A paper-based sensor for rapid detection of lipopolysaccharide based on a smart phone

Abstract

The application belongs to the technical field of paper-based sensor preparation and detection application, and discloses a paper-based sensor capable of rapidly detecting lipopolysaccharide based on a smart phone. The paper-based biosensor capable of rapid detection is constructed based on a smart phone. The colorimetric performance of filter paper is combined with the characteristics of digital reconstruction of a smart phone App, and a large amount of dyeing agents are introduced through UATRP, so that the lipopolysaccharide paper-based sensor not only realizes instant and rapid detection, but also has a wider detection range and a lower detection limit. The linear detection range of the lipopolysaccharide concentration is 1-10 8 pg / mL, and the detection limit is 0.9172 pg / mL (S / N=3).

Description

Technical Field

[0001] This invention belongs to the field of paper-based sensor preparation and detection application technology. This invention relates to a paper-based sensor for detecting lipopolysaccharides and its preparation method. Specifically, this invention relates to a paper-based sensor that introduces a large amount of amino dye Nyl Blue chloride based on ultrafast visible light-induced atom transfer radical polymerization, which can be rapidly detected by a smartphone mobile application device. Background Technology

[0002] Lipopolysaccharide (LPS) is a major structural component of the outer membrane of all Gram-negative bacteria. LPS consists of amphiphilic, negatively charged glucosamine phospholipids (called lipid A) and hydrophilic polysaccharide chains. The latter comprises an oligosaccharide core directly covalently linked to lipid A and distal O-antigen chains of repeating oligosaccharide units. The presence of LPS in the blood can trigger a cascade of excessive immune responses, leading to fever, sepsis, organ failure, and ultimately death. Furthermore, because LPS contamination of food, drinking water, pharmaceuticals, and wound dressings poses a serious threat to human health, the point-of-care detection of low-abundance LPS is of great significance in clinical diagnosis, healthcare, food safety, environmental monitoring, pharmaceutical production, and biomedical research.

[0003] Currently, amoeboid cell lysate (LAL)-based analytical techniques have become the gold standard for traditional LPS analysis due to their extremely high sensitivity (or low detection limit) and selectivity for LPS. However, they suffer from low reproducibility, are relatively expensive and time-consuming, especially when used in the low LPS concentration range. In recent years, the development of new detection methods for lipopolysaccharides (LPS) has attracted increasing attention, but paper-based sensors capable of rapidly detecting LPS are still rare.

[0004] Most paper-based sensors currently on the market are colorimetric, relying on coarse color changes for qualitative analysis. Therefore, quantitative analysis in the paper-based sensor field still needs improvement. Due to numerous objective factors affecting the paper surface, paper-based sensors have a narrow detection range and a high detection limit. Furthermore, widely used paper-based sensors or test strips are primarily designed for detecting common diseases, while the detection of trace disease biomarkers in the body is still in its early stages of development.

[0005] Previous biosensors mostly required the use of equipment for quantitative analysis and detection, and lacked portability, high cost, and low user-friendliness. Even portable biosensors often had production costs that were beyond the reach of ordinary people, thus making them unsuitable for large-scale market production.

[0006] Current specialized disease screenings involve numerous screening items, high costs, slow analysis speeds, cumbersome testing methods, and the need for specialized technical personnel. Therefore, there is a strong societal demand for affordable and portable testing methods. Summary of the Invention

[0007] To overcome the shortcomings of existing technologies, this invention provides a paper-based sensor for rapid detection of lipopolysaccharides (LPS) based on smartphones, along with its preparation method and application. The sensor introduces a large amount of dyeing agent onto the paper surface using carbon quantum dot-catalyzed UATRP, and utilizes the colorimetric properties of filter paper and a smartphone app to digitally reconstruct and detect LPS.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution:

[0009] A paper-based sensor is composed of qualitative filter paper, chitosan (CS), acetic acid, sodium hydroxide, glutaraldehyde (GA), and aptamer (Apt).

