A paper-based microfluidic sensor array platform and its preparation method and application

Abstract

The present invention discloses a paper-based microfluidic sensor array platform and its preparation method and application. The present invention combines paper-based microfluidic technology, nanotechnology and fluorescence sensing technology to prepare a fluorescence sensing detection platform for rapid detection and identification of multiple biothiols. The paper-based microfluidic sensor array platform of the present invention is a "signal off-on" type. After the biothiol receptor is introduced into the paper-based chip, the fluorescence of the carbon quantum dots is quenched, and after the biothiol is added, the fluorescence will be restored. The present invention utilizes the difference in fluorescence intensity produced by the reaction of multiple biothiol receptors with different biothiols, and establishes recognition modes of different biothiols according to the change in fluorescence intensity. A low-cost, high-efficiency multi-channel paper-based microfluidic sensor array is established through inkjet printing technology. The complex multidimensional data is converted into a simple visual map through a multivariate analysis method to accurately distinguish single and mixed biothiols.

Description

Technical Field

[0001] The present invention relates to the field of biosensor technology, and in particular to a paper-based microfluidic sensor array platform and a preparation method and application thereof. Background Art

[0002] Biothiols, such as cysteine, homocysteine, and glutathione, play vital roles in various physiological functions and diseases due to their intrinsic redox and nucleophilic properties. In general, abnormal levels of biothiols in the body are associated with many chronic and degenerative diseases, such as cancer, Alzheimer's disease, cardiovascular disease, and neurodegenerative diseases. Therefore, monitoring changes in biothiols levels is of great significance in fields such as medical research and disease diagnosis.

[0003] Paper-based microfluidic devices have been widely developed and applied for point-of-care testing due to their simplicity, disposable and low cost. Hydrophobic barriers are prepared by printing on paper-based materials to form hydrophilic areas. Samples can be spontaneously transported to the detection area through the capillary force provided by the paper-based fiber structure without any external driving force, thus achieving compact and rapid detection. So far, paper-based microfluidic devices have been combined with various detection techniques, including colorimetry, electrochemistry, fluorescence, chemiluminescence, etc. Among them, fluorescence-based paper-based microfluidic devices are gradually becoming a new development trend, benefiting from the advantages of portability, integration and sensitivity.

[0004] Compared to traditional sensors with high selectivity, sensor arrays are composed of a series of non-selective sensing units, which mimic the olfactory or taste system of mammals. Sensor arrays produce non-specific responses to analytes, and their responses are processed by multivariate analysis (linear discriminant analysis, hierarchical cluster analysis, or principal component analysis) to obtain a unique fingerprint for identifying the target. Sensor arrays do not require the laborious synthesis of specific recognition receptors and have advantages in diversity, anti-interference, and simultaneous detection and discrimination of a series of compounds with similar structures or properties.

[0005] At present, the method of detecting biothiols is still to use sensors with high selectivity. Each sensor detects a specific type of biothiols, and the operation is complicated when detecting multiple biothiols. At present, there are also sensors that can quantitatively detect multiple biothiols, but they can only detect biothiols and cannot specifically determine what type of biothiols they are. The detection method still has inconveniences. Summary of the invention

[0006] The purpose of the present invention is to overcome the above-mentioned deficiencies in the prior art and to provide a paper-based microfluidic sensor array platform and a preparation method and application thereof.

[0007] The first object of the present invention is to provide a method for preparing a paper-based chip.

[0008] A second object of the present invention is to provide a paper-based chip.

[0009] The third object of the present invention is to provide an application of the paper-based chip in preparing a paper-based microfluidic device.

[0010] A fourth object of the present invention is to provide a paper-based microfluidic device.

[0011] A fifth object of the present invention is to provide an application of the paper-based microfluidic device in preparing a device for detecting biothiols.

[0012] A sixth object of the present invention is to provide a paper-based microfluidic sensor array platform.

[0013] In order to achieve the above object, the present invention is implemented by the following scheme:

[0014] A method for preparing a paper-based chip comprises the following steps:

[0015] S1: activating hydroxyl groups on the glass fiber paper, washing to obtain activated glass fiber paper, and then modifying amino groups on the activated glass fiber paper to obtain pretreated glass fiber paper;

[0016] S2: reacting a solution containing carbon quantum dots, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride with the pretreated glass fiber paper in a dark environment to obtain glass fiber paper modified with carbon quantum dots;

[0017] S3: mixing a biothiol receptor, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and reacting the mixture with the carbon quantum dot-modified glass fiber paper in a dark environment to obtain a paper-based chip, wherein the biothiol receptor is a Michael receptor.

[0018] Preferably, the preparation method of the carbon quantum dots in step S2 is: preparing an aqueous solution containing citric acid and urea, heating at 160-200° C., centrifuging, removing solids, collecting the supernatant, and dialyzing to obtain carbon quantum dots.

[0019] More preferably, the mass ratio of citric acid to urea is 0.5-1.1:1, the heating time is 11-13 hours, the centrifugation is 8000-10500 rpm for 10-12 minutes, the molecular weight cutoff of the dialysis bag is 500-1000, and the dialysis time is 30-50 hours.

[0020] Most preferably, the mass ratio of citric acid to urea is 1:1, the heating temperature is 200° C., the heating time is 12 h, the centrifugation is 10000 rpm for 10 min, the molecular weight cutoff of the dialysis bag is 500, and the dialysis time is 48 h.

