Method for determining formaldehyde in urine based on colorimetric method and application of method in early-stage biomarker detection of Alzheimer disease

Abstract

The invention discloses a method for determining formaldehyde in urine based on a colorimetric method and application of the method in early-stage biomarker detection of Alzheimer's disease. The method for determining the formaldehyde in the urine based on the colorimetric method comprises the following steps: treating a urine sample by using activated carbon before detecting the urine sample to be detected, and then determining the formaldehyde in the urine sample by using the colorimetric method.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical detection, and specifically relates to a method for determining formaldehyde in urine based on colorimetry and an application of the method in detecting early biomarkers of Alzheimer's disease. Background Art

[0002] Changes in human health, including many diseases such as stroke, cancer, multiple sclerosis, and age-related neurological dementia, are likely to affect the normal metabolism of formaldehyde in the human body, leading to elevated levels of formaldehyde in human urine. Therefore, monitoring urine formaldehyde levels is of great significance for health care work. However, urine samples are complex and diverse, and contain many interfering substances that affect the detection and quantification of formaldehyde. Separation or removal of these interfering substances is a prerequisite for the determination of urine formaldehyde. Currently, some commonly used standard methods include gas chromatography (GC), high performance liquid chromatography (HPLC), and fluorescence high performance liquid chromatography (Fluo-HPLC). In the latter case, formaldehyde is bound to a certain fluorescent probe (such as 2,4-dinitrophenylhydrazine (DNPH) or ampicillin) and then separated and detected by elution from HPLC. However, these chromatographic methods require complex and expensive instruments and are not suitable for routine applications. In fact, to date, there is no simple and effective method to reliably detect and quantify the formaldehyde content in urine. Summary of the invention

[0003] The present invention aims to overcome the deficiencies of the prior art and provide a method for determining formaldehyde in urine based on colorimetry and its application in the detection of early biomarkers of Alzheimer's disease.

[0004] In order to achieve the above object, the technical solution provided by the present invention is:

[0005] The method for determining formaldehyde in urine based on colorimetry is to treat the urine sample with activated carbon to remove interfering substances before detecting the urine sample to be tested, and then determine the formaldehyde content in the urine sample by colorimetry.

[0006] Preferably, the method is to treat the urine sample with activated carbon before testing the urine sample to be tested, then add the urine sample treated with activated carbon to a cuvette containing MBTH solution for reaction, then add FeCl3 solution containing HCl to the cuvette for reaction, and read the absorbance A 628 The formaldehyde concentration in the urine sample is quantified by referring to the formaldehyde standard solution; or, the method is to treat the urine sample with activated carbon before detecting the urine sample to be tested, then add the urine sample treated with activated carbon to the well plate, add MBTH solution to the well plate for reaction, then add FeCl3 solution containing HCl to the well plate for reaction, and read the absorbance A 628The formaldehyde concentration in the urine sample is quantified by referring to a formaldehyde standard solution; the activated carbon is animal activated carbon, preferably, the size of the activated carbon is 20-100 mesh, and more preferably, the activated carbon is activated carbon prepared from coconut shells.

[0007] Preferably, the mass-to-volume ratio of the activated carbon to the urine sample is 0.1:100 to 0.3:100, preferably 0.2:100, and the unit of the mass-to-volume ratio is g / μL.

[0008] Preferably, the urine sample is treated with activated carbon by adding Milli-Q water, CaCl2 solution and urine sample to a container containing activated carbon; the mass volume ratio of the activated carbon to Milli-Q water is 0.1:400 to 0.3:400, preferably 0.2:400, and the unit of the mass volume ratio is g / μL; the mass volume ratio of the activated carbon to the CaCl2 solution is 0.1:500 to 0.3:500, preferably 0.2:500, and the unit of the mass volume ratio is g / μL.

[0009] Preferably, the concentration of CaCl2 in the CaCl2 solution is 15-25mM, preferably 20mM, and the CaCl2 solution contains isopropanol, and the volume percentage content of the isopropanol in the CaCl2 solution is 15-25%, preferably 20%.

[0010] Preferably, the activated carbon is used to treat the urine sample for 20-50 min, preferably 30 min, and then the activated carbon is separated from the urine sample.

[0011] Preferably, the reaction time of adding the urine sample to the cuvette containing MBTH solution is 0.5-1.5 h, preferably 1 h, and the reaction time of adding the FeCl3 solution containing HCl is 0.5-1.5 h, preferably 1 h.

[0012] Preferably, in the cuvette, the volume ratio of the MBTH solution to the urine sample treated with activated carbon is 40:200 to 60:200, preferably 50:200; the volume ratio of the FeCl3 solution containing HCl to the urine sample treated with activated carbon is 40:200 to 60:200, preferably 50:200; in the well plate, the urine sample treated with activated carbon is divided into two types: undiluted solution and diluted solution, the diluted solution is a urine sample and a diluent prepared in a volume ratio of 1:1, the diluent is an isopropanol aqueous solution, and the The volume percentage of isopropanol diluent in water is 5-20%, preferably 10%. After the urine sample or diluted urine sample treated with activated carbon is added to the well plate, the MBTH solution is added for reaction. The volume ratio of the MBTH solution to the urine sample or diluted urine sample treated with activated carbon is 20:140 to 40:140, preferably 30:140. The volume ratio of the FeCl3 solution containing HCl to the urine sample treated with activated carbon is 20:140 to 40:140, preferably 30:140.

[0013] Preferably, the MBTH concentration in the MBTH solution is 35-45 mM, preferably 40 mM; the HCl concentration in the FeCl3 solution containing HCl is 80-120 mM, preferably 100 mM, and the FeCl3 concentration is 70-90 mM, preferably 80 mM.

[0014] The above-mentioned method based on colorimetry for determining formaldehyde in urine can be used to detect early biomarkers of Alzheimer's disease.

