Method and system for detecting macromolecules in water

Abstract

The present application relates to a method and system for detecting macromolecules in water. The present application utilizes the interaction between particles and polymers under flow conditions, especially the high sensitivity of particle retention to macromolecular substances, to detect macromolecular information in water through the retention of particles at microchannel obstacles. The detection method of the present application has high sensitivity, short time consumption, low cost and small implementation difficulty compared with conventional chromatographic methods. In addition, due to the specificity of particle-polymer interaction, it is not easily disturbed by other small molecular substances compared with TOC, UV254, COD and other methods, and has high specificity.

Description

Technical Field

[0001] This invention belongs to the field of trace analysis, specifically relating to a method and system for detecting macromolecules in water. Background Technology

[0002] Water quality testing is crucial for drinking water safety, irrigation systems, precision manufacturing, and laboratory cleanliness. Among these, the detection of macromolecules is of paramount importance. For example, even extremely low concentrations of natural organic matter and biomolecules in surface water can lead to membrane fouling during water treatment and promote in-situ accumulation of microplastics. When synthetic polymers are used industrially as flocculants, oil displacement agents, and soil conditioners, long-term accumulation in the environment can lead to biotoxicity. Biopharmaceutical and semiconductor manufacturing processes are extremely sensitive to trace amounts of macromolecules; even minute residues can significantly impact product quality.

[0003] Existing macromolecule detection methods can be divided into three categories according to the detection target: First, total quantity measurement, such as TOC (total organic carbon content), which cannot specifically identify macromolecules (e.g., as described in the literature YOON G, PARK SM, YANG H, et al. Selection criteria for oxidation method in total organic carbon measurement[J]. Chemosphere, 2018, 199: 453-458); Second, specific component measurement, such as UV254 (aromatic functional group absorption) or COD (oxidizable organic matter), which only targets specific molecular characteristics and also cannot specifically identify macromolecules (e.g., in the literature WEISHAAR JL, AIKEN GR, BERGAMASCHI BA, et al. Evaluation of specific ultraviolet absorbance as an indicator of the chemical composition and reactivity of dissolved organic carbon [J]. Environmental Science & Technology, 2003, 37(20): 4702-4708, and in the literature KOLB M, BAHADIR M, TEICHGRABER B. Determination of Chemical oxygen demand (COD) using an alternative wetchemical method free of mercury and dichromate [J]. Water Research, 2017, 122: 645-654); thirdly, separation and identification, such as gel permeation chromatography or high performance liquid chromatography, combined with detectors such as refractive index and mass spectrometry, can effectively separate and identify macromolecules with high sensitivity, but the system cost is expensive, operation is complex, detection is time-consuming, and the molecular weight range is usually limited (e.g., liquid chromatography can generally only be used for 10...). 4Molecules below Da (e.g., those described in the literature KAWASAKI N, MATSUSHIGE K, KOMATSU K, et al. Fast and precise method for HPLC–size exclusion chromatography with UV and TOC (NDIR) detection: Importance of multiple detectors to evaluate the characteristics of dissolved organic matter [J]. Water Research, 2011, 45(18): 6240-6248). Summary of the Invention

[0004] The problem the invention aims to solve

[0005] The technical problem to be solved by the present invention is to provide a method for detecting macromolecular information in water that is highly sensitive, time-saving, low-cost and easy to implement.

[0006] Solution for solving the problem

[0007] To address the aforementioned problems, the inventors conducted long-term and in-depth research and proposed utilizing the interaction between particles and polymers under flow conditions, especially the high sensitivity of particle retention to macromolecules, to detect macromolecular-related information in water by measuring the amount of particles retained at microchannel obstacles, thus completing this invention.

[0008] Specifically, the present invention solves the problems of the present invention through the following solutions.

[0009] [1]. A method for detecting macromolecules in water, comprising the following steps:

[0010] (1) Perform at least one of the following sub-steps (1-1) and (1-2):

[0011] (1-1) Inject the sample to be tested into the channel of the microfluidic chip; inject particulate suspension A into the channel of the microfluidic chip, and use a microscopic imaging device to take and record images of the inside of the microfluidic chip during the injection process, wherein the particles in the particulate suspension A have negatively charged surfaces;

[0012] (1-2) Inject the sample to be tested into the channel of the microfluidic chip; inject particulate suspension B into the microfluidic chip, and use a microscopic imaging device to take and record images of the inside of the microfluidic chip during the injection process, wherein the particles in the particulate suspension B have positively charged surfaces;

[0013] (2) Determine the detection results based on the images recorded in sub-step (1-1) and / or sub-step (1-2);

[0014] The microfluidic chip has a channel through which the sample to be tested and the particulate suspension can flow, and a sample injection port and a particulate suspension injection port connected to the channel. At least one obstacle is disposed in the channel. In two directions perpendicular to and orthogonal to the flow direction within the channel, the obstacle has the same size as the channel in one direction and a smaller size than the channel in the other direction. The wall of the microfluidic chip has a transparent area so that an image of the inside of the microfluidic chip can be captured along the direction in which the obstacle has the same size as the channel.

[0015] The sample to be tested contains water, and the particulate suspension A and the particulate suspension B contain water and particles, wherein the concentration of the particles is greater than 0.0001 wt.% and the diameter of the particles is greater than 200 nm.

[0016] [2]. According to the detection method described in [1], wherein step (1) includes sub-steps (1-1) and (1-2);

[0017] In step (2), the detection result is determined based on the images recorded in sub-steps (1-1) and (1-2).

[0018] [3]. The detection method described in [1] is a method for detecting the concentration of macromolecules in the sample to be tested, wherein...

[0019] In step (1), images of the inside of the microfluidic chip are captured and recorded at fixed intervals of 1 to 5 seconds;

[0020] Step (2) includes the following sub-steps (2-1a) and (2-2a):

[0021] (2-1a) Obtain the particle retention signal at the time of shooting from each image. When the change in particle retention signal within the interval of 10~60 s is less than 5%, stop shooting and take the last image as the final image. Obtain the particle retention signal in the equilibrium state from the final image.

[0022] (2-2a) The concentration of the macromolecule in the sample is obtained by comparing the particle retention signal under equilibrium conditions with the concentration standard curve of the macromolecule to be detected.

[0023] The concentration standard curve of the macromolecule to be detected is obtained by the following method: using a series of solutions of the macromolecule with known concentrations as standard samples, the particle retention signal of each standard sample under equilibrium state is obtained in the manner of step (1) and sub-step (2-1a), and the relationship between the particle retention signal under equilibrium state and the concentration of the macromolecule in the standard sample is fitted to obtain the concentration standard curve of the macromolecule.

[0024] [4]. The detection method described in [1] or [2] is a method for detecting the type and / or molecular weight of macromolecules in the sample to be tested, wherein...

[0025] In step (1), images of the inside of the microfluidic chip are captured and recorded at fixed intervals of 1 to 5 seconds;

[0026] Step (2) includes the following sub-steps (2-1b):

[0027] (2-1b) The particle retention amount at the time of shooting is obtained from each image. When the change in particle retention amount within the interval of 10~60 s is less than 5%, shooting is stopped and the last image is used as the final image. The particle retention amount signal in the equilibrium state is obtained from the final image.

[0028] A series of aqueous solutions of macromolecules to be tested at different concentrations were used as test samples. The particle retention signal of each test sample under equilibrium state was obtained through the above steps (1) and sub-steps (2-1b).