[0010] The specific steps of the above-mentioned paper-based sensor fabrication method are as follows:

[0011] S1. Dissolve 1.25g of CS in 50mL of acetic acid with a volume / volume of 2%, and stir with a magnetic stirrer for 3-4 hours to obtain a CS solution.

[0012] S2. Prepare a paper base (PB) on the surface of qualitative filter paper using a 3mm diameter punch. Immerse the PB in a CS solution, then remove it and place it in a petri dish. Dry it in a vacuum drying oven at 50℃ for 1 hour to obtain CS / PB. Drop 20μL of GA (0.625-10% V / V) onto the CS / PB surface and wait 7.5-120 minutes to obtain GA / CS / PB.

[0013] S3. Drop 20 μL of 2 μM Apt (purchased from Shanghai Sangon Biotech Co., Ltd.) onto GA / CS / PB and incubate for 7.5-120 minutes to obtain Apt / GA / CS / PB.

[0014] The paper-based sensor prepared by the above method is used in the detection of lipopolysaccharides, specifically as follows:

[0015] Apt / GA / CS / PB was incubated in samples containing lipopolysaccharides for 7.5–120 minutes. LPS and Apt were prepared via specific base pair recognition. To eliminate the influence of exposed aldehyde groups due to lack of reaction with amino Apt, 0.25–4 M ethylamine hydrochloride (EACl) was selected as a blocking agent. 20 μL of EACl was incubated on LPS / Apt / GA / CS / PB for 0.75–12 hours to obtain EACl / LPS / Apt / GA / CS / PB. Most monosaccharide residues in the LPS polysaccharide chain contain at least one cis-diol site. Since cis-diol can selectively crosslink with boric acid groups, BPBA / EACl / LPS / Apt / GA / CS / PB is obtained by incubation in a 0.0625-1mM 4-(bromomethyl)phenylboronic acid (BPBA) solution prepared from 10mM pH=7.4 PBS containing 2% (v / v) N,N-dimethylformamide for 7.5-120 minutes. BPBA / EACl / LPS / Apt / GA / CS / PB was immersed in a carbon quantum dot-catalyzed UATRP polymerization solution. 1 mL of the polymerization solution contained 5-80 mM copper bromide, 2.7-43.2 mM methacrolein (MLA), 4.3-68.8 μM tris(2-pyridinemethyl)amine (TPMA), 0.25-4 mg carbon quantum dots (CQDs), and 0.36 mL deionized water. The mixture was activated on a modified electrode surface for 2-32 minutes at room temperature under LED light to prepare PLMA / LPS / Apt / GA / CS / PB. The carbon quantum dots were prepared by dissolving 1 g of o-phenylenediamine in 100 mL of deionized water, adding the solution to a reaction vessel, and heating at 200 °C for 8 h. After the reaction vessel cooled naturally to room temperature, the mixture was filtered through a 0.22 μm membrane and freeze-dried to obtain the final product. Finally, PLMA / LPS / Apt / GA / CS / PB was dissolved in 0.000025-0.0004M Nell Blue Chloride (CNB) solution for 5-80 minutes to obtain CNB / PLMA / LPS / Apt / GA / CS / PB.

[0016] CNB / PLMA / LPS / Apt / GA / CS / PB were placed in a dark box. Under the same height and light intensity, images were taken with a smartphone, and the RGB values ​​were obtained using the "ColorCare" app. No specific smartphone was required, but an iPhone 12 was preferred. The detection results were calculated based on a linear regression equation: R / (R+G+B)=-0.016logc(pg / mL)+0.385, with a correlation coefficient R0. 2 =0.997.

[0017] The advantages of this invention compared to the prior art are:

[0018] The LPS paper-based sensor of this invention has a wider detection range and lower detection limit compared to other paper-based sensors, and is highly operable. Furthermore, this method features low background signal, high selectivity, avoids false positive interference, is relatively simple to operate, and is cost-effective. Moreover, LPS paper-based sensors are relatively rare, and this method has great application potential in rapid LPS detection and analysis.

