CN109655514A - A kind of method of quick detection beef freeze thawing degree - Google Patents
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Abstract
The invention discloses a kind of methods of quickly detection beef freeze thawing degree, this method judges beef freeze thawing degree by the protoferriheme loss amount that electrolyte grid-control grapheme transistor (SGGT) sensor detects beef in frozen-thaw process, include the following steps: that (1) makes electrolyte grid-control grapheme transistor (SGGT), and assembles content of hemachrome detection sensor；(2) determination sensor channel current corresponds to the real-time response relationship of content of hemachrome；(3) beef sample freeze thawing treatment to be detected obtains ferroheme fluid sample；(4) the ferroheme fluid sample is detected using the sensor, and corresponds to the hemoglobin loss amount of beef in the real-time response Relation acquisition frozen-thaw process of content of hemachrome according to sensor channel electric current；(5) beef sample freeze thawing degree is judged according to the hemoglobin loss amount.The present invention for the identification of different number of freezing and thawing beef have many advantages, such as response quickly, can real-time detection, sample treatment it is simple.
Description
Technical field
Measurement techniques for quality detection of meat detection field of the present invention, is related to beef freeze thawing degree detecting method more particularly to a kind of utilize is electrolysed
The method that matter grid-control grapheme transistor (SGGT) quickly detects different number of freezing and thawing beef.
Background technique
Currently, beef available on the market is divided into cold fresh meat and chilled meat substantially.It is stored for a long time by the way of freezing
On the one hand beef extends the shelf-life of beef, expand sale radius, is on the other hand also beneficial to electricity market price and maintains price.So
And the ice crystal generated in meat refrigerating process and the salinity of generation increase, it will reduce the quality of meat products, such as cause group
Knit damage, protein denaturation, Oxidation of Fat and Oils, color deterioration etc..It is endless to be particularly due to the cold chain of meat products in the circulation process
It is kind, lead to chilled meat multigelation, it is easier to pollute meat products, or even rotten.And cold fresh meat is due to without freezing,
Therefore it will be better than chilled meat on flavor, nutrition and processing characteristics.But since cold fresh meat and freeze thawing meat are in shape, color and line
Reason etc. is similar, so being difficult to be distinguished.In recent years, there is a large amount of freeze thawing beef as fresh in the market
The fraud that beef is sold.
In view of this, although at present there is it is some based on composition transfer after chilled meat freeze thawing (such as enzymatic activity, moisture,
DNA, institutional framework etc.) carry out taxonomic history method.Chinese patent literature CN107389656A discloses a kind of for characterizing
The method of beef fat quality comparison during multigelation, this method is by carrying out multigelation processing to beef, to defrosting
Beef afterwards carries out fat-extraction, recycles laser Raman spectrometer to carry out Raman spectroscopy scans to the fat of extraction, by dividing
Raman spectrum is analysed, realizes the characterization for beef fat quality comparison during multigelation.However, this method needs complexity
The deficiencies of sample treatment, detection device are expensive, and there are detections to lack real-time, equipment investment is big.Chinese patent literature
CN103760193A discloses a kind of poultry number of freezing and thawing method for quick identification, which is based on meat cell after freezing and defrosting
Film receives destruction, and capacitor effect reduces this principle, by establishing neural network discrimination model, by the resistance that Different Kinds of Meat is numerous and disorderly
The different feature united analysis of robust, subsequent measurement only need to be acquired characteristic frequency point information, and required equipment is simple.But by
Need to establish neural network model in this method, the accuracy of the complexity of model foundation process and the model finally established is led
Causing the use of this method has limitation.
As it can be seen that for chilled meat, the especially detection of the freeze thawing degree of freezing beef, there are detection process in the prior art
The problems such as complicated, required equipment cost is high.
Summary of the invention
In order to solve the above technical problems, the invention proposes a kind of method of quickly detection beef freeze thawing degree, this method
Based on the Electrochemical Detection platform of electrolyte grid-control grapheme transistor, the protoferriheme ingredient in beef diffusate is carried out
Quick real-time detection, and be the freeze thawing that main index carrys out forecast assessment beef with the protoferriheme content in beef diffusate
Degree has the characteristics that test method is simple and quick, equipment investment is few.