[0015] The present invention will be further described below:

[0016] The present invention has developed a convenient and reliable colorimetric method for detecting and quantifying urine formaldehyde concentration. First, each urine sample to be tested is treated with a suitable activated carbon to remove all major interfering substances while maintaining a minimum loss of urine formaldehyde, and then the urine formaldehyde is quantified with a reagent, which may also include some other low molecular weight aldehydes. The reagents used are 3-methyl-2-benzothiazolinone hydrazone (MBTH) and FeCl3 / HCl. In addition, the composition of the main interfering substances is also determined, which are various organic derivatives containing various types of dihydroxybenzenes. Under the detection conditions of the present invention, the reactivity of these interfering substances far exceeds that of formaldehyde, producing brown-red species, which seriously interferes with the formaldehyde determination. However, these interfering substances can be effectively adsorbed and removed by activated carbon. Preliminary data of the present invention show that there are some good correlations between the urine formaldehyde content and the urine Aβ protein content detected by the Aβ protein-specific detection strip, which is expected to make the urine formaldehyde detection of the present invention a convenient and non-invasive biomarker for early detection of Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : Main reaction flow chart for the detection of formaldehyde using MBTH–FeCl3 / HCl reagent.

[0018] Figure 2 : Kinetics of condensation reaction of MBTH reagent with formaldehyde or urine sample: Where A is the time course of condensation reaction of MBTH with the following species in a 96-well plate: 30μM (row 1) and 15μM (row 2) formaldehyde standard solutions; 5% urine sample U-xx#3227 before treatment with activated carbon (rows 3-4); the same urine sample U-cc#3227 after treatment with activated carbon (rows 5-6); Note 1: The condensation reaction time of each column solution is indicated above the figure; Note 2: Each colorless condensation reaction product is subsequently induced by FeCl3 / HCl reagent One-hour oxidative coupling colorimetric reaction detection quantitative; Note 3: The solutions shown in the 12th column are formaldehyde standard series samples after standard detection steps; B is the visible spectrum of the solutions from left to right in the first row of the plate; C is the visible spectrum of the solutions from left to right in the third row of the plate; D is the visible spectrum of the solutions from left to right in the fifth row of the plate; E is the correlation diagram of the absorbance at the wavelength position shown by the dotted line in the spectra of the above solutions B, C, and D and the condensation reaction time of each solution; F is the spectrum of the formaldehyde standard series sample detection solution shown in the 12th column of the plate; G is the linear calibration curve of the formaldehyde standard series sample detection concentration shown in the 12th column.

[0019] Figure 3: Kinetics of oxidative coupling reaction initiated by FeCl3 / HCl colorimetric reagent: wherein A is the change in the visible spectrum of a 10 μM formaldehyde standard solution after one hour of condensation reaction with the subsequent oxidative coupling reaction time; B is the change in the visible spectrum of a 4% urine sample (U-xx#330) before treatment with activated carbon after one hour of condensation reaction with the subsequent oxidative coupling reaction time; C is a correlation diagram between the absorbance at the wavelength position indicated by the dotted line in the above A and B solution spectra and the oxidative coupling reaction time; D is the change in the visible spectrum of an 8% identical urine sample (U-cc#330) treated with activated carbon after one hour of condensation reaction with the subsequent oxidative coupling reaction time; E is a correlation diagram between the absorbance at the wavelength position indicated by the dotted line in the above A and D solution spectra and the oxidative coupling reaction time; F is the color of the final solution shown in the above A, B, D spectra. Note 1: The oxidative coupling reaction time in the figure is calculated from the time when the FeCl3 / HCl reagent is added to each solution after one hour of condensation reaction; Note 2: The kinetic experiment was carried out in a cuvette, and the background reference solution was an aqueous solution containing MBTH, which had almost no absorption and was stable in the range of 350-800nm; Note 3: The light yellow FeCl3 has a strong absorption in the range of <500nm. As a result of the oxidation-reduction of FeCl3 with MBTH and other components in the urine sample, the spectra of all solutions in the range of <500nm first increase and then decrease with the progress of the oxidative coupling reaction; due to the instability of the colored products of the interfering substances, for untreated urine samples, the spectrum first increases and then decreases, covering the entire visible spectrum.

[0020] Figure 4 :Effect of phosphate on formaldehyde determination using MBTH-FeCl3 reagent; The experimental steps and conditions refer to the standard procedure, and the sample formaldehyde (10μM) standard is added with different concentrations of phosphate buffer (pH 7.4), but the added phosphate is neutralized by adding an equivalent amount of HCl at the same time as the FeCl3 color reagent. The inset shows the final color of the 10mM formaldehyde standard analysis solution containing different concentrations of phosphate.

[0021] Figure 5 : Correlation between urine formaldehyde concentration and Αβ test results of test strip No. 2 (A) and test strip No. 6 (B), and between different elderly groups (C), analysis of variance followed by Kruskal-Wallis test, ± red lines indicate the mean standard deviation of each group.

[0022] Figure 6 :The urine formaldehyde level was positively correlated with the urine PO4 concentration (A), but had no significant correlation with the Mini-Mental State Examination (MMSE) (B), centiloid (C), and PET amyloid imaging scores (D). DETAILED DESCRIPTION

[0023] 1. Materials and Methods Chemical reagents: 3-Methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate (MBTH.HCl.H2O) was purchased from Sigma. It is dissolved in high-purity water (18.2M) to form a 40mM (i.e., 9.4mg / mL) working solution. After air oxidation, the solution may turn brown and partially lose efficacy within 2-3 days at room temperature, but if stored at 4°C, it can usually remain colorless for 2-3 weeks. If the solution is prepared with deoxygenated purified water containing one equivalent of HCl and stored in a sealed tube, the shelf life at room temperature can be extended to more than 6 months, but excess HCl must be neutralized before use. The formaldehyde standard solution can be prepared by gradually diluting a 100mM formaldehyde stock solution (containing 10% MeOH) with 10% isopropanol. The stock solution is prepared from an original stock solution containing 37.1% HCHO (13.1M) and 13.1% MeOH (Ajax Finechem, Thermo Fisher Scientific; batch number 395324). FeCl3·6H2O was dissolved in 1.0M HCl to obtain a 0.8M FeCl3 stock solution (containing 1.0M HCl) and then diluted 10 times with high purity water to obtain the working solution. Isopropyl alcohol (>99.9%; Honeywell Chemicals) was used as a diluent for urine formaldehyde determination at a concentration of 10%. Its contribution to the background absorbance at 628 nm is negligible. The activated carbon used (20-100 mesh, from animals) was purchased from BDH Chemicals. Various equivalent or even better activated carbons can also be obtained from other suppliers, but they must be pre-tested to ensure that the background contribution of the blank control is small and the solution after treatment with the selected activated carbon must be close to neutral.