[0019] This invention utilizes a smartphone to construct a paper-based biosensor for rapid detection. Combining the colorimetric properties of filter paper with the digital reconstruction capabilities of a smartphone app, and introducing a large amount of dye via UATRP, the lipopolysaccharide paper-based sensor achieves not only instantaneous and rapid detection but also a wider detection range and a lower detection limit. The linear detection range for lipopolysaccharide concentration is 1-10. 8 The detection limit was 0.9172 pg / mL (S / N = 3). Attached Figure Description

[0020] Figure 1 These are scanning electron microscope (SEM) images of BPBA / EACl / LPS / Apt / GA / CS / PB(A) and PLMA / LPS / Apt / GA / CS / PB(B) of Embodiment 1 of the present invention at the same magnification.

[0021] Figure 2 This is the X-ray photoelectron spectrum of PLMA / LPS / Apt / GA / CS / PB in Embodiment 1 of the present invention.

[0022] Figure 3 This describes the comparison of the sensor prepared in Example 1 of this invention with LPS and interfering substances (sodium 1,2-dipalmitoylphosphatidyl(1-propanetriol) salt (DPPG), cholesterol (TC), bovine serum albumin (BSA), adrenaline (AD), uric acid (UA), dopamine (DA), glucose (Glu), D-galactose (D-Gal), D-mannose (D-Man), saccharin (Sac), ascorbic acid (AA), citrate (Cit)) and blank color development, as well as the comparison of ΔR / (R+G+B) values.

[0023] Figure 4 This invention relates to CNB / PLMA / LPS / Apt / GA / CS / PB prepared using lipopolysaccharide solutions of different concentrations under the conditions of Example 1. Data (A) of the actual samples directly captured by an iPhone 12 and data collected by an app, as well as the corresponding working curves (B) (plotted as R / (R+G+B) versus the logarithm of LPS concentration). Detailed Implementation

[0024] The present invention is described in detail below through specific embodiments, but this does not limit the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can all be obtained commercially.

[0025] Example 1

[0026] 1. Fabrication of paper-based sensors:

[0027] Dissolve 1.25 g of CS in 50 mL of 2% acetic acid and stir with a magnetic stirrer for 3-4 hours to obtain a CS solution. Prepare PB on the surface of qualitative filter paper using a 3 mm diameter perforator. Immerse the PB in the CS solution, then place it in a petri dish and dry it in a vacuum drying oven at 50 °C for 1 hour to obtain CS / PB. Then, drop-cast 20 μL of 2.5% GA (v / v) onto the CS / PB surface and wait for 30 minutes to obtain GA / CS / PB. Drop-cast 20 μL of 2 μM Apt onto GA / CS / PB and incubate for 30 minutes to obtain Apt / GA / CS / PB.

[0028] 2. Application of paper-based sensors in the detection of lipopolysaccharides:

[0029] Apt / GA / CS / PB was incubated in a lipopolysaccharide-containing sample for 30 minutes. LPS and Apt were prepared via specific base pair recognition. To eliminate the influence of exposed aldehyde groups due to lack of interaction with amino Apt, 1 MEACl was selected as the blocking agent. 20 μL of EACl was incubated on LPS / Apt / GA / CS / PB for 3 hours to obtain EACl / LPS / Apt / GA / CS / PB. Most monosaccharide residues in the LPS polysaccharide chain contain at least one cis-diol site. Since cis-diol can selectively crosslink with borate groups, BPBA / EACl / LPS / Apt / GA / CS / PB was obtained by incubation for 30 minutes in a 0.25 mM BPBA solution prepared from 10 mM pH 7.4 PBS containing 2% (v / v) N,N-dimethylformamide. Then, BPBA / EACl / LPS / Apt / GA / CS / PB was immersed in a carbon quantum dot-catalyzed UATRP solution. 1 mL of the polymerization solution contained 20 mM copper bromide, 10.8 mM MLA, 17.2 μM TPMA, 1 mg CQDs, and 0.36 mL deionized water. Activation was performed on the modified electrode surface under LED light at room temperature for 8 minutes to obtain PLMA / LPS / Apt / GA / CS / PB. The carbon quantum dots were prepared by dissolving 1 g of o-phenylenediamine in 100 mL of deionized water, adding it to the reactor, and heating at 200 °C for 8 hours. After the reactor cooled naturally to room temperature, the mixture was filtered through a 0.22 μm membrane and freeze-dried. Finally, PLMA / LPS / Apt / GA / CS / PB was dissolved in 0.000025–0.0004 M CNB solution for 20 minutes to obtain CNB / PLMA / LPS / Apt / GA / CS / PB.