[0021] Preferably, in step S1, the hydroxyl groups on the glass fiber paper are activated with hydrochloric acid, and the amino groups on the activated glass fiber paper are modified with a mixed solution of ethanol and water containing aminopropyltriethoxysilane;

[0022] In step S2, the mass ratio of the carbon quantum dots to N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:248-252:123-127, and the reaction time is 4-7 hours;

[0023] In step S3, the masses of N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are the same as those used in preparing glass fiber paper modified with carbon quantum dots, and the reaction time is 2 to 3.5 hours.

[0024] More preferably, in step S1, the glass fiber paper is soaked with hydrochloric acid, and the hydrochloric acid is washed away to obtain the glass fiber paper treated with hydrochloric acid; and the glass fiber paper soaked with hydrochloric acid is soaked with a mixed solution of ethanol and water containing aminopropyltriethoxysilane to obtain the pretreated glass fiber paper;

[0025] More preferably, in step S1, the concentration of the hydrochloric acid is 0.18-0.22M, and the immersion time is 15-30min; the volume ratio of the aminopropyltriethoxysilane to ethanol and water is 0.19-0.21:1, the volume ratio of ethanol to water is 0.9-1.1:1, and the immersion time of the ethanol and water mixed solution containing aminopropyltriethoxysilane is 3-4.5h;

[0026] More preferably, in step S1, the concentration of the hydrochloric acid is 0.2 M and the soaking time is 30 min.

[0027] More preferably, in step S1, the volume ratio of aminopropyltriethoxysilane to ethanol and water is 0.2:1, the volume ratio of ethanol to water is 1:1, and the immersion time of the mixed solution of ethanol and water containing aminopropyltriethoxysilane is 4 hours.

[0028] More preferably, in step S2, the mass ratio of the carbon quantum dots to N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:250:125, and the reaction time is 6 hours.

[0029] More preferably, in step S3, the masses of N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are the same as those used in preparing glass fiber paper modified with carbon quantum dots, and the reaction time is 3 h.

[0030] Preferably, the biothiol acceptor in step S3 is 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzoic acid, 4-(2,2-dicyanovinyl)benzoic acid and / or (E)-4-(2-nitrovinyl)benzoic acid.

[0031] More preferably, the mass ratio of the carbon quantum dots to 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzoic acid is 1:374-416, the mass ratio of the carbon quantum dots to 4-(2,2-dicyanovinyl)benzoic acid is 1:331-368, and the mass ratio of the carbon quantum dots to (E)-4-(2-nitrovinyl)benzoic acid is 1:230-386.

[0032] More preferably, the mass ratio of the carbon quantum dots to 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzoic acid is 1:435, the mass ratio of the carbon quantum dots to 4-(2,2-dicyanovinyl)benzoic acid is 1:355, and the mass ratio of the carbon quantum dots to (E)-4-(2-nitrovinyl)benzoic acid is 1:240.

[0033] A paper-based chip is prepared by the preparation method.

[0034] The paper-based chip is used in preparing a paper-based microfluidic device.

[0035] A paper-based microfluidic device, characterized in that it is composed of a top paper layer and a bottom paper layer, the top paper layer and the bottom paper layer are detachably connected, the top paper layer is composed of a hydrophilic region and a hydrophobic region, the hydrophilic region is composed of a sample inlet and a detection unit connected by a hydrophilic channel, and each hydrophilic region contains more than two detection units;

[0036] The bottom paper layer is fixed with two or more paper-based chips according to claim 5, each paper-based chip is modified with a different biothiol receptor;

[0037] The detection unit of the top paper layer corresponds to the position of the paper-based chip of the bottom paper layer.

[0038] Preferably, the biothiol acceptor is two or three of 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzoic acid, 4-(2,2-dicyanovinyl)benzoic acid or (E)-4-(2-nitrovinyl)benzoic acid;

[0039] The concentration of the 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzoic acid is 1.8 to 2.0 mM, the concentration of the 4-(2,2-dicyanovinyl)benzoic acid is 1.8 to 2.0 mM, and the concentration of the (E)-4-(2-nitrovinyl)benzoic acid is 1.2 to 2.0 mM.

[0040] More preferably, the concentration of the 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzoic acid is 2.0 mM, the concentration of the 4-(2,2-dicyanovinyl)benzoic acid is 1.8 mM, and the concentration of the (E)-4-(2-nitrovinyl)benzoic acid is 1.2 mM.

[0041] Preferably, an observation port is also provided on the top paper layer.

[0042] More preferably, the top paper layer and the bottom paper layer are riveted together by rivets to form a paper-based microfluidic device.

[0043] More preferably, the top paper layer is circular filter paper, the hydrophilic regions are distributed in a dendrite-like manner, and the sample inlet and the circular detection unit are both circular.

[0044] More preferably, each top paper layer has 1 to 4 hydrophilic areas, each hydrophilic area has a sample inlet located in the middle of the top paper layer, and each hydrophilic area has 2 to 3 detection units located at the edge of the top paper layer. When the sample solution to be tested is dripped into the sample inlet, due to the capillary action of the hydrophilic area, the sample solution to be tested enters multiple detection units simultaneously through the hydrophilic channel.

[0045] Most preferably, each top paper layer has 4 hydrophilic areas and each hydrophilic area has 3 detection units.

[0046] More preferably, the bottom paper layer is a circular filter paper of the same size as the top paper layer, and the bottom paper layer is affixed with a circular paper-based chip as a sensing unit corresponding to the properties, size and position of the detection unit of the top paper layer.

[0047] More preferably, each hydrophilic area and its 2 to 3 detection units corresponding to 2 to 3 paper-based chips constitute a detection group. The other areas of the bottom paper layer are hydrophobic areas. After the sample solution reaches the detection unit, it penetrates into the paper-based chip of the bottom paper layer. The paper-based chip is consistent in shape and size with the observation port.

