A general preparation method of hydrogen-bonded organic framework composite film test paper with visual detection of biological small molecule performance

Abstract

The application discloses a preparation method of a general hydrogen-bonded organic framework composite film test paper with visual detection of biological small molecules, which adopts green and environment-friendly agar as a film test paper base and encapsulates enzyme / enzyme-like hydrogen-bonded organic framework materials as a framework carrier in the composite film test paper. The application is based on a cascade reaction of biological catalysis, and for the first time, the non-toxic edible agar prepared film base is combined with the encapsulated enzyme / enzyme-like hydrogen-bonded organic framework materials, so as to prepare the hydrogen-bonded organic framework composite film test paper for visual detection of biological small molecules. The test paper not only has excellent characteristics of visual detection of biological small molecules, but also has strong stability and flexibility. The composite film test paper in the application has simple synthesis process, low cost, strong operability, and can be stored and carried conveniently, and the detection of biological small molecules can be realized at any time and anywhere without instruments, so that the application has wide application prospect.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biochemical sensing, in particular to a preparation method of a universal hydrogen-bond organic framework composite film test paper with visual detection of biological small molecule performance. BACKGROUND

[0002] Many biological small molecules are indispensable in human metabolism and play an important function, and detection of the biological small molecules can help patients monitor a certain index of a disease and prevent various diseases and complications. In recent years, hydrogen-bond-organic framework materials as a new type of material have advantages of diversity of construction groups, adjustable size, easy recycling, and enrichment of substrates, and therefore, the hydrogen-bond-organic framework materials have caused in-depth research.

[0003] Traditional detection of biological small molecules in the human body generally needs to be analyzed and detected in a hospital by means of instruments, is complicated and cumbersome, and has poor operability. The application develops a universal green and environment-friendly composite film test paper, for the first time mixes nontoxic edible agar and porous hydrogen-bond organic framework materials encapsulating proteins, and applies the hydrogen-bond organic framework materials to detection of biological small molecules. In addition to high sensitivity and high stability, the hydrogen-bond organic framework materials can realize visual detection of biological small molecules at any time and anywhere without any instrument equipment, are convenient and fast, and are easy to operate, and solve the dependence of traditional detection on instruments and the limitation of a complicated process. SUMMARY

[0004] The application provides a preparation method of a universal hydrogen-bond organic framework composite film test paper with visual detection of biological small molecule performance, and the preparation method is easy, low in cost, high in stability, and recyclable.

[0005] In order to solve the above technical problems, the technical scheme adopted by the application is as follows:

[0006] The application provides a preparation method of a universal hydrogen-bond organic framework composite film test paper with visual detection of biological small molecule performance, and the preparation method is easy, low in cost, high in stability, and recyclable.

[0007] 1) Preparation of a hydrogen-bond organic framework material encapsulating enzymes / enzymes dispersion solution:

[0008] In a dispersion solution environment, the hydrogen-bond organic framework material encapsulating enzymes / enzymes and a TMB solution are uniformly mixed, the mixed solution is stirred, and a layered homogenized dispersion solution is obtained.

[0009] 2) Preparation of agar film substrate:

[0010] Agar powder was added to the dispersion solution, heated and stirred until a clear and transparent solution was formed. The mixture was then poured into a petri dish, and after the solution solidified, it was freeze-dried to obtain a dry and loose film substrate. (The bottom surface of the film substrate in the petri dish is attached to the bottom of the inner cavity of the petri dish, and the side of the film substrate facing upwards in the petri dish is defined as the loose A side).

[0011] 3) Preparation of hydrogen-bonded organic framework composite thin film test paper:

[0012] The hydrogen-bonded organic framework material dispersion of the encapsulated enzyme / enzyme-like material prepared in step 1) is coated onto the loose surface A of the agar film substrate prepared in step 2), and after freeze-drying, hydrogen-bonded organic framework composite film test paper for detecting small biological molecules is obtained.

[0013] Furthermore, the dispersion solution mentioned in step 1) is any one or more of water, PBS buffer, Tris-HCl solution, and HAc-NaAc buffer.

[0014] Furthermore, the enzyme / enzyme encapsulated in step 1) is any combination of GOx and Au-BSA, GOx and Hemin-BSA, UOx and Au-BSA, UOx and Hemin-BSA, XOD and Au-BSA, XOD and Hemin-BSA, β-Gal, GOx and Au-BSA, β-Gal, and GOx and Hemin-BSA.

[0015] Furthermore, the hydrogen-bonded organic framework material mentioned in step 1) is HOF-FAFU-1, whose building blocks are any one or more combinations of 3,3'5,5'-tetra(4-carboxyphenyl)-4,4'-dihydroxybiphenyl, 3,3'5,5'-tetra(4-carboxyphenyl)-4,4'-diaminobiphenyl, and 3,3'5,5'-tetra(4-carboxyphenyl)-4,4'-methoxybiphenyl.