[0030] Place CNB / PLMA / LPS / Apt / GA / CS / PB in a dark box, take photos with a smartphone under the same height and light intensity, and obtain the RGB values ​​through the "Color" App.

[0031] Figure 1 SEM images of BPBA / EACl / LPS / Apt / GA / CS / PB and PLMA / LPS / Apt / GA / CS / PB at 3 μm after gold sputtering. Figure 1 As can be seen in A, after modification with GA, Apt, LPS, EAC1, and BPBA, the shape becomes wavy. Figure 1 B Comparison and Figure 1 A clearly visible broccoli-shaped polymer protrusions from the smooth surface indicate that polymerization has indeed occurred on the surface of PLMA / LPS / Apt / GA / CS / PB, and the surface is covered by polymer, proving that PLMA / LPS / Apt / GA / CS / PB has been successfully prepared.

[0032] PLMA / LPS / Apt / GA / CS / PB were further characterized using XPS, and the results are as follows: Figure 2 As shown, distinct characteristic peaks appear at 532.4, 399.5, 284.8, 133.5, and 67.2 eV, which are attributed to the characteristic peaks of O1s, N1s, C1s, P2p, and Br3d, respectively. Figure 2 The inset shows a high-resolution spectrum of Br 3d. The presence of nitrogen indicates that CS has been coated onto the PB surface, and the presence of phosphorus indicates that the paper-based LPS / Apt / GA / CS / PB has been successfully prepared. Even after EACl completely seals the aldehyde groups of GA, the continued presence of abundant carbon and oxygen proves the successful preparation of PLMA / LPS / Apt / GA / CS / PB, with a large number of exposed aldehyde groups in MLA. The presence of Br indicates that Br atoms are exposed on the polymer-modified filter paper surface, indicating that UATRP has been successfully implemented. Figure 1 This further demonstrates the successful preparation of PLMA / LPS / Apt / GA / CS / PB.

[0033] To demonstrate that the CNB / PLMA / LPS / Apt / GA / CS / PB constructed in this experiment has specific selectivity for LPS, under the same conditions, a blank (without identifying the analyte) and the same concentration as LPS (1.0 × 10⁻⁶) were used. 8 Paper bases were prepared using DPPG, TC, BSA, AD, UA, DA, Glu, D-Gal, D-Man, Sac, AA, and Cit (pg / mL). The color comparisons of the actual samples (blank, LPS, and interfering substances) with the RGB (i.e., ΔR / (R+G+B)) comparisons of LPS and interfering substances excluding the blank are shown below. Figure 3 As shown in the figure, there is a significant difference between the RGB values ​​of these interfering substances after removing the blank and those of LPS after removing the blank. This is likely due to the high specificity and affinity between LPS and its Apt. Apt cannot recognize the interfering substances and therefore shows no obvious color reaction, while LPS shows a significant color change after recognizing Apt. Figure 3 The results show that the CNB / PLMA / LPS / Apt / GA / CS / PB paper-based sensor has good anti-interference ability and specific selectivity for LPS.