[0048] Most preferably, each hydrophilic region and three paper-based chips corresponding to its three detection units constitute a detection group.

[0049] When the top paper layer and the bottom paper layer are riveted together, the side of the bottom paper layer on which the paper-based chip is attached is located inside the paper-based microfluidic device.

[0050] The paper-based microfluidic device is used in the preparation of a device for detecting biological thiols. The device for detecting biological thiols is a device for detecting the type and / or content of biological thiols.

[0051] A paper-based microfluidic sensor array platform comprises the paper-based microfluidic device, a fluorescence detection device and a computer.

[0052] When in use, after the sample solution to be tested is dripped into the sample inlet, due to the capillary action of the hydrophilic area, the sample solution to be tested passes through the hydrophilic channel and enters multiple detection units at the same time. After reaching the detection unit, the sample solution to be tested penetrates into the paper-based chip of the bottom paper layer of the paper-based microfluidic device to form a paper-based chip to be detected. The observation port of the paper-based microfluidic device is overlapped with the paper-based chip to be detected, and the paper-based microfluidic device is placed in a fluorescence detection device. The fluorescence detection device emits ultraviolet light, which is irradiated on the paper-based chip to be detected through the observation port to detect the fluorescence intensity of the sample to be tested on the paper-based chip to be detected. After completing the detection of one of the detection groups, that is, the detection of 3 detection units in the same hydrophilic area, the paper-based microfluidic device is rotated to continue to detect the fluorescence intensity of the next detection group.

[0053] The computer is connected to the fluorescence detection device circuit to collect and process the fluorescence intensity data of the sample to be tested. The fluorescence spectrum standard data of multiple biological thiols to be tested are processed by the computer through the origin software from multiple dimensions, the difference in fluorescence intensity change ((F-F0) / F) is calculated, and a visualized standard spectrum is obtained through linear discriminant and hierarchical cluster analysis. Combined with the data and position of the standard biological thiol, the object to be tested that is close to the position and data of the standard biological thiol contains the corresponding type of biological thiol, thereby determining the type of biological thiol contained in the object to be tested.

[0054] The diversity of electrophilicity in biothiol receptors and the diversity of nucleophilicity in biothiols make each biothiol receptor react with 6 different types of biothiols at different rates, and the extent to which carbon quantum dots recover fluorescence is different, resulting in unique fluorescence reactions.

[0055] If only the same biothiol receptor is targeted, different biothiols may produce results with similar fluorescence intensities, making it difficult to distinguish the types of biothiols; however, for multiple biothiol receptors, different biothiols produce multiple fluorescence intensities after reaction, and then from multiple dimensions (each biothiol receptor is used as a dimension), the types of biothiols can be clearly distinguished. Although the structures and reactivity of the six biothiols are similar, the differences in the reactions of the three biothiol receptors with biothiols can still be used to accurately distinguish the types of biothiols. At the same time, the present invention is also used to detect other types of biothiols other than (MPA), cysteine ​​(Cys), dithiothreitol (DTT), dimercaptosuccinic acid (DMSA), homocysteine ​​(Hcy) and glutathione (GSH).

[0056] Compared with the prior art, the present invention has the following beneficial effects:

[0057] The present invention discloses a paper-based microfluidic sensor array platform and its preparation method and application. The present invention combines paper-based microfluidic technology, nanotechnology and fluorescence sensing technology to prepare a fluorescence sensing detection platform for rapid detection and identification of multiple biothiols. The paper-based microfluidic sensor array platform of the present invention is a "signal off-on" type. After the biothiol receptor is introduced into the paper-based chip, the fluorescence of the carbon quantum dots is quenched, and after the biothiol is added, the fluorescence will be restored. The present invention utilizes the difference in fluorescence intensity generated by the reaction of multiple biothiol receptors with different biothiols, and establishes recognition modes of different biothiols according to the change in fluorescence intensity. A low-cost, high-efficiency multi-channel paper-based microfluidic sensor array is established through inkjet printing technology. The complex multidimensional data is converted into a simple visual map through a multivariate analysis method, and single and mixed biothiols are accurately distinguished, effectively solving the problems of cumbersome operation, large instrument volume and high cost. As a field testing tool, it is expected to play an important role in disease diagnosis, daily physiological monitoring, environmental pollution detection, food safety assessment and other aspects. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1The fluorescence spectra of different concentrations of biothiol receptors reacting with carbon quantum dots and the correlation diagram of fluorescence quenching efficiency and the concentration of biothiol receptors. A is the fluorescence spectrum of biothiol receptor 1 (TR1); B is the fluorescence spectrum of biothiol receptor 2 (TR2); C is the fluorescence spectrum of biothiol receptor 3 (TR3). D is the correlation diagram of biothiol receptor 1 (TR1), E is the correlation diagram of biothiol receptor 2 (TR2), and F is the correlation diagram of biothiol receptor 3 (TR3). The error bars represent the standard deviation of three parallel tests; the curves in each fluorescence spectrum are from top to bottom, respectively, 0.001, 0.01, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 and 2.0 mM biothiol receptors.

[0059] Figure 2 The fluorescence spectra of CQDs-TR1 after reacting with different concentrations of biothiols, where A is MPA, B is Cys, C is DTT, D is DMSA, E is GSH, and F is Hcy; the curves in each figure are 0, 10, 50 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 μM biothiols from bottom to top.

[0060] Figure 3 The fluorescence spectra of CQDs-TR2 after reacting with different concentrations of biothiols, where A is MPA, B is Cys, C is DTT, D is DMSA, E is GSH, and F is Hcy; the curves in each figure are 0, 10, 50 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 μM biothiols from bottom to top.