[0024] Urine samples were collected by the Australian Imaging Biomarkers and Lifestyle (AIBL) Research Center from participants with Alzheimer's disease (AD), mild cognitive impairment (MCI), and normal cognition (CN). Urine was centrifuged to remove the precipitate and then aliquoted and stored at -80°C.

[0025] Urine phosphate determination: The malachite green phosphate assay kit provided by Sigma was used for colorimetric determination in a 96-well plate. In short, each urine sample was diluted 50 times with double distilled water (dd-H2O) after removing the precipitate (Note: the precipitate contains almost no phosphate), and then 40 μL was transferred into a 96-well plate well containing 80 μL dd-H2O. The sample was further diluted 3×3 times in sequence to form a set of four 80 μL sample solutions, with total dilution factors of 150, 450, 1,350 and 4,050, respectively. Then 20 μL of the kit detection solution was added to each sample well, and the color of the solution immediately turned green with a maximum absorption wavelength of 620 nm. After incubating the reaction mixture for about 30 minutes, the A of each plate well was recorded.620 The PO4 concentration in each well was quantified by reference to a set of phosphate (PO4, charge omitted for simplicity) standards (0–40 μM) measured under the same conditions. Each sample was measured in duplicate, and the A values ​​within the valid calibration range (0.15–0.90) were selected. 620 The average urine PO4 concentration was calculated from the readings. Table S1 gives an example of data processing.

[0026] Urine sample pretreatment: Each collected urine sample can be stored at 4°C for up to 24 hours, or frozen for long-term storage. If there is a precipitate in the thawed urine sample, it can be removed by centrifugation, but the present invention confirms that the urine precipitate contains almost no formaldehyde, and whether the precipitate is removed or not has little effect on the measurement results. Add 400 μL of Milli-Q water, 500 μL of 20 mM CaCl2 solution containing 20% ​​isopropanol, and 100 μL of urine sample to a 1.5 mL sample tube containing about 0.2 g of animal activated carbon (BDH Chemicals), that is, each activated carbon-treated sample contains 10 mM CaCl2, 10% isopropanol and 10% of the urine sample stock solution. Mix the sample and activated carbon thoroughly (preferably on a rotating stirrer) for about 30 minutes, and then separate them from the activated carbon by centrifugation (or let it stand until separation or pass the activated carbon-treated sample through a disposable small filter). The background control experiment is a blank solution without urine.

[0027] Urine formaldehyde determination in 96-well plate: This operation was performed in duplicate for each sample directly in the wells of a 96-well plate. 140 and 70 μL of each sample treated as above were pipetted into two different wells, and 70 μL of 10% isopropanol was added to the second well to produce a 1:1 dilution. MBTH reagent (30 μL 40 mM) was then added and mixed thoroughly to initiate the condensation reaction between MBTH and formaldehyde to produce a colorless azine intermediate (see Figure 1 ; Note: Strictly speaking, urine samples also contain other forms of aldehydes RCHO, where R can be various organic groups of different sizes, but formaldehyde should be the main form of aldehydes in urine. For simplicity, the present invention uses formaldehyde to represent all forms of detectable aldehydes). After incubating the condensation reaction mixture at room temperature for about one hour, 30 μL of 80 mM FeCl3 reagent containing 100 mM HCl was added to promote the rapid oxidation of MBTH and further induce an oxidative coupling color reaction between the oxidized MBTH and the pre-formed azine to produce a blue formazan product. The maximum absorption value of this product is about 628 nm, which can be used to detect and quantify the formaldehyde content in the solution ( Figure 1 After the reaction is carried out for about one hour, the absorbance value of each well solution at 628 nm (A 628) and quantified formaldehyde content with reference to a set of formaldehyde standards (0–40 μM) under the same assay conditions. To ensure reliable subtraction of background A 628 Absorption, formaldehyde standard solution and blank solution both contain 10% isopropanol. Within the experimental error range, the A of the blank solution with and without activated carbon treatment 628 The readings should be similar, otherwise, another activated carbon should be selected. It is also recommended to record the full solution spectrum of each sample solution in the range of 350–800 nm in order to analyze in detail the overall changes in the solution spectrum under different solution compositions and measurement conditions. In order to avoid possible sample loss during the measurement process, the reaction microplate should be routinely sealed with a sealing film during the reaction incubation period.

[0028] The urine formaldehyde content can be calculated from the A of each sample solution according to the following formula (1): 628 Original data to estimate:

[0029] [Urine formaldehyde concentration] = [(A 628 - Background) / Slope] x Total Dilution Factor (1)

[0030] The background term in formula (1) is the A of 10% isopropanol solution under the same measurement conditions. 628 Detection background, slope is A 628 Linear calibration correlation with formaldehyde standard solution concentration (μM). Table S2 gives an example of data processing. Determination of urine formaldehyde in cuvette: This operation is directly performed in a disposable semi-micro cuvette with a 10 mm optical path. To each cuvette pre-filled with 50 μL of 40 mM MBTH, 300 μL of H2O (or 10% isopropanol) and 200 μL of urine sample treated with activated carbon as described above are added. After one hour of reaction, 50 μL of 80 mM FeCl3 reagent containing 100 mM HCl is added and mixed thoroughly to promote the formation of blue formaldehyde product. After another hour of reaction, read A 628 The data were used to quantify the urine formaldehyde concentration by referring to the formaldehyde standard. The blank reference solution contained MBTH but not FeCl3. Although the light yellow FeCl3 has only a weak absorption at 628nm, it has a large absorption in the range less than 500nm. And due to the complex redox reaction between FeCl3 and MBTH and some components in urine, the absorption spectrum in this range is unstable and in a dynamic reaction state, which complicates the spectral analysis.