[0016] Further, in step 1), the concentration of the hydrogen-bonded organic framework material for encapsulating the enzyme / enzyme-like substance is greater than 0.01 mM; the TMB solution concentration is greater than 0.1 mM; the stirring speed is greater than 100 rpm / min; and the stirring time is greater than 1 min. Preferably, the concentration of the hydrogen-bonded organic framework material for encapsulating the enzyme / enzyme-like substance is 0.1-10 mM; the TMB solution concentration is 0.1-10 mM; the stirring speed is 100-1000 rpm / min; and the stirring time is 1-30 min.

[0017] Furthermore, in step 2), the concentration of agar powder is greater than 1 mg / mL; the heating temperature is greater than 70℃, the heating and stirring time is greater than 0.1 h; and the freeze-drying time after the mixed solution solidifies is greater than 2 h. Preferably, the concentration of agar powder is 10-100 mg / mL; the heating temperature is 80-120℃, the heating and stirring time is 0.1-2 h; and the freeze-drying time after the mixed solution solidifies is 4-12 h.

[0018] Further, in step 3), the diameter of the agar film substrate is greater than 1 mm, the coating amount of the hydrogen-bonded organic framework material dispersion for encapsulating the enzyme / enzyme-like material is greater than 0.5 mL, and the freeze-drying time is greater than 2 hours. Preferably, the diameter of the agar film substrate is 10-50 mm, the coating amount of the hydrogen-bonded organic framework material dispersion for encapsulating the enzyme / enzyme-like material is 0.5-10 mL, and the freeze-drying time is 4-12 h.

[0019] Furthermore, the coating method described in step 3) can be any one of drip coating, spin coating, spray coating, or printing.

[0020] Preferably, when applying the coating using a spin coating method in step 3), the specific steps are as follows:

[0021] Place the agar film substrate on the spin coater with the loose A side of the film facing upwards, and align the center of the film substrate with the axis of rotation of the spin coater.

[0022] Next, the film substrate is rotated at high speed using a spin coater, which is divided into two stages. In each stage, the homogenization solution of the composite material prepared in step 1) is titrated on the A side of the rotating film substrate.

[0023] Finally, freeze-drying is performed to obtain the composite film test paper for detecting small biological molecules.

[0024] Furthermore, the first stage of high-speed rotation has a rotation speed of 500-1000 rpm / min and a rotation time of 36-54 s, while the second stage has a rotation speed of 1000-6000 rpm / min and a rotation time of 60-120 s.

[0025] This invention provides a composite film test strip that is convenient, environmentally friendly, allows for immediate detection, and is easy to operate, thereby enabling qualitative and quantitative detection of small biological molecules, including glucose, uric acid, lactose, and xanthine.

[0026] The beneficial effects of this invention are as follows:

[0027] 1) This invention is the first to utilize agar, which is inexpensive, widely available, non-toxic, pollution-free, edible, green and environmentally friendly, as the base of the thin film test paper. It is combined with a porous hydrogen-bonded organic framework material for encapsulating enzymes / enzymes to produce a hydrogen-bonded organic framework material composite thin film test paper for the visual detection of small biological molecules.

[0028] 2) The thin film test paper preparation method provided by the present invention is simple, stable and repeatable, effectively solving the dependence on instruments and complicated process of traditional detection, and has broad application prospects.

[0029] 3) The composite film test strip for detecting small biological molecules prepared by this invention has the characteristics of simple operation, excellent visualization detection of small biological molecules, good mechanical properties and flexibility, low cost, and green environmental protection. It can be used as a general-purpose test strip sensor for detecting small biological molecules.

[0030] 4) The composite film test strip prepared by this invention can detect small biological molecules such as glucose, uric acid, lactose, and xanthine in real time, thereby realizing qualitative and quantitative analysis of small biological molecules, which has great potential for practical application in the fields of biology and medicine. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 This is a SEM image of the nano-GOx-Au-BSA@HOF-FAFU-1-OH prepared in Example 1 of this invention;

[0033] Figure 2 This is a SEM image of the agar film substrate surface in the comparative example;

[0034] Figure 3 This is a SEM image of the surface of the glucose detection test strip prepared in Example 1 of the present invention;

[0035] Figure 4 This is a schematic diagram of the contact angle of the agar film substrate surface in the comparative example;

[0036] Figure 5 This is a schematic diagram of the contact angle of the glucose detection test strip prepared in Example 1 of the present invention;

[0037] Figure 6 This is a schematic diagram of the mechanical properties of the glucose detection test strip prepared in Example 1 of the present invention;

[0038] Figure 7 This is a schematic diagram of the water absorption properties of the glucose detection test paper prepared in Example 1 of the present invention;

[0039] Figure 8 The graphs shown in Example 14 of this invention are: absorbance of the sensor solution for glucose solutions of different concentrations (left side) and the relationship between glucose concentration and absorbance (right side).

