WO2024141981A1 - Method and device for the detection of phenylalanine in biological samples - Google Patents

Abstract

The invention relates to a method for the detection of phenylalanine in biological samples and to the corresponding reading device. More specifically, the invention refers to an enzymatic detection method and the detection device comprises a disposable sensor within which both the enzymatic recognition reaction and the colorimetric transduction of phenylalanine through the formation of metal nanostructures with characteristic optical absorption properties take place.

Description

Method and device for the detection of phenylalanine in biological samples

Technical field

The present invention relates to a method for detecting phenylalanine in biological samples and the corresponding reading device.

More specifically, the invention refers to an enzymatic detection method and the detection device comprises a disposable sensor within which take place both the enzymatic recognition reaction and the colorimetric transduction of phenylalanine through the formation of nanostructured materials based of metal nanoparticles, for instance gold nanoparticles (Au°) with characteristic optical absorption properties.

Known art

Hyperphenylalaninemias (HPA) are a group of hereditary diseases, due to the malfunction of the hydroxylase phenylalanine enzyme (PAH), which catalyzes the first reaction of the metabolism of the essential amino acid phenylalanine (also indicated in the following with Phe), with the consequent accumulation of the latter in the blood. Mutations in the corresponding gene that codes for the PAH enzyme are the cause of most cases of hyperphenylalaninemia. The associated phenotypes vary from classical phenylketonuria (PKU), to which the most serious form of the disease is associated, to mild HPA.

The affected subjects, who appear absolutely normal in the first months of life, develop, progressive especially neurological damages. The classical development includes encephalopathy, convulsions, anomalous behavior with hyperactivity, autistic or schizophrenic signs, eczema and slightly pigmented skin.

The neurological damage, due to the accumulation of phenylalanine, can be prevented with the introduction of a diet poor in this amino acid immediately after birth. Diet therapy therefore, at present, represents the most effective therapy, which must be suitably modulated on the basis of the blood phenylalanine values.

At present there are no systems on the market that allow to perform a home-testing monitoring of Phe levels in PKU patients. The official procedure provides: sampling of a sample through dried-blood-spot, shipping of the samples to the reference laboratory, execution of the chemical-clinical analyzes with MS-MS techniques, reporting and communication to the patient. This standard process involves the use of laboratory reagents and instrumentation that can only be used by highly qualified personnel and provides for a minimum response time to the patient from 4 to 6 days. Consequently, the optimization of the food diet takes place very late.

EP1618207 describes a colorimetric test that uses organic dyes such as methylene blue, bengala pink, azure c or a tetrazolium salt.

The main disadvantage of the test is the interference of the various reagents especially when they are concentrated (dried on a solid phase) and their stability.

In fact, in the patent document there are three calibration lines: in solution, a calibration line is shown in the 0-250 pM range; in aqueous gelatin, a straight line is shown in the range 2,500 to 5,000 pM; on the dry gelatin film, a straight line is shown in the range from 1 ,000 to 15,000 pM. The decrease in analytical performance in terms of LOD (Limit of Detection) and sensitivity of the method by passing from the ideal conditions (homogeneous solution) to dry film is evident.

In addition, blood is always prepared externally to the system with laborious methods that guarantee effective purification of the plasma but cannot be integrated into a portable device.

In addition, as indicated in EP1618207, colorimetric reagents on dry film are stable for a period of only 3 days. There are no specificity tests.

US6468416 describes a sensor for L-phenylalanine in which all the reagents necessary for the determination of L-phenylalanine are integrated together, thus allowing a quick and convenient quantification without resorting to equipment, devices or special techniques. The sensor is built by combining L-Phenylalanine dehydrogenase, with the nicotinamide adenine dinucleotide (oxidized form - NAD+) or the nicotinamide adenine dinucleotide phosphate (oxidized form - NADP+) as coenzyme and electronic mediator, which are used as reagents. The transduction is electrochemical, obtained with a system of electrodes and the quantification of L-phenylalanine is obtained with an electrochemical device. The disadvantages of the technique described are: less sensitivity both with standard Phe samples (250 pM), and with real blood samples (1000 pM). There are no specificity tests. Compared to electrochemical measures, the methodology proposed in the present invention shows better sensitivity. The calibration lines obtained with standard solutions cover the interval 50-2000 pM and real samples of patients affected by PKU have been tested in the interval 40-1000 pM.