[0029] Step (2) also includes the following sub-steps (2-2b):

[0030] (2-2b) The relationship between the particle retention signal at equilibrium obtained in sub-step (2-1b) and the concentration of macromolecules in the sample is fitted based on the following formula to obtain the coefficient k and the power-law exponent n:

[0031]

[0032] Among them, I d The signal represents the particle retention rate under equilibrium conditions, where C is the concentration of macromolecules in the sample, k is the coefficient, and n is the power law exponent.

[0033] By comparing the coefficients k and power law exponent n with the calibration database, the types and / or molecular weights of macromolecules can be obtained.

[0034] The calibration database is obtained through the following method:

[0035] (i) Prepare a series of aqueous solutions of known concentrations as standard samples using macromolecules of known molecular weight and known species, and obtain the corresponding coefficient k and power law exponent n of the macromolecules of known molecular weight and known species in the manner of step (1) and sub-steps (2-1b) and (2-2b);

[0036] (ii) Using a variety of macromolecules with known molecular weights and known types, the coefficient k and power law exponent n corresponding to each macromolecule are obtained in accordance with the method in step (i), thereby obtaining a calibration database.

[0037] [5]. According to the detection method described in [4], wherein,

[0038] Before performing step (1), the following steps are also included:

[0039] (1a) Injecting an aqueous solution of the macromolecule to be detected into the channel of the microfluidic chip.

[0040] (2a) Injecting a particulate suspension A into a microfluidic chip, and using a microscopic imaging device to capture and record images of the inside of the microfluidic chip during the injection process, wherein the particles in the particulate suspension A have negatively charged surfaces;

[0041] (3a) Inject an aqueous solution of the macromolecule to be detected into the channel of the microfluidic chip;

[0042] (4a) Injecting particulate suspension B into a microfluidic chip, and using a microscopic imaging device to capture and record images of the inside of the microfluidic chip during the injection process, wherein the particles in the particulate suspension B have positively charged surfaces;

[0043] The electrical properties and aggregation state of the macromolecules are determined based on the images recorded in steps (2a) and (4a).

[0044] [6]. The detection method according to any one of [1] to [5], wherein the particles are one or more selected from polymer microspheres and inorganic particles; preferably, the particles are fluorescently modified particles.

[0045] [7]. The detection method according to any one of [1] to [5], wherein the volume of the sample to be tested or macromolecular solution injected into the microfluidic chip is 0.05 mL or more.

[0046] [8]. The detection method according to any one of [1] to [5], wherein the ratio U of the injection flow rate U of the particulate suspension A and the particulate suspension B to the hydraulic diameter dc of the obstacle is 10 or more, wherein U is a value in m / s and dc is a value in m.

[0047] [9]. A system for performing any of the detection methods described in [1] to [8], comprising the microfluidic chip, the injection pump and the microscopic imaging device.

[0048]

[10] . According to the system described in [9], the obstacle is cylindrical in shape, and the height direction of the cylinder is the same as the direction in which the obstacle and the channel have the same dimensions.

[0049] The effects of the invention

[0050] The detection method of this invention has high sensitivity (up to the ppb level), short processing time (response time is typically less than 1 min), lower cost, and easier implementation compared to conventional chromatographic methods. Furthermore, due to the specificity of particle-polymer interactions, it is less susceptible to interference from other small molecules compared to methods such as TOC, UV254, and COD, exhibiting high specificity. Attached Figure Description

[0051] Figure 1 This is a schematic diagram of a preferred embodiment of the detection method and detection system of the present invention;

[0052] Figure 2 (a) is a schematic diagram of the preparation of the sample to be tested in Example 1;

[0053] Figure 2 (b) is the final image recorded in Example 1;

[0054] Figure 3 (a) is the normalized equilibrium retention strength I in Example 2. e Fitted curve of the relationship between the macromolecule concentration C and the macromolecule concentration C;

[0055] Figure 3 (b) shows the curve of normalized retention intensity I as a function of particle injection time in Example 2, as well as the fitting result and characteristic response time of the corresponding pseudo-first-order kinetic model.

[0056] Figure 4 (a) is the curve showing the change of normalized retention intensity I with injection time in Example 3;

[0057] Figure 4 (b) represents the normalized equilibrium retention strength I in Example 3. e Relationship with particle concentration;

[0058] Figure 4 (c) is the curve of normalized retention intensity I versus injection time in Example 4;

[0059] Figure 4 (d) represents the normalized equilibrium retention strength I in Example 4. eRelationship with the injection flow rate of the particulate suspension;

[0060] Figure 5 The final image recorded in Example 5;

[0061] Figure 6 (a) is the normalized equilibrium retention strength I in Example 6. e The curve showing the relationship between molecular weight and macromolecule molecular weight;

[0062] Figure 6 (b) represents the normalized equilibrium retention strength I in Example 6. e The relationship curve between the concentration of macromolecules in the sample and the corresponding fitting curve. Detailed Implementation

[0063] The present invention will now be described in detail. The description of the technical features described below is based on representative embodiments and specific examples of the present invention, but the present invention is not limited to these embodiments and specific examples.

[0064] Unless otherwise expressly stated, the “molecular weight” described in this specification refers to the weight-average molecular weight, which can be determined by gel permeation chromatography (GPC) using polystyrene as a standard.

[0065] In this instruction manual, "room temperature" refers to a temperature in the range of 15 to 35 °C, such as 20 to 30 °C. Unless otherwise explicitly stated, the operations described in this instruction manual are performed at room temperature.

[0066] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0067] In this specification, the numerical range indicated by "above" or "below" refers to the numerical range that includes the stated number.

[0068] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0069] In this specification, the terms "optionally" or "optionally" are used to indicate the use or non-use of certain substances, components, procedures, application conditions, etc.

[0070] All unit names used in this manual are international standard unit names, and unless otherwise stated, the "%" indicates weight or mass percentage.

[0071] In this specification, references to "preferred embodiments," "implementation methods," etc., mean that a specific element (e.g., feature, structure, property, and / or characteristic) related to that embodiment is included in at least one of the embodiments described herein, and may or may not be present in other embodiments. Furthermore, it should be understood that the elements may be combined in any suitable manner in various embodiments.

[0072] <Detection Method>

[0073] One objective of this invention is to provide a method for detecting macromolecules in water, comprising the following steps:

[0074] (1) Perform at least one of the following sub-steps (1-1) and (1-2):

[0075] (1-1) Inject the sample to be tested into the channel of the microfluidic chip; inject particulate suspension A into the channel of the microfluidic chip, and use a microscopic imaging device to take and record images of the inside of the microfluidic chip during the injection process, wherein the particles in the particulate suspension A have negatively charged surfaces;

[0076] (1-2) Inject the sample to be tested into the channel of the microfluidic chip; inject particulate suspension B into the channel of the microfluidic chip, and use a microscopic imaging device to take and record images of the inside of the microfluidic chip during the injection process, wherein the particles in the particulate suspension B have positively charged surfaces.