[0034] Apt / GA / CS / PB were soaked in LPS of different concentrations (1, 10). 1 10 2 10 3 10 4 10 5 10 6 10 7 10 8In pg / mL), LPS / Apt / GA / CS / PB modified with different concentrations were then polymerized by UATRP initiated by BPBA for 8 min. PLMA / LPS / Apt / GA / CS / PB was then immersed in 0.0001M CNB, and the results are as follows: Figure 4 The actual sample image of A is shown. Finally, the RGB values ​​were obtained using a color sampling app on a smartphone. The RGB results for each of the five samples are shown below. Figure 4 The data acquisition section of App B is shown in the figure. As can be seen from the figure, with the increase of LPS concentration, the color gradually changes from dark green to lighter and then to straw yellow (the filter paper is yellow after polymerization, and turns green after reacting with blue CNB). This is likely because as LPS gradually decreases, the number of aldehyde groups introduced by polymerization decreases, and therefore the number of CNB groups that combine with the aldehyde groups of MLA also decreases, thus the color changes according to the trend shown in the figure. Using the RGB values ​​presented by the App, a graph is plotted between R / (R+G+B) and the logarithm of the LPS concentration to obtain the working curve for LPS detection of this modified filter paper. Figure 4 B). For example Figure 4 As shown in B, the signal response range of CNB / PLMA / LPS / Apt / GA / CS / PB for detecting LPS is 1 to 10. 8 pg / mL, the linear regression equation is R / (R+G+B)=-0.016logc(pg / mL)+0.385, and the correlation coefficient R 2 =0.997. Based on the working curve, the detection limit can be calculated to be 0.9172 pg / mL (S / N = 3).

[0035] Example 2:

[0036] 1. Fabrication of paper-based sensors:

[0037] Dissolve 1.25 g of CS in 50 mL of 2% acetic acid and stir with a magnetic stirrer for 3-4 h to obtain a CS solution. Prepare PB on the surface of qualitative filter paper using a 3 mm diameter perforator. Immerse the PB in the CS solution, then place it in a petri dish and dry it in a vacuum drying oven at 50 °C for 1 h to obtain CS / PB. Then, drop-cast 20 μL of 0.625% GA (v / v) onto the CS / PB surface and incubate for 7.5 minutes to obtain GA / CS / PB. Drop-cast 20 μL of 2 μM Apt onto GA / CS / PB and incubate for 7.5 minutes to obtain Apt / GA / CS / PB.

[0038] 2. Application of paper-based sensors in the detection of lipopolysaccharides:

[0039] Apt / GA / CS / PB was incubated for 7.5 minutes in a lipopolysaccharide-containing sample. LPS and Apt were prepared via specific base pair recognition to obtain LPS / Apt / GA / CS / PB. To eliminate the influence of exposed aldehyde groups due to lack of interaction with amino Apt, 0.25M EACl was selected as the blocking agent. 20 μL of EACl was incubated on LPS / Apt / GA / CS / PB for 0.75 hours to obtain EACl / LPS / Apt / GA / CS / PB. Most monosaccharide residues in the LPS polysaccharide chain contain at least one cis-diol site. Since cis-diol can selectively crosslink with borate groups, BPBA / EACl / LPS / Apt / GA / CS / PB was obtained by incubation for 7.5 minutes in a 0.0625 mM BPBA solution containing 2% (v / v) N,N-dimethylformamide in 10 mM pH 7.4 PBS. Then, BPBA / EACl / LPS / Apt / GA / CS / PB was immersed in a carbon quantum dot-catalyzed UATRP solution. 1 mL of the polymerization solution contained 5 mM copper bromide, 2.7 mM MLA, 4.3 μM TPMA, 0.25 mg CQDs, and 0.36 mL deionized water. Activation was performed on the modified electrode surface under LED light at room temperature for 2 minutes to obtain PLMA / LPS / Apt / GA / CS / PB. The carbon quantum dots were prepared by dissolving 1 g of o-phenylenediamine in 100 mL of deionized water, adding it to the reaction vessel, and heating at 200 °C for 8 h. After the reaction vessel cooled naturally to room temperature, the mixture was filtered through a 0.22 μm membrane and freeze-dried. Finally, PLMA / LPS / Apt / GA / CS / PB was dissolved in 0.000025 M CNB solution for 5 minutes to obtain CNB / PLMA / LPS / Apt / GA / CS / PB.

[0040] Place CNB / PLMA / LPS / Apt / GA / CS / PB in a dark box, take photos with a smartphone under the same height and light intensity, and obtain the RGB values ​​through the "Color" App.

