CZ30353U1 - A device and a kit for detection of methanol poisoning - Google Patents

Description

The invention relates to a device for the determination of methanol and formic acid in samples of blood serum, blood plasma, other body fluids and alcoholic beverages in order to detect intoxication of the organism after ingestion of methanol. The device according to the invention can be manufactured in a portable form.

The invention further relates to a kit of reagents suitable for use with said apparatus.

BACKGROUND OF THE INVENTION

Methanol intoxication is a rare but very dangerous poisoning. It can occur accidentally or in local epidemics (eg methanol affair in 2012 in the Czech Republic, when more than 40 people died from the effects of methanol consumption). Rapid analysis and identification of toxic chemicals in body fluids during acute poisoning is absolutely crucial as it allows the administration of an effective antidote and subsequent effective therapy. In methanol intoxication, both ingested methanol and its metabolites, especially highly toxic formic acid, may be present in the body. Serum formic acid levels in healthy subjects range from 0.07 to 1.2 mmol / l, with typical concentrations being less than 0.25 mmol / l. During severe methanol intoxication, this level may be increased by 10 to 100 times. Patients reach the health care facilities at different stages of poisoning, so it is important to determine both the concentration of methanol itself and formic acid. Diagnosis of this poisoning itself is difficult if there is no indication of ingestion of the harmful substance. Establishing a diagnosis is also more difficult in isolated poisoning than during an ongoing epidemic of intoxication. Currently, there is no portable analytical instrument that is able to quickly determine the content of methanol and formic acid in blood plasma or serum, or other body fluids. Determination of the blood plasma or blood serum methanol level is carried out in specialized laboratories by gas chromatography. Formic acid can also be determined by gas chromatography with a flame ionization detector [Wallage. H. R., Watterson. J. H., J. Anal. Toxicol. 2008, 32, pp. 241-247] or by mass spectrometric detector [Rasanen. I., Viinamaki. J., Vuori. E., Ojanpera.

I., J. Anal. Toxicol. 2010, 34, pp. 113-121], but it is necessary to convert it to its more volatile form by means of esterification, which is time consuming. For determination of formic acid it is also possible to use enzymatic methods, with the necessity to equip the workplace with a spectrophotometer or an automatic analyzer.

The determination of methanol and ants is usually only available in selected toxicology laboratories or in the biochemical laboratories of larger hospitals. Transporting blood samples to a clinical laboratory almost always means a delay. If the ingestion of methanol is highly probable, waiting for the result of the toxicological examination cannot delay the initiation of treatment of intoxication. On the other hand, a rapid, available screening of elevated levels of both methanol and formic acid in serum would greatly facilitate the elucidation of the cause of suspected poisoning.

The proposed portable device, which is able to quickly determine the concentrations of the above substances from a drop of blood plasma or serum, outside the biochemical laboratory, is of great benefit for medical diagnosis.

The essence of the technical solution

The object of the present invention is an analytical apparatus for determining the concentration of methanol and formic acid in a sample, for example in blood serum, blood plasma, optionally in other body fluids, or to determine the concentration of methanol in alcoholic beverages.

The analytical apparatus thus comprises at least one excitation optical element emitting radiation having a wavelength in the range of 300 to 600 nm, at least one reaction vessel, at least one sensing optical element, the reaction vessel being arranged between the excitation and sensing optical element, the control unit and the power supply. control module.

-1 CZ 30353 Ul

For example, the excitation optical element may be a light emitting diode (LED). In a preferred embodiment, the excitation optical element may further comprise a plastic, glass or quartz optical fiber for collimating the emitted radiation. The excitation optical element serves to excite the reaction mixture in the reaction vessel.

The sensing optical element may be, for example, a phototransistor or a photodiode. In a preferred embodiment, the sensing optical element may further comprise a plastic, glass or quartz optical fiber for collimating the collected radiation.

The reaction vessel preferably comprises reagents such as the alcohol oxidase kit (EC 1.1.3.13), formaldehyde dehydrogenase (EC 1.2.1.46), nicotinamide adenine dinucleotide and optionally a phosphate buffer at pH 7.6; or a set of format dehydrogenase (EC 1.2.1.2), nicotinamide adenine dinucleotide and optionally PBS IX buffer pH 7.4. Particularly for field use, it may be advantageous if the reagents in the reaction vessel are already prepared and mixed, and the reaction vessel may, for example, be provided with a sealing cap. For analysis, simply open the vial and add sample and buffer to the reaction mixture.

The reaction vessels may be, for example, plastic micro-volumetric cells, spectrophotometric cuvettes, or reaction micro-containers with mobilized reagents.

The power module preferably comprises a mobile voltage source, such as batteries or solar panels, variable resistors, and a voltage stabilizer. This power module allows the device to be designed as portable, independent of mains power. The power module supplies voltage and current to the excitation optical element and the sensing optical element.

The control unit controls the switching of the functions of the excitation optical element and the sensing optical element (e.g., via the excitation circuit and the sensing circuit), records the measured signal and evaluates it (the result can then be displayed on the LCD, the LED) or eg laptop, computer.

