CN109856102A - A kind of hypoxanthic biosensor of detection aquatic products - Google Patents
A kind of hypoxanthic biosensor of detection aquatic products Download PDFInfo
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- CN109856102A CN109856102A CN201910259960.2A CN201910259960A CN109856102A CN 109856102 A CN109856102 A CN 109856102A CN 201910259960 A CN201910259960 A CN 201910259960A CN 109856102 A CN109856102 A CN 109856102A
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Abstract
The present invention relates to a kind of hypoxanthic biosensor of detection aquatic products and its applications, belong to food analysis field.The present invention is based on the catalytic activity of platinum nanometer (Pt NPs), construct a kind of biological sensor, for detecting the content of hypoxanthine Hx in aquatic products.The sensor-based system proposed is made of xanthine oxidase XOD, o-phenylenediamine OPD and Pt NPs, is reacted in the presence of XOD with oxygen according to Hx, and uric acid and H are generated2O2, Pt NPs can be catalyzed OPD and H2O2Oxidation reaction generate 2, the 3- azophenlyene diamines of hair yellow fluorescence, propose it is a kind of quickly, the detection method of easy, sensitive aquatic products freshness.Preparing for Pt NPs in the new detecting method is simple and easy to get, and the simplicity analysis to aquatic products freshness may be implemented, and has easy to operate, sensitivity and the high feature of selectivity.Use easy to spread.
Description
Technical field
The freshness that aquatic products are detected the present invention relates to a kind of detection hypoxanthic biosensor of aquatic products, belongs to
Food analysis field.
Background technique
The freshness of aquatic products is to evaluate the important indicator of its quality.Due to its property full of nutrition, aquatic products are in enzyme
It is easy to rotten in storage and selling period under the collective effect of microorganism.However, aquatic products freshness assessment be it is complicated,
Because the corruption of meat is related to different microorganisms, physical chemistry and biochemical reaction.Traditionally, there are two types of methods to assess aquatic products
Freshness: (1) by the sensory testing of Multimode Control organoleptic feature, and (2) chemistry and biochemical measurement target organism index
Concentration.Although former approach has proved to be quickly, it is expensive and sometimes unreliable, because in the initial rank of degradation
It is difficult to assess the nuance of freshness of meat before section.Later approach causes many concerns, and numerous studies attempt
By the concentration of the freshness of meat and Cucumber (such as biogenic amine, trimethylamine, volatile amine, ATP catabolite and count of bacteria)
Or exists and connect.Hypoxanthine Hx is often used as the index of reaction aquatic products freshness, develops various biologies in recent years
Sensor, including electrochemiluminescence and optical sensor, because biosensor has such as simply, quickly divide for measuring Hx
Many advantages, such as analysis and high sensitivity, however, material requested synthesizes comparatively laborious, detection cycle in the methods of most of reports
It is long, and sensor to prepare itself more complicated.
Summary of the invention
In view of above-mentioned deficiency in the prior art, it is an object of the present invention to provide the fluorescence of a kind of precise and high efficiency point
Analysis method detects the freshness of aquatic products.In various biosensors, fluorescence analysis is highly sensitive and anti-interference due to it
The Inherent advantages such as performance, it has also become one of most powerful bioanalysis and diagnostic tool.The present invention is mainly based upon Pt NPs mould
Intend the biological sensor of the catalytic activity building of peroxidase to achieve the purpose that the freshness for detecting aquatic products.Pass through
2, the 3- azophenlyene diamines of the product generation generation fluorescence of Pt NPs catalysis o-phenylenediamine OPD and xanthine oxidase XOD and Hx, 2,
3- azophenlyene diamines has fluorescent absorption peak at 580nm.The concentration of Hx in aquatic products is detected according to fluorescence intensity.
In order to achieve the purpose that realize above-mentioned detection method, the invention adopts the following technical scheme:
The hypoxanthic biosensor of detection aquatic products is catalyzed the product H of OPD and XOD and Hx by Pt NPs2O2
2, the 3- azophenlyene diamines that is fluoresced of reaction, the fresh of aquatic products is detected according to the fluorescence intensity of 2,3- azophenlyene diamines
Degree.
The preparation method of Pt NPs in the detection hypoxanthic biosensor of aquatic products, specifically: 1mL is dense
The chloroplatinic acid aqueous solution that degree is 16 mM and the citric acid three sodium solution that 1 mL concentration is 40 mM are added in 38 mL water and in room
Temperature lower stirring 30 minutes.Then, the sodium borohydride solution that 200 μ L concentration are 50 mM is added into mixture.Stand 1 hour
Afterwards, the brown suspension of Pt NPs is obtained, 4 DEG C are held it in front of use.
