Preparation method and application of multicolor colorimetric sensor for monitoring glucose in sweat

Abstract

The invention discloses a preparation method and application of a multicolor colorimetric sensor for monitoring glucose in sweat, based on a localized surface plasma effect of a gold nanorod (Au NR), 3, 3 ', 5, 5'-tetramethyl benzidine (TMB) is adopted to etch the Au NR, and the multicolor colorimetric real-time monitoring glucose sensor is simply, quickly and sensitively constructed. Characterization of multicolor colorimetric sensing can be completed by a transmission electron microscope (TEM) and an ultraviolet spectrophotometer (UV-Vis). The sensor realizes real-time monitoring of glucose in sweat, and has the advantages of high sensitivity, rapid detection, naked eye analysis and the like.

Description

[0001] The present invention specifically relates to a method for preparing a multicolor colorimetric sensor capable of detecting changes in glucose concentration in sweat in real time and with high sensitivity, and belongs to the field of biological applications of nanomaterials. Background Art

[0002] Traditional diagnosis and health assessment methods rely on outpatient visits and annual laboratory tests, which are inconvenient and expensive, and the analysis results are delayed. It cannot meet the rapidly growing demand for healthcare. Therefore, new diagnostic and treatment technologies are urgently needed to achieve home health monitoring to meet the growing demand for healthcare. The use of wearable chemical sensors provides continuous and non-invasive tracking of the dynamically changing chemical composition of various biofluids (such as sweat, tears, saliva, and interstitial fluid), thereby providing molecular-level analytical monitoring of the wearer's health status and providing the possibility of providing continuous and comprehensive medical monitoring.

[0003] Sweat glands are widely distributed in the human skin. The sweat they secrete is a biological fluid with rich biomolecules including electrolytes, small molecules and proteins. The fluctuations of these biomolecules reflect the physiological state of the human body. The diverse physiological signals brought by various biomarkers and the characteristics of easy non-invasive collection have made sweat detection widely concerned. Glucose in sweat is related to blood sugar metabolism, and it plays an important role as a target detection molecule for diabetes. At present, the means for detecting glucose in sweat include electrochemical sensing, colorimetric sensing, etc.

[0004] When incident light irradiates precious metal (such as Au, Ag, Cu, etc.) nanoparticles, if the frequency of the incident photon is consistent with the vibration frequency of the precious metal nanoparticles when transmitting to the electrons, then the nanoparticles have a strong absorption effect on the incident photon and produce a scattering phenomenon, resulting in a plasma resonance effect (SPR), which is manifested as a strong resonance absorption peak in the spectrum. Studies have found that the solution color of these precious metal nanoparticles is highly correlated with their shape, size and composition. The different shapes and sizes of precious metal nanoparticles determine their different resonance frequencies, resulting in different scattering and absorption peaks. By changing these factors, the SPR absorption peak will move to a shorter (blue shift) or longer (red shift) wavelength. Based on this unique plasma resonance effect, precious metal nanoparticles have been widely used in signal amplification and transduction of nanosensors, including surface enhanced Raman spectroscopy, colorimetry and fluorescence sensing. At present, the colorimetric detection and analysis of glucose in sweat is still a single-color dye, which is not convenient for naked eye observation, and professional instrument analysis is not accurate enough, so it has great limitations. Summary of the invention

[0005] In order to overcome the defects of the prior art and obtain a highly sensitive sweat glucose biosensor that can be semi-quantitatively analyzed by the naked eye, the present invention designs a multicolor sensor based on Au NR, which has the advantages of being label-free, highly sensitive, having a wide linear range, fast detection speed, and being able to semi-quantitatively analyze sweat glucose in real time without the need for professional instruments.

[0006] The preparation process is as follows:

[0007] 1. Preparation of a multicolor colorimetric sensor for monitoring glucose in sweat

[0008] First, ~80 nm Au NRs were synthesized using the seed growth method, prepared as follows:

[0009] Step 1. Synthesize particles

[0010] (1) Seed synthesis

[0011] Mix 10 mL 0.1 mol / L hexadecyltrimethylammonium bromide (CTAB) and 0.25 mL 0.1 mol / L chloroauric acid (HAuCl4), stir evenly at 800 r / min at 30°C, then quickly add 0.6 mL 0.01 mol / L fresh ice-cold sodium borohydride (NaBH4) to the mixed solution, stir vigorously at 1200 r / min at 30°C, and then let the mixed solution stand in a 30°C water bath for 30 min before use.