The DOI document: 10.33774/Chemrxiv-2021 -4942B describes a preclinical biosensor to detect phenylalanine photometrically in whole blood samples. An enzymatic mixture, selective for L-Phe, is immobilized on a UV transparent well and the amount of used cofactor is monitored at 340 nm. The signal transduction is optical with 340 nm spectrophotometric reading. The main disadvantage of the method presented in DOI: 10.33774/Chemrxiv-2021 -4942B is linked to the spectral region of observation used (340 nm) which is largely influenced by the components present in the biological matrix, such as bilirubin, hemoglobin, some vitamins etc. In order to have good analytical performances in the present invention, a reading region free from these interferences is used (usually wavelength > 550 nm), performing a filtration of the blood sample.

It would be of great benefit and comfort to develop sensors capable of revealing specific plasma metabolites in real time in a similar way to what has been done for instance with glucose. These sensors would allow an immediate evaluation of Phe blood level, facilitating the management, treatment and control of connected metabolic disorders. Therefore, the need to have a system that involves reduced analysis times, is easy to use, low cost and effective in monitoring the pathology is felt.

If not specifically excluded in the detailed description that follows, what is described in this chapter is to be considered as an integral part of the detailed description of the invention.

Summary of the invention

It is an object of the present invention a sensor for the determination of the levels of L-phenylalanine (L-Phe) in the biological fluid of a human subject. The transduction of the signal takes place colorimetrically when the product of the enzymatic reaction is combined with a metal precursor giving rise to colored metal nanostructures that absorb in the visible area.

Another object of the invention is a reader of the color intensity developed on the sensor with metal nanostructures, said intensity being related to the levels of L-phenylalanine present in the sample under examination.

Further object of the invention is an equipment for the determination of L-Phe which comprises:

- A sensor comprising at least:

• a filtering surface;

• a heat-sensitive reacting surface comprising at least one electron transporting cofactor and at least a metabolic enzyme, said heat-sensitive surface being able to melt at a temperature above about 35-38°C and below about 70-75°C; and

• a reacting membrane placed below the heat-sensitive surface, which comprises metal precursors such as gold compounds;

- a reader comprising a light source connected to a light detector; and

- computerized means for the analysis of the data provided by the reader comprising at least one processor, a storage memory and means for viewing the data. Further object is an analytical method for the determination of the values of L-Phe in a biological sample of a human subject, said method comprising:

• contacting an amount of a body fluid sample with the sensor of the invention;

• quantifying the concentration value of L-Phe in the sample;

• comparing the concentration value of L-Phe in the sample with the normal interval of the concentration of L-Phe in the blood, which for a healthy subject is about 0.5-2 mg/dl. HPA subjects can present concentration values well above the threshold value (up to about 20 mg/dl). This invention shows a good performance in the analytical interval from about 0 to about 25 mg/dl (0-1513.4 pM) of L-Phe.

Still further object is a kit for the determination of L-Phe in a biological sample comprising: a sensor, a reader, optionally a lancing device and related lancets, reagents and containers to conduct the test, for instance buffer aliquots, doser/s), sampling means such as micropipette/s and a set of instructions, optionally accessible remotely through electronic means.

Brief description of the figures

Further objects, purposes and advantages of the present invention will be evident from the detailed description that follows, also referring to examples of realization of the same (and its variants) and by the attached drawings, provided in pure explanatory and non-limiting way, wherein:

Fig. 1 schematically illustrates a non-limiting realization of the sensor of the invention;

Fig. 2 schematically illustrates a non-limiting implementation of the reader of the invention;

Figure 3 illustrates the scheme of the enzymatic recognition reaction (1 ) and the colorimetric transduction reaction (2);

Figure 4 illustrates the sensitivity test performed with concentrations of L-Phe in the interval from 50 to 2000 pM; Figure 5 illustrates the specificity tests performed with some of the main analytes present in human blood with concentrations equal to 200 pM;

Figure 6 shows the absorption spectra with and without polymer (agarose).