[0040] Figure 9 The images show the color development effect of the composite film test paper on glucose solutions of different concentrations in Example 15 of this invention (left side) and the relationship between glucose concentration and brightness value (G+B) / 2R (right side). (In the color development images, the glucose concentration from left to right is 0.5mM→100mM.) Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] Example 1:

[0043] The preparation method of the composite film test strip for glucose detection encapsulated with HOF-FAFU-1-OH and GOx and Au-BSA provided in this embodiment is as follows:

[0044] (1) Preparation of HOF-FAFU-1-OH:

[0045] In 10 mL of PBS buffer solution, 20 mg of HOF-FAFU-1-OH material encapsulating GOx and Au-BSA and 0.3 mM TMB solution were mixed evenly. The mixture was placed on a stirrer and stirred at 500 rpm / min for 10 min to obtain a layered and homogenized dispersion.

[0046] (2) Preparation of agar film substrate:

[0047] Add 1g of agar powder to 50mL of ultrapure water, heat and stir at 100℃ for 1h to form a clear and transparent solution. Pour the mixture into a petri dish and freeze-dry for 8h after the solution has solidified to obtain a dry and loose agar film substrate.

[0048] (3) Preparation of HOF-FAFU-1-OH encapsulated thin-film test paper for glucose detection using GOx and Au-BSA:

[0049] A 25 mm diameter agar film substrate was placed on a spin coater with a 22 mm diameter suction cup, with the loose A-side of the film facing upwards. The center of the film substrate was aligned concentrically with the axis of the spin coater. The spin coater then rotated the film substrate at high speed in two stages: the first stage was at 500 rpm / min for 36 seconds, and the second stage was at 3000 rpm / min for 80 seconds. During each stage, a homogenizing solution of the composite material was titrated onto the A-side of the rotating film substrate. Afterwards, the substrate was freeze-dried for 8 hours to obtain a glucose detection film strip coated with HOF-FAFU-1-OH encapsulated GOx and Au-BSA.

[0050] See Figure 1 As can be seen from the SEM image, the size of the HOF-FAFU-1-OH prepared in this case has reached the nanoscale.

[0051] See Figure 3 As can be seen from the SEM images, the HOF-FAFU-1-OH encapsulating GOx and Au-BSA is embedded in the film surface and exhibits extremely strong water absorption (see [link to SEM image]). Figure 5 and 7 ).

[0052] See Figure 6 Mechanical property tests on the glucose detection film paper showed that it could maintain its structural integrity after repeated stretching, squeezing, folding, and rubbing, indicating that it has good flexibility.

[0053] Example 2:

[0054] The preparation method of the composite film test strip for glucose detection encapsulated with HOF-FAFU-1-OH and Hemin-BSA provided in this embodiment is as follows:

[0055] (1) Preparation of HOF-FAFU-1-OH:

[0056] In 10 mL of PBS buffer solution, 20 mg of HOF-FAFU-1-OH material encapsulating GOx and Hemin-BSA and 0.3 mM TMB solution were mixed evenly. The mixture was placed on a stirrer and stirred at 500 rpm / min for 10 min to obtain a layered and homogenized dispersion.

[0057] (2) Preparation of agar film substrate:

[0058] Add 1g of agar powder to 50mL of ultrapure water, heat and stir at 100℃ for 1h to form a clear and transparent solution. Pour the mixture into a petri dish and freeze-dry for 8h after the solution has solidified to obtain a dry and loose agar film substrate.

[0059] (3) Preparation of HOF-FAFU-1-OH encapsulated thin-film test paper for glucose detection using GOx and Hemin-BSA:

[0060] A 25 mm diameter agar film substrate was placed on a spin coater with a 22 mm diameter suction cup, with the loose A-side of the film facing upwards. The center of the film substrate was aligned concentrically with the axis of the spin coater. The spin coater then rotated the film substrate at high speed in two stages: the first stage was at 500 rpm / min for 36 seconds, and the second stage was at 3000 rpm / min for 80 seconds. During each stage, a homogenization solution of the composite material was titrated onto the A-side of the rotating film substrate. Afterwards, the substrate was freeze-dried for 8 hours to obtain a glucose detection film strip coated with HOF-FAFU-1-OH encapsulated GOx and Hemin-BSA.