The radiation from the excitation light source changes its properties as it passes through the reaction vessel, and the radiation thus transmitted is detected by a sensing optical element in the form of an electrical voltage. The measured electrical voltage may preferably be amplified in the sensing electrical circuit by an amplifying element, such as a transistor or amplifier, and further processed.

The advantage of the device is its full portability, it can be powered by conventional AA batteries and can be used in the field, outside a specialized laboratory.

The present technical solution enables the measurement of the reaction mixture by direct measurement of the optical properties of the resulting products. Unlike conventional analytical instruments used to determine methanol or formic acid, its advantage is the rapid determination of the exact concentration of these substances in the field, for example in the patient, in the emergency vehicle, etc. The concentration of both analytes is known within 5 to 10 minutes.

The present invention can be further modified by the addition of a microcontroller (e.g. PIC 18F26K22) that allows digital signal resetting, includes programmable indicator LEDs to indicate signal level and signal the severity of intoxication depending on the concentration of toxic substances in the body fluid sample. termination of measurement and output connector for connection to external device for continuous signal / data acquisition.

In a preferred embodiment, the apparatus of the present invention may comprise at least two reaction vessels for differential measurement of the optical signal, then typically also comprise the same number of excitation and sensing optical elements.

The present invention further comprises a kit of reagents for use in an analytical instrument, the kit comprising alcohol oxidase (EC 1.1.3.13), formaldehyde dehydrogenase (EC 1.2.1.46) and nicotinamide adenine dinucleotide, optionally also a phosphate buffer at pH 7.6; or contains format dehydrogenase (EC 1.2.1.2), nicotinamide adenine dinucleotide and optionally PBS IX buffer pH 7.4.

-2EN 30353 Ul

The technical solution is further elucidated in the examples without limiting the scope of protection in any way.

Clarification of drawings

Figure 1 shows a schematic embodiment of the invention according to Example 1.

Figure 2 shows a detail of the electronic connection of the power supply module of the invention according to example 1. Figure 3 shows a detail of the electronic connection of the sensing electronic circuit of the invention according to example 1.

Figure 4 shows a schematic embodiment of the invention according to Example 2.

Figure 5 shows a detail of the electronic wiring of the power supply module of the invention according to Example 2.

Figure 6 shows a detail of the electronic circuit of the sensing electronic circuit of the invention according to Example 2.

Figure 7 shows the results of monitoring the output signal when determining formic acid in the embodiment of Example 1.

Figure 8 shows the results of the output signal monitoring in the methanol determination of the embodiment of Example 2.

Example of technical solution implementation

Example 1

One example of a technical solution of the system is shown schematically in Figure 1. This embodiment is generally comprised of a power module / circuit ii, an excitation light source 12, an optional additional light collimation element 13, a reaction vessel 14, an optional additional element 15 for collimating light collecting A sensing element 16 and a sensing electrical circuit 17 are connected to the sensing electrical circuit. An LCD display 18 is connected to display the relevant measured quantity (current, voltage, concentration) or an optional connector for connection to a continuous data acquisition device (AD converter, computer, notebook). .

The supply module ϋ, according to Figure 2, constituting the supply circuit consists of a voltage source 21, which may be, for example, a battery (conventional, rechargeable Li-ion) or a solar panel, a voltage stabilizer 22 and a control variable resistor 23, e.g. , varistor. The excitation light source according to Figure 1 is a LED 12 with a wavelength in the range of 300 to 600 nm. The optional additional light collimation element 13, 15 may be, for example, an optical fiber of suitable diameter (0.5-1.5 mm) and properties (radiation permeability, multimode / single mode) made of plastic, glass or quartz. The sensing electrical element 16 of Figure 1 may be, for example, a phototransistor, a photodiode (silicon, Ge, etc.), which may include an additional electrical circuit for converting current to voltage and an amplifying element.

The sensing electrical circuit 17 in this example of the technical solution of Figure 3 consists of a voltage stabilizer 31 for the amplifying element 32 (transistor, operational amplifier) to amplify the measured signal, and three resistors 33, 34, 35. The measured, amplified and otherwise preconditioned signal it is led to an LCD display 18, or to an output connected to a continuous data acquisition device (AD converter, computer, notebook).

Example 2

Another embodiment of the apparatus is shown schematically in Figure 4. This embodiment is generally comprised of a power module / circuit 41, two excitation light sources 421, 422 of equal wavelength, an optional additional light collimation element 43, two reaction vessels 441, 422, an optional additional light collimating element 45, a pair of optical sensing elements 461, 462, and a sensing electrical circuit 47. An LCD display 48 displaying the relevant measurand (current, voltage, concentration) or an optional connector for connection is attached to the sensing electrical circuit. to a device for continuous data acquisition (AD converter, computer, notebook).