[0012] (2) Synthesis of 80 nm Au NR

[0013] 10 mL 0.1 mol / L CTAB was mixed with 0.5 mL 0.1 mol / L HAuCl4, and 0.06 mL 0.01 mol / L silver nitrate (AgNO3) was added. The mixture was stirred at 800 r / min at 30 °C, and then 0.55 mL 0.1 mol / L ascorbic acid (AA) was added. Finally, 0.02 mL seed solution was added. The mixture was stirred vigorously at 1200 r / min for 30 s and placed in a 30 °C water bath for 24 h. The TEM of the prepared 80 nm gold rod and the solution were shown in the attached figure. Figure 1 shown.

[0014] Step 2. Prepare wearable sensor

[0015] The device pattern mold is prepared by photolithography, and the PDMS film (channel layer) with the mold pattern is obtained by spin coating and heat curing; the PDMS film with the corresponding opening pattern (sieve layer, liquid reservoir layer, bottom layer) is obtained by laser cutting. Each layer of PDMS is hydrophilic, and each layer of PDMS is encapsulated after adding the color-developing base liquid to the liquid reservoir layer. Figure 2 shown.

[0016] An application of a multicolor colorimetric sensor for monitoring glucose in sweat, specifically for the detection of glucose: first prepare 0.02mM TMB solution, 0.2mg / mL glucose oxidase solution, and 0.2mg / mL horseradish peroxidase solution, then dilute glucose solutions of different concentrations in sequence, then take 100μL of the prepared Au NR solution and the above prepared solution, mix them in a 2mL centrifuge tube, shake them thoroughly, and incubate them at room temperature for 30min. The obtained Au NR solution shows rich colors visible to the naked eye, as shown in the attached figure. Figure 3 As shown, collect UV-Vis spectrum data, as shown in the attached Figure 4 Specifically, the data acquisition range is set to 300-950nm, and then the data acquisition time interval is set to 0.5s, and the curves of Au NR with different etching degrees are scanned. The relationship curve between the absorption wavelength peak of Au NR under different glucose concentrations is obtained. The glucose concentration and wavelength have a good linear relationship, as shown in the attached figure. Figure 5 As shown in the figure, the linear relationship between different glucose concentrations and absorbance is shown in the figure. The glucose concentration has a good linear relationship with the absorption peak of the longitudinal plasma band, as shown in the figure. Figure 6 As shown. In addition, after the UV-Vis test is completed, the Au NR solution is centrifuged to remove the supernatant, and the precipitate is mixed with deionized water and centrifuged again. The above operation is repeated 2-3 times. The obtained Au NR solution presents rich colors visible to the naked eye. 7μL of different color solutions are taken and dropped on the copper mesh, and evaporated naturally or heated in an oven until the copper mesh is completely dry. The morphology of Au NR with different etching degrees and the relationship between its length and glucose concentration are obtained by TEM characterization. The linear range is 1-0.01mM, and the minimum detection limit is 0.01mM. Different concentrations of glucose have a good linear relationship with the wavelength peak and absorption intensity. The experimental results show that as the glucose concentration increases, the etching degree is higher, and the length of Au NR becomes shorter until it becomes a gold ball.

[0017] Glucose of different concentrations was injected into the prepared wearable sensor for reaction. After 30 minutes, a photo was taken to obtain the color corresponding to each concentration of glucose and a color reference card was constructed, which can be used for semi-quantitative analysis of glucose concentration in sweat by naked eye.

[0018] Beneficial Effects

[0019] In this study, Au NR was used as a multicolor colorimetric sensing substrate and real-time monitoring of sweat glucose. Glucose can be detected by combining Au NR with TMB. 2+The UV-Vis curve after etching reaction, TEM image characterization, and reference colorimetric card are used to achieve this. The sensor has the advantages of high sensitivity, wide linear range, fast detection speed, and can semi-quantitatively and continuously analyze sweat glucose in real time without professional instruments. In addition, the linear relationship between glucose concentration and wavelength peak and absorption intensity can be obtained by linear fitting. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 TEM and solution images of the 80 nm gold rod prepared in the present invention.

