| Biogenic amines (BA) present in foods as endogenous substances. In general, biogenic amines would transform into low activity substance by biological oxidation of amine oxidase, but there will be a potential danger for people if the biogenic amines intake exceeds the limit value the people normal metabolizing. Meanwhile, BA is formed upon degradation of proteins and amino acids by decarboxylase-positive microorganisms, so their levels are a good index of food decomposition. In addition, putrescine, cadaverine, and histamine have been confirmed useful chemical indicators of bacterial spoilage of food. Therefore, it is important to establish a method to detect the content of biogenic amines in foods in order to protect food safety.The analytical methods used for separation and quantification of biogenic amines are mainly based on chromatography, which has high sensitivity and wide detection range. However, these methods require extensive sample pre-treatments and expensive equipments. In contrast, enzyme biosensor provides a fast and convenient way to detect the value of biogenic amines in foods.In this paper, a novel Microfluidic chip-Chemiluminescence-Enzyme biosensor was designed and assembled to monitor the total biogenic amines content in foods. Commercial diamino oxidase (DAO from Porcine kidney E.C.1.4.3.22) was adopted as biological recognition element of the sensor, immobilized on CNBr-activated Sepharose 4B connecting with chemiluminescence reagent (potassium ferricyanide, luminal) immobilized on anion exchange resin and microfluidic chip, aiming to test a total value of biogenic amines. The results are as follows:1.In this section, the CL detection system has been developed by connecting the microfluidic chip and CL reagent column in the flow path. On the basis of previous studies, the chemiluminescence reagents were immobilized on D201 resin to replace liquid solution. The results proved that the relative CL intensity reached a maximum value when the mass ratio of luminal and potassium ferricyanide in column was 2：1,the NaOH strength was 0.1 mol/L, and the concentration of elution (Na3PO4:NaOH=1:1)was 3×10(-3)mol/L. Meanwhile, the flow velocity (pump1) was set 0.20mL/min. Moreover, passing through proving, the immobilized LC reagents had good precision (RSD=..69%) and reproducibility, which could retain its performance when using more than 70 times and be preserved for a long time.2. CNBr-activated Sepharose 4B was used as a matrix for immobilization of DAO. The coupling ability of CNBr-activated Sepharose 4B to amine groups was based on a nucleophilic addition reaction. The results showed that the protein coupling ratio became a maximum value (67.33%), when 1.0U DAO was immobilized 6 hours and 44.91% enzyme activity recovery has been obtained by calculating. In addition, the catalytic performances of immobilized DAO under different conditions, including catalysis temperature and pH have been analyzed. The fittest catalytic conditions of immobilized DAO were 37℃ and pH7.2, which were same with the free DAO. Moreover, comparing to free DAO, the tolerance of immobilized DAO was better, which was determined by the variety of fluctuation on curve.3.In our work, at first time, an enzyme sensor coupled high sensitivity chemiluminescence (CL) was designed and assembled for the determination of biogenic amines. In this section, the condition optimization of the novel biosensor including enzyme activity in column and flow velocity of pump2 has been taken into account. The study indicate that when the activity of enzyme column was set 2.7U, and the flow velocity of pump2 reached 0.25ml/min, the relative CL intensity presented an optimum value. Meanwhile, the standard curves of three biogenic amines have been established. The LOD of enzyme sensor were 0.5μM for histamine and tyramine, and 0.7μM for putrescine with a linear range up from 3 to 500μM.The limit of detection range for histamine and tyramine were from 2 to 100μM and 3 to 200μM. In addition, Operational precision was tested at 30±2℃ with seven total measurement cycles. The relative standard deviations (RSD) for successive determinations were all less than 10%. The immobilized DAO could retain its enzymatic activity up to 30 days when stored in 0.1M phosphate buffered (pH 7.2) at 4℃ with a 5% decreasing. At last, the biosensor has been applied to determine biogenic amines content in vegetable, fish and red wine with satisfactory results. The results were also compared to efficient and traditional HPLC method and showed similar conclusion. |
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