CN215931679U

CN215931679U - Colloidal gold test paper for rapidly detecting vitamin B1 in food

Info

Publication number: CN215931679U
Application number: CN202121736157.2U
Authority: CN
Inventors: 汤新; 刘刚
Original assignee: Shenzhen University
Current assignee: Shenzhen University
Priority date: 2021-07-28
Filing date: 2021-07-28
Publication date: 2022-03-01
Anticipated expiration: 2031-07-28

Abstract

The utility model discloses colloidal gold test paper for rapidly detecting vitamin B1 in food, which comprises a supporting pad 1, a nitrocellulose membrane 2, a conjugate pad 3, a water absorption pad 4 and a sample pad 7; the nitrocellulose membrane 2 is located on the supporting pad 1, the water absorption pad 4 is connected above one end of the nitrocellulose membrane 2, the conjugate pad 3 is connected above the other end of the nitrocellulose membrane, and the sample pad is connected above the other end of the conjugate pad, which is characterized in that a detection line 6 coated with a VB1 antibody is arranged in the middle of the nitrocellulose membrane 2, two sides of the detection line 6 are respectively provided with a control line 5 in parallel, and the conjugate pad 3 is coated with a gold-labeled vitamin B1 antibody and a control line conjugate.

Description

Colloidal gold test paper for rapidly detecting vitamin B1 in food

Technical Field

The utility model belongs to the field of food industry analysis, and particularly relates to colloidal gold test paper for detecting vitamin B1.

Background

Vitamin B1(VB1) is also known as thiamine or anti-neuritis. It mainly exists in two forms of thiamine hydrochloride and thiamine nitrate, and the chemical names are respectively 3- [ (4-amino-2-methyl-5-pyrimidyl) methyl]-5- (2-hydroxyethyl) -4-methyl-thiazole hydrochloride and 4-methyl-3- [ (2-methyl-4-amino-5-pyrimidinyl) methyl]-5- (2-hydroxyethyl) thiazole; is divided intoSub-formulae are respectively C₁₂H₁₇ClN₄OS HCl and C₁₂H₁₇N₅O₄S, molecular weight 337.29. VB1 is a water-soluble vitamin, takes part in sugar catabolism process in the form of coenzyme, and has the functions of maintaining normal functions of heart, nerve and digestive system and promoting development. VB1 deficiency can lead to several serious diseases, such as: causing various neuroinflammations, edema, beriberi, etc., and further causing discomfort or death. Outbreaks of VB1 deficiency with inadequate food processing and cooking, resulting in excessive loss of VB1 and severe inadequate intake, have been reported, as in 2004 where a formula was forcibly recalled in israel, which caused 3 infants to die and 10 infants to become sick, as the milk had no vitamin B1 added, resulting in impaired infant brain development. According to the third nutrition survey result in China, compared with the standard established by the Chinese society of nutrition, the VB1 intake amount per capita in China is obviously insufficient. VB1 is widely present in brown rice, yeast, cereals, liver, meat, beans, VB1 related medicines (VB1 tablet, composite VB1 tablet, VB1 oral liquid, etc.) and infant feeding formula milk powder. Therefore, qualitative and quantitative analysis of VB1 is of great significance in science such as clinical science and food science.

The prior art method of detecting VB1 is as follows: the silicotungstic acid gravimetric method has low requirements on instruments, but has troublesome and time-consuming operation, and the ultraviolet spectrophotometry has simple operation, does not need a standard substance, but is greatly influenced by whether the wavelength is accurately measured by the instruments. The non-aqueous titration method and the potentiometric titration method are simple to operate and high in accuracy, but a toxic reagent mercury acetate is used in the non-aqueous titration method to cause pollution; GB 5009.84-2016 (determination of vitamin B1 in food national safety standard food) implemented in 2017 integrates and replaces GB/T5009.84-2003 (determination of thiamine (vitamin B1) in food), GB 541311-2010 (determination of vitamin B1 in food safety national standard infant food and dairy products), GB/T7628-2008 (determination of vitamin B1 in grains), and GB/T9695.27-2008 (determination of vitamin B1 content in meat and meat products), and adds a high-efficiency liquid chromatography method as a first method and takes a fluorescence photometry method as a second method. Wherein, the fluorescence spectrophotometry has high operation and complex operation; the high performance liquid chromatography (improved reversed phase high performance liquid chromatography, high performance liquid chromatography-fluorescence detection method) has the disadvantages of expensive instrument, complex pretreatment, high operation cost and long analysis time, and can not realize the requirements of simple operation and rapid detection.