-3GB 30353 UI

The power supply circuit 41 (according to Figure 5) consists of a voltage source 51, which may be, for example, a battery (conventional, rechargeable Li-ion) or a solar panel, a voltage stabilizer 52, two control variable resistors 531, 532, e.g. , varistors. The excitation light sources according to Figure 4 are LEDs 421, 422 with the same wavelength in the range of 300 to 600 nm. The optional additional light collimation element 43, 45 may be, for example, an optical fiber of suitable diameter (0.5 to 1.5 mm) and properties (radiation permeability, multimode / singlemode) made of plastic, glass or quartz. The sensing electrical elements 461, 462 of Figure 4 may be, for example, phototransistors, photodiodes (silicon, Ge, etc.), etc., which may include an additional electrical circuit for converting current to voltage and an amplifying element.

The sensing electrical circuit 47 in this example of the technical solution of Figure 6 is formed by a voltage stabilizer 61 for the amplifier element 62 (transistor, operational amplifier) that serves to amplify the measured reference signal, potentiometers 63 and reference voltage source 64 for signal reset and resistors 65 resistors may be the same or different). The measured, amplified and otherwise pre-treated signal is fed to the LCD display 48, or to an output connected to a continuous data acquisition device (AD converter, computer, notebook).

Example 3

The technical solution of Example 1 was used to determine the concentration of formic acid in blood serum samples. In these experiments, reaction vessel 14 was filled with a reaction mixture consisting of 16.5 volumes of format dehydrogenase (EC 1.2.1.2, 27.6 g / L), 33.5 volumes of nicotinamide adenine dinucleotide (6.25 g / L) dissolved in PBS 1X buffer, pH 7.4. To this solution was added 5 volumes of blood plasma sample containing various concentrations of formic acid shown in Figure 7, which shows the measured results. The results demonstrate a sensitive determination of formic acid in blood serum within minutes. Example 4

The technical solution of Example 2 was used to determine the concentration of methanol in blood serum samples. In these experiments, both reaction vessels 441, 442 were filled with the same reaction mixture consisting of 100 parts by volume of alcohol oxidase (EC 1.1.3.13, 1.1 g / l), 600 parts by volume of formaldehyde dehydrogenase (EC 1.2.1.46, 1 g / l) and 600 volumes of nicotinamide adenine dinucleotide (5 mM) in 0.1 M phosphate buffer pH 7.6. To this solution was added 20 volumes of a blood plasma sample containing various concentrations of methanol shown in Figure 7, which shows the measured results. The results demonstrate a sensitive determination of methanol within minutes.

Industrial applicability

The apparatus and kit of the present invention can be used to determine the concentration of methanol and formic acid in body fluid and alcoholic beverage samples.

PROTECTION REQUIREMENTS

Claims (9)

An analytical instrument for determining the concentration of methanol and formic acid in a sample, characterized in that it comprises at least one excitation optical element (12, 421, 422) emitting radiation having a wavelength in the range 300 to 600 nm, at least one scanning optical element (16). 461, 462), at least one reaction vessel (14, 441, 442) disposed between the excitation (12, 421, 422) and the sensing optical element (16, 461, 462), the control unit and the power module (11, 41). The analytical apparatus of claim 1, wherein the excitation optical element (12, 421, 422) further comprises a plastic, glass or quartz optical fiber (13, 43) for collimating the emitted radiation. -4EN 30353 Ul The analytical instrument of claim 1 or 2, wherein the sensing optical element (16, 461, 462) further comprises a plastic, glass or quartz optical fiber (15, 45) for collimating the radiation collected. Analytical instrument according to any one of the preceding claims, characterized in that the reaction vessel (14, 441, 442) is selected from the group consisting of plastic micro-volume cuvettes, spectrophotometric cuvettes, reaction micro-vials with immobilized reagents. Analytical instrument according to claim 4, characterized in that the reaction vessel (14, 441, 442) comprises a set of reagents selected from the group of: alcohol oxidase, formaldehyde dehydrogenase, nicotinamide adenine dinucleotide and optionally a phosphate buffer at pH 7.6; and kits: format dehydrogenase, nicotinamide adenine dinucleotide, and optionally PBS IX buffer pH 7.4. Analysis apparatus according to any one of the preceding claims, characterized in that the power module (11, 41) comprises a mobile voltage source (21, 51), preferably batteries or solar panels, variable resistors (23, 531, 532) and a voltage stabilizer. (22,52). An analysis device according to any one of the preceding claims, characterized in that the control unit comprises an integrated circuit adapted to digitally reset the signal and comprising a programmable indicator LED for indicating the level of the signal and signaling the severity of intoxication as a function of toxic substances in the body fluid sample. for emitting an acoustic signal signaling completion of the measurement, and an output connector for connection to an external continuous signal / data acquisition device. Analytical instrument according to any one of the preceding claims, characterized in that it comprises at least two reaction vessels (441, 442) for differential measurement of the optical signal, at least two excitation optical elements (421, 422) and at least two sensing optical elements (461, 462). ). Reagent kit for use in an analytical instrument according to any one of the preceding claims, characterized in that it contains alcohol oxidase, formaldehyde dehydrogenase and nicotinamide adenine dinucleotide, optionally also a phosphate buffer having a pH of 7.6; or it contains the format dehydrogenase, nicotinamide adenine dinucleotide and optionally also PBS IX buffer pH 7.4. 5 drawings