[0077] (2) Determine the detection results based on the images recorded in sub-step (1-1) and / or sub-step (1-2);

[0078] The microfluidic chip has a channel through which the sample to be tested and the particulate suspension can flow, and a sample injection port and a particulate suspension injection port connected to the channel. At least one obstacle is disposed in the channel. In two directions perpendicular to and orthogonal to the flow direction within the channel, the obstacle has the same size as the channel in one direction and a smaller size than the channel in the other direction. The wall of the microfluidic chip has a transparent area so that an image of the inside of the microfluidic chip can be captured along the direction in which the obstacle has the same size as the channel.

[0079] The sample to be tested contains water, and the particulate suspension A and particulate suspension B contain water and particles, wherein the concentration of the particles is greater than 0.0001 wt.% and the diameter of the particles is greater than 200 nm.

[0080] The detection method of this invention is based on particle-macromolecule interactions under flow conditions. Since macromolecules readily adhere to solid surfaces, the surface of obstacles can effectively enrich macromolecules in the sample during microchannel flow. After the particle suspension is injected, the aggregated macromolecules (including macromolecules that are originally aggregated and those aggregated by particle-macromolecule electrostatic interactions) are in a physically weakly cross-linked state, between a chemically cross-linked gel state and a dispersed monomolecular state in solution. They possess a loose network structure and viscoelastic fluidization behavior, allowing them to spontaneously extend downstream of the obstacle, forming ribbon-like structures to capture particles, thereby visualizing the presence of macromolecules.

[0081] The detection method of this invention can effectively perform qualitative and / or quantitative analysis of macromolecules in water, and can obtain one or more information on the presence, state (aggregate / dissolved state, charged state), concentration, type, and molecular weight of macromolecules in water. The various aspects of this invention are described in detail below.

[0082] The features of the embodiments described below are applicable to other embodiments without contradicting each other or departing from the inventive concept, and they can be combined with each other arbitrarily.

[0083] <Implementation Method 1>

[0084] The first embodiment of the present invention provides a qualitative detection method for macromolecules in water, which can obtain information on the presence or absence of macromolecules in water (whether the water contains macromolecules or not). In the detection method of this embodiment, in step (1), at least one of sub-steps (1-1) and (1-2) is performed; in step (2), the detection result is determined based on the images recorded in sub-steps (1-1) and / or (1-2).

[0085] In this embodiment, the presence of macromolecular substances in the sample is directly determined by whether a band-like structure is formed in the microfluidic chip.

[0086] In step (1), there is no particular restriction on the order of sub-steps (1-1) and (1-2). Either one can be performed first. If a band-like structure is observed in the recorded image, it can be determined that the sample to be tested contains macromolecular substances. If no band-like structure is observed, the other sub-step in sub-steps (1-1) and (1-2) is performed. If a band-like structure is observed in the recorded image, it can be determined that the sample to be tested contains macromolecular substances. If no band-like structure is observed, it can be determined that the sample to be tested does not contain macromolecular substances.

[0087] <Implementation Method Two>

[0088] The second embodiment of the present invention provides a qualitative detection method for macromolecules in water, which can acquire information on the presence and state (aggregation / dissolution state, charged state) of macromolecules in water. In the detection method of this embodiment, in step (1), sub-steps (1-1) and (1-2) are performed; in step (2), the detection result is determined based on the images recorded in sub-steps (1-1) and (1-2).

[0089] In this embodiment, the presence and state information of macromolecular substances in the sample to be tested are directly determined by whether a strip structure is formed in the microfluidic chip in sub-steps (1-1) and (1-2).

[0090] Specifically, the presence and state information of macromolecular substances in the sample to be tested can be determined based on whether there are banded structures in the images recorded in sub-steps (1-1) and (1-2), as shown in Table 1 below.

[0091] Table 1

[0092]

[0093] Since particle retention requires the formation of a large molecular aggregate network, for well-dispersed and dissolved macromolecules, electrostatic interaction can trigger the aggregation of macromolecules on the surface of the obstacle and capture a large number of particles only when the particles and macromolecules have opposite charges. For aggregated macromolecules (such as flocculent or gel-like macromolecules), they have already formed a network structure, so they can capture any type of particles after adhering to the surface of the obstacle.

[0094] <Implementation Method 3>

[0095] A third embodiment of the present invention provides a method for the quantitative detection of macromolecules in water, which can obtain concentration information of macromolecules in water. Specifically, this embodiment is a method for detecting the concentration of known macromolecular substances (whose type and molecular weight are known).

[0096] In this embodiment, step (1) includes either sub-step (1-1) or (1-2). Specifically, sub-step (1-1) or (1-2) is performed based on the properties (charge properties) of the macromolecule to be detected. More specifically, the particles are selected based on the charge and aggregation state of the macromolecule to be detected. If the macromolecule to be detected is positively charged (cationic polymer), sub-step (1-1) is performed; if the macromolecule to be detected is negatively charged (anionic polymer), sub-step (1-2) is performed; and if the macromolecule to be detected forms aggregates in water, either sub-step (1-1) or (1-2) can be performed.

[0097] In step (1), i.e., when performing sub-steps (1-1) or (1-2), images of the inside of the microfluidic chip are captured and recorded at fixed intervals of 1 to 5 seconds. Specifically, starting from the injection of the particle suspension, multiple images are captured and recorded at fixed intervals of 1 to 5 seconds (e.g., 2 seconds, 3 seconds, 4 seconds, etc.) using a microscopic imaging device.

[0098] In step (2), the multiple images obtained in step (1) are processed and the results are compared with the concentration standard curve of the macromolecule to be detected, so as to obtain the concentration of the macromolecule in the sample to be tested.

[0099] Specifically, step (2) includes the following sub-steps (2-1a) and (2-2a):

[0100] (2-1a) Obtain the particle retention signal at the time of shooting from each image. When the change in particle retention signal within the interval of 10~60 s is less than 5%, stop shooting and take the last image as the final image. Obtain the particle retention signal in the equilibrium state from the final image.

[0101] (2-2a) The concentration of the macromolecule in the sample is obtained by comparing the particle retention signal under equilibrium state with the concentration standard curve of the macromolecule to be detected.

[0102] In some implementations, the particle retention signal is obtained by converting the recorded image into a grayscale image, corresponding to image matrix A; denoising image matrix A and removing background flowing particles, and then superimposing the grayscale intensities. (where (i,j) are the coordinates of each point in the image matrix A) Calculate the particle retention intensity as the particle retention amount signal.

[0103] In some implementations, the particle retention signal obtained from each image is compared with the particle retention signal obtained from previously captured and recorded images. When the change in the particle retention signal within a 10–60 s interval (e.g., 20 s, 30 s, 40 s, 50 s) is less than 5%, the detection system is considered to have reached a steady state, and capturing is stopped, with the last image used as the final image. The particle retention signal in the equilibrium state is obtained from the final image.

[0104] After obtaining the particle retention signal under equilibrium conditions, it is compared with the concentration standard curve of the macromolecule being tested to obtain the concentration information of the macromolecule in the sample to be tested.

[0105] Preferably, the particle retention intensity is normalized, for example, by normalizing it using the cross-sectional area of ​​the obstacle, to obtain a normalized retention intensity, which serves as the particle retention amount signal. More preferably, the particle retention intensity under equilibrium conditions is normalized to obtain a normalized particle retention intensity under equilibrium conditions, which serves as the particle retention amount signal under equilibrium conditions. This signal is then compared with a standard curve of the detected macromolecule's concentration to obtain the concentration information of the detected macromolecule.