The Immune colloidal gold technology (Immune colloidal gold technology) is a novel Immune labeling technology which applies colloidal gold as a tracer marker to antigen and antibody. The colloidal gold is prepared from chloroauric acid (HAuCl)₄) Under the action of reducing agent such as white phosphorus, ascorbic acid, sodium citrate, trisodium citrate, tannic acid, etc., the gold particles are polymerized into specific size, and become a stable colloidal state due to electrostatic action, and are called colloidal gold. The colloidal gold has negative charge in weak alkali environment, can form firm combination with the positive charge group of protein molecules, and does not influence the biological characteristics of the protein because the combination is electrostatic combination. Colloidal gold can bind to many other biological macromolecules in addition to proteins. According to some physical properties of colloidal gold, such as high electron density, particle size, shape and color reaction, and immune and biological characteristics of the conjugate, colloidal gold is widely used in the fields of immunology, histology, pathology and cell biology.

The colloidal gold-based visual detection provides a novel technical platform for qualitative and quantitative analysis of VB 1. The colloidal gold particles have a high electron density, and when macromolecules bound to the colloidal gold particles are aggregated in a large amount at the corresponding ligands, red or pink spots visible to the naked eye are formed. The colloidal gold test strip fixes various reaction reagents on the test strip in a strip, a sample solution moves from one end of the test strip to the other end through the capillary action, and an object to be detected in a sample reacts specifically on a chromatography material aiming at a receptor of the object to be detected. The sample to be detected combined with the colloidal gold is intercepted and gathered in a certain area of the chromatographic material, and the color development can be visually obtained through visual inspection. The utility model utilizes the principle of colloidal gold, and develops the colloidal gold for rapidly detecting VB1 by mutually combining colloidal gold particles coupled with BSA or OVA and a monoclonal mouse anti-VB 1 antibody fixed on a Nitrocellulose (NC) membrane.

SUMMERY OF THE UTILITY MODEL

The utility model provides the colloidal gold test paper for rapidly detecting the vitamin B1 in food, which has the advantages of convenience, rapidness, simple operation, accurate result and the like.

The technical scheme of the utility model is as follows:

a colloidal gold test paper for rapidly detecting vitamin B1 in food comprises a supporting pad 1, a nitrocellulose membrane 2, a conjugate pad 3, a water absorption pad 4 and a sample pad 7; the nitrocellulose membrane 2 is located on the supporting pad 1, the water absorption pad 4 is connected above one end of the nitrocellulose membrane 2, the conjugate pad 3 is connected above the other end of the nitrocellulose membrane, and the sample pad is connected above the other end of the conjugate pad, which is characterized in that a detection line 6 coated with a VB1 antibody is arranged in the middle of the nitrocellulose membrane 2, two sides of the detection line 6 are respectively provided with a control line 5 in parallel, and the conjugate pad 3 is coated with a gold-labeled vitamin B1 antibody and a control line conjugate.

Further, a fixing pad 8 is arranged on the sample pad 7. The fixing gasket 8 can be made of single-sided adhesive.

Further, the control line 5 is coated with an anti-DNP antibody, and the control line conjugate is DNP-BSA labeled colloidal gold, wherein BSA refers to bovine serum albumin.

Further, the utility model comprises a shell 9 for wrapping the colloidal gold test paper, and windows for exposing the sample pad 7, the detection line 6 and the two control lines 5 are respectively arranged on the shell. The housing is preferably a plastic housing.

The colloidal gold test paper for rapidly detecting the vitamin B1 in the food is prepared by a double-antibody sandwich method by adopting a two-way lateral flow immunoreaction principle. When VB1 in the sample and the BSA-VB1 colloidal gold conjugate on the coupling pad are chromatographically flowed on the nitrocellulose membrane at the same time during the test, and when VB1 and the BSA-VB1 colloidal gold conjugate in the sample are chromatographed to a detection area (VB1 detection line) on the nitrocellulose membrane, VB1 antibody coated on the nitrocellulose membrane in advance reacts and is fixed on the detection line of the nitrocellulose membrane. The more complex on the detection line, the higher the optical density value on the strip. Meanwhile, in the detection process, the control line conjugate is chromatographed on the nitrocellulose membrane along with the sample, and when the control line is chromatographed to the nitrocellulose membrane, DNP-BSA colloidal gold reacts with the anti-DNP antibody coated on the nitrocellulose membrane in advance so as to be fixed on the control line. Since the gold colloids were red, the DNP-BSA gold colloids immunocomplexes in anti-DNPs immobilized on a nitrocellulose membrane control line would appear red. When VB1 is not present in the sample, only two control lines are shown. When the sample contains VB1, the detection line is clearly displayed. Further, after the reaction is finished, a colloidal gold card reader (Guangdong Dayuanzhou food safety science and technology, Inc.) can be used for concentration detection, and the result is displayed.