[0061] Figure 4 The fluorescence spectra of CQDs-TR3 after reacting with different concentrations of biothiols, where A is MPA, B is Cys, C is DTT, D is DMSA, E is GSH, and F is Hcy; the curves in each figure are 0, 10, 50 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 μM biothiols from bottom to top.

[0062] Figure 5 Schematic diagram of the paper-based microfluidic device (μPAD). 1-μPAD, 13-rivets.

[0063] Figure 6 Schematic diagram of the structure of the top paper layer of the paper-based microfluidic device (μPAD). 11-top paper layer, 111-hydrophilic area, 112-hydrophobic area, 113-observation port, 1111-sample inlet, 1113-detection unit, 1112-hydrophilic channel.

[0064] Figure 7 Schematic diagram of the bottom paper layer of the paper-based microfluidic device (μPAD). 121-Paper-based chip.

[0065] Figure 8 The state of the paper-based microfluidic device (μPAD) after the sample solution to be tested is added.

[0066] Fig. 9 The paper-based microfluidic device (μPAD) is placed in a fluorescence detection device.

[0067] Fig.10 Schematic diagram of the connection between the paper-based microfluidic device (μPAD) and the fluorescence detection device.

[0068] Fig.11 Schematic diagram of the paper-based microfluidic sensor array platform for the detection of biothiols.

[0069] Fig.12 Flowchart of the paper-based microfluidic sensor array platform for the detection of biothiols.

[0070] Fig.13 The detection results of 6 biothiols with a concentration of 10 μM. A is a typical score map; B is a cluster heat map; C is a box plot.

[0071] Fig.14 A typical score plot of an analyte containing DMSA and Cys biothiol.

[0072] Fig.15 The following is a typical score graph for an analyte containing Cys, DMSA and GSH biothiols. T1 is: 60% Cys-20% DMSA-20% GSH; T 2 For: 20% Cys-60% DMSA-20% GSH; T 3 It is: 20% Cys-20% DMSA-60% GSH.

[0073] Fig.16 Scoring of the identification criteria for six biothiols in fetal bovine serum. DETAILED DESCRIPTION

[0074] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0075] Example 1 Preparation of carbon quantum dots, biothiol receptors and paper chips

[0076] 1. Experimental Methods

[0077] 1. Preparation of carbon quantum dots (CQDs)

[0078] 1g of citric acid (CA) and 1g of urea were dissolved in 10mL of water by hydrothermal method to obtain a mixed solution, which was then transferred to a 25mL Teflon-lined autoclave, heated at 180°C for 12h, and centrifuged at 10000rpm for 10min to remove the solids in the mixed solution. The centrifuged supernatant was collected and dialyzed in a dialysis bag (MWCO of 500) for 48h. The dialysate was deionized water. The sample in the dialysis bag was freeze-dried to obtain a solid phase, which was carbon quantum dots.

[0079] 2. Preparation of biological thiol receptors

[0080] (1) Preparation of biological thiol receptor 1:

[0081] A solution of 4-aminobenzoic acid (0.9 g) and succinic anhydride (718.61 mg) in 10 mL of dioxane was stirred at 100°C for 10 min to obtain reaction mixture 1. 2.98 g of ammonium sulfate and 0.93 mL of DMSO were added to reaction mixture 1 and heated at 100°C for 5 h to obtain reaction mixture 2. Reaction mixture 2 was filtered with a cotton plug and dioxane in reaction mixture 2 was removed in vacuo to obtain a crude product. The crude product was dissolved in ethyl acetate and treated with 0.1 mol / L dilute hydrochloric acid, saturated NaHCO 3 After washing, add anhydrous Na 2 SO 4 The product was dried and dehydrated, the solvent was evaporated under vacuum conditions, and recrystallized to obtain 1.1 g of 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzoic acid, i.e., biothiol receptor 1.

[0082] (2) Preparation of biothiol acceptor 2: To CH4-aminobenzoic acid at 0°C 3 5 mmol of PhI(OAc) was added to the CN solution 2 and 6mmol malononitrile, the 4-aminobenzoic acid is 5.0mmol, the CH 3 CN volume was 20 mL to obtain mixture a. At 20-25 °C and N 2 The mixture was stirred under an atmosphere of 45 min to obtain a reaction mixture. The progress of the stirring reaction was monitored by TLC. After the stirring reaction was completed, the solvent of the reaction mixture was evaporated under vacuum and 5 mL of saturated NaHCO 3 The reaction mixture was quenched with 10 mL of EtOAc as an extracting solution to extract the biothiol receptor 2. The organic layers were combined, washed with saturated aqueous NaCl solution, and washed with Na 2SO 4 The product was dried and concentrated under reduced pressure, and recrystallized to obtain 0.8 g of 4-(2,2-dicyanovinyl)benzoic acid, i.e., biothiol acceptor 2.

[0083] (3) Preparation of biothiol receptor 3: In a 25 mL Schlenk tube, add 4 mmol of (E)-4-(2-carboxyvinyl)benzoic acid, 4.8 mmol of Cu(NO 3 ) 2 and 20 mL of CH 3 CN to obtain mixture b, which was sealed and then stirred at 110°C in air for 8 h. The progress of the reaction was monitored by TLC. After the stirring reaction was completed, 30 mL of water was added to obtain mixture c. The biothiol receptor 3 in mixture c was extracted with dichloromethane (3x20 mL), and the combined organic layers were successively treated with 30 mL of saturated NaHCO 3 The solution was washed with 20 mL of water and then 2 SO 4 The residue was dried on a plate and concentrated using a rotary evaporator. The concentrate was purified by recrystallization to obtain 0.5 g of (E)-4-(2-nitrovinyl)benzoic acid, i.e., biothiol receptor 3.