Definitions

The term "about" or "around" as used here when referring to a measurable value such as a quantity, a time duration, and the like, is intended to enclose variations of ± 20%, ± 10%, ± 5%, ± 1 %, or ± 0.1 % to a specified value, where these variations are appropriate to perform the methods described.

As used here, the terms "Electronic means" indicate any physical memorization that uses electronic technologies such as a hard drive, ROM, RAM, flash memory, non-volatile memory, or any means substantially and functionally equivalent.

In some embodiments, the information about a threshold or reference sample of body fluid are stored for instance in a computer readable memorization support.

As used here, the term "light source" refers to any device that emits electromagnetic radiation with wavelengths compatible with the color transition of the colorimetric transduction reaction. In some embodiments, a filter is used together with the light source.

As used here, the term "light detector" refers to any device capable of detecting or quantifying the presence of electromagnetic radiation in the wavelengths compatible with the color transition of the colorimetric transduction reaction. In some embodiments, the device described here comprises one or a plurality of light detectors. These light detectors can comprise one out of a combination of photodiodes, CMOS cameras (active pixels sensors based on complementary transistors couples with a semi- conductive oxide metal junction) or CCD (charging coupling sensors, Charge-Coupled Device), or spectrophotometers. In some embodiments, a filter is used together with the light detector. As used here, the term "metabolic enzyme" or "phenylalanine dehydrogenase", also indicated as PDH, indicates the enzyme that converts L-Phe in phenylpyruvate in the presence of an oxidizing agent (e.g. NAD+ or NADP+) as indicated in the reaction of Figure 3. Such an enzyme can comprise its functional fragment or its recombinant form or a fusion protein or a non-enzyme molecular system PDH-Mimics.

As used here, the term "functional fragment" means any portion of a polypeptide that is sufficient to preserve at least a partial biological function that is similar or substantially similar to the Wild Type polypeptide function on which the fragment is based.

As used here, the term "PDH-Mimics" indicates those synthetic compounds that mimic the catalytic site of the PHD enzyme, where by PHD enzyme the portions of it are also meant. In accordance with what is indicated for the term "metabolic enzyme" for enzymes, among the PDH- Mimics compounds can be mentioned as not limiting examples organic molecules such as cyclodextrins and calixarenes, nanomaterials such as graphene oxide and metal-organic structures with portions that mimic the catalytic site of the PHD enzyme [1 ].

As used here, the terms "co-factor", "oxidant agent(s)” and "electron transporter" indicate any substance (organic or inorganic) that is able to transport electrons in the redox process of the invention. More specifically, the wordings indicate the compounds that are used in combination with the metabolic enzyme for the oxidation of L-Phe to phenylpyruvate. Such compounds can be for instance, but they are not limited to: NAD+, NADP+, FAD (flavin adenine dinucleotide), cytochromes a, b, c, cytochrome a3 and cytochrome P450.

As used here, the terms "body fluid/s" indicate any fluid isolated from a subject comprising, but not necessarily limited to, a sample of whole blood, a sample of serum or plasma, a sample of urine, a sample of mucus, a saliva sample, a sample of sweat.

As used here, the term "specificity" indicates the ability of the method to discriminate the presence of a certain analyte in a complex matrix. As used here, the term "nanostructure/s" indicates any organic, inorganic, or organic-inorganic hybrid material of size < 100 nm.

Detailed description of the invention

The present invention relates to an analytical method and corresponding reading system for the quantitative determination of L- phenylalanine (L-Phe) in biological samples and in particular for monitoring phenylalanine levels in biological samples in patients with hyperphenylalaninemia. The method is sensitive, specific, easy to use and integrated in a single device.

The method and the device can be used at home, in the hospital, or by the clinician to measure L-phenylalanine and can contribute to an effective and fast follow-up for patients with phenylketonuria (PKU) or to follow the effectiveness of the PKU patients’ therapy.