Drawings

FIG. 1 is a schematic structural diagram of a colloidal gold test strip for rapidly detecting vitamin B1 in food according to the present invention.

Wherein, 1: supporting pad, 2: nitrocellulose membrane, 3: conjugate pad, 4: absorbent pad, 5: control wire, 6: and a detection line, 7, a sample pad, 8, a fixed gasket.

Fig. 2 is a schematic structural diagram of another embodiment of the colloidal gold test strip for rapidly detecting vitamin B1 in food according to the present invention.

Wherein, 7: sample pad, 6: detection line, 5: control line, 9: a housing.

Detailed Description

The colloidal gold test paper or test paper box for rapidly detecting VB1 in the utility model is described in detail below with reference to the accompanying drawings, and is not to be construed as limiting the utility model. The materials and reagents used in the following examples are all generally commercially available unless otherwise specified. The utility model is applicable to: VB1 qualitative and quantitative detection in cereals, rice, livers, meats, beans, fruits, vegetables, VB1 medicines (VB1 tablets, composite VB1 tablets, VB1 oral liquid and the like) infant formula milk powder and the like.

[ example 1 ] preparation of test sample

Preparation of a detection sample:

1. preparation of solid powder samples (e.g. milk powder or capsule-type powder)

Adding 50ml warm water into 1g of milk powder (or capsule medicine powder) to completely dissolve, diluting with 0.01mol/LPBS (pH7.4) buffer solution according to a volume ratio of 1:1, filtering with 4 layers of filter paper if turbid, placing 4ml in a 5ml centrifuge tube, centrifuging at 5000r/min for 5min, taking 100 μ l of supernatant to detect on prepared test paper, and directly observing C, T line appearance after 10 min.

2. Preparation of fresh milk samples

Diluting with 0.01mol/L PBS (pH7.4) buffer solution at a volume ratio of 1:1, filtering with 4 layers of filter paper if turbid, placing 4ml in a 5ml centrifuge tube, centrifuging at 5000r/min for 5min, detecting 200 μ L of supernatant on prepared test paper, and directly observing C, T line development after 10 min.

3. Fresh fruits, vegetables, legumes: taking about 500g, crushing the sample by a crusher, sieving the crushed sample by a 20-mesh sieve to prepare homogenate or powder with consistent uniformity, namely homogenate or solid test sample, and immediately measuring or storing in a refrigerator in a refrigerating way.

Weighing 1g (accurate to 0.01g) homogenate or solid sample, placing in a 5ml centrifuge tube, adding 4ml 0.01mol/L PBS (pH7.4) solution, vortex for 5min with a vortex instrument, centrifuging for 5min at 5000r/min, taking 100 μ L supernatant, detecting on prepared test paper, and directly observing C, T line appearance after 10 min.

4. Fresh liver, meat: taking 250g of liver or meat, homogenizing the sample by a homogenizer or a pulverizer to prepare homogenate with consistent uniformity, namely a homogenate sample, taking 3g of the homogenate sample, placing the homogenate sample in a 100ml triangular flask, 6ml of ethyl acetate, then oscillating and mixing in a constant temperature oscillator for 30min, centrifuging for 10min at 5000r/min, sucking 6ml of supernatant fluid, placing the supernatant fluid in a rotary evaporator for evaporation at 45 ℃, dissolving the remainder by 1.5ml of isooctane and chloroform (v: v 2/3), adding 1ml of 0.01mol/L PBS (pH7.4) solution for oscillation for 10min, centrifuging for 10min at 5000r/min, sucking 100 mu L of supernatant fluid for detection on prepared test paper, and directly observing the line development condition of C, T after 10 min.