To achieve the above object, the technical solution adopted by the present invention are as follows:
A kind of method of quick detection beef freeze thawing degree, the detection method pass through electrolyte grid-control grapheme transistor
(SGGT) the protoferriheme loss amount of beef judges beef freeze thawing degree in sensor detection frozen-thaw process, the detection method packet
Include following steps:
(1) electrolyte grid-control grapheme transistor (SGGT) is made, and assembles content of hemachrome detection sensor；
(2) determination sensor channel current corresponds to the real-time response relationship of content of hemachrome；
(3) beef sample freeze thawing treatment to be detected obtains ferroheme fluid sample
(4) the ferroheme fluid sample is detected using the sensor, and corresponded to according to sensor channel electric current blood red
The hemoglobin loss amount of beef in the real-time response Relation acquisition frozen-thaw process of cellulose content；
(5) beef sample freeze thawing degree is judged according to the hemoglobin loss amount.
According to preferably technical solution, in the step (2), (4), the voltage VG for being applied to transistor gate is greater than
Dirac point voltage.
According to preferably technical solution, in the step (2), measurement channel current corresponds to the real-time sound of content of hemachrome
Curve is answered, and calculates the response of the normallized current under various concentration, to establish the Working calibration curve of sensor.
According to preferably technical solution, under conditions of grid voltage VG=0.3V, the working stamndard of the sensor is bent
Line is y=0.07956x+0.54315；Under conditions of grid voltage VG=0.7V, the Working calibration curve of the sensor is
Y=0.1598x+1.13765.
According to preferably technical solution, in the step (4), hemoglobin loss rate I is utilizedDamageHemoglobin loss amount is characterized,
IDamage=CExudation/CAlways, wherein CExudationIndicate the content of hemachrome in beef freeze thawing diffusate, CAlwaysIndicate protoferriheme total in beef
Content.
It is bent according to protoferriheme loss late and number of freezing and thawing relationship in the step (5) according to preferably technical solution
Line judges beef sample freeze thawing degree.
According to preferably technical solution, in the step (1), in the source electrode and leakage for transferring graphene to patterned substrate
Before between pole, source electrode and drain electrode electrode surface is pre-processed.
According to preferably technical solution, the pretreatment is plasma cleaning.
Compared with prior art, the beef freeze thawing journey disclosed by the invention based on solution grid-control grapheme transistor sensor
The detection method of degree is adsorbed on single-layer graphene surface by protoferriheme, to change graphene doped level, generates electricity
Flow the variation of signal.This method can realize the high sensitivity detection for the protoferriheme in beef diffusate and beef, lead to
The ratio for crossing two parameters obtains the loss late of protoferriheme in beef, and then is inferred to degree of the beef by freeze thawing.This
Invention has response quickly for the identification of different number of freezing and thawing beef, can real-time detection, sample treatment be simple, cost of manufacture
The advantages that low, without complicated electrode modification, operating voltage low (generally less than 1V).
Detailed description of the invention
Fig. 1 is the method flow diagram that existing acidification acetone method detects ferroheme；
Fig. 2 is the SGGT assembly structure diagram that the present invention makes；
Fig. 3 is the method flow diagram of quick detection beef freeze thawing degree proposed by the present invention；
Fig. 4 is the SGGT flow chart that the present invention makes；
Fig. 5 is grid voltage VGChannel current I under conditions of=0.7VDSVersus time curve；
Fig. 6 is grid voltage VGThe Working calibration curve of inventive sensor under conditions of=0.7V；
Fig. 7 is grid voltage VGChannel current I under conditions of=0.3VDSVersus time curve；
Fig. 8 is grid voltage VGThe Working calibration curve of inventive sensor under conditions of=0.3V；
Fig. 9 is protoferriheme loss late and number of freezing and thawing corresponding relationship curve；
Figure 10 is that the Working calibration curve of inventive sensor and traditional spectrophotometry measure curve comparison relational graph.