[0084] 3. Preparation of paper chips

[0085] (1) Soak Φ7 mm glass fiber paper in 0.2 M hydrochloric acid solution for 30 min and wash with deionized water to remove excess hydrochloric acid.

[0086] (2) Soak the glass fiber paper soaked in hydrochloric acid in step (1) in 20 mL of ethanol / water (1:1, v / v) containing 200 μL of aminopropyltriethoxysilane for 4 h, and then fix it on a culture dish to prevent it from floating.

[0087] (3) Add 1 mL of carbon quantum dots (1 mg / mL), 5 mL of 50 mg / mL of N-hydroxysuccinimide and 5 mL of 25 mg / mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to the culture dish of step (2), and react with the paper in the dark for 6 hours. The carbon quantum dots are modified on the paper surface through an amide reaction between the carboxyl groups of the carbon quantum dots and the amino groups on the paper surface.

[0088] (4) Wash the paper after the reaction in step (3) with deionized water for 3 times, then add 1 mL of the biothiol receptor (biothiol receptor 1, biothiol receptor 2 or biothiol receptor 3) prepared in this example, 5 mL of N-hydroxysuccinimide with a concentration of 50 mg / mL and 5 mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride with a concentration of 25 mg / mL in a culture dish, and react with the paper in the dark for 3 hours, the amino groups of the carbon quantum dots react with the carboxyl groups on the surface of the biothiol receptor to modify the biothiol receptor on the surface of the carbon quantum dots. The obtained paper after the reaction is a paper-based chip loaded with carbon quantum dots and biothiol receptors (CQDs-TRs), which is used as a sensing unit, and the paper-based chip is stored at 4°C.

[0089] Example 2 Effect of Biothiol Receptor Concentration on Detection

[0090] 1. Experimental Methods

[0091] The biothiol receptor was combined with carbon quantum dots to generate a fluorescent probe, and the effect of different biothiol receptor concentrations on the fluorescence intensity of the fluorescent probe was detected by fluorescence titration.

[0092] Experimental steps: First, 0.1 mL (1 g / mL) of the carbon quantum dot solution prepared in Example 1 was added with 0.1 mL of different concentrations of biothiol receptors 1, 2 and 3 prepared in Example 1, and the experimental concentrations of each biothiol receptor were 0.001, 0.01, 0.05, 0.1, 0.2, 0.4, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8 and 2.0 mM, respectively. Then, 0.4 mL of N-hydroxysuccinimide (50 mg / mL) and 0.4 mL of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (25 mg / mL) were added and reacted for 3 hours. The amino groups on the carbon quantum dots reacted with the amino groups on the biothiol receptors to modify the biothiol receptors, and then fluorescence detection was performed.

[0093] 2. Experimental Results

[0094] like Figure 1 A. Figure 1 B and Figure 1 As shown in Figure C, as the concentration of the biothiol receptor increases, the fluorescence intensity of the carbon quantum dots gradually decreases, indicating that the three biothiol receptors prepared in Example 1 effectively quench the fluorescence of the carbon quantum dots. Figure 1 D. Figure 1 E and Figure 1As shown in Figure F, compared with the three biothiol receptors at the same concentration, biothiol receptor 3 has the most effective quenching effect. The optimal concentrations of biothiol receptor 1, biothiol receptor 2, and biothiol receptor 3 are 2.0 mM, 1.8 mM, and 1.2 mM, respectively, and their quenching reaction reaches about 90% at these concentrations.

[0095] Example 3 Interaction between biothiol receptor (TR) and biothiols

[0096] 1. Experimental Methods

[0097] The biothiols to be tested include: 3-mercaptopropionic acid (MPA), cysteine ​​(Cys), dithiothreitol (DTT), dimercaptosuccinic acid (DMSA), homocysteine ​​(Hcy) and glutathione (GSH).

[0098] Biothiol receptors combined with carbon quantum dots (CQDs-TRs): The carbon quantum dots prepared in Example 1 are respectively combined with biothiol receptors (TR) 1 to 3 prepared in Example 1 to generate probes, namely CQDs-TR1, CQDs-TR2 and CQDs-TR3.

[0099] The fluorescence titration method was used to detect the change in fluorescence intensity of the fluorescent probes generated after the above 6 biothiols reacted with the 3 biothiol receptors prepared in Example 1 respectively.

[0100] The specific experimental steps are as follows: first, 0.1 mL (1 g / mL) of the carbon quantum dot solution prepared in Example 1 was added with 0.1 mL, 2.0 mM of biothiol receptor 1, 0.1 mL, 1.8 mM of biothiol receptor 2, and 0.1 mL, 1.2 mM of biothiol receptor 3, followed by the addition of 0.4 mL of N-hydroxysuccinimide (50 mg / mL) and 0.4 mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (25 mg / mL), and the reaction was continued for 3 h. The amino groups on the carbon quantum dots reacted with the amino groups on the receptors to modify the receptors. Then, different concentrations (0, 10, 50 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 μM) of biothiols were added for detection.

[0101] 2. Experimental Results

[0102] like Figure 2 As shown, CQDs-TR1 and different concentrations of biothiol MPA ( Figure 2 A) Cys( Figure 2 B) DTT( Figure 2 C) DMSA ( Figure 2 D) Hcy Figure 2 E) and GSH ( Figure 2F) Fluorescence spectrum after the reaction. As the concentration of biothiols increases, the fluorescence intensity of CQDs-TR1 gradually increases. Different biothiols have different degrees of inhibition on the fluorescence intensity of CQDs-TR1.

