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    • 专利摘要:
    Portable device for the detection, diagnosis and monitoring of tyrosinemia. The invention consists of the design of a portable, rapid and reliable device for the detection, diagnosis and monitoring of tyrosinemia using very small volumes of biological samples in an in situ manner. This device has integrated a potentiostat attached to an electrical system capable of transforming the electrochemical signal produced on a disposable electrode, constituted by conductive nanomaterials, in a signal easy to interpret by the user. This microdevice would represent an alternative method for neonatal screening, which would allow a rapid quantification of tyrosine in biological samples in the early detection programs of metabolic diseases. In addition, it could be incorporated into the daily life of a patient with tyrosinemia to improve their quality of life, being able to control tyrosine levels at any time by performing a simple analysis and thus avoid complications associated with the disease. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2679375A1
    申请号:ES201700139
    申请日:2017-02-23
    公开日:2018-08-24
    发明作者:Alberto ESCARPA MIGUEL；Maria Cristina GONZALEZ MARTIN；Laura GARCIA CARMONA；María MORENO GÚZMAN
    申请人:Universidad de Alcala de Henares UAH；
    IPC主号:
    专利说明:

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    PORTABLE DEVICE FOR DETECTION. DIAGNOSIS AND FOLLOW-UP
    OF TYROSINEMIA
    SECTOR OF THE TECHNIQUE
    The invention is part of the Analytical Chemistry sector and particularly electrochemical measurement devices for the development of miniaturized sensors.
    STATE OF THE TECHNIQUE
    Tyrosinemia is a rare metabolic disease with an autosomal recessive genetic basis characterized by defects in the genes encoding enzymes of the catabolic pathway of tyrosine (population incidence less than 1: 100,000). There are different types of tyrosinemia, depending on which enzyme is the genetic defect. Type I thiosinemia is characterized by the deficiency of fumaroyl acetoacetate hydrolase, this type of tyrosinemia has the most serious effects of the disease, since in the absence of treatment it causes liver failure, painful neurological seizures, mental retardation, rickets and hepatocarclnoma that cause The sick person does not usually exceed 2 years of life. Type II and III tyrosinemia are characterized by a failure in the enzyme tyrosine aminotransferase and 4-hydroxyphenylpyruvate dioxygenase, respectively. The incidence of tyrosinemia in these cases is unknown, since there are only isolated cases. These types of tyrosinemia are not as severe as those of type I. Mental retardation occurs occasionally and to a milder degree, in addition to this symptomatology they present eye damage and skin lesions (CR Scott, Am. J. Med. Genet., 2006, 42C: 121-126).
    In recent years, the implementation of analytical techniques for screening in hospitals for the detection of tyrosinemia within the neonatal screening program has grown. In it, metabolic diseases have a great weight because the detection and early treatment prevent neurological damage, reduce morbidity and mortality and reduce the possible disabilities associated with these diseases. However, neonatal screening programs differ between countries and, more strikingly, between different autonomous communities. Currently, the diagnosis is carried out by quantifying tyrosine in blood impregnated in paper in the programs of early detection of metabolic diseases.
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    In recent years some communities have incorporated automated tandem mass spectrometry (MS-MS) as a method of neonatal metabolic screening. The MS-MS measures many different molecules in a single test, however, the application of this technique is a great cost. It should also be noted that screening methods are not diagnostic procedures, and it is necessary to carry out confirmation tests to corroborate the disease and, where appropriate, apply a treatment (GM Calderón López, F. Jiménez Parrilla, A. Losada Martínez, Protocol of the Spanish Association of Pediatrics, 2008, 44).
    Once neonatal screening has been performed, diagnostic tests are performed for positive cases. They are currently based on three conventional studies:
    1. Clinical symptomatology. Study the different clinical manifestations that present the diversity of patients.
    2. Biochemical tests. It is based mainly on the quantification of amino acids, analysis of organic acids by gas chromatography-mass spectrometry and enzymatic determinations. This last study is based on the quantification of the activity of the enzymes involved in this metabolic pathway.
    3. Genetic studies. It is possible to detect mutations using simple molecular biology techniques. More than 30 different mutations have been described depending on the origin of their parents.
    In high-risk families, prenatal diagnosis can be carried out using different techniques:
    1. Quantification of succinllacetone in amniotic fluid.
    2. Measurement of fumaroyl acetoacetase hydrosllase in corial hairiness.
    3. Analysis of mutations in corial hairiness or amniocytes, in the case where the mutations of the parents are characterized.
    All these diagnostic tests, whether confirmation or prenatal diagnosis imply major limitations such as sampling and the volume required, the time spent and the necessary instrumentation. The performance of all the studies entails the waiting of the different biochemical tests with the following complication of the symptomatology due to the time factor, so important in the diagnosis, since as
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    It was commented earlier, a rapid detection can avoid different damages in the three target organs: liver, kidney and peripheral nervous system.
