CN1415967A - Device for detecting inorganic arsenic in drinking water based on enzyme inhibition reaction - Google Patents

Abstract

This invention relates to a researching device testing inorganic As content in water base no acetycholinesterase suppressino reaction composed of peristaltic pump, film reactor, detector, signal analysis system and analysis reagent packet. The acetylcholinesterase in dry powder is absorbed on cellulose acetate membrane and analyzed into free state enzyme to react with arsenous radical, its reactivity is suppressed to get the concentration of arsenous radical by determining suppression rate of enzyme reactivity then to realize the determination of whole inorganic As content in water by recover the arsenic radical to arsenous radical by the recover reagent with detect limitation: 20ppb, linear sphere: 50-1000 ppb.

Description

Based on inorganic arsenic pick-up unit in the potable water of enzyme inhibitory reaction

The invention belongs to the analytical test field, relate to a kind of development that detects the device of inorganic arsenic content in the water based on the acetylcholinesterase inhibitory reaction.This device is mainly by peristaltic pump, film reactor, and detecting device, Signal Analysis System, and the analytical reagent bag is formed.During detection, in the film reactor of particular design, the acetylcholinesterase that is absorbed and fixed on the cellulose acetate film with dry powder form resolves to the free state enzyme, and with sample in arsenous anion reaction, its activity inhibited.Can draw arsenous anion concentration in the sample by the inhibiting rate of measuring enzymatic activity.Arsenate in the water is reduced to arsenous anion can realizes in the water total inorganic arsenic Determination on content by going back original reagent.This device is limited to 20ppb to the detection of inorganic arsenic in the water, and the range of linearity is 50-1000ppb.This device can be used as the scene of inorganic arsenic content in potable water or the underground water and detects.

The background in present technique field and current situation are roughly as follows: the low dosage arsenic that potable water causes is exposed to ubiquity all over the world.The detection of current arsenic mainly relies on AAS, AFS, ICP-MS, modern large-sized analytic instrument such as ICP-AES.Though these instruments have very high sensitivity and accuracy, owing to costing an arm and a leg and being difficult to miniaturization, so can only will in the laboratory, detect after the sample preparation.Because it is being the rural area of drinking water source with underground water that arsenic poisoning mostly occurs, the water source that need to detect is many and disperse, and the migration susceptibility of arsenate ion in underground water is very strong, even for the water well, the concentration of arsenic is also among gradually changing, so only rely on large-sized analytic instrument to be difficult to the arsenic content in the potable water is carried out effective monitoring, for this reason, press for development of miniaturized in-situs tester device and method.Using maximum in-situ check and test methods at present is chemical reagent bar method.The cardinal principle of this method is by add reductibility reagent in sample solution, by series of chemical arsenic is reduced to arsine gas, detected capture of arsine gas then, and with the reaction of top developer, measure the concentration of arsenic in the original aqueous solution at last by colorimetric.The advantage of this method is easy to be quick, and has commercialization detector bar or kit to sell, and shortcoming is detection sensitivity poor (100ppb), and the hypertoxic arsine gas that reaction produces might constitute a threat to operator's health.Similar device of the present invention does not appear in the newspapers as yet at present.The document of relevant this respect can be referring to 1.D.Chakraborti et al, and Talanta 2002,58:3-222.A.M.Featherstone, et al., Anal.Chim.Acta 2000,409:2153.Stoytcheva, et al., Electroanalysis, 1998,10:994-9984.A.Hussam, et al., Environ.Sci.Technol., 2000,33 (20): 3686-36885.D.K.Nordstrom, Sicence 2002,296:2143-2145

The purpose of this invention is to provide a kind of based on the inhibiting Flow Injection Analysis device of arsenous anion to acetylcholinesterase.This apparatus structure is simple, be easy to carry, with low cost, the scene that can be used for water source arsenic content is detected, its stability and repeatability can satisfy requirement of quantitative test.