[0106] Concentration standard curve of the macromolecule to be detected

[0107] The concentration standard curve of the macromolecule to be detected is obtained by the following method: using a series of solutions of the macromolecule with known concentrations as standard samples, the particle retention signal under equilibrium state of each standard sample is obtained in the manner of step (1) and sub-step (2-1a), and the relationship between the particle retention signal under equilibrium state and the concentration of the macromolecule in the standard sample (based on the Freundlich equation) is fitted to obtain the concentration standard curve of the macromolecule.

[0108] Preferably, the particle retention intensity of each standard sample under equilibrium state is normalized using the cross-sectional area of ​​the obstacle to obtain the normalized particle retention intensity of each standard sample under equilibrium state, which is used as the particle retention amount signal under equilibrium state. The relationship between the normalized particle retention intensity and the concentration of macromolecules in the standard sample is then fitted to obtain the concentration standard curve of the macromolecule.

[0109] Preferably, the series of aqueous solutions of the macromolecule at known concentrations serving as standard samples are multiple aqueous solutions of different concentrations ranging from 0.0001 mg / L to 1 g / L. From the perspective of detection accuracy, it is preferable to have 3 or more aqueous solutions, more preferably 4 or more, and even more preferably 5 or more. From the perspective of detection cost, the number of aqueous solutions is typically 20 or less, preferably 15 or less, for example, 10 or less.

[0110] The concentrations of the various aqueous solutions can vary at equal or unequal intervals within these ranges, as long as they differ from each other. From the perspective of detection accuracy, if the concentrations of the aqueous solutions are sorted from smallest to largest, then among two adjacent aqueous solutions, the larger concentration is more than five times the smaller concentration.

[0111] This embodiment can be used to detect test samples with concentrations in the range of 0.0001 mg / L to 1 g / L.

[0112] <Implementation Method Four>

[0113] The fourth embodiment of the present invention provides a method for the (quantitative) detection of macromolecules in water, which can obtain information such as the type and molecular weight of the macromolecule being detected. Specifically, this embodiment is a method for detecting the type and / or molecular weight information of unknown macromolecular substances.

[0114] In some implementations, if the charge / aggregation state of the macromolecule is unknown, its charge / aggregation state is determined as described in Implementation Method 2 above, and then a suitable particle suspension is selected based on this information, as described in Implementation Method 3 above, i.e., step (1-1) or (1-2) is selected.

[0115] In some implementation schemes, a series of aqueous solutions of the macromolecules to be detected at different concentrations are first prepared as test samples.

[0116] Preferably, the series of aqueous solutions of the macromolecules to be detected at different concentrations are multiple aqueous solutions with concentrations ranging from 0.0001 mg / L to 1 g / L. From the perspective of detection accuracy, it is preferable to have 3 or more aqueous solutions, more preferably 4 or more, and even more preferably 5 or more. From the perspective of detection cost, the number of aqueous solutions is usually 20 or less, preferably 15 or less, for example, 10 or less.

[0117] The concentrations of the aqueous solutions used as test samples can cover all or part of the above range; for example, the concentrations of multiple aqueous solutions can be between 0.001 mg / L and 1 mg / L. The concentrations of the aqueous solutions can vary at equal or unequal intervals within these ranges, as long as they are different from each other. From the perspective of detection accuracy, if the concentrations of the aqueous solutions are sorted from smallest to largest, then among two adjacent aqueous solutions, the larger concentration is more than five times the smaller concentration.

[0118] In this embodiment, the particle retention signal under equilibrium state for each sample is obtained according to the description in Embodiment 3 above. Then, the relationship between the obtained particle retention signal under equilibrium state and the concentration of macromolecules in the sample is fitted based on the following formula to obtain the coefficient k and the power law exponent n:

[0119]

[0120] Among them, I d The signal represents the particle retention rate under equilibrium conditions, where C is the concentration of macromolecules in the sample, k is the coefficient, and n is the power law exponent.

[0121] By comparing the coefficients k and the power law exponent n with the calibration database, the types and / or molecular weights of macromolecules can be obtained.

[0122] Calibration database

[0123] The calibration database was obtained through the following methods:

[0124] (i) Prepare a series of aqueous solutions of known concentrations as standard samples using macromolecules of known molecular weight and known species, and obtain the coefficient k and power law exponent n corresponding to the macromolecules of known molecular weight and known species in the manner of step (1) and sub-steps (2-1b) and (2-2b), respectively;

[0125] (ii) Using a variety of macromolecules with known molecular weights and known types, the coefficient k and power law exponent n corresponding to each macromolecule are obtained in accordance with the method in step (i), thereby obtaining a calibration database.

[0126] The particle retention signal depends on the amount of macromolecules accumulated on the obstacle surface and the interaction between macromolecules and particles. Each type of macromolecule has a different adsorption tendency and adsorption mode on the obstacle surface, and the chain length and spatial morphology both affect the strength of the interaction between it and the flowing particles. Therefore, different types and molecular weights of macromolecules correspond to different Ig values. d The -C relationship is determined by the coefficient k and the power law exponent n.

[0127] In this embodiment, when performing the detection of the sample to be tested and obtaining the calibration database, the particle retention signal in the equilibrium state can be normalized using the cross-sectional area of ​​the obstacle, as described in Embodiment 3.

[0128] The various aspects of the above-described embodiments are described in detail below.

[0129] particulate suspension

[0130] Unless otherwise specified, "particle suspension" in this specification refers to particle suspension A and / or particle suspension B.

[0131] Unless otherwise specified, “particles” in this specification refers to the particles contained in particle suspension A and / or particle suspension B.

[0132] A particulate suspension contains water and particles, in which the particles are suspended in the water in a substantially uniform manner, forming a suspension.

[0133] In the particulate suspension used in this invention, the concentration of particles is 0.0001 wt.% or more, preferably 0.001 wt.% or more, and even more preferably 0.01 wt.% or more. As an upper limit, it can be, for example, 1.0 wt.% or less, or 0.1 wt.% or less.

[0134] In the particulate suspension used in this invention, the diameter of the particles is 200 nm or more, preferably 500 nm or more, and even more preferably 700 nm or more; the upper limit can be, for example, less than 10 μm, less than 5 μm, etc.

[0135] The particles used in this invention have negatively or positively charged surfaces, which facilitates the formation of suspensions and allows them to interact with macromolecules to form ribbon-like aggregate structures.

[0136] The particles used in this invention can be one or more selected from polymer microspheres and inorganic particles. Examples of polymer particles include polystyrene microspheres, poly(meth)acrylamide microspheres, poly(meth)acrylate microspheres, poly(meth)methyl methacrylate microspheres, polyethylene particles, polypropylene particles, and polyvinyl chloride particles. Examples of inorganic particles include silica particles, metal oxide particles, and metal particles. These particles can be unmodified (usually negatively charged) or electrically modified (negatively or positively charged), for example, they can be amino-modified (positively charged).

[0137] Preferably, fluorescently modified particles, such as fluorescently modified polystyrene microspheres, are used to improve the accuracy of subsequent image recognition and reduce detection errors.

[0138] In the embodiments described above, the ratio U / dc of the injection flow rate U (m / s) of the particulate suspension to the hydraulic diameter dc (m) of the obstacle is preferably 10 or more, more preferably 100 or more, more preferably 10000 or more. Here, the ratio refers to a numerical ratio.