    Additionally, this disease lacks proper treatment, which means that patients and their families are forced to strictly control the daily intake of protein content to avoid high concentrations of tyrosine. When this amino acid is in high concentrations, it is degraded by alternative catabolic pathways that generate carcinogenic products. The few treatments that currently exist to treat tyrosinemia are based on blocking the activity of enzymes found in these alternative pathways, and which are responsible for reducing the generation of toxic products derived from abnormal tyrosine synthesis. Consequently, although high concentrations of carcinogenic products are avoided, high levels of tyrosine are still maintained. For this reason, it is extremely necessary to carry out, in addition to treatment, a very strict control of the levels of tyrosine / carcinogenic products in order to adjust the dose of the drugs administered and at the same time the amount of protein ingested per day. Thus, the only way to achieve an adequate benefit / risk balance for each patient is to carry out periodic analyzes of the tyrosine concentration in biological fluids, which currently goes through its HPLC analysis coupled to ultraviolet detection visible in centers. specific clinical analysis, a process that in its entirety involves several days with the consequent problem in the regulation of protein content and drugs.
    Due to the great problem caused by the delay in the quantification of tyrosine levels, the need for tools that allow the decentralization of the analysis, using low sample consumption and, more importantly, reducing the analysis time, is evident. Thus, the fact of obtaining portable tools for the detection of tyrosine creating potential point-of-care tools, would allow real-time monitoring by the patient at home or by the medical staff in consultation for regulation of the dose of drug and / or protein content.
    In this sense, electrochemical techniques are very attractive due to their inherent properties, such as high sensitivity, precision, simple operation,
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    fast and low cost analysis as well as the possibility of minlaturization (J. Tashkhourian, M. Daneshi, S.F. Nami-Ana, Anal Chim Acta., 2016, 902: 89-96). There is currently no established device that performs this type of decentralized analysis. However, there are research papers capable of detecting tyrosine, however none of them have all the features necessary to develop a point-of-care at the same time, which is why there is possibly no commercial device to date. Below are the works found in the scientific literature in the last six years for the determination of tyrosine using electrochemical methods:
    . M. Hasanzadeh, A. karimzadeh, N. Shadjou, A. Mokhtarzadeh, L. Bageri, S. Sadeghi, S. Mahbub. Mat. Sci. Eng. C. (2016) 62, 814-830.
    - F. Tadayon, S. Vaheda; H. Bagheri. Mat. Sci. Eng. C. (2016) 68, 805-813.
    - Ó. A. Yokus, F. Kardas, O. Akyildmm, T. Eren, N. Atar, M. L. Yola. Sensors and Actuat. B, (2016) 233, 47-54.
    - S. Shrestha, R. J. Mascarenhas, O. J. D'Souza, A. K. Satpati, Z. Mekhallf, Dhason A., P. Mariis. J. Electroanal. Chem. (2016) 778, 32-40.
    - H. R. Zare, B. Moradiyan, Z. Shekari, A. Benvidi. Measurement (2016) 90, 510-518.
    - O.J. D'Souza, R.J. Mascarenhas, A. K. Satpati, L. V. Aiman, Z. Mekhalif. Ionios (2016) 22, 405-415.
    - F. Chekina, S. Bagherib. Elektrokhimiya (2016) 52, 201-208.
    - M. Taei, F. Hasanpour, H. Salavati, S.H. Banitaba, F. Kazemi. Mat. Sci. Eng. C. (2016) 59, 120-128.
    - R. Jarosová, J. Rutherford, G. M. Swain. Analyst (2016) 141, 6031-6041.
    - J. Tashkhourian, M. Daneshi, S.F. Nami-Ana
    - Analytica Chimica Acta (2015) 902, 1-8.
    - A. K. Bhakta, R. J. Mascarenhas, O. J. D'Souza, A. K. Satpati, S. Detriche, Z. Mekhalif, J. Dalhalle, Materials Science & Engineerlng C (2015) 1873, 328-37.
    - M. Rahman, N. Siraj Lopa, K. Kim, J.-J. Read. Journal of Electroanalytical Chemistry (2015) 754, 87-93.
    - M. L. Yola, T. Eren, N. Atar. Sensors and Actuators. B: Chemical (2015) 201, 149157.
    - N. Baig and A. Kawde, Anal. Methods (2015) 7, 9535-9541.
    - S. Zhu, J. Zhang, X. Zhao, H. Wang, G. Xu, J. You. Microchim Acta (2014) 181,445451.
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    - E. Molaakbari, A. Mostafavi, H. Beitollahi. Electroanalysis (2014) 26, 2252-2260.
    - S. Chitravathi, B E. Kumara Swamy, G.P. Mamatha, B.N. Chandrashekar J. Mol. Liq. (2012) 172, 130-135.