The analytical method is based on an enzymatic reaction (for instance phenylalanine dehydrogenase/ NAD(P)+ dependent) highly specific for L-Phe, as confirmed by several studies reported in literature [2- 5], and by a transduction colorimetric reaction based on the formation of metal nanostructures (such as those with Au or Ag, Pd, Ni, Pt) or of metal alloys (such as Ni/Co) mediated by a cofactor such as NADH or NADPH as a reducing agent. The relevant enzymes, in this case, the phenylalanine dehydrogenase (PheDH or PDH), its functional fragment or its recombinant form or a fusion protein or a non-enzyme molecular system PDH-mimics, have the NAD+ or NADP+ species as cofactors or other electrons transporters as indicated above. In its reduced form obtained from this reaction, the cofactor reacts with a metal precursor, such as AuCI⁴’, in the presence of polymer and surfactants forming nanostructures, characterized by a plasmonic absorption of localized surface resonance (LSPR) in the visible region.

The degree and intensity of the color transition are correlated to the concentration of nanostructures and the levels of L-Phe in the biological liquid under examination. More specifically and according to an exemplary and non-limiting aspect of the present invention, L-phenylalanine dehydrogenase (L-PDH) converts L-phenylalanine into phenylpyruvate, with the concomitant production of an equivalent amount of NADH/NADPH, which in turn reduces a metal precursor (for instance gold) to give nanostructures which, in the presence of polymer, give a characteristic color that can be optically measured.

The analytical method is based on the enzymatic detection and optical transduction of the levels of L-Phe in biological samples of patients with hyperphenylalaninemia or healthy individuals. The transduction takes place by optical way through the in situ formation of metal nanostructures, for instance gold, with coloring that depends and can be correlated to the amount of L-phenylalanine in the sample.

As part of the invention, the metals that can be used to obtain nanostructures can be chosen between, but are not limited to: silver gold, platinum, palladium, copper, cobalt and alloys such as nickel/cobalt, gold/silver and gold/platinum in various proportions.

Metals are used in their oxidized form, which will then be reduced in the presence of the electron transporter. The oxidized form will be in the form of metal precursors such as, for instance, by way of a non-limiting example, gold, silver, palladium, platinum salts to obtain the relative metal nanostructures of Ag°, Pd°, Pt°.

The characteristics necessary for the choice of metals and alloys that can be used as part of the present invention are the following:

- Metals must be reduced in the presence of the cofactor, for instance NADH or NADPH, in its reduced state;

- Experimental conditions (reagent concentration, reaction times and reaction temperature) must be such that metal structures are formed with nanometric dimensions

- Metal nanostructures must develop at a temperature of 70-75°C and be characterized by an optical absorption or localized surface plasmonic resonance absorption in the range from 500 to 1500 nm. This characteristic absorption is shown well by Au° absorption spectra (Figure 6).

The expert in the field, based on his knowledge of chemical kinetics and reading the technical information contained in this description, is able to develop the conditions for obtaining metal nanoparticles characterized by an optical absorption or localized surface plasmonic resonance absorption that is inside the range 500-1500 nm.

The integration of the analytical method with the modular sensor is made possible thanks to the presence of a polymer layer having a melting point between the reactions of enzymatic sensing (around 36-38°C) and the colorimetric transduction reaction (around 70-75°C).

The polymer layer (indicated in the following as a heat-sensitive polymer 2) separates the enzymatic reaction from the reagents of the colorimetric transduction. The latter are deposited on a membrane, for instance of nitrocellulose (indicated in the following as a reacting membrane 3).

Inside the sensor, the filtration of the biological sample is performed, so that the filtered plasma is collected in the surface of the 2nd heatsensitive polymeric layer 2, containing the reagents for the enzymatic reaction (PDH, NAD(P)+ and buffer). Heating at 36-38°C, typically for about 30 min, converts L-Phe into the biological sample in phenylpyruvate with the formation of NADH or NADPH. A subsequent heating at about 70- 75°C causes the melting of the heat-sensitive polymeric layer 2, allowing the mixing of the reducing agent NADH or NADPH and the melted polymer with the colorimetric transduction reagents deposited on the reacting membrane 3. In these conditions on the membrane metal nanostructures are formed, preferably gold, optically detectable by absorption preferably in the 550-800 nm area. The intensity of this coloring is proportional to the Phe levels present in the biological sample.