Specific embodiment
A specific embodiment of the invention is described in detail below in conjunction with attached drawing.
The study found that by the beef of different number of freezing and thawing, since the ice crystal generated in refrigerating process is for beef tissue
And the destruction of cell membrane, there is also difference for the protoferriheme content being lost in diffusate.Based on this, the present invention attempts to pass through
The freeze thawing degree such as the number of freezing and thawing for extracting the content of hemachrome in freeze thawing beef diffusate to judge beef, and then to the product of beef
Matter is assessed.
A variety of prior arts existing for the detection of ferroheme itself.However, conventional hematin test method is often
Need complicated sample treatment, expensive detection device, and professional operator.Such as shown in FIG. 1 pass through is acidified third
The method flow of ketone method detection ferroheme.This method needs first to collect sample to be measured in the market, is crushed homogenate, uses acid
Change the ferroheme in acetone extraction sample, after hatching a period of time at dark, centrifuging and taking supernatant measures it and is divided angle value, most
Content of hemachrome is calculated by light splitting angle value afterwards.Although the ferroheme that this method can relatively accurately measure in sample contains
Amount, but it needs the instrument and equipment of complicated sample treatment and profession, and can not be measured in real time, cannot be quick
Testing result out.
In view of this, the invention proposes a kind of using solution grid-control grapheme transistor as the electrochemistry side of sensor
Method, the real-time detection of Lai Jinhang freeze thawing beef.
Solution grid-control grapheme transistor (SGGT:Solution-Gated Graphene Transistor), refers to electrolysis
Matter grid-control grapheme transistor is a kind of field effect transistor with grapheme transistor, and what is connect with channel is electrolysis
Matter solution, rather than insulating layer itself are equivalent to the combination of sensor and amplifier.Since SGGT has at low cost, production
Simply, the advantages such as operating voltage is low, are widely used in field of biosensors such as DNA, glucose.
The present invention has developed the electrolyte grid-control grapheme transistor that can detecte protoferriheme, is assembled into sensor
Structural schematic diagram it is as shown in Figure 2.Referring to Fig. 2, which includes being formed in substrate of glass 6
Grid 1, source electrode 2, drain electrode 3 and the single-layer graphene channel 4 being covered between source electrode 2 and drain electrode 3, are mounted on dress for transistor
In the container for having phosphate buffer 5 (PBS:Phosphate Buffered Saline), make the phosphate buffer 5
The insulating layer between graphene-channel 4 and grid 1 is served as electrolyte solution, grid voltage passes through graphene and electrolyte interface
Electric double layer be applied to graphene-channel.
When protoferriheme is added in the phosphate buffer 5, protoferriheme is adsorbed on by π-π interaction
Graphene-channel surface causes graphene doped level to change, and channel current can also change therewith, and then pass through ditch
Road curent change determines protoferriheme concentration.The grapheme transistor that the present invention makes shows dipole characteristic: working
When grid voltage is higher than Dirac point voltage, channel current rises with protoferriheme concentration and is risen；It is low in active gate voltage
When Dirac point voltage, channel current rises and upper decline with protoferriheme concentration.
As shown in figure 3, detection method proposed by the present invention mainly comprises the steps that sample treatment, sensor production, height
Iron hematin test, assessment Quality Beef.Wherein, it should be readily apparent to one skilled in the art that sample treatment and sensor production step
Suddenly without stringent sequencing.Detection method proposed by the present invention will be specifically described below.
Firstly, carrying out the production of solution grid-control grapheme transistor and the processing of beef sample.
The present invention makes the production process of solution grid-control grapheme transistor as shown in figure 4, mainly including the following steps:
(1) substrate is made.Use glass cutter that glass slide is cut into the small sheet glass of 1cm × 1cm as substrate, by glass base
Acetone, ethyl alcohol, deionized water is successively used to be cleaned by ultrasonic 20-30 minutes in bottom.