5. Taking about 100g of other solid samples with low water content, such as grains (rice) with water content of about 15%, crushing the samples by a crusher, sieving the samples by a 20-mesh sieve to obtain powder with consistent uniformity, namely a solid sample, and immediately measuring or storing the solid sample in a refrigerator for storage.

Weighing 1g (accurate to 0.01g) of solid sample, placing the solid sample in a 5ml centrifuge tube, adding 4ml of 0.01mol/LPBS (pH7.4) solution, whirling for 5min by a vortex instrument, centrifuging for 5min at 5000r/min, taking 100 mu l of supernatant, detecting on prepared test paper, and directly observing C, T line development conditions after 10 min.

Example 2 preparation of colloidal gold test paper for rapidly detecting VB1

Preparing a colloidal gold test paper structure shown in figure 1, which comprises a supporting pad 1 and a nitrocellulose membrane 2 positioned on the supporting pad, wherein a water absorption pad 4 is connected above one end of the nitrocellulose membrane 2 in a stacking manner, a conjugate gasket 3 is connected above the other end of the nitrocellulose membrane in a stacking manner, a sample pad 7 is connected above one end of the conjugate gasket, which is far away from the nitrocellulose membrane 2, in a stacking manner, and a fixing pad 8 is bonded above the other end of the sample pad; the fixing pad is made of single-sided adhesive, a detection line 6 in the middle of the nitrocellulose membrane 2 is coated with a monoclonal mouse anti-VB 1 antibody, control lines 5 are arranged on two sides of the detection line respectively, a gold-labeled BSA-VB1 and a control line conjugate are coated on the conjugate pad 3, the control line 5 is coated with an anti-DNP antibody, and the control line conjugate is colloidal gold labeled by DNP-BSA. Wherein, the nitrocellulose membrane 2 is used for fixing the coated antibody in the colloidal gold test paper and is also the place where the immunoreaction occurs; test line 6 was performed by using a coating buffer (carbonic acid buffer CBS, pH 9.60.1mol/L) for monoclonal mouse anti-VB 1 antibody and weighing 0.75g of Na₂CO₃，1.466g NaHCO₃The volume is adjusted to 500ml with distilled water.]Diluting to 0.2-0.5mg/ml, preferably 0.2mg/ml, taking 1ul/cm to scribe on the nitrocellulose membrane 2, and drying to obtain; the control line 5 is prepared by diluting the anti-DNP antibody to a concentration of 0.2-0.5mg/ml, preferably 0.2mg/ml, using 3% methanol, 0.1% Tween-20 in 0.01mol/L PBS (pH7.4) buffer, streaking 1ul/cm onto the nitrocellulose membrane 2, and drying.

The material of the conjugate pad 3 is a glass fiber filter membrane, the glass fiber filter membrane used for preparing the conjugate pad is placed into a pre-blocking buffer (0.01 mol/L PBS (pH7.4) buffer containing 0.1% BSA, 5% sucrose, 0.5% PEG20000 and 0.1% Tween-20) to be soaked for 5 minutes and then taken out, after drying, a Biodot coating instrument is used for coating gold-labeled BSA-VB1 and a control line conjugate on the conjugate pad 3, the coating amount is 1ul/mm, and the conjugate pad is dried, so that the conjugate pad is obtained. Preferably, the pre-blocking buffer of the conjugate pad 3 comprises 0.8% casein, 4% bovine serum albumin, 0.007% boric acid, 0.1% PEG.

The sample pad 7 may provide a primary filtration of the liquid sample. Soaking the sample pad in 141 blocking solution (BSA with mass fraction of 0.2% -0.4%, 0.01mol/L PBS (pH7.4) buffer solution) for 5min, and drying at 37 deg.C for 4.5 h. Preferably, the 141 confining liquid comprises: BSA with the mass fraction of 0.4 percent and 0.01mol/L PBS (pH7.4) buffer solution, and the components are stuck on a supporting pad 1 according to the structure shown in figure 1 to obtain the colloidal gold test paper for rapidly detecting VB 1.