All this is performed within the sensor that is modular, miniaturized, is achievable as a disposable element and is connected to a reader. The sensor presents all the necessary reagents for the analysis pre- loaded (reagents-on-board) and allows the execution of the in-situ blood filtration. The enzymatic recognition mechanism, colorimetric transduction and reading take place in a single reactor and in sequential step, without any action/intervention of the operator. The organic fluid can be filtered inside the sensor and the filtration product is subjected to a first enzymatic recognition reaction; the reaction product is therefore detected through a colorimetric reaction based on the formation of metal nanostructured materials (Au° nanoparticles) in a polymer matrix.

The sensor is made by inserting a heat-sensitive polymer layer that separates a first area in which the enzymatic reaction is developed from a second area in which the colorimetric transduction reaction is conducted. The two reactions, enzymatic and colorimetric, are conducted at different temperatures.

The intensity of the developed color, spectrophotometrically detectable, is related to the level of L-Phe in the biological sample.

The biological sample is a body fluid, preferably whole blood or serum/plasma or urine.

The reader which allows to perform the appropriate temperature cycles for the development of the enzymatic reaction and the colorimetric reaction, performs colorimetric reading for absorption in reflection of electromagnetic radiation within the sensor.

This reader comprises a light source (such as a LED) and a light detector (such as a photodiode), capable of correlating the variation of light radiation with an electrical signal, corresponding to a concentration value of L-Phe in the sample. This electrical signal is converted into numerical value and sent to a system suitable for viewing, which can be local (for instance a liquid crystal display) or remote (for instance a software on a smartphone).

According to an invention embodiment, the reader is connected via Bluetooth with a tablet where an app that is used to pick up and collect all the analysis data is installed, up to the final result. With particular reference to figures 1 and 2, in which a non-limiting implementation form of the invention is illustrated, they are shown in sequence:

• the sensor A which comprises:

- a compartment for sampling or a sample holder compartment 6, which preferably is separable and removable

- the walls 5 of the sample holder compartment 6

- a first filtering layer 1 or filtering membrane 1 , which constitutes the bottom of the sample holder compartment 6

- a second layer or heat-sensitive membrane 2 or heat-sensitive polymer 2

- a third reaction layer 3 or reacting membrane 3, typically white, for instance nitrocellulose

- a reading window 4

- an external envelope 7 surrounding layers 2, 3 and 4.

• the reader B which comprises:

- a housing 8 for the sensor A

- a heating element 9

- a light source 10

- a light detector 11

- an area of transduction, processing and data transmission 12

Inside the sensor and below the sample holder compartment 6 are placed, in sequence, the filtering membrane 1 , below which the heatsensitive membrane 2 is placed, below which the reacting membrane 3 is placed below which the reading window 4 is placed. The membranes 2, 3 and 4 are all substantially in contact and placed above the reading window 4.

On the reacting membrane 3 the colorimetric reaction takes place with formation of colored metal nanostructures. According to an embodiment of the invention, membranes 2, 3 and the reading window 4 can be joined together along their perimeter in order to facilitate the positioning on reader B and the consecutive performance of enzymatic and colorimetric reactions followed by spectrophotometric reading.

The filtering membrane 1 is positioned on the bottom of compartment 6 and above the membrane 2.

In the operations of the reading system, after the sampling of the biological fluid, the sample holder compartment 6 is removed and the 2, 3 membrane and reading window 4 complex is placed in contact with reader B for the processing of L-Phe levels.

More specifically:

- The first filtering layer 1 or filtering membrane 1 is used to separate the products of interest (to be examined) from the interfering cells and therefore allows the permeation of the plasma and all its constituents, comprising phenylalanine, but prevents the passage of interfering species such as red blood cells, white blood cells and platelets. This filtering membrane 1 (typically in diameter about 0.5 cm housed in a removable compartment) can comprise polymer membranes such as poly (ether sulphones), Nation, polytetrafluoroethylene, cellulose acetate, cellulose, polypropylene, cellulose ether or other dialysis membranes or filtering membranes known in themselves.