[0103] like Figure 3 As shown, CQDs-TR2 and different concentrations of biothiol MPA ( Figure 3 A) Cys( Figure 3 B) DTT( Figure 3 C) DMSA ( Figure 3 D) Hcy Figure 3 E) and GSH ( Figure 3 F) Fluorescence spectrum after the reaction. As the concentration of biothiols increases, the fluorescence intensity of CQDs-TR2 gradually increases. Different biothiols have different degrees of inhibition on the fluorescence intensity of CQDs-TR2.

[0104] like Figure 4 As shown, CQDs-TR3 and different concentrations of biothiol MPA ( Figure 4 A) Cys( Figure 4 B) DTT( Figure 4 C) DMSA ( Figure 4 D) Hcy Figure 4 E) and GSH ( Figure 4 F) Fluorescence spectrum after the reaction. As the concentration of biothiol increases, the fluorescence intensity of CQDs-TR3 gradually increases. Different biothiols have different degrees of inhibition on the fluorescence intensity of CQDs-TR3.

[0105] The biothiol receptors 1 to 3 prepared in Example 1 of the present invention are all self-synthesized Michael receptors, which can trigger the light-induced electron transfer effect and quench the fluorescence of carbon quantum dots.

[0106] Biothiols have high nucleophilicity and when reacting with biothiol acceptors, they destroy the photoinduced electron transfer effect, resulting in partial recovery of fluorescence. The -SH groups in the six biothiols react with the electrophilic groups in the biothiol acceptors through Michael addition reactions, thereby destroying the photoinduced electron transfer effect between carbon quantum dots and biothiol acceptors.

[0107] The diversity of electrophilicity in biothiol receptors and the diversity of nucleophilicity in biothiols make each biothiol receptor react with 6 different types of biothiols at different rates, and the extent to which carbon quantum dots recover fluorescence is different, resulting in unique fluorescence reactions.

[0108] If only the same biothiol receptor is targeted, different biothiols may produce results with similar fluorescence intensities, making it difficult to distinguish the types of biothiols; however, for multiple biothiol receptors, different biothiols produce multiple fluorescence intensities after reaction. If we look at them comprehensively from multiple dimensions (each biothiol receptor is used as a dimension), we can clearly distinguish the types of biothiols. Although the structures and reactivities of the six biothiols are similar, the differences in the reactions between the three biothiol receptors and biothiols can still be used to accurately distinguish the types of biothiols. Using the unique fluorescence response pattern produced by each biothiol as a "fingerprint", it can be distinguished by linear discriminant analysis and hierarchical clustering analysis within the range of 5 to 100 μM.

[0109] Example 4 Preparation of a paper-based microfluidic sensor array platform for detecting biothiols

[0110] 1. Preparation of paper-based microfluidic device (μPAD)

[0111] The paper-based microfluidic device is composed of filter paper, designed using Solid-Works 2014 3D CAD software, and a hydrophobic border is printed on the filter paper using low-cost, easy-to-operate inkjet printing technology (Supporting Information). The paper-based microfluidic device is composed of two layers of filter paper.

[0112] like Figure 5 , which is a schematic diagram of a paper-based microfluidic device 1 , a top paper layer 11 and a bottom paper layer 12 are riveted together by rivets 13 to form the paper-based microfluidic device 1 .

[0113] like Figure 6 As shown, it is a schematic diagram of the structure of the top paper layer 11 of the paper-based microfluidic device 1. The top paper layer 11 is a circular filter paper, which is composed of a hydrophilic area 111, a hydrophobic area 112 and an observation port 113 distributed in a dendrite shape. There are four hydrophilic areas 111, and the circular sample inlet 1111 and the circular detection unit 1113 in each hydrophilic area 111 are connected by a hydrophilic channel 1112.

[0114] Each hydrophilic region 111 has a sample inlet 1111 located in the middle of the top paper layer 11, and each hydrophilic region 111 has three detection units 1113 located at the edge of the top paper layer 11. When the sample solution to be tested is dripped into the sample inlet 1111, due to the capillary action of the hydrophilic region 111, the sample solution to be tested passes through the hydrophilic channel 1112 and enters multiple detection units 1113 at the same time.

[0115] like Figure 7As shown, it is a schematic diagram of the structure of the bottom paper layer 12 of the paper-based microfluidic device 1, wherein the bottom paper layer 12 is a circular filter paper of the same size as the top paper layer 11, and the bottom paper layer 12 is affixed with 12 circular paper-based chips 121 as sensing units, corresponding to the properties, size and position of the detection unit 1113 of the top paper layer 11. The three paper-based chips 121 corresponding to each hydrophilic area 111 and its three detection units 1113 are a detection group. The other areas of the bottom paper layer 12 are hydrophobic areas. After the sample solution reaches the detection unit 1113, it penetrates into the paper-based chip 121 of the bottom paper layer 12 of the paper-based microfluidic device 1. The paper-based chip 121 is consistent in shape and size with the observation port 113.

[0116] When the top paper layer 11 and the bottom paper layer 12 are riveted together, the side of the bottom paper layer 12 on which the paper-based chip 121 is attached is located inside the paper-based microfluidic device 1 .

[0117] The three paper-based chips 121 of each detection group are respectively the paper-based chip loaded with carbon quantum dots and biothiol receptor 1 (CQDs-TR1 / Paper sensing unit), the paper-based chip loaded with carbon quantum dots and biothiol receptor 2 (CQDs-TR2 / Paper sensing unit) and the paper-based chip loaded with carbon quantum dots and biothiol receptor 3 (CQDs-TR3 / Paper sensing unit) prepared in Example 1. During preparation, the concentrations of the biothiol receptor 1, biothiol receptor 2 and biothiol receptor 3 are 2.0 mM, 1.8 mM and 1.2 mM respectively.