[0139] Typically, the injection flow rate of particulate suspension needs to meet the following requirements. ,here U is the maximum shear stress on the surface of the obstacle (in Pa), where μ is the viscosity of the particle suspension (in Pa·s), and U is the average injection velocity (in m / s). , For the injected flow (m 3 / s), The width of the channel is (m). Let m be the channel depth (m), where m is a coefficient determined by the ratio of the channel depth to the cylinder diameter (m > 1, which can be obtained by theoretical calculation or numerical simulation of the three-dimensional flow field). The critical shear stress of macromolecular aggregates (in the literature) Generally in the order of 0.1–10Pa (refer to DJABOUROV M, NISHINARI K, ROSS-MURPHY S B. Physical gels from biological and synthetic polymers [M]. Cambridge: Cambridge University Press, 2013, and STOODLEY P, LEWANDOWSKI Z, BOYLE JD, et al. Structural deformation ofbacterial biofilms caused by short-term fluctuations in fluid shear: An insitu investigation of biofilm rheology [J]. Biotechnology and Bioengineering, 1999, 65(1): 83-92, and LEE SH, SECCHI E, KANG P K. Rapid formation of bioaggregates and morphology transition to biofilm streamers induced by pore-throat flows [J]. Proceedings of the National Academy of Sciences, 2023, 120(14): e2204466120).

[0140] As a general estimate, μ is taken as the viscosity of water (1 mPa·s (10⁻⁶ mPa·s)). -3 Pa·s), take m = 10 (which is on the same order of magnitude as m = 15 in the example), If the Pa is 0.1, then the average injection velocity and the cylinder diameter satisfy U / d c When the value is greater than 10, the stable formation of banded aggregates can usually be guaranteed. As a conservative estimate, let μ be the viscosity of water (1 mPa·s), and let m = 1 (theoretically m > 1). If the Pa is 10, then the average injection velocity and the cylinder diameter satisfy U / d c > 10 4 At that time, it can certainly ensure the stable formation of the ribbon-like aggregates.

[0141] Sample to be tested

[0142] In the detection method of this invention, the sample to be tested contains water and optionally macromolecules. As used herein, "macromolecules" refers to compounds with an index-average molecular weight of 500 or higher, including but not limited to polymers.

[0143] The detection method of this invention can effectively detect various types of macromolecules, including synthetic macromolecules as well as natural and biological macromolecules. Synthetic macromolecules include, but are not limited to, cationic polymers, anionic polymers, and nonionic polymers, specifically including cationic poly(meth)acrylamide, anionic poly(meth)acrylamide, nonionic poly(meth)acrylamide, poly(methacrylic acid), polyvinyl alcohol, and polyvinylpyrrolidone. Natural and biological macromolecules include alginate, xanthan gum, cellulose and its derivatives, serum proteins, and humic acid.

[0144] When the sample to be tested contains macromolecules (such as in embodiments two, three, and four above, and in part of embodiment one), the concentration of macromolecules in the sample to be tested is 0.0001 mg / L to 1 g / L, preferably 0.001 mg / L to 1 mg / L.

[0145] In step (1) of the detection method of the present invention, the injection volume of the sample to be tested is 0.01 mL or more, preferably 0.1 mL or more, even more preferably 0.5 mL or more, and even more preferably 1 mL or more. The upper limit can be, for example, 50 mL or less, 10 mL or less, etc.

[0146] In step (1) of the detection method of the present invention, the injection flow rate of the sample to be tested is 10. -8 For speeds above m / s, 10 is preferred. -4 above m / s; the upper limit is, for example, 10. -1 Below m / s, 10 -2 Below m / s, etc.

[0147] microfluidic chip

[0148] In this invention, the microfluidic chip has a channel through which the sample to be tested and the particulate suspension can flow, and a sample injection port and a particulate suspension injection port connected to the channel. At least one obstacle is disposed within the channel. In two directions perpendicular to and orthogonal to the flow direction within the channel, the obstacle has the same size as the channel in one direction (this direction is also referred to herein as the depth of the channel) and a smaller size than the channel in the other direction. The wall of the microfluidic chip has a transparent area so that images of the interior of the microfluidic chip can be captured along the direction in which the obstacle has the same size as the channel.

[0149] Preferably, the channels of the microfluidic chip are substantially linear, meaning that the flow pattern of the sample and particulate suspension does not change macroscopically when flowing through the channels.

[0150] The microfluidic chip used in this invention has two injection ports on one side of its channel, which are used for injecting the sample to be tested and the particulate suspension, respectively, to avoid premature premixing of the two and affecting the detection results.

[0151] The microfluidic chip used in this invention can also be provided with one or more outlets for the outflow of test samples and particulate suspensions. The outlets are preferably located on the side opposite the inlet.

[0152] Preferably, the microfluidic chip of the present invention is fabricated by PDMS photolithography.

[0153] Preferably, the channel length of the microfluidic chip is 20~50 mm.

[0154] Preferably, the cross-section of the channel in the microfluidic chip can be circular or polygonal (e.g., quadrilateral, pentagonal, hexagonal, etc.), and is more preferably rectangular. When the cross-section of the channel is rectangular, the longer side of the rectangle is preferably 0.5~10 mm (preferably 1~5 mm), and the shorter side is 10~200 μm (preferably 20~70 μm).

[0155] Preferably, the cross-sectional area of ​​the channels in the microfluidic chip is 0.005~0.2 mm. 2 .

[0156] Preferably, the obstacle is a cylinder, and more preferably, the direction of the height of the cylinder is the same as the direction of the channel.

[0157] The walls of the microfluidic chip have transparent areas, which can be part or all of the walls of the microfluidic chip. The transparent areas enable images of the inside of the microfluidic chip to be captured along the direction where the obstacle and the channel have the same size.

[0158] <Detection System>

[0159] Another object of the present invention is to provide a system for the detection method of the present invention, comprising a microfluidic chip, an injection pump, and a microscopic imaging device.

[0160] For information on the microfluidic chip in the detection system of this invention, please refer to the description above.

[0161] The syringe pump is configured to communicate with two injection ports of the microfluidic chip, thereby injecting the test sample and particulate suspension into the channels of the microfluidic chip. Preferably, the syringe pump is equipped with a metering device for quantifying the test sample and particulate suspension.

[0162] The microscopic imaging device is used to capture images of the inside of a microfluidic chip. It is preferably equipped with one or more of the following: fluorescence excitation / receiver, transmission light source, and reflection light source. The pixel resolution of the output image is not less than the particle size used.

[0163] Preferably, the detection system of the present invention also includes an image processing device to process the captured and recorded images to obtain the required data such as particle retention signal.

[0164] Example

[0165] The schematic diagram of the microfluidic detection system used in the following embodiments is shown below. Figure 1 As shown. The system includes a microchip with an internal obstacle structure (fabricated by photolithography using PDMS material, with a channel length of 3.5 mm, a width of 2 mm, a depth of 40 μm, and a cylinder with a diameter of 50 μm at the center of the microchannel), a constant flow syringe pump (Harvard Pump 11 Elite) and a microsyringe (Hamilton), and a microscopic imaging device (microscope system is Nikon SMZ18, magnification of 13.5x, image resolution of 0.22 μm / pixel, and image capture time interval of 2 s).

[0166] Example 1

[0167] This embodiment studies the qualitative detection of macromolecules in unknown samples.