The application of the detection hypoxanthic biosensor of aquatic products freshness in detection water, specifically: it will
Hx and xanthine oxidase XOD are placed in 37oC shaking table reacts 30min, and Pt NPs is added and reacts 30min, adds OPD reaction
30min, the last fluorescence intensity at 580nm, the detection method time-consuming is short, easy to operate.
Wherein, by Pt NPs, H2O2It is reacted with the OPD of various concentration, the optimal concentration for obtaining OPD is 2 mM, by Pt
NPs, H2O2Different time is reacted with OPD, obtaining optimum reacting time is 30min.
Deficiency compared with prior art, advantages of the present invention:
1. the biological sensor that the catalytic activity of the invention using Pt NPs constructs is by Pt NPs catalysis OPD and by XOD
The H generated with Hx2O2The fluorescence power of generation just may know that the concentration of Hx.Detection work can be completed in 10-15min.
2. Pt NPs used in the present invention is modified as stabilizer on surface by citric acid, pass through sodium borohydride reduction chlorine
Platinic acid obtains, and preparation process is simple and quick.
3. the present invention does not need complicated instrument and cumbersome operation in the detection process, every time Pt NPs needed for detection
Amount it is few (50 μ L), testing cost is low.
4. the range of linearity of present invention detection Hx is 8-2500 μM, the high sensitivity of detection, selectivity is good, and detection is limited to
2.88μM。
5. measuring method of the invention, can measure the concentration of Hx in aquatic products by fluorescent absorption peak, this method is sensitive
Degree is high, and specificity is good, and detection process is easy, and economic cost is low, and stability is good, use easy to spread.
Detailed description of the invention
Fig. 1 is that Pt NPs is catalyzed OPD and H2O2Reaction generates the fluorescent absorption spectrum of 2,3- azophenlyene diamines；
Fig. 2 is the feasibility analysis to the sensor；
Fig. 3 is the optimization of OPD concentration and reaction time in reaction process；
Fig. 4 is the curve graph of the corresponding different fluorescence intensities of the Hx of various concentration；
Fig. 5 is the canonical plotting of Hx；
Fig. 6 is the interference experiment of Hx detection.
Specific embodiment
In order to make content of the present invention easily facilitate understanding, With reference to embodiment to of the present invention
Technical solution is described further, but the present invention is not limited only to this.
Embodiment 1:
The chloroplatinic acid aqueous solution that 1mL concentration is 16 mM and the citric acid three sodium solution that 1 mL concentration is 40 mM are added to 38
In mL water and it is stirred at room temperature 30 minutes.Then, it is molten that the sodium borohydride that 200 μ L concentration are 50 mM is added into mixture
Liquid.After standing 1 hour, the brown suspension of Pt NPs is obtained, Pt NPs average grain diameter obtained is about 2.4 nm.Above procedure
Used in all glasswares pass through chloroazotic acid immersion, and thoroughly cleaned, dried with ultrapure water.
Fig. 1 and Fig. 2 show the verifying to the sensor feasibility, and individual Pt NPs does not have fluorescent absorption peak, when adding
Enter OPD and H2O2Solution has fluorescent absorption peak at 580nm later, and works as H2O2When concentration is high, the intensity of absorption peak is dropped
It is low.Apparent downward trend can be just presented in the fluorescence intensity of solution when only having in Fig. 2 while Hx and XOD is added；Wherein a cylindricality is
Pt NPs+OPD+500 μM Hx+XOD, b cylindricality is+OPD+XOD+50 μM of H of Pt NPs2O2, c cylindricality be Pt NPs+
OPD+Hx+50 μM H2O2.Fig. 3 is the reaction condition optimization to the sensing system, using control variate method, it is determined that OPD's
Optium concentration is 2 Mm, and reaction Best Times are 30min.
Under optimum experimental condition, 50 are added after the Hx of various concentration is reacted 30min in 37 degrees Celsius of shaking tables with XOD
μ LPt NPs, then be catalyzed reaction 30min after carry out fluorescent absorption detection, the relationship of obtained Hx concentration and fluorescence intensity such as Fig. 4,
Shown in Fig. 5, observe that FL intensity is gradually quenched with the increase of Hx concentration, and FL intensity relative to Hx concentration in 8-2500
It is linear, related coefficient 0.990 in the range of μM.Using this method, the detection limit (LOD) of Hx (S/N=3, wherein
S represents sensitivity, and N represents noise) to calculate separately be 2.88 μM.