    - Y. Fan, J.-H. Liu, H.-T. Lu, Q. Zhang. Microchim Acta (2011) 173, 241-247.
    - O. J. D'Souza, R. J. Mascarenhas, A. K. Satpatic, I. N.N. Namboothiri, S. Detriche, Z. Mekhalif, J. Delhallee. RSC Adv, 5 (2015) 91472-91481.
    - S. M. Ghoreishi, M. Behpour, M. Delshad, A. Khoobi. Cent. Eur. J. Chem. (2012) 10, 1824-1829.
    - D. B. Gunasekara, M. K. Hulvey, S. M. Lunte. Electrophoresis (2011) 32, 832-837
    - X. L¡, Z. Chen, F. Yang, J. Pan, Y. Li. J. Sep. Sci. (2013) 36, 1590-1596.
    - J. Narang, N. Chauhan, S. Pundir, C. S. Pundir. Bioprocess Biosyst Eng. (2013) 36, 1545-1554.
    - A. Babaei, M. Zendehdel, B. Khalilzadeh, M. Abnosi. Chin J. Chem. (2010) 28, 1967-1972.
    - M.-Y. Wei, L.-H. Guo, P. Famouri. Trends Anal. Chem. (2012) 39, 130-148.
    - Jagriti Narang, Nidhi Chauhan, Shikha Pundir, C. S. Pundir. Bioprocess Biosyst Eng (2013) 36, 1545-54.
    M. Arvand, T. M. Gholizadeh. Colloids and Surfaces B: Biointerfaces (2013) 103, 8493.
    • S. M. Ghoreishi, M. Behpour, N. Jafari, A. Khoobi. Analytical Letters, (2013) 46, 299311.
    X. Tang, Y. Liu, H. Hou, T. You. Talanta (2010) 80, 2182-2186.
    Conventional electrodes are used in most of the reviewed works, which due to their intrinsic characteristics make the possibility of becoming a portable and user-friendly device very difficult for non-specialized users. On the other hand, very few methods are validated with samples from real patients so, although they are capable of measuring tyrosine, they have not been validated for the diagnosis and / or follow-up of tyrosinemia. In addition, the fact that the method of the present invention employs an electrode based on nanomaterials entails a reduction of fouling that comes from the biological matrix, allowing measurements to be carried out without any prior treatment of the samples, which is essential in point-of-care devices.
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    In this sense, none of the works found in the literature meets, at the same time, all the necessary criteria to constitute an adequate point-of-care: portability, low sample consumption, low analysis time, on-site measurement and ease of Management by non-specialized users (absence of sample treatment and simple measures).
    For all this it is proposed the creation of an integrated device, capable of quantifying tyrosine levels in a reliable and simple way in less than a minute, without any sample treatment and easy to use to be applied in diagnosis and monitoring of tyrosinemia , both by the healthcare staff in consultation, and by the patient himself at home.
    DESCRIPTION OF THE INVENTION
    The present invention consists in the development of state-of-the-art detection systems using nanomaterials for tyrosine sensing in tyrosinemia using a short time, low amount of sample, in a very simple way and at a low cost.
    This system is capable of making measurements in different biological fluids without any pretreatment and using a small amount of sample. The meter uses a non-invasive method capable of monitoring a patient in situ, even from his own home.
    This system consists of:
    - A tyrosinemia sensor: working electrode, which consists of a modified band of nanomaterials that can be adapted to any size and shape, an auxiliary conductive nanomaterial electrode and a silver reference electrode. All electrodes are integrated to be able to measure using a drop of the sample, which means very little expense of this (50 pil).
    - Tyrosinemia measuring device: it is an electronic device based on a miniaturized potentiostat that carries an associated software that transforms an electrochemical signal into a digital, numerical and / or luminous signal, easily interpreted by a non-specialized user.
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    DESCRIPTION OF THE FIGURES
    To complete this description, and in order to help a better understanding of the characteristics of the Invention, a set of drawings is attached as an integral part of said description, where, as an illustration and not limitation, the following has been represented :
    Figure 1 shows several designs for an electrode system of drop measurement based on three electrodes: working electrode based on nanomaterlales (1.1), auxiliary electrode of carbon material (1.2) and silver reference electrode (1.3), being the Figure 1A a design aimed at adult patients and Figure 1B a design aimed at pediatric patients.
    Figure 2 represents a diagram of the portable sensor, which is composed of a tyrosine measuring device (2.1), drop electrode (2.2), actual drop sample of 50 pl (2.3).
    MODE OF REALIZATION
    The device consists of two elements: on the one hand an instrument to carry out the measurement and its interpretation, and on the other a disposable electrode system where the measurement will be carried out using a drop of sample.