[0168] The particulate suspension used in this embodiment has a concentration of 0.05 wt.% and is prepared with purified water. The particles are green fluorescent polystyrene microspheres with a diameter of 1 μm, manufactured by Tianjin Bestlite Chromatography Technology Development Center. Their surface charge is unmodified and naturally negatively charged (product number 7-3-0100).

[0169] Sample to be tested: such as Figure 2 As shown in (a), two 100 mL samples of Milli-Q ultrapure water were placed in clean wide-mouth bottles. One sample was sealed and placed in a sterile environment (sealed sterile group); the other sample was placed in the laboratory air environment and covered only with aluminum foil with small holes (covered to reduce evaporation, with small holes left to contact the air environment to achieve the effect of natural contamination) (air exposure group). Both samples were kept in the dark for one week.

[0170] Experimental method: First, 1.5 mL of the sample to be tested was injected into the microchannel at a constant flow rate of 50 μL / min. Then, a particle suspension was injected at a constant flow rate of 50 μL / min. After 1 min, the final image was recorded using a microscopic imaging device, as shown below. Figure 2 As shown in (b).

[0171] Depend on Figure 2As can be seen from (b), for the sealed sterile group, particles are almost not retained on or near the barrier surface; for the air-exposed group, particles are significantly retained, forming the ribbon-like aggregate structure shown in the figure; using TOC (total organic carbon content) detection (the detection instrument is the TOC-L CPH from Shimadzu Corporation of Japan, based on the combustion catalytic oxidation method), the organic carbon content of the two groups was < 0.1 mg / L (below the detection limit) and 0.97 mg / L, respectively, further confirming that there is macromolecular pollution from contact with the air environment in the air-exposed group, and the detection method of the present invention is effective.

[0172] Example 2

[0173] This embodiment studies the quantitative detection of known macromolecules in water.

[0174] Sample to be tested: Prepared at a concentration of 10 -4 ~10 2 mg / L (10-fold dose per group, 7 groups total) cationic polyacrylamide (molecular weight 5×10⁻⁶) 6 A copolymer of Da, acrylamide (AM) and acryloyloxyethyltrimethylammonium chloride (DMC), with an ionicity of 30%, was used as the test sample.

[0175] The experimental method for macromolecule detection is as follows: First, 2.5 mL of the sample to be tested is injected into the microchannel at a flow rate of 50 μL / min, followed by injection of the particle suspension at a flow rate of 50 μL / min (same as in Example 1); images are recorded using a microscopic imaging device at 2-second intervals.

[0176] The above 7 groups of test samples with different concentrations were used to conduct experiments, and the experiment was repeated 3 times for each group of test samples.

[0177] Based on the recorded images, the normalized retention intensity I of the particles was calculated using the following method: The recorded RGB images were converted to grayscale using the image processing toolkit in Matlab software, corresponding to image matrix A. Gaussian filtering was applied to the image matrix for noise reduction, and the grayscale values ​​of regions with brightness lower than the minimum brightness of the retained particles were set to 0 to eliminate the influence of background flowing particles. Then, based on the cross-sectional area of ​​the obstacle, the normalized retention intensity I was calculated using the following formula:

[0178]

[0179] Where A is the image matrix, dc is the cylinder diameter, and (i, j) are the matrix coordinates, corresponding to any position in the image.

[0180] When the relative change of the normalized particle retention intensity I within a 30-second interval is less than 5%, the retention is considered to have reached a steady state, and this is taken as the final image. The normalized retention intensity obtained from the final image is defined as the normalized equilibrium retention intensity I. e Normalized equilibrium retention intensity I e The relationship between the macromolecule concentration C and the fitted relationship was obtained as follows: Figure 3 As shown in (a). By Figure 3 As can be seen from (a), at a concentration of only 10 -3 Even under conditions of mg / L (1 ppb) or lower, the detection method of the present invention can still output a stable signal, indicating that the detection method of the present invention has extremely high detection sensitivity. Based on the fitting of the relationship between retention intensity I and macromolecular concentration C, a power exponent of 0.56 was obtained (fitting R0). 2 The coefficient is 0.989, indicating that the retention response can be described and quantified by the Freundlich equation; for known macromolecules, the concentration of the sample to be tested can be directly calculated through the calibration curve.

[0181] For samples with concentrations of 1 mg / L, 0.1 mg / L, and 0.001 mg / L, the curves showing the change in normalized retention intensity I as a function of particle injection time (time from initial moment to final steady state) are as follows: Figure 3 As shown in (b). By Figure 3 As can be seen from (b), during the particle injection process, the particle retention first increases rapidly, then tends to stabilize and reach an equilibrium state.

[0182] Figure 3 The curve in (b) is the fitting result of the pseudo-first-order dynamic model of the following equation.

[0183]

[0184] Where t is time, I e The normalized equilibrium retention strength is given by k1, where k1 is the pseudo-first-order kinetic rate constant, and I is the normalized equilibrium retention strength. e Both k1 and k2 are determined by fitting, by Figure 3 As can be seen from (b), the fitted curve follows the same trend as the data points. Based on pseudo-first-order dynamic fitting, the characteristic response time is defined as... Efficiency is measured quantitatively, and the statistical results are as follows: Figure 3 As shown in (c), by Figure 3 As can be seen from (c), the response time is less than 1 minute at any concentration, indicating that the detection method of the present invention also has the advantage of rapid measurement.

[0185] Example 3

[0186] This embodiment studies the effect of particle concentration in particulate suspension on the detection results.

[0187] Test sample: Prepare an aqueous solution of cationic polyacrylamide with a concentration of 1 mg / L. The cationic polyacrylamide used is the same as in Example 2.

[0188] Particle suspensions: Six particle suspensions were prepared with particle concentrations of 0.5 wt.%, 0.05 wt.%, 0.005 wt.%, 0.001 wt.%, 0.0005 wt.%, and 0.00005 wt.%, respectively. The particles were the same as those in Example 1.

[0189] Experimental method: First, 2.5 mL of the sample to be tested was injected into the microchannel at a flow rate of 50 μL / min. Then, the particle suspension was injected at a flow rate of 50 μL / min. Images were recorded using a microscopic imaging device at 2-second intervals. The normalized retention intensity I and the normalized equilibrium retention intensity I were obtained from the recorded images according to the method described in Example 2 above. e Experiments were conducted using the six different concentrations of particle suspensions mentioned above.

[0190] In experiments using concentrations of 0.5 wt.%, 0.05 wt.%, 0.005 wt.%, and 0.00005 wt.%, the normalized retention intensity I changed with injection time as follows: Figure 4 As shown in (a), the normalized equilibrium retention intensity I was obtained from experiments using particle suspensions of six different concentrations. e The relationship with particle concentration is as follows Figure 4 As shown in (b).

[0191] like Figure 4 As can be seen from (a) and (b), as the particle concentration decreases, the response time gradually increases, and the particle retention strength in the equilibrium state decreases slightly, but the differences are small. However, when the particle concentration decreases to 0.00005 wt.%, the response curve shifts significantly, and the particle retention strength in the equilibrium state becomes extremely low. This is because for macromolecular solutions in the dissolved state, the interaction strength between particles and polymers determines the aggregation strength. When the particle concentration is too low, the interaction is weak, and even if the injection time is extended, the banded aggregation structure is difficult to form.