[0021] Figure 2 This is a diagram showing the structure of the wearable device in the present invention, the actual object, and the color rendering effect.

[0022] Figure 3 It is a multicolor reference colorimetric chart of the relationship between Au NR color and glucose concentration in the present invention.

[0023] Figure 4 It is an absorption curve diagram of the Au NR spectrum under the action of different concentrations of glucose in the present invention.

[0024] Figure 5 is a linear relationship diagram between different glucose concentrations and Au NR wavelengths in the present invention.

[0025] Figure 6 It is a linear relationship diagram between different glucose concentrations and Au NR absorption peaks in the present invention. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is specifically described below in conjunction with embodiments.

[0027] Example 1

[0028] First, prepare 0.02mM TMB solution, 0.2mg / mL glucose oxidase solution, and 0.2mg / mL horseradish peroxidase solution, add 0.01mM glucose solution, take 100μL of the prepared Au NR solution and the above prepared solution and mix them in a 2mL centrifuge tube, shake well and incubate at room temperature for 30min, collect UV-Vis spectrum data, as shown in the attached figure. Figure 4 As shown, the absorbance value is 1.17, and the longitudinal absorption peak position is 788.31nm. After the UV-Vis test, the Au NR solution is centrifuged to remove the supernatant, and the precipitate is mixed with deionized water and centrifuged again. The above operation is repeated 3 times. The obtained Au NR solution is pink (attached) visible to the naked eye. Figure 3 7 μL of the solution was dropped on the copper mesh, and evaporated naturally or heated in an oven until the copper mesh was completely dry. The morphology of the Au NR etching extent and its length were obtained by TEM characterization, which was 65 nm.

[0029] Example 2

[0030] First, prepare 0.02mM TMB solution, 0.2mg / mL glucose oxidase solution, and 0.2mg / mL horseradish peroxidase solution, add 0.07mM glucose solution, take 100μL of the prepared Au NR solution and the above prepared solution and mix them in a 2mL centrifuge tube, shake well and incubate at room temperature for 30min, collect UV-Vis spectrum data, as shown in the attached figure. Figure 4 As shown, the absorbance value is 0.98, and the longitudinal absorption peak position is 745.7nm. After the UV-Vis test is completed, the Au NR solution is centrifuged to remove the supernatant, and the precipitate is mixed with deionized water and centrifuged again. The above operation is repeated 3 times. The obtained Au NR solution presents an orange-red color visible to the naked eye (attached Figure 3 7 μL of the solution was dropped on the copper mesh and evaporated naturally or heated in an oven until the copper mesh was completely dry. The morphology of the Au NR etching extent and its length were obtained by TEM characterization, which was 55 nm.

[0031] Example 3

[0032] First, prepare 0.02mM TMB solution, 0.2mg / mL glucose oxidase solution, and 0.2mg / mL horseradish peroxidase solution, add 0.17mM glucose solution, take 100μL of the prepared Au NR solution and the above prepared solution and mix them in a 2mL centrifuge tube, shake well and incubate at room temperature for 30min, collect UV-Vis spectrum data, as shown in the attached figure. Figure 4 As shown, the absorbance value is 0.62, and the longitudinal absorption peak position is 682.2nm. After the UV-Vis test is completed, the Au NR solution is centrifuged to remove the supernatant, and the precipitate is mixed with deionized water and centrifuged again. The above operation is repeated 3 times. The obtained Au NR solution is green (attached) visible to the naked eye. Figure 3 7 μL of the solution was dropped on the copper mesh, and evaporated naturally or heated in an oven until the copper mesh was completely dry. The morphology of the Au NR etching extent and its length were obtained by TEM characterization, which was 45 nm.