[0031] Condensation reaction kinetics: Figure 1The first step reaction shown is a condensation reaction between formaldehyde and MBTH, and the product is a colorless azine intermediate that cannot be directly detected. However, after the addition of the FeCl3 / HCl reagent, the condensation reaction is not only inhibited by the acidic conditions caused by the addition of HCl, but also quenched by the rapid oxidation and exhaustion of MBTH after the addition of excess FeCl3 (see Figure 1 ). Therefore, the first step of the condensation reaction kinetics can be completed by detecting the final blue product. The experiment was carried out in a 96-well plate according to the steps described above, and the condensation reaction time range was set from the shortest about 20 seconds (i.e. <0.4min) to about 24h, and the color development reaction time after adding the FeCl3 / HCl reagent was fixed at one hour.

[0032] Oxidative coupling reaction kinetics: The experiment was conducted in two steps: first, the first condensation reaction was carried out in a 1.5 mL sample tube to obtain the desired colorless azine intermediate, and then the product was quantitatively transferred into a disposable cuvette containing FeCl3 / HCl reagent, mixed quickly and measured by A 628 Follow the kinetics of the oxidative coupling reaction (see Figure 1 ). The specific steps are as follows: add 300μL H2O (or 10% isopropanol), 50μL 40mM MBTH and 200μL 20μM formaldehyde standard solution or 200μL 10-20% urine sample treated with or without activated carbon as described above to a 1.5mL sample tube. After reacting for about an hour, transfer the solution quantitatively into a semi-micro disposable cuvette. The cuvette is pre-loaded with 50μL of 80mM FeCl3 reagent containing 100mM HCl. After quick pipetting and mixing, monitor the A 628 The oxidative coupling reaction kinetics were followed for about one hour. The blank reference solution was MBTH aqueous solution without FeCl3 / HCl.

[0033] Statistical analysis: Analyses were performed using the PRISM program according to references (①Ai, L., Wang, J., Li, T., Zhao, C., Tang, Y., Wang, W., Zhao, S., Jiang, W., Di, Y., Fei, X., Luo, H., Li, H., Luo, W., Yu, Y., Lin, W., He, R. and Tong, Z. (2018) Arapid and sensitive fluorescence method for detecting urineformaldehyde in patients with Alzheimer's disease. Annals of Clinical Biochemistry. 56, 210-218; ②Wang, Y., Pan, F., Xie, F., He, R. and Guo, Q. (2022) Correlation Between Urine Formaldehyde and Cognitive Abilities in the Clinical Spectrum of Alzheimer's Disease. Frontiers in Aging Neuroscience. 14).

[0034] 2. Results and Analysis

[0035] Urinary phosphate determination: In an acidic medium, PO4 anions react rapidly with excess molybdate to form giant molybdate phosphate anions. This anion interacts with malachite green dye to form a green complex with strong absorbance at a wavelength of 620nm, which can be quantified very sensitively. Its detection sensitivity is in the μM range, while the average PO4 concentration in human urine is estimated to be in the tens of mM range. Therefore, urine samples must be diluted heavily to effectively quantify the PO4 concentration therein. However, actual urine PO4 concentrations vary greatly, ranging from as low as 2-3mM to as high as 50-60mM in this study. In order to be able to reliably determine the PO4 concentration in various urine samples in just one round of experiments, for each urine sample, the present invention generally prepares a set of four urine samples with different dilution multiples for determination and analysis. However, this one-size-fits-all dilution method will inevitably produce some sample solutions that are too dilute or too concentrated, and its A 620 The reading will be outside the valid calibration range of the PO4 standard (0.15–0.90; A 620>0.9 solution is prone to precipitation). We judge these readings as invalid readings and usually exclude them. The present invention performs at least two replicates of each sample. We found that for each dilution series of each urine sample, at least two of the four urines with different dilution multiples can produce valid A 620 The average recovery of PO4 added to 10 different urine samples was (102±5)%.

[0036] Formaldehyde determination principle: The overall reaction process of using MBTH and FeCl3 / HCl reagent to detect formaldehyde is as follows: Figure 1 The determination process can be divided into two reaction stages: (1) MBTH hydrazine attacks the aldehyde carbonyl (R-CHO) with nucleophilicity to generate the condensation product azine (reaction step i); (2) MBTH is oxidized by FeCl3 to generate MBTH active cation (reaction step ii), which immediately undergoes oxidative coupling reaction with the azine generated in the condensation reaction step (i) to generate blue cation formazan (reaction step iii), the concentration of which can usually be measured by A 628 According to the reported extinction coefficient of 65 mM cm -1 , the detection sensitivity of this method for formaldehyde is in the low μM range.

[0037] In fact, all reagents currently used to detect formaldehyde carry an active hydrazine group; Figure 1 The condensation reaction type shown in step (i) is the common basic reaction for all current formaldehyde detection. This step is a nucleophilic reaction, so it is more favorable in a neutral medium, but it will be inhibited to varying degrees in an acidic medium. However, the MBTH oxidation step (ii) and the oxidative coupling step (iii) dominated by FeCl3 can only be carried out in a weakly acidic medium (pH ~ 2), because the FeCl3 reagent is easily precipitated in the form of "rust" and loses its oxidizing ability when pH>3. More importantly, the reaction step (ii) proceeds very quickly, so that the excess FeCl3 reagent required for the reaction step (iii) can quickly consume the reduced MBTH through the fast reaction step (ii), thereby quickly quenching the condensation reaction of step (i). Therefore, in order to ensure the completion of the slower condensation reaction step, the detection process must be divided into two stages, that is, the reaction step (i) is carried out separately from the reaction steps (ii, iii).

[0038] Stability of MBTH reagent: The prepared MBTH aqueous solution is not very stable and will gradually oxidize into many other different substances over time, especially in neutral or weakly alkaline media. Therefore, when stored at room temperature, the colorless MBTH aqueous solution will gradually turn brown and produce precipitation within a few days, but if stored at 4°C, its stability can be extended to 1-2 weeks. However, weakly acidic media can inhibit the oxidation rate of MBTH by air. The shelf life of a 10-100mM MBTH solution containing an equal concentration of HCl can exceed 6 months at room temperature, especially if the oxygen in the HCl solution is blown away with N2, but the HCl added to the reagent must be neutralized with NaOH before use.

