, lateral, and dorsal parts of one Mackerel. The storage conditions in Figure 6a are 30 °C for 0 (fresh condition), 10, 15, 18, and 24 h. Since the sensor responds to ammonia, TMA, and DMA with different sensitivities, we convert the response (the current variation ratio) into an effective ammonia concentration (noted as Eff. NH3 concentration) as shown in the y axis in Figure 6a. In the first 10 h, there is no significant increase of the Ef f. NH3 concentration. When storage time increases from 15 to 24 h, the Eff. NH3 concentration apparently increases. The increase of the Ef f. NH3 concentration in samples from the ventral parts is much more significant than those from lateral parts or dorsal parts. This result agrees well with the previous reports that the ventral parts exhibits higher TVB-N then the lateral parts and the dorsal parts.21,22 In the following experiments, we only use ventral parts of the fish samples. In Figure 6b, we change the storage temperature from 22 and 25 to 30 °C. It is known that the spoilage of fish is attributed to the reduction of TMAO with the enzymes produced by 535
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that in marine fish.23 Hence, the generated amount of TMA and DMA in freshwater fish is also expected to be low. In a previous report, when using the chromatography−mass spectrometric method, the amount of TMA and DMA in marine fish is 40 times larger than that in freshwater fish.23 Here, when we compares the Ef f. NH3 concentration for the three kinds of fish in Figure 7, we observe that Eff. NH3 concentrations from Beltfish and Mackerel are quite similar to each other. The Ef f. NH3 concentration from Tilapia, however, exhibits a rather high value, 500 ppb, after 24 h storage time. The value is much higher than those for Beltfish (318 ppb) and Mackerel (218 ppb). One plausible explanation for the unexpected high response in Tilapia may be the high level of ammonia generated from Tilapia. In our sensor, as shown in Figure 5a−c, the sensitivity to 500 ppb ammonia is about 1.9 times higher than that to 500 ppb DMA and is about 3.8 times higher than that to 500 ppb TMA. In Tilapia, the low concentration of TMAO delivers low concentrations of TMA and DMA, but fish spoilage also generates ammonia. Hence, using our sensor, the high Eff. NH3 concentration may be primarily due to the ammonia from the spoiled Tilapia. To study the summarized effect of TMA, DMA, and ammonia, in the following section, we compare the Ef f. NH3 concentration to the conventional TVB-N value for the three kinds of fishes. Comparing Sensor Response with TVB-N Value. In this section, all samples are tested by two methods for comparison. One is the gas sensing response using our proposed method shown in Figure 3a−c. The other is the conventional titration TVB-N testing as shown in Figure 3a,d−f. It is noted that the TVB-N testing is done in the Food Industry Research and Development Institute by using their public food safety evaluation service. To avoid any sample variation, two samples taken from the ventral position of one fish are compared. To reduce the variation due to storage time, the testing of the two
bacteria. Increasing the storage temperature activates the bacteria and speeds up the fish spoilage. As shown in Figure 6b, Ef f. NH3 concentration successfully reflects such a temperature-enhanced spoilage process. After confirming that our sensor can reflect the expected influences of storage time, storage temperature, and fish parts, we then start to check the sensor response to three different kinds of fish. As shown in Figure 7, we prepare samples from
Figure 7. Effective ammonia concentration of Tilapia, Beltfish, and Mackerel as a function of storage time in 25 °C.
Tilapia (Oreochromis mossambicus), Beltfish (Trichiurus lepturus), and Mackerel while the storage temperature is fixed at 25 °C and the fish part is the ventral part. For all three kinds of fishes, the Eff. NH3 concentration increases significantly when the storage time increases. A high response is obtained when the storage time is higher than 24 h. With identical 24 h storage time, the Ef f. NH3 concentration for Tilapia is much higher than those for Beltfish and Mackerel. This is an unexpected and interesting result. Tilapia used in this study is the freshwater fish while Beltfish and Mackerel are marine fishes. It is known that in freshwater fish the amount of TMAO is much less than
Figure 8. Effective ammonia concentration as a function of TVB-N. The r represents the correlation coefficient. Fish conditions are as follows: (a) Mackerel with 22, 25, and 30 °C storage temperature. The r is individually calculated. (b) Mackerel with all the sample conditions in (a). (c) Beltfish with 25 °C storage temperature. (d) Combination of the data from Mackerel and from Beltfish with 25 °C storage temperature. 536
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Figure 9. (a) TVB-N values and (b) Ef f. NH3 concentration (at 22, 25, and 30 °C) as a function of storage time.