[0033] Example 4

[0034] First, prepare 0.02mM TMB solution, 0.2mg / mL glucose oxidase solution, and 0.2mg / mL horseradish peroxidase solution, add 0.23mM glucose solution, take 100μL of the prepared Au NR solution and the above prepared solution in a 2mL centrifuge tube, shake thoroughly, and incubate at room temperature for 30min, collect UV-Vis spectrum data, as shown in the attached figure. Figure 4As shown, the absorbance value is 0.46, and the longitudinal absorption peak position is 651.88nm. After the UV-Vis test is completed, the Au NR solution is centrifuged to remove the supernatant, and the precipitate is mixed with deionized water and centrifuged again. The above operation is repeated 3 times. The obtained Au NR solution is blue (attached) visible to the naked eye. Figure 3 7 μL of the solution was dropped on the copper mesh and evaporated naturally or heated in an oven until the copper mesh was completely dry. The morphology of the Au NR etching extent and its length were obtained by TEM characterization, which was 35 nm.

[0035] Example 5

[0036] First, prepare 0.02mM TMB solution, 0.2mg / mL glucose oxidase solution, and 0.2mg / mL horseradish peroxidase solution, add 0.2973mM glucose solution, take 100μL of the prepared Au NR solution and the above prepared solution and mix them in a 2mL centrifuge tube, shake well and incubate at room temperature for 30min, collect UV-Vis spectrum data, as shown in the attached figure. Figure 4 As shown, the absorbance value is 1.17, and the longitudinal absorption peak position is 630.22nm. After the UV-Vis test, the Au NR solution is centrifuged to remove the supernatant, and the precipitate is mixed with deionized water and centrifuged again. The above operation is repeated 3 times. The obtained Au NR solution is purple (attached) visible to the naked eye. Figure 3 7 μL of the solution was dropped on the copper mesh, and evaporated naturally or heated in an oven until the copper mesh was completely dry. The morphology of the Au NR etching extent and its length were obtained by TEM characterization, which was 25 nm.

[0037] Example 6

[0038] First, prepare 0.02mM TMB solution, 0.2mg / mL glucose oxidase solution, and 0.2mg / mL horseradish peroxidase solution, add 0.3mM glucose solution, take 100μL of the prepared Au NR solution and the above prepared solution and mix them in a 2mL centrifuge tube, shake well and incubate at room temperature for 30min, collect UV-Vis spectrum data, as shown in the attached figure. Figure 4 As shown, the absorbance value is 0.24, and the longitudinal absorption peak position is 615.44nm. After the UV-Vis test is completed, the Au NR solution is centrifuged to remove the supernatant, and the precipitate is mixed with deionized water and centrifuged again. The above operation is repeated 3 times. The obtained Au NR solution is red (attached) visible to the naked eye. Figure 3 7 μL of the solution was dropped on the copper mesh, and evaporated naturally or heated in an oven until the copper mesh was completely dry. The morphology of the Au NR etching extent and its length were obtained by TEM characterization, which was 15 nm.

[0039] Example 7

[0040] The device pattern mold was prepared by photolithography. Four smooth glasses were prepared to cast PDMS and the PDMS was evenly and evenly spin-coated on the glass sheets by rotating at a speed of 500rpm / s for 10s. The PDMS was then placed in an oven and dried at 65°C for 5h. The PDMS on the mold was peeled off for backup. The PDMS on the remaining four glasses was cut into corresponding patterns with a laser at a power of 70w and a frequency of 50pHz. After each layer of PDMS was hydrophilically treated, a color developing base liquid was dripped into the reservoir layer, and then the PDMS layers were encapsulated to complete the preparation. Glucose was injected into the inlet of the device for reaction for 30min, and photos were taken and recorded, and a colorimetric reference card was constructed.

[0041] Example 8

[0042] Prepare the device pattern mold by photolithography, prepare four smooth glasses to cast PDMS and rotate it continuously at 500rpm / s for 10s to evenly and evenly spin-coat the PDMS on the glass sheets, and then put it in an oven to dry and solidify at 65℃ for 9h. Peel off the PDMS on the mold and keep it for later use. Cut the PDMS on the remaining four glasses into corresponding patterns with a laser at 70w power and 50pHz frequency. After each layer of PDMS is hydrophilic, add the color developing base liquid to the liquid reservoir layer, and then encapsulate each layer of PDMS to complete the preparation, as shown in the attached Figure 2 As shown, glucose was injected into the device inlet and reacted for 30 minutes before taking photos and recording, and a colorimetric reference card was constructed.