Example 3 VB1 rapid detection by using colloidal gold test paper

According to the preferred colloidal gold test paper for rapidly detecting VB1, as shown in figure 1, the colloidal gold test paper comprises a supporting gasket 1, a nitrocellulose membrane 2 positioned on the supporting gasket, a water absorption pad 4 is connected above one end of the nitrocellulose membrane in a stacked manner, a conjugate pad 3 is connected above the other end of the nitrocellulose membrane in a stacked manner, a sample pad 7 is connected above one end of the conjugate pad far away from the nitrocellulose membrane 2 in a stacked manner, a fixed pad 8 is bonded above the other end of the sample pad, the fixed pad is single-sided adhesive, a detection line 6 in the middle of the nitrocellulose membrane 2 is coated with a monoclonal mouse anti-VB 1 antibody, control lines 5 are respectively arranged on two sides of the detection line, and gold-labeled BSA-VB1 and a control line conjugate are coated on the conjugate pad 3. Preferably, the control line 5 is coated with an anti-DNP antibody and the control line conjugate is DNP-BSA labeled colloidal gold. The detection is carried out according to the following steps: 1) the sample to be tested is aspirated, and the sample is returned to room temperature if the sample is stored at low temperature. 2) The sample to be tested was added to the sample pad 7 and reacted for 10 min. 3) And (3) directly observing or placing the colloidal gold test paper in a colloidal gold card reader (Guangdong Dayuanzhou food safety science and technology Co., Ltd.) to judge the result.

Embodiment 4. the colloidal gold test paper box of the utility model is used for rapidly detecting VB1

Taking another colloidal gold test paper box for rapidly detecting VB1, as shown in FIG. 2, the colloidal gold test paper box comprises a supporting gasket 1 and a nitrocellulose membrane 2 positioned on the supporting gasket, wherein one end of the nitrocellulose membrane is connected with a water absorption pad 4, the other end of the nitrocellulose membrane is connected with a conjugate pad 3, the conjugate pad is connected with a sample pad 7, a fixed pad 8 is bonded above the other end of the sample pad, the fixed pad is made of single-sided adhesive, a detection line 6 in the middle of the nitrocellulose membrane 2 is coated with a monoclonal anti-VB 1 antibody, two sides of the detection line are respectively provided with a control line 5, and the conjugate pad 3 is coated with gold-labeled BSA-VB1 and a control line conjugate. Preferably, the control line 5 is coated with an anti-DNP antibody and the control line conjugate is DNP-BSA labeled colloidal gold. The colloidal gold test paper further comprises a shell 9, and the shell wraps the test paper and exposes the sample pad 7, the detection line 6 on the nitrocellulose membrane 2 and the two control lines 5. The detection is carried out according to the following steps: 1) the collected VB1 sample to be tested needs to be restored to the room temperature if the sample is stored at low temperature. 2) The sample to be tested was added to the sample pad 7 and reacted for 10 min. 3) And (3) directly observing or placing the colloidal gold test paper in a colloidal gold card reader (Guangdong Dayuanzhou food safety science and technology Co., Ltd.) to judge the result.

Claims

1. A colloidal gold test paper for rapidly detecting vitamin B1 in food comprises a supporting pad (1), a nitrocellulose membrane (2), a conjugate pad (3), a water absorption pad (4) and a sample pad (7); nitrocellulose membrane (2) is located on supporting pad (1), connect water absorption pad (4) above nitrocellulose membrane (2) one end, couple gasket (3) is connected to the other end top, sample pad (7) is connected to couple gasket other end top, its characterized in that, set up in the middle of nitrocellulose membrane (2) and be wrapped up in detection line (6) that have the VB1 antibody, detection line (6) both sides are equipped with control line (5) respectively, couple gasket (3) coating gold-labeled vitamin B1 antibody and control line couple.

2. The colloidal gold test paper for rapidly detecting vitamin B1 in food according to claim 1, wherein a fixing pad (8) is arranged above the sample pad (7).

3. The colloidal gold test strip for rapid detection of vitamin B1 in foods according to claim 1, wherein the control line (5) is coated with an anti-DNP antibody.

4. The colloidal gold test strip for rapid detection of vitamin B1 in foods of claim 1, wherein the control line conjugate is DNP-BSA labeled colloidal gold.

5. The colloidal gold test paper for rapidly detecting vitamin B1 in foods as claimed in claim 2, wherein the fixing pad (8) is a single-sided adhesive.

6. The colloidal gold test strip for rapidly detecting vitamin B1 in food according to claim 1 or 2, further comprising a housing (9) for enclosing the colloidal gold test strip, wherein windows for exposing the sample pad (7), the detection line (6) and the two control lines (5) are respectively provided.