[0039] Calibration of formaldehyde standard solution: according to the reaction process Figure 1 The reaction steps (i, ii, iii) outlined above allow for the easy and reliable determination of formaldehyde in simple formaldehyde solutions using MBTH and FeCl3 / HCl reagents ( Figure 2 A, F, G). In a medium with a pH of 5-8, formaldehyde and excess MBTH rapidly undergo a condensation reaction (step i), and the quantitative reaction time is about 30 min ( Figure 2 B, E (i)). Then, FeCl3 reagent containing HCl was added to acidify the reaction medium to pH ~ 2, so that the excess FeCl3 could induce and complete the reaction steps (ii, iii) in about 10 minutes ( Figure 3 A, C(i)). The final solution was sky blue ( Figure 2 A, 3F), with a characteristic absorption maximum at about 628 nm. Further increasing the reaction time of any or both assay steps only resulted in slight fluctuations in the absorbance intensity at 628 nm, indicating that the reaction process Figure 1 The colorless intermediate azine (B) and the final blue product formazan (D) are stable. Figure 2 F and G show the conventional formaldehyde standard solution detection spectrum and the corresponding A 628 A linear calibration curve was obtained with respect to formaldehyde concentration.

[0040] Sample pretreatment is a prerequisite for urine formaldehyde detection: human urine is rich in more than 3,000 metabolites, including formaldehyde and many other components. They may react with MBTH (or any other similar reagent containing a reactive hydrazine group) in some way. In fact, the direct determination of formaldehyde in untreated urine samples using MBTH and FeCl3 / HCl reagents produces very different results, and the color of the solution may vary from green to purple-brown ( Figure 2 A, 3F). Their visible solution spectra show additional absorption bands, with the main absorption band appearing at ∼500 nm in most cases ( Figure 2 C, 3B). This absorption band is 628Worse still, the excess FeCl3 reagent required for the test rapidly oxidizes these interfering species (let’s call them species X) and renders them more reactive than formaldehyde in competing with the MBTH reagent ( Figure 2 B, C, E (i-iii)). Therefore, direct determination of formaldehyde in untreated urine samples is severely interfered by species X. Obviously, a prerequisite for meaningful urine formaldehyde determination using MBTH-FeCl3 reagent is that the urine sample must be pretreated to remove or minimize these interfering species X while minimizing the loss of urine formaldehyde.

[0041] Determine the composition of X species: The reactivity and spectral characteristics of these X interfering species are similar to species such as phenol, catechol and catecholamines known to exist in human urine. Suitable oxidants can cause them to undergo rapid oxidative coupling reactions with MBTH to produce reddish products. (For current information on the detection of catecholamines in urine or blood, please refer to: https: / / my.clevelandclinic.org / health / diagnostics / 22144-catecholamines-test). In order to confirm the composition of these interfering species, the present invention selected a representative member of these compounds, 1,2-dihydroxybenzene (i.e., catechol), to react under formaldehyde determination conditions. Although no direct reaction of catechol with MBTH was detected, it was found that the addition of FeCl3 reagent immediately triggered a rapid oxidative coupling reaction between MBTH and oxidized catechol to produce a brown-red solution, whose solution spectrum is similar to the X species in the urine sample. Utilizing the difference in the reactivity of X substance and formaldehyde to MBTH and FeCl3 reagent, the present invention can also design suitable experimental procedures and conditions to directly determine the content of X species in untreated urine samples.

[0042] Removal of phosphate: It is worth noting that urine is rich in minerals, especially PO4 ions. The present invention confirms that if the concentration of PO4 ions in urine samples is too high, it may damage the oxidation function of FeCl3 and affect the test results ( Figure 4 ). This may be due to the formation of Fe(III)-phosphate complexes, which leads to a decrease in the reduction potential of FeCl3. Therefore, as the first step of pretreatment, CaCl2 is added to the original urine diluent to precipitate and remove PO4 ions in the urine. The present invention confirms that excessive CaCl2 has little effect on subsequent formaldehyde determination. Comparative experiments before and after CaCl2 treatment confirm that more than 99% of PO4 ions in urine can be removed by this procedure.

[0043] Removal of urine protein: It has been reported that urine protein may be the main interfering component in urine formaldehyde determination, so urine samples are often pretreated with trichloroacetic acid (TCA). However, for conventional urine formaldehyde determination, this procedure is cumbersome and time-consuming because trichloroacetic acid makes urine samples strongly acidic, and the acidified sample must be neutralized before the first condensation reaction with MBTH. The present invention tested a simpler procedure, that is, passing the diluted urine sample in pH ~ 8 buffer through a disposable anion exchange column. It is well known that most proteins are acidic and are expected to be removed by anion exchange columns in pH ~ 8 solutions, while formaldehyde is not. For example, the present invention has demonstrated that bovine serum albumin (BSA) can be easily removed by anion exchange columns. However, the present invention found that this treatment procedure did not help the determination of urine formaldehyde, and the main X interfering species remained unchanged. It is generally believed that the protein content in healthy human urine is very low. Obviously, urine protein may not be the main interfering component in urine formaldehyde determination, and other organic species with large content in urine, such as various hydroquinones as mentioned above, are more likely to be the main interfering substances.

[0044] Removal of organic interferences: It is well known that activated carbon can effectively remove a wide range of organic matter from solutions. In fact, this study showed that after appropriate treatment of urine samples with activated carbon, all major interfering species responsible for absorption at 500 nm can be completely removed and urine formaldehyde content can be consistently detected under a fixed set of assay conditions ( Figure 2 , 3 ). Controlled comparative experiments showed that activated carbon can effectively remove a wide range of organic substances, including all organic molecules containing aromatic rings, such as hydroquinone and bicinchoninic acid (BCA), as well as all tested peptides and most proteins. Interestingly, activated carbon has limited adsorption capacity for bovine serum albumin, which is probably due to the compact structure of bovine serum albumin and the high hydrophilicity of its molecular surface. However, the same treatment of formaldehyde solution with animal activated carbon (purchased from BDH) also resulted in a loss of 20-30% of the added formaldehyde, which is consistent with the formaldehyde recovery results of 30 representative urine samples treated with the same activated carbon. The average formaldehyde recovery of these 30 urine samples was (70±5)%. However, this small and proportional absorption of formaldehyde by activated carbon does not prevent the distinction and quantification of the relative formaldehyde content of different urine samples. On the other hand, some of the latest experimental data of the present invention show that activated carbon prepared from coconut shells can increase formaldehyde recovery to 90-100%, while also effectively removing X interfering species in urine samples.