[0192] Example 4

[0193] This embodiment studies the effect of particulate suspension injection flow rate on detection results.

[0194] Test sample: Prepare an aqueous solution of cationic polyacrylamide with a concentration of 1 mg / L. The cationic polyacrylamide used is the same as in Example 2.

[0195] Particle suspension: concentration of 0.05 wt.%, particle type is the same as in Example 1.

[0196] Experimental method: First, 2.5 mL of the sample to be tested was injected into the microchannel at a flow rate of 50 μL / min, followed by the injection of a particle suspension. Images were recorded using a microscopic imaging device at 2-second intervals. The normalized retention intensity I and the normalized equilibrium retention intensity I were obtained from the recorded images according to the method described in Example 2 above. e The injection flow rates of the particulate suspension were set to 0.5 μL / min, 5 μL / min, 10 μL / min, 20 μL / min, 50 μL / min, and 250 μL / min, respectively, for six groups of experiments.

[0197] The normalized retention intensity I as a function of injection time in experiments with injection flow rates of 0.5 μL / min, 5 μL / min, 50 μL / min, and 250 μL / min for particulate suspensions is shown below. Figure 4 As shown in (c), the normalized equilibrium retention intensity I was obtained from experiments conducted at six different injection flow rates. e The relationship with the injection flow rate of the particulate suspension is as follows: Figure 4 As shown in (d).

[0198] Depend on Figure 4 As shown in (c) and (d), the response time gradually increases with decreasing particle injection flow rate, and the particle retention strength in the equilibrium state decreases slightly, but the differences are small. However, when the injection flow rate decreases to 0.5 μL / min, the response curve shifts significantly, and the particle retention strength in the equilibrium state becomes extremely low. This is because the formation of the banded aggregate structure depends on fluid shearing, i.e. ,in The maximum shear stress on the obstacle surface is given by μ, the viscosity of the particle suspension is 1 mPa·s, U is the average injection velocity (U = Q / WH, where Q is the injection flow rate, W is the channel width (2 mm), and H is the channel depth (40 μm)). c The diameter of the cylinder (50 μm), m = 15, was obtained from flow numerical simulations of the same structure as the microchip (simulation settings: μ = 1 mPa·s, U = 2×10⁻⁶). -3 m / s, d c = 50 μm, thus = 0.6 Pa, substituting into the formula, we get m = 15). Let be the critical shear stress of the macromolecular aggregate (taken as 0.1 Pa); substituting... The calculated critical flow velocity is 3.3 × 10⁻⁶. -4 The flow rate is m / s, which corresponds to a critical injection flow rate of 1.7 μL / min, consistent with experimental results.

[0199] Example 5

[0200] In this embodiment, the detection of different types of macromolecules is studied.

[0201] The following samples were used in this embodiment:

[0202] Cationic polyacrylamide aqueous solution: a copolymer of acrylamide (AM) and acryloyloxyethyltrimethylammonium chloride (DMC), with a molecular weight of 5 × 10⁻⁶. 6 Da, with an ionization degree of 30%, is a macromolecule in a dissolved state and has a concentration of 1 mg / L;

[0203] Anionic polyacrylamide aqueous solution: a copolymer of acrylamide (AM) and acrylic acid (AA), with a molecular weight of 5 × 10⁻⁶. 6 Da, with an ionization degree of 30%, is a macromolecule in a dissolved state and has a concentration of 1 mg / L;

[0204] Naturally aged nonionic polyacrylamide solution: Acrylamide (AM) polymer, molecular weight 18×10 6 After being prepared into a solution, Da was placed in a clean, wide-mouthed bottle, sealed, and protected from light for two months to allow for natural aging. During this process, the macromolecules were in an aggregated state, and the concentration was 1 mg / L.

[0205] The following particulate suspension is used in this embodiment:

[0206] Negatively charged particle suspension: The particles are green fluorescent polystyrene microspheres with a diameter of 1 μm, manufactured by Tianjin Bestlite Chromatography Technology Development Center. They are naturally negatively charged with unmodified surfaces, product number 7-3-0100, and the particle concentration is 0.05 wt.%.

[0207] Positively charged particle suspension: The particles are green fluorescent polystyrene microspheres with a diameter of 1 μm, manufactured by Tianjin Bestlite Chromatography Technology Development Center, with amino-modified surface, positively charged, product number 7-31-0100, and particle concentration of 0.05 wt.%.

[0208] Experimental Method: First, 2.5 mL of the test sample was injected into the microchannel at a flow rate of 50 μL / min, followed by the injection of a particle suspension at a flow rate of 50 μL / min. Six sets of experiments were conducted using cross-combinations of the three test samples and two particle suspensions. The final images were obtained in the same manner as in Example 4, as shown below. Figure 5 As shown.

[0209] Depend on Figure 5It can be seen that when particles and macromolecules have opposite electrical charges, electrostatic interactions can trigger the aggregation of macromolecules on the obstacle surface, capturing a large number of particles. For aggregated macromolecules (such as flocculent or gel-like structures), they have already formed a network structure, thus they can capture any type of particle after adhering to the obstacle surface. This indicates that the detection method of the present invention can effectively detect three types of macromolecules.

[0210] Example 6

[0211] In this embodiment, the influence of the molecular weight of macromolecules on the detection results is studied.

[0212] Samples to be tested: Samples with molecular weights of 2×10⁻⁶ were used. 5 5×10 5 5×10 6 8×10 6 18×10 6 Da's cationic polyacrylamide (a copolymer of acrylamide (AM) and acryloyloxyethyltrimethylammonium chloride (DMC), with an ionicity of 30%) was prepared into aqueous solutions with concentrations of 1 mg / L and 0.1 mg / L, respectively, to obtain 10 test samples.

[0213] Experimental method: First, 2.5 mL of the sample to be tested was injected into the microchannel at a flow rate of 50 μL / min. Then, a particle suspension was injected at a flow rate of 50 μL / min (same as in Example 1). The final image was obtained in the same manner as in Example 4, and the normalized equilibrium retention intensity I was obtained. e Experiments were conducted using the 10 test samples mentioned above, and the normalized equilibrium retention strength I was determined. e The relationship with the molecular weight of macromolecules is as follows: Figure 6 As shown in (a).

[0214] Depend on Figure 6 As can be seen from (a), at the same concentration, the normalized equilibrium retention intensity I e The normalized equilibrium retention intensity I increases with increasing molecular weight because larger molecular weights correspond to longer chain structures, making it easier to form aggregate networks and trap particles; for the same molecular weight of the test sample, the normalized equilibrium retention intensity I increases with higher concentrations. e The concentration is higher because at higher concentrations, the sample contains more macromolecules, which make it easier to form aggregate networks and capture particles.

[0215] Example 7

[0216] Samples to be tested: Samples with molecular weights of 5 × 10⁻⁶ were used. 6 Da and 5×10 5Da's cationic polyacrylamide (a copolymer of acrylamide (AM) and acryloyloxyethyltrimethylammonium chloride (DMC), with an ionicity of 30%) was prepared at a concentration of 10. -4 ~10 2 Fourteen samples were obtained by preparing an aqueous solution of mg / L (10 times per group, 7 groups in total).