(2) pattern-making polarizing electrode.After being dried up substrate of glass with high pure nitrogen, substrate of glass is consolidated by high temperature gummed tape
It is scheduled on mask plate, deposits upper patterned chrome gold electrode in substrate of glass using magnetron sputtering.Chromium is first deposited when depositing electrode
Layer, thickness about 8-10nm, preferably 10nm；Redeposited layer gold, with a thickness of 90-120nm, preferably 100nm.The effect master of de-posited chromium layer
If avoiding gold electrode from the breakage that falls off occur to increase the adhesiveness of gold with substrate of glass.
(3) single-layer graphene is prepared.Using CH4(40SCCM) and H2The mixed gas of (20SCCM) passes through chemical vapor deposition
Product (CVD) synthesizes single-layer graphene at 1000 DEG C, and makees catalysis substrate with the Cu foil of 25 μ m-thicks.Again by poly-methyl methacrylate
Ester (PMMA) film is spin-coated on graphene.In order to make PMMA on graphene spin coating it is more uniform, using two-step method spin coating:
800rpm at a slow speed before this, spin coating 10-12 seconds, then be quick 2000rpm, spin coating 20-25 seconds.It is then immersed in etching solution
(CuSO4: HCl:H2O=10g:50mL:50mL it in), to etch Cu substrate, and is washed with distilled water 3-5 times.
(4) graphene shifts.Source electrode and drain electrode electrode surface is located in advance using chemically or physically method before transfer
Reason, to improve the detection sensitivity of electrode.The method of Selection utilization plasma cleaning of the present invention pre-processes the electrode.
Then, the method shifted using wet process, is transferred graphene between the source electrode of patterned substrate and drain electrode, and at 120 DEG C
Annealing 15-20 minutes.It impregnates 1-1.5 hours finally, the substrate after annealing is put into 50-60 DEG C of acetone, more renews
Acetone after impregnate again, repeat 3-4 times.
(5) device encapsulates.The siloxane layer for connecting the metal wire waterproof of source electrode, drain and gate is subjected to waterproof sealing
Protection, causes short circuit to prevent from contacting in sensor test process with electrolyte.
After electrolyte grid-control grapheme transistor completes, it is thus necessary to determine that sensor is bent for the response of ferroheme
Line.Specific step is as follows:
(1) sensor is assembled.Sensor is assembled according to structure shown in Fig. 2, will have grid 1, source electrode 2, drain electrode 3 three
The transistor of electrode and single-layer graphene channel 4 is mounted on the small of the phosphate buffer (0.1M, pH value=7.4) equipped with 10mL
In beaker.
(2) channel current real-time response curve (IDS-t) is measured.Pass through two of Labview software control in the present invention
2400 multifunctional power ammeter of Keithley carries out real-time current monitoring, in fixed source voltage VDS=0.05V and grid voltage
VGUnder conditions of=0.7V, channel current I is measuredDSIt changes with time.Until channel current IDSWhen not changing substantially, with
This current value is initial current I0, then start the protoferriheme solution that various concentration is added into phosphate buffer, often
Minor tick 10min records the raised current value I of sensor under various concentrationn, measurement result is as shown in Figure 5.
(3) the normallized current response under various concentration: NCR=(I is calculatedn-I0)/I0.With pair of protoferriheme concentration
For numerical value as abscissa, the Working calibration curve for establishing inventive sensor is y=0.1598x+1.13765.Research and development discovery, this
The detection limit of invention sensor can achieve 10nM, and the range of linearity is 100nM-10 μM, and concrete outcome is as shown in Figure 6.
According to another embodiment of the present invention, the said determination channel current real-time response curve the step of in (2), at it
In the case that his condition is constant, in grid voltage VGChannel current I is measured under conditions of=0.3VDSIt changes with time, finally
Measurement result is as shown in Figure 7.Accordingly, this hair is established using the logarithm of protoferriheme concentration as abscissa in step (3)
The Working calibration curve of bright sensor is y=0.07956x+0.54315.The detection of sensor is limited to 100nM, and the range of linearity is
300nM-10 μM, concrete outcome is as shown in Figure 8.