[0061] Example 3:

[0062] The preparation method of the composite film test strip for glucose detection encapsulated with HOF-FAFU-1-NH2 and Au-BSA provided in this embodiment is as follows:

[0063] (1) Configuration of HOF-FAFU-1-NH2:

[0064] In 10 mL of PBS buffer solution, 20 mg of HOF-FAFU-1-NH2 material encapsulating GOx and Au-BSA and 0.3 mM TMB solution were mixed evenly. The mixture was placed on a stirrer and stirred at 500 rpm / min for 10 min to obtain a layered and homogenized dispersion.

[0065] (2) Preparation of agar film substrate:

[0066] Add 1g of agar powder to 50mL of ultrapure water, heat and stir at 100℃ for 1h to form a clear and transparent solution. Pour the mixture into a petri dish and freeze-dry for 8h after the solution has solidified to obtain a dry and loose agar film substrate.

[0067] (3) Preparation of HOF-FAFU-1-NH2-encapsulated thin-film test paper for glucose detection using GOx and Au-BSA:

[0068] A 25 mm diameter agar film substrate was placed on a spin coater with a 22 mm diameter suction cup, with the loose A-side of the film facing upwards. The center of the film substrate was aligned concentrically with the axis of the spin coater. The spin coater then rotated the film substrate at high speed in two stages: the first stage was at 500 rpm / min for 36 seconds, and the second stage was at 3000 rpm / min for 80 seconds. During each stage, a homogenizing solution of the composite material was titrated onto the A-side of the rotating film substrate. Afterwards, the substrate was freeze-dried for 8 hours to obtain a glucose detection film strip coated with HOF-FAFU-1-NH2 encapsulated GOx and Au-BSA.

[0069] Example 4:

[0070] The preparation method of the composite film test strip for glucose detection encapsulated with HOF-FAFU-1-NH2 and Hemin-BSA provided in this embodiment is as follows:

[0071] (1) Configuration of HOF-FAFU-1-NH2:

[0072] In 10 mL of PBS buffer solution, 20 mg of HOF-FAFU-1-NH2 material encapsulating GOx and Hemin-BSA and 0.3 mM TMB solution were mixed evenly. The mixture was placed on a stirrer and stirred at 500 rpm / min for 10 min to obtain a layered and homogenized dispersion.

[0073] (2) Preparation of agar film substrate:

[0074] Add 1g of agar powder to 50mL of ultrapure water, heat and stir at 100℃ for 1h to form a clear and transparent solution. Pour the mixture into a petri dish and freeze-dry for 8h after the solution has solidified to obtain a dry and loose agar film substrate.

[0075] (3) Preparation of HOF-FAFU-1-NH2-encapsulated thin-film test paper for glucose detection using GOx and Hemin-BSA:

[0076] A 25 mm diameter agar film substrate was placed on a spin coater with a 22 mm diameter suction cup, with the loose A-side of the film facing upwards. The center of the film substrate was aligned concentrically with the axis of the spin coater. The spin coater then rotated the film substrate at high speed in two stages: the first stage was at 500 rpm / min for 36 seconds, and the second stage was at 3000 rpm / min for 80 seconds. During each stage, a homogenizing solution of the composite material was titrated onto the A-side of the rotating film substrate. Afterwards, the substrate was freeze-dried for 8 hours to obtain a glucose detection film strip coated with HOF-FAFU-1-NH2 encapsulated GOx and Hemin-BSA.

[0077] Example 5:

[0078] The preparation method of the composite film test strip for uric acid detection encapsulated with HOF-FAFU-1-OH and UOx and Au-BSA provided in this embodiment is as follows:

[0079] (1) Preparation of HOF-FAFU-1-OH:

[0080] In 10 mL of PBS buffer solution, 20 mg of HOF-FAFU-1-OH material encapsulating UOx and Au-BSA and 0.3 mM TMB solution were mixed evenly. The mixture was placed on a stirrer and stirred at 500 rpm / min for 10 min to obtain a layered and homogenized dispersion.

[0081] (2) Preparation of agar film substrate:

[0082] Add 1g of agar powder to 50mL of ultrapure water, heat and stir at 100℃ for 1h to form a clear and transparent solution. Pour the mixture into a petri dish and freeze-dry for 8h after the solution has solidified to obtain a dry and loose agar film substrate.

[0083] (3) Preparation of HOF-FAFU-1-OH encapsulated UOx and Au-BSA thin-film test strips for uric acid detection:

[0084] A 25 mm diameter agar film substrate was placed on a spin coater with a 22 mm diameter suction cup, with the loose A-side of the film facing upwards. The center of the film substrate was aligned concentrically with the axis of the spin coater. The spin coater then rotated the film substrate at high speed in two stages: the first stage was at 500 rpm / min for 36 seconds, and the second stage was at 3000 rpm / min for 80 seconds. During each stage, a homogenization solution of the composite material was titrated onto the A-side of the rotating film substrate. Afterwards, the substrate was freeze-dried for 8 hours to obtain a uric acid detection film strip coated with HOF-FAFU-1-OH encapsulated with UOx and Au-BSA.