Claims

1. A method for preparing a multicolor colorimetric sensor for monitoring glucose in sweat, characterized in that: The method comprises the following steps: (1) seed synthesis of synthesized particles; (2) synthesis of 80 nm Au NR; and (3) preparation of TMB and enzyme solution, exploration and verification of different pH conditions, and preparation of wearable sensors.

2. The preparation method according to claim 1, characterized in that The step 1 of synthesizing particles (1) is specifically as follows: Weigh 2.4 mg of TMB powder and dissolve it in 1 mL of dimethyl sulfoxide (DMSO), and dilute it with artificial sweat to 0.02 mM for later use; weigh 2 mg of glucose oxidase and 2 mg of horseradish peroxidase and dissolve them in 1 mL of artificial sweat, and then dilute them to 0.2 mg / mL for later use; take 1 M glucose solution and dilute it to 1-0.01 mM for later use; adjust the pH of artificial sweat to about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, and 7.5 with hydrochloric acid and set aside; spin-coat dimethylsiloxane (PDMS).

3. The preparation method according to claim 1, characterized in that: The step 2 of preparing the wearable sensor specifically comprises: preparing a device pattern mold by photolithography, spin coating and heating to cure to obtain a PDMS film with a mold pattern, namely, a channel layer; laser cutting to obtain a PDMS film with corresponding opening patterns, namely, a sieve layer, a liquid storage layer, and a bottom layer, performing a hydrophilic treatment on each PDMS layer, and encapsulating each PDMS layer after dripping a color-developing base liquid on the liquid storage layer.

4. An application of a multicolor colorimetric sensor for monitoring glucose in sweat, characterized in that: The specific test for glucose is as follows: (1) Glucose oxidase solution and horseradish peroxidase solution were prepared, and then glucose solutions of different concentrations were diluted in sequence. The prepared Au NR solution and the prepared solutions were mixed in a centrifuge tube, and the mixture was fully shaken and mixed, and then incubated at room temperature, and then UV-Vis spectrum data was collected; the data collection range was set first, and then the data collection time interval was set; (2) Scan a blank sample as a baseline, the blank sample is deionized water, and then scan the curves of Au NR with different etching degrees, export the data and draw a wavelength-absorption relationship curve in origin, obtain the relationship curve between the wavelength peak and absorbance of Au NR under different glucose concentrations, and perform linear processing on the curve to obtain the linear relationship between the peak value and absorbance and the glucose concentration; (3) After the UV-Vis test, the Au NR solution was centrifuged to remove the supernatant, and the precipitate was mixed with deionized water and centrifuged again; the obtained Au NR solution showed rich colors visible to the naked eye, and different color solutions were dropped on the copper mesh, and evaporated naturally or heated in an oven until the copper mesh was completely dry. The morphology of Au NR with different etching degrees and the relationship between its length and glucose concentration were obtained by TEM characterization; different concentrations of glucose had a good linear relationship with the wavelength peak and absorption intensity; the experimental results showed that as the glucose concentration increased, the etching degree was higher, and the length of Au NR became shorter until it became a gold ball; (4) Glucose of different concentrations is injected into the prepared wearable sensor for reaction, and photos are taken to obtain the color corresponding to each concentration of glucose. A color reference card is constructed for semi-quantitative analysis of the glucose concentration in sweat by naked eye.

5. The use according to claim 4, characterized in that The glucose oxidase solution in (1) is 0.02 mM TMB solution, 0.2 mg / mL, and the horseradish peroxidase solution is 0.2 mg / mL. 100 μL of each is mixed in a 2 mL centrifuge tube, and the mixture is fully shaken and incubated at room temperature for 30 min. The data acquisition range is set to 300-950 nm, and the data acquisition time interval is 0.5 s.

6. The use according to claim 4, characterized in that In the step (3), the Au NR solution is centrifuged to remove the supernatant, the precipitate is mixed with deionized water and centrifuged again, and the operation is repeated 2-3 times; 7 μL of the different color solutions are dropped onto the copper mesh.

7. The use according to claim 4, characterized in that The linear range of (3) is 1-0.01 mM, and the minimum detection limit is 0.01 mM.

8. The use according to claim 4, characterized in that In (4), the reaction time is 30 min.

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