It can be seen that although the grapheme transistor that the present invention makes shows dipole characteristic, in active gate voltage
(VG=0.7V) be higher than Dirac point voltage when, sensor can obtain higher detection accuracy, and the range of linearity of sensor
It is wider.
Since protoferriheme content difference initial in different beef samples will cause the difference of initial diffusate, and seep
Other compositions also can generate interference to protoferriheme content detection result in liquid out, therefore only with the judgement of protoferriheme content
Number of freezing and thawing there may be large error.In order to reduce above-mentioned disturbing factor, accuracy in detection is improved, the present invention passes through calculating
The ratio I of protoferriheme content and protoferriheme content total in beef sample in diffusateDamage=CExudation/CAlways, and with the value
IDamageTo represent the protoferriheme loss late of beef.
According to preferred embodiment, the present invention is prepared by the following IDamage:
(1) actual sample is handled.
Actual sample pretreatment: removing fascia for rump steak, be cut into i fritter, 3≤i≤5, and volume about 5cm × 5cm ×
4cm.By above-mentioned sample Vacuum Package in small packaging bag, and K1, K2, Ki ... are marked in order.Above-mentioned sample is distinguished
Carry out 1-i freeze thawing treatment, Freezing-Melting Condition are as follows: -20 DEG C of freezings are for 24 hours；4 DEG C thaw for 24 hours.
(2) ferroheme extracts
For every a sample of label K1, K2, Ki ..., proceed as follows respectively:
The acquisition of leachate: 20g beef sample is taken to be added in the phosphate buffer of 20mL, 0.1M, after mixing
It is extracted 30-40 minutes at dark, is centrifuged 10-15min under the conditions of 4 DEG C of temperature, revolving speed 10000g, takes supernatant through glass fibers
After tieing up Suction filtration device filtering, diffusate sample is obtained.
The extraction of total protoferriheme in beef: 5g beef sample is taken to be added to the phosphate buffer of 20mL, 0.1M
In, using high speed disperser 10000rpm homogeneous 30-40 seconds, it is placed in the dark place of being protected from light and is incubated for 1-1.5 hours, in temperature 4 DEG C, turn
It is centrifuged 10-15min under the conditions of fast 10000g, takes supernatant after the filtering of glass fibre Suction filtration device, obtains total protoferriheme
Fluid sample.
(3) measurement of protoferriheme loss.In fixed source voltage VDS=0.05V and grid voltage VGThe item of=0.7V
Under part, channel current I is measuredDSIt changes with time.Until channel current IDSWhen not changing substantially, it is with this current value
Initial current I0, be then added into phosphate buffer every a sample K1, K2, Ki ... diffusate sample or total high-speed rail
Ferroheme fluid sample records raised channel current In, and calculate normallized current response, then sensing according to the present invention
Device is in VDS=0.05V, VGThe working curve (referring to Fig. 6) of=0.7V obtains the diffusate sample of every a sample K1, K2, Ki ...
Protoferriheme content C in productExudationAnd total protoferriheme content C in beefAlways, and calculate every a sample K1, K2,
The protoferriheme loss late I of Ki ...Damage=CExudation/CAlways。
Since the number of freezing and thawing of above-mentioned every a sample K1, K2, Ki ... are different, and then protoferriheme damage can be obtained
Mistake rate IDamageWith the relation curve of number of freezing and thawing.As shown in figure 9, the present invention determines the relation curve of i=3.As can be seen that with
The increase of number of freezing and thawing, protoferriheme loss late are also increasing.Accordingly, we can be according to the damage of protoferriheme in beef
Vector judges the freeze thawing degree of beef.
Figure 10 is shown to be lost according to the number of freezing and thawing and protoferriheme of traditional acidification acetone spectrophotometry measurement
The number of freezing and thawing and protoferriheme loss late that relation curve (being represented by dotted lines) and the use present invention between rate measure are therewith
Between relation curve (being indicated with solid line).It can be seen that two curves is very close, therefore also demonstrate of the invention feasible
Property.