[0041] Example 3:

[0042] 1. Fabrication of paper-based sensors:

[0043] Dissolve 1.25 g of CS in 50 mL of 2% acetic acid and stir with a magnetic stirrer for 3-4 hours to obtain a CS solution. Prepare PB on the surface of qualitative filter paper using a 3 mm diameter perforator. Immerse the PB in the CS solution, then place it in a petri dish and dry it in a vacuum drying oven at 50 °C for 1 hour to obtain CS / PB. Then, drop 20 μL of [V / V value missing] onto the CS / PB surface.

[0044] 1.25% GA was added, and after 15 minutes, GA / CS / PB was obtained. 20 μL of 2 μM Apt was drop-cast onto GA / CS / PB and incubated for 15 minutes to obtain Apt / GA / CS / PB.

[0045] 2. Application of paper-based sensors in the detection of lipopolysaccharides:

[0046] Apt / GA / CS / PB was incubated in a lipopolysaccharide-containing sample for 15 minutes. LPS and Apt were prepared via specific base pair recognition. To eliminate the influence of exposed aldehyde groups due to lack of interaction with amino Apt, 0.5% MEACl was selected as the blocking agent. 20 μL of EACl was incubated on LPS / Apt / GA / CS / PB for 1.5 hours to obtain EACl / LPS / Apt / GA / CS / PB. Most monosaccharide residues in the LPS polysaccharide chain contain at least one cis-diol site. Since cis-diol can selectively crosslink with borate groups, BPBA / EACl / LPS / Apt / GA / CS / PB was obtained by incubation for 15 minutes in a 0.125 mM BPBA solution prepared from 10 mM pH 7.4 PBS containing 2% (v / v) N,N-dimethylformamide. Then, BPBA / EACl / LPS / Apt / GA / CS / PB was immersed in a carbon quantum dot-catalyzed UATRP solution. 1 mL of the polymerization solution contained 10 mM copper bromide, 5.4 mM MLA, 8.6 μM TPMA, 0.5 mg CQDs, and 0.36 mL deionized water. Activation was performed on the modified electrode surface under LED light at room temperature for 4 minutes to obtain PLMA / LPS / Apt / GA / CS / PB. The carbon quantum dots were prepared by dissolving 1 g of o-phenylenediamine in 100 mL of deionized water, adding it to the reaction vessel, and heating at 200 °C for 8 h. After the reaction vessel cooled naturally to room temperature, the mixture was filtered through a 0.22 μm membrane and freeze-dried. Finally, PLMA / LPS / Apt / GA / CS / PB was dissolved in 0.00005 M CNB solution for 10 minutes to obtain CNB / PLMA / LPS / Apt / GA / CS / PB.

[0047] Place CNB / PLMA / LPS / Apt / GA / CS / PB in a dark box, take photos with a smartphone under the same height and light intensity, and obtain the RGB values ​​through the "Color" App.

[0048] Example 4:

[0049] 1. Fabrication of paper-based sensors:

[0050] Dissolve 1.25 g of CS in 50 mL of 2% acetic acid and stir with a magnetic stirrer for 3-4 h to obtain a CS solution. Prepare PB on the surface of qualitative filter paper using a 3 mm diameter perforator. Immerse the PB in the CS solution, then place it in a petri dish and dry it in a vacuum drying oven at 50 °C for 1 h to obtain CS / PB. Then, drop-cast 20 μL of 5% GA (v / v) onto the CS / PB surface and incubate for 60 minutes to obtain GA / CS / PB. Drop-cast 20 μL of 2 μM Apt onto GA / CS / PB and incubate for 60 minutes to obtain Apt / GA / CS / PB.