    The electrochemical measurement is carried out on an electrode support, which contains a carbon working electrode based exclusively on nanomaterials. This system is composed of three electrodes: working electrode based on nanomaterials (1.1), auxiliary electrode made of carbon material (1.2) and silver reference electrode (1.3). Notwithstanding the intrinsic versatility of the previously described device, it is possible to modify the configuration of the electrode support, being possible to use any design that maintains an area of 0.2 cm2 in the working electrode, so that it is adaptable to any dimension and shape, thus providing designs Attractions for different audiences. Likewise, all three electrodes that make up this system can create more attractive designs for children (Figure 1B), thus facilitating their participation in the follow-up of the disease.
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    The main device consists of a miniaturized potentiostat with dimensions such that the device is portable and easy to use (approximate dimensions 7x15x1 cm), in addition it would consist of software capable of interpreting the analytical signal obtained, all integrated within an allantean device. protective. In turn, it would contain a screen to provide the value of the tyrosine concentration found and two lights, a green one that would indicate a normal tyrosine value in the analyzed sample and a red one, which would indicate an abnormal value. On the other hand it would have a simple interface to facilitate the use by non-specialized personnel, with buttons to perform the measurements (on / off and start / stop). An outline of this device and the measurement process is shown in Figure 2.
    The fact that the working electrode is based on nanomaterials allows to significantly reduce the fouling given by the biological matrix, which leads to the possibility of carrying out measurements without diluting the sample and without applying any other additional treatment. Its dimensions are such that the electrode would be disposable and easy to use allowing its coupling to the measuring system (potentiostat). In addition, its production is simple and the unit could be marketed at a low cost, which would facilitate its application as a point-of-care tool and therefore its use in situ by the non-specialized user, by attaching this disposable electrode to the main device through a slot (electrical contact with the potentiostat).
    The measurement process would begin with the coupling of a new (disposable) electrode on the device. This software will have a threshold value established, above which the person would encounter abnormal tyrosine levels and below which the person would be within normal levels. If you want to perform a quantitative analysis, it must be calibrated by using an external standard in order to create a specific calibration for the electrode in question and provide reliable and quantifiable measurements. This will be arranged in boats marked with numbers from 1 to 3, and each will contain a tyrosine pattern with concentrations between 25 pM and 1000 pM. The user will dispose a drop of each can on the electrode consecutively: first the number 1, after finishing the measurement number 2 and finally the number 3. This drop must be in sufficient quantity to cover the system of three electrodes completely and in no case will exceed 50pl.
    Once the device for the electrode to be used has been calibrated, a real sample of blood, plasma or urine (without any treatment) is placed on top of the electrode, just like in the case of the standards, completely covering the three electrodes, approximately 50 pl. Once ready and after pressing the start button, the measurement will begin and after a few seconds the tyrosine value contained in this sample will appear on the screen of the device, in the desired concentration unit. Additionally, a light incorporated in the device will turn on, being red in case of abnormal levels and green in cases of normal levels. This fact constitutes, not only an easy link for monitoring by non-specialized users, but also an effective diagnostic and screening tool. If the device is only going to be used for this purpose, external calibration would not be necessary.
    权利要求:
    Claims (7)
    [1]
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    1. A portable device for the detection, diagnosis and monitoring of tyrosinemia comprising:
    to. A potentiostat
    b. An electronic system that transforms the electrochemical measurement into an easily interpreted digital signal.
    C. A digital display that indicates tyrosine levels.
    d. A disposable micro device that integrates a working electrode, an auxiliary electrode and a reference electrode.
    and. An opening for coupling a disposable electrode.
    F. On / Off and Start / Stop buttons.
    g. A green light and a red light.
    [2]
    2. The device according to claim 1, characterized in that it detects tyrosine levels in different biological fluids such as urine, blood and plasma without any treatment of the sample.
    [3]
    3. The device according to claim 1, characterized in that the integrated disposable electrode consists exclusively of filtered conductive nanomaterials that act as a working electrode using a sample drop as the object of measurement.
    [4]
    The device according to claim 1, characterized in that the integrated disposable electrode is composed of a working electrode based exclusively on nanomaterials with an approximate area of 0.2 cm2, a silver reference electrode and an auxiliary electrode; adaptable to any dimension and shape.
    [5]
    5. The device according to claim 1, characterized in that it detects, diagnoses and tracks tyrosinemia with a drop of biological fluid without treatment by simply pressing a button and performing an amperometric measurement in situ.
    [6]
    6. The device according to claims 1 and 5, characterized in that the amperometric measurement is transformed into a light signal, easy to understand by the user,
    which will be green when there is a normal tyrosine value and red when it is an abnormal value.
    [7]
    7. The device according to claim 1, characterized in that it can be transported by the user and thus be able to carry out measurements and track tyroslnemla in situ in
    Anywhere and at any time.
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    优先权:
    申请号 | 申请日 | 专利标题
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