[0085] Example 6:

[0086] The preparation method of the composite film test strip for uric acid detection encapsulated with HOF-FAFU-1-OH and Hemin-BSA provided in this embodiment is as follows:

[0087] (1) Preparation of HOF-FAFU-1-OH:

[0088] In 10 mL of PBS buffer solution, 20 mg of HOF-FAFU-1-OH material encapsulating UOx and Hemin-BSA and 0.3 mM TMB solution were mixed evenly. The mixture was placed on a stirrer and stirred at 500 rpm / min for 10 min to obtain a layered and homogenized dispersion.

[0089] (2) Preparation of agar film substrate:

[0090] Add 1g of agar powder to 50mL of ultrapure water, heat and stir at 100℃ for 1h to form a clear and transparent solution. Pour the mixture into a petri dish and freeze-dry for 8h after the solution has solidified to obtain a dry and loose agar film substrate.

[0091] (3) Preparation of HOF-FAFU-1-OH encapsulated UOx and Hemin-BSA thin-film test strips for uric acid detection:

[0092] A 25 mm diameter agar film substrate was placed on a spin coater with a 22 mm diameter suction cup, with the loose A-side of the film facing upwards. The center of the film substrate was aligned concentrically with the axis of the spin coater. The spin coater then rotated the film substrate at high speed in two stages: the first stage at 500 rpm / min for 36 seconds, and the second stage at 3000 rpm / min for 80 seconds. During each stage, a homogenizing solution of the composite material was titrated onto the A-side of the rotating film substrate. Afterwards, the substrate was freeze-dried for 8 hours to obtain a uric acid detection film strip coated with HOF-FAFU-1-OH encapsulated with UOx and Hemin-BSA.

[0093] Example 7:

[0094] The preparation method of the composite film test strip for uric acid detection using HOF-FAFU-1-NH2 encapsulated UOx and Au-BSA provided in this embodiment is as follows:

[0095] (1) Configuration of HOF-FAFU-1-NH2:

[0096] In 10 mL of PBS buffer solution, 20 mg of HOF-FAFU-1-NH2 material encapsulating UOx and Au-BSA and 0.3 mM TMB solution were mixed evenly. The mixture was placed on a stirrer and stirred at 500 rpm / min for 10 min to obtain a layered and homogenized dispersion.

[0097] (2) Preparation of agar film substrate:

[0098] Add 1g of agar powder to 50mL of ultrapure water, heat and stir at 100℃ for 1h to form a clear and transparent solution. Pour the mixture into a petri dish and freeze-dry for 8h after the solution has solidified to obtain a dry and loose agar film substrate.

[0099] (3) Preparation of HOF-FAFU-1-NH2-encapsulated thin-film test strips for detecting uric acid containing UOx and Au-BSA:

[0100] A 25 mm diameter agar film substrate was placed on a spin coater with a 22 mm diameter suction cup, with the loose A-side of the film facing upwards. The center of the film substrate was aligned concentrically with the axis of the spin coater. The spin coater then rotated the film substrate at high speed in two stages: the first stage was at 500 rpm / min for 36 seconds, and the second stage was at 3000 rpm / min for 80 seconds. During each stage, a homogenization solution of the composite material was titrated onto the A-side of the rotating film substrate. Afterwards, the substrate was freeze-dried for 8 hours to obtain a uric acid detection film strip coated with HOF-FAFU-1-NH2 encapsulated UOx and Au-BSA.

[0101] Example 8:

[0102] The preparation method of the composite film test strip for uric acid detection using HOF-FAFU-1-NH2 encapsulated UOx and Hemin-BSA provided in this embodiment is as follows:

[0103] (1) Configuration of HOF-FAFU-1-NH2:

[0104] In 10 mL of PBS buffer solution, 20 mg of HOF-FAFU-1-NH2 material encapsulating UOx and Hemin-BSA and 0.3 mM TMB solution were mixed evenly. The mixture was placed on a stirrer and stirred at 500 rpm / min for 10 min to obtain a layered and homogenized dispersion.

[0105] (2) Preparation of agar film substrate:

[0106] Add 1g of agar powder to 50mL of ultrapure water, heat and stir at 100℃ for 1h to form a clear and transparent solution. Pour the mixture into a petri dish and freeze-dry for 8h after the solution has solidified to obtain a dry and loose agar film substrate.