[0217] Experimental method: First, 2.5 mL of the sample to be tested was injected into the microchannel at a flow rate of 50 μL / min. Then, a particle suspension was injected at a flow rate of 50 μL / min (same as in Example 1). The final image was obtained in the same manner as in Example 4, and the normalized equilibrium retention intensity I was obtained. e Experiments were conducted using the 14 test samples mentioned above, and the normalized equilibrium retention strength I was determined. e The relationship between the concentration of macromolecules in the test sample and the fitting curve are as follows: Figure 6 As shown in (b), for a molecular weight of 5 × 10 5 Da, concentration of 10 -4 No effective signal was detected in the test sample at a concentration of mg / L.

[0218] Depend on Figure 6 As can be seen from (b), the concentration-normalized equilibrium retention intensity I of macromolecules at different molecular weights e The curves all exhibit an ideal power-law relationship, and the power-law exponent increases as the molecular weight decreases (i.e., due to the weakening of the aggregate structure, it tends to accumulate more linearly). These differences suggest that molecular weight identification can be achieved through testing at multiple concentrations by establishing a calibration database for different molecular weights.

[0219] Industrial availability

[0220] The detection method and detection system of this invention can be widely used in water quality testing, macromolecular substance analysis and other fields.

Claims

1. A method for detecting macromolecules in water, characterized in that: Includes the following steps: (1) Perform at least one of the following sub-steps (1-1) and (1-2): (1-1) Inject the sample to be tested into the channel of the microfluidic chip; inject particulate suspension A into the channel of the microfluidic chip, and use a microscopic imaging device to take and record images of the inside of the microfluidic chip during the injection process, wherein the particles in the particulate suspension A have negatively charged surfaces; (1-2) Inject the sample to be tested into the channel of the microfluidic chip; inject particulate suspension B into the microfluidic chip, and use a microscopic imaging device to take and record images of the inside of the microfluidic chip during the injection process, wherein the particles in the particulate suspension B have positively charged surfaces; (2) Determine the detection results based on the images recorded in sub-step (1-1) and / or sub-step (1-2); The microfluidic chip has a channel through which the sample to be tested and the particulate suspension can flow, and a sample injection port and a particulate suspension injection port connected to the channel. At least one obstacle is disposed in the channel. In two directions perpendicular to and orthogonal to the flow direction within the channel, the obstacle has the same size as the channel in one direction and a smaller size than the channel in the other direction. The wall of the microfluidic chip has a transparent area so that an image of the inside of the microfluidic chip can be captured along the direction in which the obstacle has the same size as the channel. The sample to be tested contains water, and the particulate suspension A and the particulate suspension B contain water and particles, wherein the concentration of the particles is greater than 0.0001 wt.% and the diameter of the particles is greater than 200 nm.

2. The detection method according to claim 1, characterized in that, Step (1) includes sub-steps (1-1) and (1-2); In step (2), the detection result is determined based on the images recorded in sub-steps (1-1) and (1-2).

3. The detection method according to claim 1, characterized in that, It is a method for detecting the concentration of macromolecules in a sample, wherein... In step (1), images of the inside of the microfluidic chip are captured and recorded at fixed intervals of 1 to 5 seconds; Step (2) includes the following sub-steps (2-1a) and (2-2a): (2-1a) Obtain the particle retention signal at the time of shooting from each image. When the change in particle retention signal within the interval of 10~60 s is less than 5%, stop shooting and take the last image as the final image. Obtain the particle retention signal in the equilibrium state from the final image. (2-2a) The concentration of the macromolecule in the sample is obtained by comparing the particle retention signal under equilibrium conditions with the concentration standard curve of the macromolecule to be detected. The concentration standard curve of the macromolecule to be detected is obtained by the following method: using a series of solutions of the macromolecule with known concentrations as standard samples, the particle retention signal of each standard sample under equilibrium state is obtained in the manner of step (1) and sub-step (2-1a), and the relationship between the particle retention signal under equilibrium state and the concentration of the macromolecule in the standard sample is fitted to obtain the concentration standard curve of the macromolecule.

4. The detection method according to claim 1 or 2, characterized in that, It is a method for detecting the types and / or molecular weight of macromolecules in a sample to be tested, among which... In step (1), images of the inside of the microfluidic chip are captured and recorded at fixed intervals of 1 to 5 seconds; Step (2) includes the following sub-steps (2-1b): (2-1b) The particle retention amount at the time of shooting is obtained from each image. When the change in particle retention amount within the interval of 10~60 s is less than 5%, shooting is stopped and the last image is used as the final image. The particle retention amount signal in the equilibrium state is obtained from the final image. A series of aqueous solutions of macromolecules to be tested at different concentrations were used as test samples. The particle retention signal of each test sample under equilibrium state was obtained through the above steps (1) and sub-steps (2-1b). Step (2) also includes the following sub-steps (2-2b): (2-2b) The relationship between the particle retention signal at equilibrium obtained in sub-step (2-1b) and the concentration of macromolecules in the sample is fitted based on the following formula to obtain the coefficient k and the power-law exponent n: Among them, I d The signal represents the particle retention rate under equilibrium conditions, where C is the concentration of macromolecules in the sample, k is the coefficient, and n is the power law exponent. By comparing the coefficients k and power law exponent n with the calibration database, the types and / or molecular weights of macromolecules can be obtained. The calibration database is obtained through the following method: (i) Prepare a series of aqueous solutions of known concentrations as standard samples using macromolecules of known molecular weight and known species, and obtain the corresponding coefficient k and power law exponent n of the macromolecules of known molecular weight and known species in the manner of step (1) and sub-steps (2-1b) and (2-2b); (ii) Using a variety of macromolecules with known molecular weights and known types, obtain the coefficient k and power law exponent n corresponding to each macromolecule in accordance with the method in step (i), thereby obtaining a calibration database.

5. The detection method according to claim 4, characterized in that, Before performing step (1), the following steps are also included: (1a) Injecting an aqueous solution of the macromolecule to be detected into the channel of the microfluidic chip. (2a) Injecting a particulate suspension A into a microfluidic chip, and using a microscopic imaging device to capture and record images of the inside of the microfluidic chip during the injection process, wherein the particles in the particulate suspension A have negatively charged surfaces; (3a) Inject an aqueous solution of the macromolecule to be detected into the channel of the microfluidic chip; (4a) Injecting particulate suspension B into a microfluidic chip, and using a microscopic imaging device to capture and record images of the inside of the microfluidic chip during the injection process, wherein the particles in the particulate suspension B have positively charged surfaces; The electrical properties and aggregation state of the macromolecules are determined based on the images recorded in steps (2a) and (4a).

6. The detection method according to any one of claims 1 to 5, characterized in that, The particles are selected from one or more of polymer microspheres and inorganic particles; preferably, the particles are fluorescently modified particles.

7. The detection method according to any one of claims 1 to 5, characterized in that, The volume of the sample or macromolecular solution injected into the microfluidic chip is 0.05 mL or more.

8. The detection method according to any one of claims 1 to 5, characterized in that, The ratio U / dc of the injection flow rate U of the particulate suspension A and particulate suspension B to the hydraulic diameter dc of the obstacle is 10 or more, where U is a value in m / s and dc is a value in m.

9. A system for performing the detection method according to any one of claims 1 to 8, characterized in that, It includes the microfluidic chip, the injection pump, and the microscopic imaging device.

10. The system according to claim 9, characterized in that, The obstacle is cylindrical in shape, and the height direction of the cylinder is the same as the dimension of the passage.

Images