[0118] like Figure 8 As shown, the state of the paper-based microfluidic device 1 after the sample solution to be tested is added dropwise.

[0119] 2. Preparation of a Paper-based Microfluidic Sensor Array Platform for Detecting Biothiols

[0120] like Fig. 9 and Fig.10 As shown, the paper-based microfluidic device 1 is placed in the experimental table of the fluorescence detection device 2 to detect samples in a dark environment. The fluorescence detection device 2 is provided with a through hole for emitting ultraviolet light to emit ultraviolet light to irradiate the paper-based chip 121. The experimental table of the fluorescence detection device 2 is prepared by 3D printing.

[0121] like Fig.11As shown, the paper-based microfluidic sensor array platform for detecting biothiols of the present invention is composed of three parts, namely: a paper-based microfluidic device 1, a fluorescence detection device 2 and a computer 3 for signal processing. The computer 3 is connected to the fluorescence detection device 2 circuit to collect and process the fluorescence intensity data of the sample to be tested. The computer 3 processes the fluorescence spectrum standard data of the biothiols from multiple dimensions, and obtains a visualized standard spectrum through linear discriminant and cluster analysis.

[0122] like Fig.12 As shown, when in use, after the sample solution to be tested is dripped into the sample inlet 1111, the observation port 113 of the paper-based microfluidic device 1 is overlapped with the paper-based chip 121 to be detected, and the paper-based microfluidic device 1 is placed in the fluorescence detection device 2. The fluorescence detection device 2 emits ultraviolet light, which is irradiated on the paper-based chip 121 to be detected through the observation port 113. The computer 3 detects and analyzes the fluorescence intensity of the sample to be tested on the paper-based chip 121 to be detected. After completing the detection of one of the detection groups, that is, the detection of the three detection units 1113 of the same hydrophilic area 111, the paper-based microfluidic device 1 is rotated to continue to detect and analyze the fluorescence intensity of the next detection group.

[0123] Example 5 Identification of biothiols using a paper-based microfluidic sensor array platform

[0124] 1. Experimental Methods

[0125] 1. Using the paper-based microfluidic sensor array platform prepared in Example 4, the six biothiols (MPA, Cys, DTT, DMSA, GSH and Hcy) in Example 3 were identified.

[0126] 15 μL of 6 biothiols with a concentration of 10 μM were used to react with the 3 sensor units in Example 4 respectively. The experiment was repeated 5 times. The fluorescence signal at 420 nm was detected and recorded by a fluorescence detection device. The initial fluorescence intensity F0 without adding biothiols and the fluorescence intensity F after the biothiols reacted were recorded. The difference in fluorescence intensity change ((F-F0) / F) was used to identify biothiols. A fluorescence reaction matrix consisting of 3 paper sheets × 6 biothiols × 5 repetitions was established. The multivariate analysis method (linear discriminant analysis LDA and hierarchical cluster analysis HCA) of origin software was used to convert complex multidimensional data into simple visualization patterns to obtain typical score maps, cluster heat maps and box plots.

[0127] 2. The paper-based microfluidic sensor array platform prepared in Example 4 was used to detect analytes containing 2 and 3 biothiols.

[0128] The test substance t contains 10 μM biothiol, which is composed of two biothiols, DMSA and Cys, wherein the test substance t1 contains 75% (7.5 μM) DMSA and 25% (2.5 μM) Cys, the test substance t2 contains 50% (5.0 μM) DMSA and 50% (5.0 μM) Cys, and the test substance t3 contains 25% (2.5 μM) DMSA and 75% (7.5 μM) Cys.

[0129] The test substance T contains 10 μM biothiols, which are composed of three types of biothiols: Cys, DMSA and GSH. Among them, the test substance T1 contains 60% (6.0 μM) Cys, 20% (2.0 μM) DMSA and 20% (2.0 μM) GSH, the test substance T2 contains 20% (2.0 μM) Cys, 60% (6.0 μM) DMSA and 20% (2.0 μM) GSH, and the test substance T3 contains 20% (2.0 μM) Cys, 20% (2.0 μM) DMSA and 60% (6.0 μM) GSH.

[0130] 15 μL of the test substance was taken to react with the three sensor units in Example 4 respectively, and the other detection steps were the same as those in Experiment 1 of this Example.

[0131] 3. Using the paper-based microfluidic sensor array platform prepared in Example 4, blind samples of 24 single biothiols and 24 biothiols mixtures containing 10 μM were tested.

[0132] 2. Experimental Results

[0133] 1. If Fig.13 As shown in A, 30 (6 biothiols × 5 repeats) typical fluorescence response patterns were divided into 6 different groups without overlap, indicating that the paper-based microfluidic sensor array platform prepared in Example 4 has good resolution for the 6 biothiols.

[0134] like Fig.13 As shown in Figure B, a clustering heat map was obtained based on the fluorescence intensity response of the paper-based microfluidic sensor array platform to the six biothiols. The clustering results showed that all biothiols were correctly assigned to their respective groups. The response of each sensing unit to the biothiols was clearly seen from the heat map. For the CQDs-TR1 / Paper sensing unit, the fluorescence intensity of GSH, HCY, and MPA did not change much, for the CQDs-TR2 / Paper sensing unit, the fluorescence intensity of GSH, DMSA, and HCY changed greatly, and for the CQDs-TR3 / Paper sensing unit, the fluorescence intensity of DTT and MPA changed greatly.

[0135] like Fig.13The box plot also depicts the differences in the responses of the three sensing units to each biothiol as shown in C. The differences in fluorescence intensity changes ((F-F0) / F) are: DTT>GSH>Hcy>MPA>Cys>DMSA.