[0051] 2. Application of paper-based sensors in the detection of lipopolysaccharides:

[0052] Apt / GA / CS / PB was incubated in a lipopolysaccharide-containing sample for 60 minutes. LPS and Apt were prepared via specific base pair recognition. To eliminate the influence of exposed aldehyde groups due to lack of interaction with amino Apt, 2MEACl was selected as the blocking agent. 20 μL of EACl was incubated on LPS / Apt / GA / CS / PB for 6 hours to obtain EACl / LPS / Apt / GA / CS / PB. Most monosaccharide residues in the LPS polysaccharide chain contain at least one cis-diol site. Since cis-diol can selectively crosslink with borate groups, BPBA / EACl / LPS / Apt / GA / CS / PB was obtained by incubation for 60 minutes in a 0.5 mM MBPBA solution of 10 mM pH 7.4 PBS containing 2% (v / v) N,N-dimethylformamide. Then, BPBA / EACl / LPS / Apt / GA / CS / PB was immersed in a carbon quantum dot-catalyzed UATRP solution. 1 mL of the polymerization solution contained 40 mM copper bromide, 21.6 mM MLA, 34.4 μM TPMA, 2 mg CQDs, and 0.36 mL deionized water. Activation was performed on the modified electrode surface under LED light at room temperature for 16 minutes to obtain PLMA / LPS / Apt / GA / CS / PB. The carbon quantum dots were prepared by dissolving 1 g of o-phenylenediamine in 100 mL of deionized water, adding it to the reaction vessel, and heating at 200 °C for 8 h. After the reaction vessel cooled naturally to room temperature, the mixture was filtered through a 0.22 μm membrane and freeze-dried. Finally, PLMA / LPS / Apt / GA / CS / PB was dissolved in 0.0002 M CNB solution for 40 minutes to obtain CNB / PLMA / LPS / Apt / GA / CS / PB.

[0053] Place the CNB / PLMA / LPS / Apt / GA / CS / PB cameras inside a dark box. Under the same height and light intensity, take photos with a smartphone and obtain the RGB values ​​using the "Color" app.

[0054] Example 5:

[0055] 1. Fabrication of paper-based sensors:

[0056] Dissolve 1.25 g of CS in 50 mL of 2% acetic acid and stir with a magnetic stirrer for 3-4 h to obtain a CS solution. Prepare PB on the surface of qualitative filter paper using a 3 mm diameter perforator. Immerse the PB in the CS solution, then place it in a petri dish and dry it in a vacuum drying oven at 50 °C for 1 h to obtain CS / PB. Then, drop-cast 20 μL of 10% GA (v / v) onto the CS / PB surface and incubate for 120 minutes to obtain GA / CS / PB. Drop-cast 20 μL of 2 μM Apt onto GA / CS / PB and incubate for 120 minutes to obtain Apt / GA / CS / PB.

[0057] 2. Application of paper-based sensors in the detection of lipopolysaccharides:

[0058] Apt / GA / CS / PB was incubated in a lipopolysaccharide-containing sample for 120 minutes. LPS and Apt were prepared via specific base pair recognition. To eliminate the influence of exposed aldehyde groups due to lack of interaction with amino Apt, 4MEACl was selected as the blocking agent. 20 μL of EACl was incubated on LPS / Apt / GA / CS / PB for 12 hours to obtain EACl / LPS / Apt / GA / CS / PB. Most monosaccharide residues in the LPS polysaccharide chain contain at least one cis-diol site. Since cis-diol can selectively crosslink with borate groups, BPBA / EACl / LPS / Apt / GA / CS / PB was obtained by incubation for 120 minutes in a 1 mM PBS solution containing 2% (v / v) N,N-dimethylformamide. Then, BPBA / EACl / LPS / Apt / GA / CS / PB was immersed in a carbon quantum dot-catalyzed UATRP solution. 1 mL of the polymerization solution contained 80 mM copper bromide, 43.2 mM MLA, 68.8 μM TPMA, 4 mg CQDs, and 0.36 mL deionized water. Activation was performed on the modified electrode surface under LED light at room temperature for 32 minutes to obtain PLMA / LPS / Apt / GA / CS / PB. The carbon quantum dots were prepared by dissolving 1 g of o-phenylenediamine in 100 mL of deionized water, adding it to the reactor, and heating at 200 °C for 8 h. After the reactor cooled naturally to room temperature, the mixture was filtered through a 0.22 μm membrane and freeze-dried. Finally, PLMA / LPS / Apt / GA / CS / PB was dissolved in 0.0004 M CNB solution for 80 minutes to obtain CNB / PLMA / LPS / Apt / GA / CS / PB.