In order to study the selectivity of sensor-based system Hx, other substrates, including glucose are tested under the same conditions
(Glu), lactic acid (LA), ascorbic acid (AA), uric acid (UA), K+, Zn2+, Mg2+, Ca2+.Use the method for proposition.Adding this
After a little substrates (800 μM), the significant decrease of fluorescence intensity is not observed, and the significant reduction (Fig. 6) of fluorescence intensity after adding Hx.
These results indicate that these interference have little influence on the performance of biosensor, the good of proposed biosensor is demonstrated
Good selectivity and anti-interference ability.
By detecting fish, the content of Hx verifies the practicability (table 1) of biosensor in shrimp and squid.From experimental result
From the point of view of, this biological sensor based on Pt NPs can be used for detecting the freshness before aquatic products go bad, to judge
Whether aquatic products are edible.As summarized in table 2, the rate of recovery is in the range of 103.94-109.00%.In addition, according to country
Standard GB/T 5009.45-1996, when Hx concentration is lower than 529 μM, aquatic products are considered fresh.The threshold value is much higher than this
The LOD of the biosensor proposed in invention.In fact, (respectively 13.70 ± 1.31 μM of Hx concentration in shrimp and fish even living
With 12.97 ± 0.66 μM) it is still higher than the LOD instruction of the biosensor.Therefore, the biosensor proposed is in assessment water
It has a good application prospect in terms of the freshness of product, is caused to be avoided by cumbersome test method by fishes and shrimps corruption
Economic loss.
Table 1: pass through the comparison for the Hx concentration that the sensor-based system and HPLC that are proposed measure in shrimp and the flesh of fish
1h represents aquatic products dead latter hour,
C1 indicates the Hx concentration detected by HPLC, and C2 is indicated by the Hx concentration of the biosensor detection proposed, and water
Product is stored in 4 DEG C after death.
Data are shown as average value ± SD(n=3)
Table 2: pass through the rate of recovery of Hx in the biosensor assay shrimp of proposition
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with repair
Decorations, are all covered by the present invention.
Claims (7)
1. a kind of preparation method for detecting the hypoxanthic biosensor of aquatic products, it is characterised in that: the biosensor
It is using citric acid as stabilizer, as obtained by the preparation of sodium borohydride reduction chloroplatinic acid for Pt NPs.
2. a kind of preparation method for detecting the hypoxanthic biosensor of aquatic products according to claim 1, feature
It is: specific steps are as follows: the chloroplatinic acid aqueous solution that 1mL concentration is 16 mM and the trisodium citrate that 1 mL concentration is 40 mM is molten
Liquid is added in 38 mL water and 30min is stirred at room temperature；Then, the boron that 200 μ L concentration are 50 mM is added into mixture
Sodium hydride solution；After standing 1h, the brown suspension of Pt NPs is obtained, 4 DEG C are held it in front of use, Pt is prepared
NPs。
3. a kind of hypoxanthic biosensor of detection aquatic products that a kind of method as described in claim 1 is prepared.
4. a kind of hypoxanthic biosensor of detection aquatic products that method as described in claim 1 is prepared is in detection water
Freshness on application.
5. application according to claim 4, it is characterised in that: by 30 μ L hypoxanthine Hx and 20 μ L xanthine oxidases
XOD is placed in 37 DEG C of shaking table reaction 30min, and 100 μ L Pt NPs are added and react 30min, it is anti-to add 50 μ L o-phenylenediamine OPD
0 ~ 60min is answered, the last fluorescence intensity of the solution after oxidation.
6. application according to claim 5, it is characterised in that: the time that o-phenylenediamine OPD reaction is added is 30min.
7. application according to claim 5, it is characterised in that: fluorescence intensity judgement of the solution at 580nm after oxidation
The concentration of hypoxanthine Hx.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111175509A (en) * | 2020-02-21 | 2020-05-19 | 福州大学 | ELISA visual detection kit constructed based on Fenton reaction and application of kit in detection of ZEN |
| CN114660053A (en) * | 2022-03-22 | 2022-06-24 | 青岛科技大学 | Preparation and application of microfluid aggregation-based fish flesh freshness detection paper-based chip |
| CN117169205A (en) * | 2023-09-08 | 2023-12-05 | 中国海洋大学 | Detection method of hypoxanthine based on colorimetric biosensor |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111175509A (en) * | 2020-02-21 | 2020-05-19 | 福州大学 | ELISA visual detection kit constructed based on Fenton reaction and application of kit in detection of ZEN |
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| CN117169205A (en) * | 2023-09-08 | 2023-12-05 | 中国海洋大学 | Detection method of hypoxanthine based on colorimetric biosensor |
| CN117169205B (en) * | 2023-09-08 | 2024-04-05 | 中国海洋大学 | Detection method of hypoxanthine based on colorimetric biosensor |
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Application publication date: 20190607 |