TMA, DMA, and ammonia. The lack of TMAO leads to a very low TMA and DMA concentration and hence a low TVB-N value. The Eff. NH3 concentration, however, may serve as a much more sensitive parameter to the spoilage status. As shown in Figure 9b, with 25 °C storage temperature, when storage time increases from 0 (fresh) to 24 h, the Ef f. NH 3 concentration exhibits a more than 10-times increase from 35 to 500 ppb. The TVB-N value, with the same spoilage condition, only changes between 10.3 and 20.3. The 10-times increase of the Eff. NH3 concentration may be a rather useful indicator to determine the freshness of the freshwater fish. It is particularly noted that the 500 ppb Eff. NH3 concentration in Beltfish and in Mackerel represents a severe spoilage status and the corresponding TVB-N value is as high as 50 mg/100 g. In Tilapia, even if there is no TMA and DMA at all, the 500 ppb ammonia should represent a spoilage status because the high ammonia concentration is generated from the decomposition of urea, amino acids, and nucleotides by urease and deaminase.15 In Figure 9b, when the temperature is reduced from 30, 25, to 22 °C, the increase of the Ef f. NH3 concentration is less obvious. However, even with 22 °C storage temperature, the Ef f. NH3 concentration reaches 200 ppb for 24 h storage time. According to the results in Mackerel and Beltfish in Figure 8d, the Eff. NH3 concentration of 200 ppb already corresponds to the quality limit as TVB-N = 28 mg/100 g. Here, in Tilapia, we may also consider that 200 ppb represents a quality limit. This implies that even though the low TMAO in Tilapia leads to a low concentration of TMA and DMA, ammonia from the decomposition of urea and amino can be easily detected by using the proposed sensor. In previous work, to propose a useful indicator to determine the freshness of freshwater fish, chromatography−mass spectrometry was used to determine TMA, DMA, and methylamine (MA) in marine fish and freshwater fish. Even the TMA concentration in trout, a freshwater fish, is very low. The increase of TMA is well correlated to fish spoilage and hence TMA is proposed to be a useful indicator to determine the freshness of freshwater fish. However, to detect the change of the very low concentration of TMA, utilization of the expensive chromatography−mass spectrometry is required. It is noted that, in the work using chromatography−mass spectrometry, ammonia is not observed since the signal of ammonia is usually overlapped with water molecules due to the similar molecular weights. Here, in our work, ammonia is proposed to possibly serve as a useful indicator to determine the freshness of freshwater fish. As previously discussed (Figure 5d), the proposed gas sensor has different sensitivities to ammonia, TMA, and DMA. When detecting ammonia, the
samples is started almost simultaneously. It is however noted that the testing using our proposed method takes only a few minutes while the testing using conventional TVB-N takes 4 h. First, we investigate the testing results of Mackerel. The samples are stored at 22, 25, and 30 °C for different times. The plot of the Ef f. NH3 concentration as a function of storage time is shown in Figure 6b. The comparison between the Ef f. NH3 concentration and TVB-N value is plotted in Figure 8a. For samples stored at 22, 25, and 30 °C, the correlation coefficient (r) between the Eff. NH3 concentration and TVB-N value is 0.998, 0.992, and 0.976, respectively. If the difference due to storage temperature is neglected, the correlation coefficient calculated from all the sample conditions is 0.932 as shown in Figure 8b. For Beltfish, the samples are stored at 25 °C from 0 (fresh) to 36 h. The plot of the Ef f. NH3 concentration as a function of storage time is shown in Figure 7. The comparison between Ef f. NH3 concentration and TVB-N value is plotted in Figure 8c with a correlation coefficient as 0.960. Since in Figure 7 the gas sensing results for Mackerel and for Beltfish are quite similar to each other, we then combine the data from Mackerel and from Beltfish with 25 °C storage temperature into Figure 8d; a correlation coefficient of 0.971 is obtained in