[0045] Selection of activated carbon: The adsorption specificity and adsorption capacity of activated carbon from different sources may vary greatly depending on the brand and particle size, so they must be tested individually before use. Ideally, the activated carbon selected will not only effectively remove interfering substances, but also have minimal adsorption of formaldehyde. Powdered activated carbon has a large surface area and has a high adsorption capacity, but it is usually difficult to separate from the treated urine sample. Large particles of activated carbon have limited adsorption capacity. As a rule of thumb, activated carbon with a size range of 20-100 mesh is suitable. In addition, in any case, the adsorption of interfering substances in urine samples by activated carbon must be far below its adsorption saturation, otherwise, the interfering substances cannot be completely removed, which will lead to an overestimation of the formaldehyde content. Using enough activated carbon under a fixed set of measurement conditions, the detected urine formaldehyde content remains basically constant. For the animal activated carbon (BDH Chemicals) used in this study, 200 μL of undiluted urine sample requires approximately the same weight of activated carbon for pretreatment. However, the actual concentration of different urine samples may vary greatly.

[0046] Adding isopropanol: For the 96-well plate measurement method, 10% isopropanol is usually added to the formaldehyde standard solution and the diluted urine sample solution. The benefit is that it can reduce the reading deviation caused by the formation of different surface menisci due to different solution components. In addition, adding 10% isopropanol to the measurement solution also helps to increase the formaldehyde recovery rate and decompose bubbles that may be generated when mixing various reagents.

[0047] Condensation reaction kinetics: Adding an excess of MBTH reagent to a urine sample treated with activated carbon will trigger a condensation reaction between MBTH and urine formaldehyde or other formaldehyde-like substances ( Figure 1 Although the condensation reaction product is colorless, the progress of the reaction can be indirectly monitored by the subsequent color development reaction steps (ii, iii) induced by the FeCl3 / HCl reagent. This theory is based on the following observations of the present invention: first, after the addition of the FeCl3 / HCl reagent, not only the first condensation reaction will be inhibited by the introduced HCl, but also the excess FeCl3 will quickly convert the MBTH required for the condensation reaction in the reaction step (ii) into the oxidized MBTH required for the reaction step (iii), thereby quenching the condensation reaction (i) and promoting the oxidative coupling reaction (iii) (see reaction flow Figure 1 ); secondly, all aldehyde-related coloring substances that absorb at 628 nm completely develop color in less than an hour and remain stable for more than 10 hours ( Figure 3 E). The reaction kinetics study of the present invention shows that the condensation reaction between MBTH and formaldehyde standard solution is completed within about 30 minutes at room temperature ( Figure 2B, E(i)). However, the equivalent reaction steps for charcoal-treated urine samples appear to consist of two types of reactions: a rapid reaction that is completed within approximately 30 minutes and a slow reaction that can take more than 24 hours ( Figure 2 D, E (iv)). Rapid reactions may be mainly attributed to formaldehyde species, while slow reactions may be related to other aldehyde species. Therefore, although the formaldehyde concentration of the pure formaldehyde sample measured in this test is not sensitive to the condensation reaction time after 30 minutes of reaction, the apparent formaldehyde concentration of urine in this test depends to a certain extent on the length of the condensation reaction time. A series of comparative experiments showed that as the first step condensation reaction time increased from 0.5h to 1h and then to 1.5h, the measured apparent formaldehyde concentration of urine increased by an average of about 7%, while the control value of the formaldehyde standard remained unchanged (see Table S2). As a compromise, the present invention selects a condensation reaction time of about 1h to maximize the formaldehyde detection rate, but it is inevitable that A 628 Some level of other aldehydes (RHCO) are included in the reading. Those with simple unsaturated R groups are reported to condense with MBTH at slower rates. Apparently, while activated carbon treatment removes most X interfering species, it is less efficient at removing some formaldehyde-like species. Increasing the reaction temperature will speed up the condensation reaction, but is not recommended for urine samples because it will disproportionately promote the slower reactions of those formaldehyde-like species, increasing their contribution to the final apparent formaldehyde reading.

[0048] Urine samples that were not treated with activated carbon contained various interfering substances. It is noteworthy that after adding the FeCl3 / HCl reagent to the untreated urine sample containing MBTH, the characteristic absorption of species X at 500nm was almost completely revealed even in the shortest mixing time after the addition of MBTH (<0.4min) ( Figure 2 A, C, E (ii)), which is consistent with the fact that these X species are directly oxidized by FeCl3 before undergoing a coupled color reaction with MBTH. For example, the typical representative of X species, hydroquinone, must first be oxidized to quinone before it can undergo a condensation reaction with MBTH to produce a brown-red product. Oxidative coupling reaction kinetics: This study was based on a colorless solution after one hour of the first condensation reaction. As mentioned above, adding an excess of FeCl3 / HCl reagent to the condensation reaction mixture will quickly consume the reduced MBTH and quench the first condensation reaction, but at the same time it will trigger a subsequent oxidative coupling color reaction (iii), causing the solution color to change. The colors of the formaldehyde standard solution and the analytical solutions of the activated carbon-treated and untreated urine samples are all different ( Figure 3 F) A related to species X 500The fastest development, reaching the maximum value in about 5 minutes, and then the intensity slightly decays ( Figure 3 B, C (ii, iii)). A related to pure formaldehyde components 628 It also develops rapidly, reaching its maximum value in less than 10 minutes ( Figure 3 A, C(i)), but the A of urine sample 628 The absorbance value takes about 20 minutes to fully develop ( Figure 3 D, E(v)). However, once fully developed, A 628 It remains stable for at least 10 hours. Therefore, the oxidative coupling reaction time is preferably between 0.5 and 1 hour.