[0136] 2. If Fig.14 As shown, it is a typical score diagram of analytes containing different concentrations of DMSA and Cys biothiol. These three analytes are completely separated, and the paper-based microfluidic sensor array platform of Example 4 of the present invention has good resolution.

[0137] like Fig.15 As shown, it is a typical score diagram of analytes containing different concentrations of Cys, DMSA and GSH biothiol. These three analytes are completely separated, and the paper-based microfluidic sensor array platform of Example 4 of the present invention has good resolution.

[0138] 3. As shown in Table 1, CQDs-TR 1 、CQDs-TR 2 and CQDs-TR 3 They are fluorescent probes obtained by reacting carbon quantum dots with biothiol receptors 1, 2 and 3 respectively. Table 1 records the fluorescence response values ​​of each fluorescent probe after reacting with the biothiol in the blind sample. The paper-based microfluidic sensor array platform of Example 4 of the present invention has accurate detection results for the biothiol receptors in the blind sample and strong specificity.

[0139] Table 1 Identification of 24 single biothiols and 24 biothiols mixtures in blind samples

[0140]

[0141]

[0142]

[0143]

[0144]

[0145] Example 6 Real sample detection

[0146] 1. Experimental Methods

[0147] The six biothiols (MPA, Cys, DTT, DMSA, GSH and Hcy) in Example 2 were added to fetal bovine serum (FBS) respectively, and the biothiols were detected using the paper-based microfluidic sensor array platform prepared in Example 4. FBS was diluted 50 times with deionized water before detection to reduce the matrix effect, and the added concentration of each biothiol was 10 μM.

[0148] 2. Experimental Results

[0149] like Fig.16 As shown, the identification standard scores of 6 biothiols in fetal bovine serum are obtained. These 6 biothiols are completely separated in fetal bovine serum.

[0150] In the subsequent analysis and detection of other real samples containing biothiols, the data of the standard biothiol detected by the paper-based microfluidic sensor array platform of Example 4 are compared with the data of the analyte. The analyte with a position and data close to the standard biothiol contains the corresponding type of biothiol.

[0151] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for preparing a paper-based chip, characterized in that: The following steps are involved: S1: activating hydroxyl groups on the glass fiber paper, washing to obtain activated glass fiber paper, and then modifying amino groups on the activated glass fiber paper to obtain pretreated glass fiber paper; S2: reacting a solution containing carbon quantum dots, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride with the pretreated glass fiber paper in a dark environment to obtain glass fiber paper modified with carbon quantum dots; S3: mixing a biothiol receptor, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and reacting the mixture with the carbon quantum dot-modified glass fiber paper in a dark environment to obtain a paper-based chip, wherein the biothiol receptor is a Michael receptor.

2. The preparation method according to claim 1, characterized in that: In step S1, hydroxyl groups on the glass fiber paper are activated with hydrochloric acid, and amino groups on the activated glass fiber paper are modified with a mixed solution of ethanol and water containing aminopropyltriethoxysilane; In step S2, the mass ratio of the carbon quantum dots to N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:248-252:123-127, and the reaction time is 4-7 hours; In step S3, the masses of N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are the same as those used in preparing glass fiber paper modified with carbon quantum dots, and the reaction time is 2 to 3.5 hours.

3. The preparation method according to claim 1, characterized in that: In step S3, the biothiol acceptor is 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzoic acid, 4-(2,2-dicyanovinyl)benzoic acid and / or (E)-4-(2-nitrovinyl)benzoic acid.

4. The preparation method according to claim 3, characterized in that: The mass ratio of the carbon quantum dots to 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzoic acid is 1:400-450, the mass ratio of the carbon quantum dots to 4-(2,2-dicyanovinyl)benzoic acid is 1:310-360, and the mass ratio of the carbon quantum dots to (E)-4-(2-nitrovinyl)benzoic acid is 1:190-240.

5. A paper-based chip, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the paper-based chip according to claim 5 in preparing a paper-based microfluidic device.

7. A paper-based microfluidic device, characterized in that: The invention comprises a top paper layer (11) and a bottom paper layer (12), wherein the top paper layer (11) and the bottom paper layer (12) are detachably connected, wherein the top paper layer (11) comprises a hydrophilic region (111) and a hydrophobic region (112), wherein the hydrophilic region (111) comprises a sample inlet (1111) and a detection unit (1113) connected via a hydrophilic channel (1112), and each hydrophilic region comprises more than two detection units (1113); The bottom paper layer (12) is fixed with two or more paper-based chips (121) according to claim 5, and each paper-based chip (121) is modified with a different biothiol receptor; The detection unit (1113) of the top paper layer (11) corresponds in position to the paper-based chip (121) of the bottom paper layer (12).

8. The paper-based microfluidic device according to claim 7, characterized in that: The biothiol acceptor is two or three of 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzoic acid, 4-(2,2-dicyanovinyl)benzoic acid or (E)-4-(2-nitrovinyl)benzoic acid; The concentration of the 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzoic acid is 1.8 to 2.0 mM, the concentration of the 4-(2,2-dicyanovinyl)benzoic acid is 1.8 to 2.0 mM, and the concentration of the (E)-4-(2-nitrovinyl)benzoic acid is 1.2 to 2.0 mM.

9. Use of the paper-based microfluidic device according to claim 7 or 8 in preparing a device for detecting biothiols.

10. A paper-based microfluidic sensor array platform, characterized in that: The paper-based microfluidic sensor array platform comprises the paper-based microfluidic device (1) according to claim 7 or 8, a fluorescence detection device (2) and a computer (3).

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