the comparison plot, revealing that the Eff. NH3 concentration represents the TVB-N value in Mackerel and in Beltfish. Specifically speaking, in fresh status, the TVB-N value is 12−18 mg/100 g and the Eff. NH3 concentration is less than100 ppb. According to the Food and Agriculture Organization (FAO), a TVB-N value of 28−35 mg/100 g is considered to be the acceptability limit for good-quality fish.2 Here in this work, a TVB-N value of 28−35 mg/100 g corresponds to the Ef f. NH3 concentration of 200− 300 ppb. Finally, we evaluate the Ef f. NH3 concentration and the TVB-N values for Tilapia, the freshwater fish. Samples are stored at 22, 25, or 30 °C. The TVB-N values and the Eff. NH3 concentration plotted as a function of storage time are shown in Figure 9a,b, respectively. We first observe the change of TVB-N values in Figure 9a. When storage temperature is 22 or 25 °C, the TVB-N value does not increase significantly even with a 24 h storage time. For 25 °C, the TVB-N values in 12 h condition (17 mg/100 g) is even higher than that in 18 h condition (16 mg/100 g). Since the change of TVB-N is only small, the fluctuation can be explained by the deviation of TVB-N analysis. Only when the storage temperature is as high as 30 °C for 18 h does the TVB-N reach the value of 36.5. This result agrees with the well-known fact that, in freshwater fish, TVB-N is usually low due to the very low TMAO concentration in freshwater fish.3 The main components in TVB-N include 537
DOI: 10.1021/acssensors.6b00829 ACS Sens. 2017, 2, 531−539
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sensor delivers a particularly large response. Such unequal sensitivity, however, does not exist in TVB-N analysis. In TVBN analysis, every ammonia molecule, TMA molecule, and DMA molecule contributes one basic nitrogen. Hence, the very low TMA and DMA in freshwater fish directly cause a low TVB-N value. The gas sensor in this work, on the other hand, exhibits a particularly high sensitivity to ammonia and hence may be an effective sensor to determine the freshness of freshwater fish. A more detailed study needs to be carried out in future work.
CONCLUSION We have presented a method to quickly determine fish freshness. The freshness detection system is composed of an ultrasensitive amine sensor, a gas tube, a desiccation cylinder to control the relative humidity, and a pump to control the gas flow rate. The amine gas sensor can detect ammonia, dimethylamine (DMA), and trimethylamine (TMA) in the ppb regime; hence the sensor can respond to the volatile gas from the raw fish meat to reflect the spoilage status. While conventional TVB-N analysis takes about 4 h to do the sample pretreatment and the titration, our sensor detects the volatile amine gas within 1 min. The proposed method effectively reflects the influence of storage temperature and fish portion (ventral, dorsal, and lateral) on the spoilage status. When detecting marine fishes such as Mackerel and Beltfish, the sensing results were well correlated to the results in TVB-N analysis. Moreover, for Tilapia (a freshwater fish) with low TMAO in meat, our method can effectively reflect the spoilage status when TVB-N analysis becomes less sensitive. The proposed sensor system is composed of solid-state sensor and a simple pump-controlled flow channel, the fabrication cost is low, and the operation is simple. The proposed fast method is promising for developing on-site real-time freshness detection both in the fish factory and in home utilization. ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.6b00829. Detail of colloidal lithography process for sensor fabrication (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (H.-W. Z.). Fax:+886-35737681. *E-mail:
[email protected] (H.-F. M.). ORCID
Hsiao-Wen Zan: 0000-0002-7685-1245 Author Contributions #
Liang-Yu Chang and Ming-Yen Chuang contributed equally to this work. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by Ministry of Science and Technology, R.O.C. under Grant 103-2221-E-009-113-MY3 and 104-2112-M-009-009-MY3. 538
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DOI: 10.1021/acssensors.6b00829 ACS Sens. 2017, 2, 531−539