[0059] Place the CNB / PLMA / LPS / Apt / GA / CS / PB cameras inside a dark box. Under the same height and light intensity, take photos with a smartphone and obtain the RGB values ​​using the "Color" app.

[0060] The embodiments described above are merely preferred embodiments of the present invention, and not all feasible embodiments of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A method for detecting lipopolysaccharides using a paper-based sensor for non-diagnostic purposes, characterized in that, Apt / GA / CS / PB was incubated in samples containing lipopolysaccharides for 7.5–120 minutes. LPS and Apt were prepared via specific base pair recognition. Using 0.25–4 M ethylamine hydrochloride as a blocking agent, 20 μL of EACl was incubated on LPS / Apt / GA / CS / PB for 0.75–12 hours to obtain EACl / LPS / Apt / GA / CS / PB. Most monosaccharide residues in the LPS polysaccharide chain contain at least one cis-diol site. 0.0625–1 mM EACl solution was prepared using 10 mM pH 7.4 PBS containing 2% V / V N,N-dimethylformamide. BPBA / EACl / LPS / Apt / GA / CS / PB was obtained by incubation in 4-(bromomethyl)phenylboronic acid solution for 7.5-120 minutes. BPBA / EACl / LPS / Apt / GA / CS / PB was then immersed in a carbon quantum dot-catalyzed UATRP polymerization solution. 1 mL of this polymerization solution contained 5-80 mM copper bromide, 2.7-43.2 mM methacrolein, 4.3-68.8 μM tris(2-pyridinemethyl)amine, 0.25-4 mg of carbon quantum dots, and 0.36 mL of deionized water. Activation was performed on the modified electrode surface for 2-32 minutes at room temperature and under LED light to obtain PLMA / LPS / Apt / GA / CS / PB. The carbon quantum dots were prepared by dissolving 1 g of o-phenylenediamine in 100 mL of deionized water and adding it to a reaction vessel. The mixture was heated at 200 °C for 8 h. After the reaction vessel naturally cooled to room temperature, 0.22... After filtration through a μm membrane and freeze-drying, PLMA / LPS / Apt / GA / CS / PB was obtained; finally, CNB / PLMA / LPS / Apt / GA / CS / PB was obtained by immersing PLMA / LPS / Apt / GA / CS / PB in 0.000025-0.0004 M Nell Blue chloride solution for 5-80 minutes. CNB / PLMA / LPS / Apt / GA / CS / PB were placed in a dark box. Under the same height and light intensity, images were taken with a smartphone, and the RGB values ​​were obtained through the "ColorCare" app. The detection results were calculated based on a linear regression equation, which is R / (R+G+B)=-0.016log c +0.385, correlation coefficient R 2 =0.997; where c is the LPS concentration, in pg / mL; The specific steps for fabricating a paper-based sensor are as follows: S1. Dissolve 1.25 g CS in 50 mL of acetic acid with a V / V of 2%, and stir with a magnetic stirrer for 3-4 h to obtain a CS solution; S2. Paper-based PB was prepared on the surface of qualitative filter paper using a 3 mm diameter punch. After immersing PB in CS solution, it was removed and placed in a petri dish. It was dried in a vacuum drying oven at 50 ℃ for 1 h to obtain CS / PB. 20 μL of GA with a V / V of 0.625-10% was dripped onto the CS / PB surface. After 7.5-120 minutes, GA / CS / PB was obtained. S3. Drop 20 μL of 2 μM Apt onto GA / CS / PB and incubate for 7.5–120 minutes to obtain Apt / GA / CS / PB; LPS is lipopolysaccharide, GA is glutaraldehyde, CS is chitosan, PB is paper base, EACl is ethylamine hydrochloride, CNB is Nell blue chloride, BPBA is 4-(bromomethyl)phenylboronic acid, and Apt is an aptamer.

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