Technical scheme of the present invention is to realize by the film reactor of a particular design and respective flow injection analytic system.Fig. 1 is the structural representation of film reactor, and wherein, A is the film reactor sectional side view, and B is an enzyme membrane part partial enlarged drawing, and C is a reactor inside surface liquid bath synoptic diagram.The each several part assembly that marked of numeral is respectively among the figure: 1, and the shell of reactor of threaded and spiral shell button; 2, the teflon supporter; 3, the plastics hoop; 4, nylon membrane; 5, semi-permeable diaphragm; 6, enzyme membrane; 7, silicone gasket.Fig. 2 is the Flow Injection Analysis systematic schematic diagram.Wherein, 1 is sample, and 2 is damping fluid, and 3 is substrate and chromogenic reagent solution, and 4 is peristaltic pump, and 5 is film reactor, and 6 is detecting device.During detection, at first with immobilization the cellulose acetate membrane of the phatidylcholine esterase film reactor shown in Figure 1 of packing into, then this reactor is inserted Flow Injection Analysis system shown in Figure 2, and with blank damping fluid balance 20 minutes, at this moment, acetylcholinesterase desorb on the cellulose acetate membrane, and between cellulose acetate membrane and semi-permeable diaphragm, form the enzyme layer of a high concentration, this helps to reduce dissociating of acetylcholinesterase-arsenous anion compound when sample determination.Behind the instrument stabilizer, inject a certain amount of enzyme reaction substrate and developer, the blank response of record instrument this moment.Get 20 ml sample solution with 25 milliliters of syringes, filter with the syringe filters that 0.45 micron miillpore filter is housed.At first in filtrate, add the pack that contains potassium iodide, arsenate in the solution is reduced to arsenous anion, add in filtrate then and comprise quantitative pralidoxime iodide, sodium ethylene diamine tetracetate, phosphatic analytical reagent bag, vibration is fully dissolved and mixing reagent.The reason of adding pralidoxime iodide in solution is very slow because of the reaction of arsenous anion under the normal condition and acetylcholinesterase; and its reaction velocity can improve 220 times after the adding pralidoxime iodide; pralidoxime iodide is again the toxinicide of organophosphorus and carbamates toxic agent simultaneously; so in solution, add the reaction velocity that pralidoxime iodide both can increase arsenous anion and acetylcholinesterase, can prevent the interference of organophosphorus and carbamates toxic agent in the sample again.In addition, because therefore the activity that some heavy metal cations also can acetylcholine esterase inhibition adds a certain amount of sodium ethylene diamine tetracetate to prevent the interference of heavy metal cation to measuring in the sample in solution.Through the flow velocity of the sample solution after the above-mentioned processing with 0.3 ml/min, by film reactor, the arsenous anion in the sample combines with acetylcholinesterase in the film reactor, makes the activity inhibited of acetylcholinesterase.Because some organophosphoruss that may exist and carbamates toxic agent and heavy metal cation be to the influence conductively-closed of acetylcholinesterase in the sample solution, thus the rate that is suppressed of acetylcholine esterase active only with solution in the concentration of arsenous anion become positive correlation.After reaction is carried out 20 minutes, inject a certain amount of enzyme reaction substrate and developer, read the response of instrument, and compare, calculate the inhibiting rate of acetylcholinesterase, can draw the content of arsenic the sample from typical curve with blank response.

Fig. 3 is based on the structural representation of the potable water inorganic arsenic pick-up unit of enzyme inhibitory reaction.Wherein, 1 is sample bottle, and 2 is the damping fluid bottle, and 3 is T-valve, and 4 is peristaltic pump, 5 substrate sampling valves, 6 is temperature controller, and 7 is heating rod, and 8 is film reactor, and 9 is detection cell, 10 is light emitting diode, and 11 is photomultiplier, and 12 is digital to analog converter, and 13 is transformer.Thus figure as can be seen, the present invention is simple in structure, be easy to carry, with low cost, is applicable to the arsenic content in rural area or open-air surface water, well water, the underground water is carried out scene detection.

In finishing process of the present invention, analysis condition to be optimized, optimal conditions is as follows: AchE concentration: 0.2u/ml, 2-PAM concentration: 1 * 10 ^-4Mol/L, the pH value: 7.4, temperature of reaction: 25 ℃, ionic strength: 0.2mol/L, reaction time: 20min.Under optimized conditions, the detection of this method is limited to 20ppb, is 50-1000ppb between linear zone.Is that sample has carried out adding and reclaims experiment with the tap water, the recovery of low concentration (50ppb) and high concentration (1000ppb) is respectively 88.1% and 95.0%, we have also investigated under the experiment condition, As (V), parathion, malathion, Fe ³⁺, Ni ²⁺, Cu ²⁺, Hg ²⁺, Zn ²⁺, Ge ²⁺, Pb ²⁺Deng the influence to measuring, the result shows, removes Cu ²⁺Inhibiting rate to AchE when 500ppb reaches outside 10%, other compound in 1ppm concentration with interior inhibiting rate to AchE all less than 10%, therefore, this method is to AsO ₃ ^3-Detection have very high specificity.

Claims (4)

1, a kind of potable water inorganic arsenic pick-up unit based on the enzyme inhibitory reaction is characterized in that using absorption method that acetylcholinesterase is fixed on the carrier film with dry powder form, is convenient to store or transportation.Immobilised enzymes desorption on the carrier film participates in reaction with the free state enzyme during detection.

2, according to claims 1 described pick-up unit, it is characterized in that using semi-permeable diaphragm or ultra filtration membrane that acetylcholinesterase is limited in the very little volume, form highly concentrated solution.

3,, it is characterized in that in sample solution adding pralidoxime iodide increasing the reaction velocity of arsenous anion and acetylcholinesterase, and prevent the interference of organophosphorus and carbamates toxic agent in the sample according to claims 1 described pick-up unit.

4,, it is characterized in that in sample solution, adding sodium ethylene diamine tetracetate to prevent the interference of heavy metal cation to measuring in the sample according to claims 1 described pick-up unit.

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