The filtering membrane 1 is housed and constitutes the back wall of the sample holder 6, which is extracted immediately after filtration, the duration of which is generally a few minutes.

- The second layer 2 is a heat-sensitive polymer film that is in solid form at room temperature, on whose surface a metabolic enzyme (PheDH or PDH) is deposited either anchored or a functional fragment or its recombinant form or a fusion protein either a non-enzymatic PDH-Mimics molecular system and the reaction cofactor chosen, preferably, between NAD+ and NADP+. This layer 2, heated to its melting temperature allows the contact of the products of the enzymatic reaction, typically NADH/NADPH with the metal precursors present on the underlying reacting membrane 3, in order to obtain a color, whose intensity will be related to the content of L-Phe present in the biological sample.

The heat-sensitive layer or heat-sensitive membrane 2 is advantageously in direct contact with the filtering membrane 1 .

- The third layer 3 or reacting membrane 3 is generally made up of a polymer material on which the reagents through which the transduction/coloring reaction that is related to the quantity of phenylalanine in the sample in question develops.

In order to increase the contrast for colorimetric reading, the transduction reagents are immobilized on a white surface, for instance of nitrocellulose. Alternatively, any material aimed at forming a layer of white non-interferent with the essay is suitable, for instance a polymer layer containing white pigments such as for instance, but not limited to, TiO2, ZnO, ZrO2, SiO2, thus allowing a good contrast of the color during the reading phase.

In the conformation reported in Figure 1 , the membrane 3 has a diameter of 0.8 cm and consists of porous nitrocellulose. The reagents for colorimetric transduction are applied or immobilized on it.

The reacting membrane 3 is visually white and provides a uniform color in the reading area.

- Membrane 4 or reading window 4, placed below layer 3 is made up of an optically transparent, biocompatible material, with high chemical stability and to biological reagents. It is made of a material transparent to the radiation used in the method of the invention and can consist of organic, inorganic or mixed material. It must have a low thermal inertia and can be constituted for instance by polycarbonate, polydimethylsiloxane. Its function can be a structural and containment function of the various layers or elements that make up the sensor, placed on the lower end of the sensor. Alternatively, membranes 2, 3 and 4 can be sealed together along their perimeter.

In a form of non-limiting implementation, the A sensor is in a cooperation relationship with the reader B. For instance, after sampling, from the A sensor, the compartment 6 is removed together with the filtering membrane 1 and the complex of membranes 2, 3 e 4 is applied on or inserted in reader B.

With particular reference to the reader, illustrated in Figure 2, the main functions of the reader are:

1 ) Sensor thermostating, to guarantee the temperatures necessary to develop the enzymatic reaction and the coloring reaction

2) Reading of the color intensity which, in an embodiment, provides a numerical result through an alphanumeric display. In a further embodiment, the light intensity reader transmits the numerical result to a device for remote display of the result.

According to a preferred embodiment, the B reader comprises: an electronic card comprising four sections: in the first there is the circuitry for the heating element 9, necessary to thermostate the reagents during the series of reactions that develop on the sensor; in the second, the circuitry for the light source 10, which in an embodiment can be composed by a light emitting diode (LED) to a specific wavelength, preferably in the range from 500 to 1500 nm, which illuminates the membrane 3; in the third there is the circuitry for the light detector 11 which receives the light reflected by the membrane and which in an embodiment can be composed of a photodiode; in the fourth, the system of data transduction, processing and transmission 12, capable of determining how much light is absorbed by the membrane itself and, by difference between the measure made at the beginning of the analysis and that made at the end of the analysis, identifies the intensity of the coloring of the membrane; the same section hosts the transmission system that sends the elaboration of the analyzes to a remote display system. The expert in the field is able to set up electronic card.

In a form of non-limiting realization, LEDs and photodiode can detect the end-color of a generated Au adduct which has a lambda max wavelength in the 550 - 800 nm interval for the determination of the reflectance.