[0049] The present invention also noted that the color of the formaldehyde standard analysis solution is slightly different from the color of the urine analysis solution treated with activated carbon ( Figure 2 A, 3F). This difference is mainly attributed to the absorption band with wavelength < 500nm ( Figure 3 A, D). The pale yellow reagent FeCl3 / HCl has a strong absorption band below 500nm, but the various redox reactions that occur with FeCl3 make this absorption band unstable and its intensity changes over time. The formaldehyde standard and urine sample have different compositions, so this absorption band will naturally be different. However, after long-term incubation, the colors of these solutions will become more similar to each other.

[0050] Optimizing the ratio of MBTH to FeCl3 / HCl reagent: The formaldehyde concentration in this assay is in the μM range, but both MBTH and FeCl3 / HCl reagents are in the mM concentration range. Such a large excess of reagent is required to ensure quantitative determination of formaldehyde in urine samples, because there may be a variety of "silent" substances (i.e., substances that undergo colorless chemical reactions) in urine that may compete with MBTH. However, in any case, the FeCl3 concentration must be much higher than the MBTH concentration (usually more than twice) to ensure that after the FeCl3 is consumed in reaction step (ii), there is still a sufficient amount of FeCl3 available for the oxidative coupling color development reaction (iii). Otherwise, the color development reaction will be hindered, resulting in an underestimation of the urine formaldehyde content. The present invention also proves that under the premise that the FeCl3 concentration is greater than twice the MBTH concentration, changing the FeCl3 concentration has little effect on the urine formaldehyde detection results; but conversely, changing the MBTH concentration has a greater impact on the results. This shows that for urine samples, the first step condensation reaction is more complicated than the subsequent oxidative coupling reaction, and therefore, the MBTH concentration used must be relatively fixed.

[0051] Formaldehyde recovery experiment: The recovery experiment is an important indicator for all quantitative analysis. The formaldehyde recovery experiment in this study was carried out by adding formaldehyde standard solution to the control blank solution or urine sample solution before activated carbon treatment. The experimental results of 30 different urine samples showed that the average recovery rate of added formaldehyde was (70±5)% when the urine samples were treated with animal activated carbon purchased from BDH Chemical Company in the UK. However, the recovery rate can be increased to (90±10)% when coconut shell activated carbon purchased from Tanerno Catalytic Technology Co., Ltd. in Changge City, China. With the continuous expansion and progress of the experiment, we are expected to find more suitable activated carbon.

[0052] Urine formaldehyde concentration: Two points must be emphasized here: First, the main purpose of the present invention is to explore a biomarker from human urine that can quickly and easily monitor human health status, rather than to quantify the absolute concentration of urine formaldehyde. In fact, the urine formaldehyde concentration detected by the present invention is an apparent concentration, which contains other aldehyde substances. The important point is that the method of the present invention can conveniently and reliably monitor the apparent formaldehyde concentration of different urine samples; secondly, there are great differences in the average urine formaldehyde concentration reported by different studies, and even the average urine formaldehyde concentration reported by the same study for the same group of urine samples using different determination methods is also very different, ranging from as low as <2μM to as high as ~300μM. Table 1 lists some other literature reported values ​​and the measured values ​​of the present invention.

[0053] Table 1 Comparison of urine formaldehyde concentrations measured in different studies

[0054]

[0055] Note 1: Data is taken from Wang, Y., Pan, F., Xie, F., He, R. and Guo, Q. (2022) Correlation Between Urine

[0056] Formaldehyde and Cognitive Abilities in the Clinical Spectrum ofAlzheimer's Disease.Frontiers in Aging

[0057] Neuroscience.14;

[0058] Note 2: Data are taken from the literature Ai, L., Wang, J., Li, T., Zhao, C., Tang, Y., Wang, W., Zhao, S., Jiang, W., Di, Y., Fei, X.,

[0059] Luo,H.,Li,H.,Luo,W.,Yu,Y.,Lin,W.,He,R.and Tong,Z.(2018)A rapid and sensitive fluorescence method

[0060] for detecting urine formaldehyde in patients with Alzheimer'sdisease.Annals of Clinical Biochemistry.56,210-218.

[0062] Correlation between different Alzheimer's disease patient groups and urine formaldehyde concentration: Hunan Qiankang Technology Co., Ltd. previously developed a urine Aβ test strip as an early screening method for Alzheimer's disease. The present invention used the #2 (designed for frozen urine) and #6 (designed for fresh urine) urine Aβ test strips with different processes to measure AIBL frozen urine and found that the urine formaldehyde concentration was positively correlated with the #2 urine Aβ test result (P<0.001; Figure 5 A, Table 2), but there was no statistical correlation with the Aβ test results of #6 test paper ( Figure 5 B) This result shows that urine formaldehyde concentration has the potential to be used as an early screening indicator.

[0063] No significant differences in urinary formaldehyde concentrations were observed between the different elderly groups ( Figure 5 C), which may be due to the small sample size (only 7 in the AD group).

[0064] Table 2 Correlation between urine formaldehyde concentration and urine Aβ test results of #2 test paper

[0065] #2 Test strips to detect Aβ results burden Positive / Negative just positive / positive Number of samples n＝49 n＝21 n＝23 n＝10 [Formaldehyde concentration] μM 90±56 178±109 164±81 272±91 P value compared with #2 (negative) P＝0.0007 P＝0.0007 P<0.0001

[0066] Correlation between urine formaldehyde concentration and PO4, Mini-Metrics and Sequential Examination (MMSE), amyloid PET, and centiloid: Overall, urine formaldehyde levels were positively correlated with urine PO4 concentration, which was expected because they were both positively correlated with urine concentration (i.e., urine color). Figure 5 The observed associations for urine formaldehyde also applied to the measured urine PO4 concentrations. There was no statistical correlation between urine formaldehyde levels and the Mini-MMSE, AβPET scores, or centiloid ( Figure 6 BD).

[0067] Table S1: Example of Excel spreadsheet data processing for PO4 quantification analysis

[0068]

[0069] annotation:

[0070] 1) For the phosphate determination of each urine sample, a set of different dilution factors (0.15k, 0.45k,

[0071] 1.35k, 4.05k), but in general, only the original data A 620 Data within the range of 0.15–0.90 (highlighted in grey) are valid readings and can be reliably used for PO4 concentration calculations; for all different urine samples, raw data from at least two dilutions met this requirement.