[0107] (3) Preparation of HOF-FAFU-1-NH2-encapsulated thin-film test strips for detecting uric acid containing UOx and Hemin-BSA:

[0108] A 25 mm diameter agar film substrate was placed on a spin coater with a 22 mm diameter suction cup, with the loose A-side of the film facing upwards. The center of the film substrate was aligned concentrically with the axis of the spin coater. The spin coater then rotated the film substrate at high speed in two stages: the first stage at 500 rpm / min for 36 seconds, and the second stage at 3000 rpm / min for 80 seconds. During each stage, a homogenizing solution of the composite material was titrated onto the A-side of the rotating film substrate. Afterwards, the substrate was freeze-dried for 8 hours to obtain a uric acid detection film strip coated with HOF-FAFU-1-NH2 encapsulated UOx and Hemin-BSA.

[0109] Example 9:

[0110] The preparation method of the composite film test strip for xanthine detection using HOF-FAFU-1-OH encapsulated XOD and Au-BSA provided in this embodiment is as follows:

[0111] (1) Configuration of HOF-FAFU-1-NH2:

[0112] In 10 mL of PBS buffer solution, 20 mg of HOF-FAFU-1-OH material encapsulating XOD and Au-BSA and 0.3 mM TMB solution were mixed evenly. The mixture was placed on a stirrer and stirred at 500 rpm / min for 10 min to obtain a layered and homogenized dispersion.

[0113] (2) Preparation of agar film substrate:

[0114] Add 1g of agar powder to 50mL of ultrapure water, heat and stir at 100℃ for 1h to form a clear and transparent solution. Pour the mixture into a petri dish and freeze-dry for 8h after the solution has solidified to obtain a dry and loose agar film substrate.

[0115] (3) Preparation of HOF-FAFU-1-OH encapsulated XOD and Au-BSA film test strips for the detection of xanthine:

[0116] A 25 mm diameter agar film substrate was placed on a spin coater with a 22 mm diameter suction cup, with the loose A-side of the film facing upwards. The center of the film substrate was aligned concentrically with the axis of the spin coater. The spin coater then rotated the film substrate at high speed in two stages: the first stage was at 500 rpm / min for 36 seconds, and the second stage was at 3000 rpm / min for 80 seconds. During each stage, a homogenizing solution of the composite material was titrated onto the A-side of the rotating film substrate. Afterwards, the substrate was freeze-dried for 8 hours to obtain a xanthine detection film strip coated with HOF-FAFU-1-OH encapsulated XOD and Au-BSA.

[0117] Example 10:

[0118] The preparation method of the composite film test strip for xanthine detection using HOF-FAFU-1-OH encapsulated XOD and Hemin-BSA provided in this embodiment is as follows:

[0119] (1) Preparation of the composite material dispersion solution:

[0120] In 10 mL of PBS buffer solution, 20 mg of HOF-FAFU-1-OH material encapsulating XOD and Hemin-BSA and 0.3 mM TMB solution were mixed evenly. The mixture was placed on a stirrer and stirred at 500 rpm / min for 10 min to obtain a layered and homogenized dispersion.

[0121] (2) Preparation of agar film substrate:

[0122] Add 1g of agar powder to 50mL of ultrapure water, heat and stir at 100℃ for 1h to form a clear and transparent solution. Pour the mixture into a petri dish and freeze-dry for 8h after the solution has solidified to obtain a dry and loose agar film substrate.

[0123] (3) Preparation of HOF-FAFU-1-OH encapsulated XOD and Hemin-BSA film test strips for the detection of xanthine:

[0124] A 25 mm diameter agar film substrate was placed on a spin coater with a 22 mm diameter suction cup, with the loose A-side of the film facing upwards. The center of the film substrate was aligned concentrically with the axis of the spin coater. The spin coater then rotated the film substrate at high speed in two stages: the first stage at 500 rpm / min for 36 seconds, and the second stage at 3000 rpm / min for 80 seconds. During each stage, a homogenizing solution of the composite material was titrated onto the A-side of the rotating film substrate. Afterwards, the substrate was freeze-dried for 8 hours to obtain a film test strip coated with HOF-FAFU-1-OH encapsulated XOD and Hemin-BSA for xanthine detection.

[0125] Example 11:

[0126] The preparation method of the composite film test strip for xanthine detection using HOF-FAFU-1-NH2 encapsulated XOD and Au-BSA provided in this embodiment is as follows:

[0127] (1) Preparation of the composite material dispersion solution:

[0128] In 10 mL of PBS buffer solution, 20 mg of HOF-FAFU-1-NH2 material encapsulating XOD and Au-BSA and 0.3 mM TMB solution were mixed evenly. The mixture was placed on a stirrer and stirred at 500 rpm / min for 10 min to obtain a layered and homogenized dispersion.