The present invention carries out the reality of freeze thawing beef using the electrochemical method of electrolyte grid-control grapheme transistor sensor
When detect.Electrolyte grid-control grapheme transistor is by three gold electrodes (source electrode, drain electrode, grid) and a graphene-channel group
At being mainly used in the Electrochemical Detection of compound in solution system.The present invention is different from traditional field effect transistor electricity consumption
Electrolyte solution replaces insulating layer and graphene-channel and gate contact, and the voltage of grid is equivalent to is applied to grid/electrolyte
With two interfaces of electrolyte/channel, there is an electric double layer in each interface, is equivalent to a capacitor, can pass through grid
Pole tension regulates and controls the variation of channel current, and each grapheme transistor is equivalent to the knot of a sensor and an amplifier
It closes.Electrolyte grid-control grapheme transistor due to operating voltage low (generally less than 1V), quickly in real time, high sensitivity, at
The advantages that this is low, easy to manufacture, can be applied the detection field in Safety of Food Quality.
Embodiment of above is only to illustrate the technical solution of the embodiment of the present invention rather than limits, although referring to above preferable
The embodiment of the present invention is described in detail in embodiment, those skilled in the art should understand that, it can be to this hair
The technical solution of bright embodiment is modified or equivalent replacement should not all be detached from the embodiment of the present invention technical solution spirit and
Range.
Claims (8)
1. a kind of method of quickly detection beef freeze thawing degree, the detection method pass through electrolyte grid-control grapheme transistor
(SGGT) the protoferriheme loss amount of beef judges beef freeze thawing degree in sensor detection frozen-thaw process, the detection method packet
Include following steps:
(1) electrolyte grid-control grapheme transistor (SGGT) is made, and assembles content of hemachrome detection sensor；
(2) determination sensor channel current corresponds to the real-time response relationship of content of hemachrome；
(3) beef sample freeze thawing treatment to be detected obtains ferroheme fluid sample；
(4) the ferroheme fluid sample is detected using the sensor, and corresponds to ferroheme according to sensor channel electric current and contains
The hemoglobin loss amount of beef in the real-time response Relation acquisition frozen-thaw process of amount；
(5) beef sample freeze thawing degree is judged according to the hemoglobin loss amount.
2. the method for quick detection beef freeze thawing degree according to claim 1, it is characterised in that: the step (2),
(4) in, the voltage VG for being applied to transistor gate is greater than Dirac point voltage.
3. the method for quick detection beef freeze thawing degree according to claim 1, it is characterised in that: in the step (2)
In, measurement channel current corresponds to the real-time response curve of content of hemachrome, and calculates the response of the normallized current under various concentration,
To establish the Working calibration curve of sensor.
4. the method for quick detection beef freeze thawing degree according to claim 3, it is characterised in that: in grid voltage VG=
Under conditions of 0.3V, the Working calibration curve of the sensor is y=0.07956x+0.54315；In grid voltage VG=0.7V
Under conditions of, the Working calibration curve of the sensor is y=0.1598x+1.13765.
5. the method for quick detection beef freeze thawing degree according to claim 1, it is characterised in that: in the step (4),
Utilize hemoglobin loss rate IDamageCharacterize hemoglobin loss amount, IDamage=CExudation/CAlways, wherein CExudationIndicate the blood in beef freeze thawing diffusate
Lycopene content, CAlwaysIndicate protoferriheme content total in beef.
6. the method for quick detection beef freeze thawing degree according to claim 5, it is characterised in that: in the step (5),
Beef sample freeze thawing degree is judged according to protoferriheme loss late and number of freezing and thawing relation curve.
7. the method for quick detection beef freeze thawing degree according to claim 1, it is characterised in that: in the step (1),
Before transferring graphene between the source electrode of patterned substrate and drain electrode, source electrode and drain electrode electrode surface is located in advance
Reason.
8. the method for quick detection beef freeze thawing degree according to claim 7, it is characterised in that: it is described pretreatment for etc.
Ion Cleaning.
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| CN112924512A (en) * | 2021-01-15 | 2021-06-08 | 合肥工业大学 | Alcohol hydrogel sensing device based on layered carbon nanosheets and method thereof |
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