This information, initially generated in analogic form by the photodiode, is transmitted to the third section of the card that converts it to digital and elaborates it to obtain a numerical value directly proportional to the concentration of L-Phe in the membrane. The expert in the field is able to carry out these operations.

The value obtained, in this embodiment, is preferably transmitted wireless to a tablet equipped with a special software for displaying the result in real time. The expert in the field is able to carry out these operations.

The electronic card also manages the thermal section, through a retro-managed mechanism, by which the temperature is regulated on the basis of the measurement of the same carried out in real time, in order to keep it in the required range, also allowing the adjustment ad lib of the reaction temperatures.

The device can be easily engineered for direct use (home) by the patient.

The device is managed by a software component or other nontransient IT product which is coded on an electronic device, and which optionally comprises instructions (such as a programmed or similar script) which, when performed, quantifies one or more concentration values of L- Phe; normalizes one or more concentration values of it on a set of control data; and shows the result to the user.

The software or IT product can be integrated into any system described here and can be digitally accessible through direct fixing to the circuit described here.

According to another aspect, the invention relates to a method for determining the level of phenylalanine in an amount of the biological fluid of a human subject.

The biological fluid is preventively filtered through a filtering membrane (membrane 1 ) which can comprise or consist of one, or a combination of various materials such as, for instance, but not limited to, glass fiber, nylon, polytetrafluoroethylene, polyester, cellulose, cellulose acetate, nitrocellulose, polycarbonate, polyvinylidene fluoride, polyether sulphones, or polysulphones, polypropylene, cellulose ether or other dialysis or filtering membranes in itself known with a retention of particles in the interval of about 2.0-15.0 pm.

Membranes can be used that load different volumes of biological liquid from 10 to 100 pl or beyond.

Membrane 1 can also comprise hemagglutinating agents, comprising but not limited to, erythrocyte antibodies, chitosan, hexadimethrine bromide, poly-L-lysine, poly-L-lysine hydrobromide, poly-D-lysine, poly-D- lysine hydrobromide, poly-DL-lysine hydrobromide, poly-L-arginine hydrochloride, poly-(allylamine hydrochloride), poly- (ethylenimine hydrochloride), diethylamino ethyl dextran, poly-(N,N-dimetyl-3,5- piperidinyl chloride), or crude or purified lectins that can agglutinate human erythrocytes efficiently such as those from Phaseolus vulgaris, Madura pomifera, Lllex europaeus, and Solanum tuberosum.

Hemagglutinating agents can be immobilized together with an inclusive polymer, such as, hydroxypropyl cellulose, hydroxyethyl cellulose, poly-(vinyl alcohol), dextran, gelatin, agarose, sodium carboxymethylcellulose, xanthan gum, polyvinylpyrrolidone, poly-(1 - vinylpyrrolidone), poly-(vinyl acetate) or poly-(methyl vinyl ether-maleic anhydride).

The filtering layer or membrane 1 has the function of retaining and not allowing the pass of the cells of the biological liquid that would interfere with the series of subsequent reactions and would alter the measure.

The method according to the invention is made in two areas overlapping with each other and separated by a heat-sensitive surface. The enzymatic reaction is conducted in the first area and the colorimetric transduction reaction is conducted in the second area.

With reference to Figure 1 , the first reaction zone comprises or substantially consists of the surface of the heat-sensitive membrane 2, suitably prepared as mentioned below; the second area comprises or consists of membrane 3, suitably prepared as mentioned below.

The method according to the invention comprises the following steps:

(a) in the first area, putting in contact an amount of the biological fluid, previously filtered, with an electron transporting cofactor and at least a metabolic enzyme and reacting enzymatically at a temperature of about 35-38°C;

(b) heating the heat-sensitive surface to its melting temperature so that the products of the enzymatic reaction of the first area come into contact with a metal precursor present in the second area;

(c) maintaining at a temperature up to about 70-75°C to develop colored nanostructures, deriving from the contact between the products of the enzymatic reaction of the first area and the metal precursor of the second area;

(d) quantifying the concentration value of phenylalanine in said biological sample by measuring the intensity of the coloring of the colored nanostructures obtained in the previous stage.