[0129] (2) Preparation of agar film substrate:

[0130] Add 1g of agar powder to 50mL of ultrapure water, heat and stir at 100℃ for 1h to form a clear and transparent solution. Pour the mixture into a petri dish and freeze-dry for 8h after the solution has solidified to obtain a dry and loose agar film substrate.

[0131] (3) Preparation of HOF-FAFU-1-NH2-encapsulated XOD and Au-BSA film test strips for the detection of xanthine:

[0132] A 25 mm diameter agar film substrate was placed on a spin coater with a 22 mm diameter suction cup, with the loose A-side of the film facing upwards. The center of the film substrate was aligned concentrically with the axis of the spin coater. The spin coater then rotated the film substrate at high speed in two stages: the first stage at 500 rpm / min for 36 seconds, and the second stage at 3000 rpm / min for 80 seconds. During each stage, a homogenizing solution of the composite material was titrated onto the A-side of the rotating film substrate. Afterwards, the substrate was freeze-dried for 8 hours to obtain a xanthine detection test strip coated with HOF-FAFU-1-NH2 encapsulated XOD and Au-BSA.

[0133] Example 12:

[0134] The preparation method of the composite film test strip for xanthine detection using HOF-FAFU-1-NH2 encapsulated XOD and Hemin-BSA provided in this embodiment is as follows:

[0135] (1) Preparation of the composite material dispersion solution:

[0136] In 10 mL of PBS buffer, 20 mg of HOF-FAFU-1-NH2 material encapsulating XOD and Hemin-BSA and 0.3 mM TMB solution were mixed evenly. The mixture was then placed on a stirrer and stirred at 500 rpm for 10 min to obtain a layered and homogenized dispersion.

[0137] (2) Preparation of agar film substrate:

[0138] Add 1g of agar powder to 50mL of ultrapure water, heat and stir at 100℃ for 1h to form a clear and transparent solution. Pour the mixture into a petri dish and freeze-dry for 8h after the solution has solidified to obtain a dry and loose agar film substrate.

[0139] (3) Preparation of HOF-FAFU-1-NH2-encapsulated XOD and Hemin-BSA film test strips for the detection of xanthine:

[0140] A 25 mm diameter agar film substrate was placed on a spin coater with a 22 mm diameter suction cup, with the loose A-side of the film facing upwards. The center of the film substrate was aligned concentrically with the axis of the spin coater. The spin coater then rotated the film substrate at high speed in two stages: the first stage was at 500 rpm / min for 36 seconds, and the second stage was at 3000 rpm / min for 80 seconds. During each stage, a homogenizing solution of the composite material was titrated onto the A-side of the rotating film substrate. Afterwards, the substrate was freeze-dried for 8 hours to obtain a film test strip coated with HOF-FAFU-1-NH2 encapsulated XOD and Hemin-BSA for xanthine detection.

[0141] Example 13:

[0142] First, prepare a 2 mg / mL suspension of HOF-FAFU-1-OH encapsulating Gox and Au-BSA with PBS. Take 1.600 mL of this suspension and add it to 0.3 mM TMB buffer, then add 0.200 mL of glucose solution of different concentrations at pH 4.0. (See attached...) Figure 8 As shown, the experimental results indicate that the sensor has good detection performance for glucose in the concentration range of 0.5mM to 100mM, and can quantitatively detect the glucose content in blood samples, which has important application value for human blood glucose detection.

[0143] Example 14:

[0144] Prepare a composite film test strip from the suspension of Example 13, and titrate 1-3 drops of blood onto the film test strip, allowing it to stand for 5-10 minutes. (Refer to Appendix) Figure 9 As shown, the experimental results indicate that the composite film test paper has good detection performance for glucose in the concentration range of 0.5mM to 100mM. It is simple, convenient, quick, and environmentally friendly.

[0145] Comparative Example:

[0146] Agar was used as the film-forming material, and no other materials were added (i.e., the composite material dispersion solution prepared in step (1) was added). Other casting components and formulations were the same as in Example 1. The SEM image of the surface of the agar film substrate obtained is shown in [reference needed]. Figure 2 The contact angle of the prepared agar film substrate is shown in [reference]. Figure 4 .