[0072] 2) The table lists a total of five representative urine samples (A, B, C, D, E) used for PO4 determination (raw data row AD) and PO4 recovery experiment after adding 10mM PO4 (raw data row EH). The determination was performed twice with reference to a set of PO4 standard solutions with [PO4] = 0, 4, 8, 16, 24, 32, 40μM.

[0073] Table S2: Example of Excel spreadsheet data processing for urine formaldehyde detection

[0074]

[0075] annotation:

[0076] 1. The raw data in the top box are the raw A of the activated carbon treated urine samples with dilution factors of 1, 2, 4, and 8. 628 The raw data in columns 1-3 are the analytical readings of the formaldehyde standards from rows A to H, which have formaldehyde concentrations of 40, 30, 20, 10, 5, 0, 0, 0 μM, respectively. For each formaldehyde standard or urine sample, three A 628 The readings correspond to the first step condensation reaction time of 0.5h, 1.0h and

[0077] The second stage oxidative coupling reaction time was 1 h for all analyzed solutions.

[0078] 2. The calibration equation Y=0.0575+0.0234X in row 22 is derived from the reaction of the formaldehyde standard in column 3 with a first condensation reaction time of 1.5 hours, but this equation is also applicable to the reaction of the formaldehyde standard at 0.5 hours and 1 hour (i.e., the data in columns 1 and 2).

[0079] 3. The data in rows 25 and 36 show the percentage of original urine in the urine samples that was treated with activated carbon, so the average urine formaldehyde concentrations found in rows 30 and 41 must be divided by these percentages to obtain the final urine formaldehyde concentrations shown in rows 31 and 42.

[0080] 4. The urine formaldehyde concentrations measured in rows 30 and 41 are the averages of solutions diluted 2 and 4 (highlighted in yellow). The data for solutions diluted 1 and 8 are not included in the averages because they are more susceptible to experimental error.

[0081] 5. The data in rows 32 and 43 are the percentage increases in urine formaldehyde concentration measured after increasing the first step reaction time from 0.5 h to 1.0 h and then to 1.5 h.

Claims

1. A method for determining formaldehyde in urine based on colorimetry, characterized in that: The method comprises the following steps: treating the urine sample with activated carbon before testing the urine sample, and then determining the formaldehyde in the urine sample by colorimetry.

2. The method for determining formaldehyde in urine based on colorimetry as claimed in claim 1, characterized in that: The method is to treat the urine sample with activated carbon before testing the urine sample to be tested, then add the urine sample treated with activated carbon into a cuvette containing MBTH solution for reaction, then add FeCl3 solution containing HCl into the cuvette for reaction, and read the absorbance A 628 The formaldehyde concentration in the urine sample is quantified by referring to the formaldehyde standard solution; or, the method is to treat the urine sample with activated carbon before detecting the urine sample to be tested, then add the urine sample treated with activated carbon to the well plate, add MBTH solution to the well plate for reaction, then add FeCl3 solution containing HCl to the well plate for reaction, and read the absorbance A 628 The formaldehyde concentration in the urine sample is quantified by referring to a formaldehyde standard solution; the activated carbon is animal or plant activated carbon.

3. The method for determining formaldehyde in urine based on colorimetry as claimed in claim 2, characterized in that: The mass-to-volume ratio of the activated carbon to the urine sample is 0.1:100 to 0.3:100, and the unit of the mass-to-volume ratio is g / μL.

4. The method for determining formaldehyde in urine based on colorimetry as claimed in claim 3, characterized in that: The method of treating the urine sample with activated carbon is to add high-purity water, CaCl2 solution and urine sample to a container containing activated carbon; the mass volume ratio of the activated carbon to high-purity water is 0.1:400 to 0.3:400, and the unit of the mass volume ratio is g / μL; the mass volume ratio of the activated carbon to the CaCl2 solution is 0.1:500 to 0.3:500, and the unit of the mass volume ratio is g / μL.

5. The method for determining formaldehyde in urine based on colorimetry as claimed in claim 4, characterized in that: The concentration of CaCl2 in the CaCl2 solution is 15-25 mM. The CaCl2 solution contains isopropyl alcohol, and the volume percentage of the isopropyl alcohol in the CaCl2 solution is 15-25%.

6. The method for determining formaldehyde in urine based on colorimetry as claimed in claim 2, characterized in that: The activated carbon is used to treat the urine sample for 20-50 minutes, and then the activated carbon is separated from the urine sample.

7. The method for determining formaldehyde in urine based on colorimetry as claimed in claim 2, characterized in that: The reaction time of the urine sample with the MBTH solution is 0.5-1.5 h, and the reaction time of the urine sample with the FeCl3 solution containing HCl is 0.5-1.5 h.

8. The method for determining formaldehyde in urine based on colorimetry as claimed in claim 2, characterized in that: In the cuvette, the volume ratio of the MBTH solution to the urine sample treated with activated carbon is 40:200 to 60:200, and the volume ratio of the FeCl3 solution containing HCl to the urine sample treated with activated carbon is 40:200 to 60:200; in the well plate, the urine sample treated with activated carbon is divided into an undiluted solution and a diluted solution, and the diluted solution is a urine sample and a diluent prepared in a volume ratio of 1:1, and the diluent is an isopropanol aqueous solution, and the volume percentage content of the isopropanol diluent in water is 5-20%. After the urine sample treated with activated carbon and the diluted urine sample are added to the well plate respectively, the MBTH solution is added for reaction, and the volume ratio of the MBTH solution to the urine sample treated with activated carbon or the diluted urine sample is 20:140 to 40:140, and the volume ratio of the FeCl3 solution containing HCl to the urine sample treated with activated carbon or the diluted urine sample is 20:140 to 40:

140.

9. The method for determining formaldehyde in urine based on colorimetry as claimed in claim 2, characterized in that: The MBTH concentration in the MBTH solution is 35-45 mM; the HCl concentration in the FeCl3 solution containing HCl is 80-120 mM, and the FeCl3 concentration is 70-90 mM.

10. Use of the method for determining formaldehyde in urine based on colorimetry according to any one of claims 1 to 9 in the detection of early biomarkers of Alzheimer's disease.

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