[0147] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a universal hydrogen-bonded organic framework composite film test strip with visual detection properties for small biological molecules, characterized in that, include: Hydrogen-bonded organic framework materials for encapsulating enzymes / enzymes were used as the framework carriers in the thin-film test strips and coated onto an agar film substrate. The specific steps are as follows: 1) Preparation of dispersions of hydrogen-bonded organic framework materials for encapsulating enzymes / enzymes: In a dispersion solution environment, the hydrogen-bonded organic framework material of the encapsulated enzyme / enzyme-like substance is mixed uniformly with a 3,3',5,5'-tetramethylbenzidine (TMB) solution. The mixture is stirred to obtain a layered and homogenized dispersion. The encapsulated enzyme / enzyme-like substance is glucose oxidase (Gox) and golden bovine serum albumin (Au-BSA), Gox and heme-bovine serum albumin (Hemin-BSA), uricase (UOx) and Au-BSA, UOx and Hemin-BSA, xanthine oxidase (XOD) and Au-BSA, XOD and Hemin-BSA. The hydrogen-bonded organic framework material is HOF-FAFU-1, which is constructed from any combination of in-BSA, β-galactosidase (β-Gal), GOx and Au-BSA, β-Gal, GOx and Hemin-BSA; the basic building block of the framework is HOF-FAFU-1, which is constructed from any combination of 3,3'5,5'-tetra(4-carboxyphenyl)-4,4'-dihydroxybiphenyl, 3,3'5,5'-tetra(4-carboxyphenyl)-4,4'-diaminobiphenyl, and 3,3'5,5'-tetra(4-carboxyphenyl)-4,4'-methoxybiphenyl. 2) Preparation of agar film substrate: Agar powder was added to the dispersion solution, heated and stirred until a clear and transparent solution was formed. The mixture was then poured into a petri dish, and after the solution solidified, it was freeze-dried to obtain a dry and loose film substrate. 3) Preparation of hydrogen-bonded organic framework composite thin film test paper: The hydrogen-bonded organic framework material dispersion for encapsulating enzymes / enzymes prepared in step 1) is coated onto the loose surface A of the agar film substrate prepared in step 2), and after freeze-drying, hydrogen-bonded organic framework composite film test paper for detecting small biological molecules is obtained.

2. The method for preparing a universal hydrogen-bonded organic framework composite film test strip with visual detection properties for small biological molecules according to claim 1, characterized in that, The dispersion solution described in steps 1) and 2) is any one or more of water, PBS buffer, Tris-HCl solution, and HAc-NaAc buffer.

3. The method for preparing a universal hydrogen-bonded organic framework composite film test strip with visual detection properties for small biological molecules according to claim 1, characterized in that, In step 1), the concentration of the hydrogen-bonded organic framework material for encapsulating the enzyme / enzyme-like substance is greater than 0.01 mM; the concentration of the TMB solution is greater than 0.1 mM; the stirring speed is greater than 100 rpm / min; and the stirring time is greater than 1 min.

4. The method for preparing a universal hydrogen-bonded organic framework composite film test strip with visual detection properties for small biological molecules according to claim 1, characterized in that, In step 2), the concentration of agar powder is greater than 1 mg / mL; the heating temperature is greater than 70 ℃, the heating and stirring time is greater than 0.1 h; and the freeze-drying time after the mixed solution solidifies is greater than 2 h.

5. The method for preparing a universal hydrogen-bonded organic framework composite film test strip with visual detection properties for small biological molecules according to claim 1, characterized in that, In step 3), the diameter of the agar film substrate is greater than 1 mm, the coating amount of the hydrogen-bonded organic framework material dispersion for encapsulating enzymes / enzymes is greater than 0.5 mL, and the freeze-drying time is greater than 2 hours.

6. The method for preparing a universal hydrogen-bonded organic framework composite film test strip with visual detection properties for small biological molecules according to claim 1, characterized in that, The coating method described in step 3) can be any one of drip coating, spin coating, spray coating, or printing.

7. The method for preparing a universal hydrogen-bonded organic framework composite film test strip with visual detection properties for small biological molecules according to claim 6, characterized in that, The coating is applied using a spin coating method, and the specific steps are as follows: Place the agar film substrate on the spin coater with the loose A side of the film facing upwards, and align the center of the film substrate with the axis of rotation of the spin coater. Next, the film substrate is rotated at high speed using a spin coater, which is divided into two stages. In each stage, the homogenization solution of the composite material prepared in step 1) is titrated on the A side of the rotating film substrate. Finally, freeze-drying is performed to obtain the composite film test paper for detecting small biological molecules; The first stage of high-speed rotation has a rotation speed of 500-1000 rpm / min and a rotation time of 36-54s. The second stage has a rotation speed of 1000-6000 rpm / min and a rotation time of 60-120s.

8. The application of a universal hydrogen-bonded organic framework composite film test strip with visual detection properties for small biological molecules according to any one of claims 1-7, characterized in that, For the qualitative and quantitative detection of small biological molecules; the small biological molecules include glucose, uric acid, xanthine and lactose.

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