The examination wavelength is comprised in the 500-1500 nm interval, preferably 550-800 nm.

The biological sample is a body fluid, preferably whole blood or serum or plasma or urine.

The electron transporting cofactor is preferably chosen between NAD+ and NADP+.

The metabolic enzyme is phenylalanine dehydrogenase or its functional fragment, its recombinant form or a fusion protein or a non- enzymatic PDH-Mimic molecular system, as defined above.

The temperature up to about 70-75°C is maintained for about 40-90 minutes, preferably 50-70 min.

The heat-sensitive surface is the heat-sensitive layer 2 illustrated in Figure 1 . The heat-sensitive polymeric layer 2 or membrane 2 consists of a heat-sensitive material with a melting point between 35 and 75°C, must be compatible with the enzymatic reactions, chemically stable and optically transparent in the regions of the visible and near infrared (390- 1500 nm). It can be made preferably, but not limited to, with a polymer chosen between agarose, carbopol, chitosan.

On the surface of the layer or membrane 2 are deposited/anchored:

- The metabolic enzyme, such as phenylalanine dehydrogenase, or similar compound having the same functions, as indicated above.

- The cofactor as indicated above, preferably, but not limited to, NAD+ or NADP+.

The metabolic enzyme and the cofactor can be prepared as a buffered aqueous solution, for instance with phosphate buffer at pH 8.5 and applied on the surface of the membrane 2.

The optimal pH for the enzymatic reaction can vary from 7.5 to 10.5, the optimal pH in the conformation of the sensor presented here is about 8.5. The buffer used can consist of any substance (such as for instance PBS, TRIS, MES etc.) that has the buffering power to the working pH of the enzyme.

In a non-limiting embodiment, the PheDH enzyme used in the present invention may have different origins comprising the forms obtained through gene recombination techniques. Non-limiting examples comprise enzymes produced by bacteria belonging to the Thermoactinomyces genus such as for instance Thermoactinomyces intermedins (ATCC 33205), bacteria belonging to the Bacillus genus such as Bacillus badius (ATCC 14574), bacteria belonging to the Sporosarcina genus such as for instance the enzyme obtained by Sigma-Aldrich (Cas N. 69403-12-9), bacteria belonging to the genus Rhodococcus such as Rhodococcus sp. M4 strain obtained by Sigma-Aldrich (Cas N. 69403-12-9).

The heat-sensitive membrane 2 has a multiple function: 1 ) having a melting temperature above the execution temperature of the enzymatic reaction (36-38°C) and a melting temperature below the execution temperature of the colorimetric transduction reaction (70-75°C), maintains separated reagents during the enzymatic reaction (conducted preferably at about 37°C), thus avoiding possible inhibitions of enzyme or non-specificity;

2) at room temperature forms a solid biocompatible surface whereon it is possible to deposit/anchor the enzyme and perform the enzymatic reaction after the filtration of the biological sample;

3) at the end of the enzymatic step a subsequent heating up to about 70-75°C melts the polymer, allowing an effective mixing between the products of the enzymatic reaction and the reagents for colorimetric transduction;

4) the presence of the polymer allows the formation of metal nanostructures on it, preferably of Au°, characterized by a plasmonic absorption of localized surface resonance (LSPR) with wavelengths greater than 500 nm, preferably greater than 550 nm, eliminating possible interference during the optical reading by the biological sample and reagents; and

5) the presence of the polymer makes the formation of metal nanostructures (preferably golden) faster, reducing analysis times.

In addition, the heat-sensitive polymeric layer 2 advantageously moves the optical absorption of metal nanostructures towards higher wavelengths eliminating possible optical interferences of the starting sample and visually appreciable even with the naked eye.

The heat-sensitive polymer 2 is placed above the reacting membrane 3. It varies from the solid state to the liquid state according to the temperature (it is solid at the temperature of the enzymatic reaction which is about 37°C and melts at the temperature of the development of the color that is maintained up to about 70-75°C). Polymer 2 can be agarose.

At the end of the development of the enzymatic reaction which, we remember, is conducted in the first area and is lasting about 30 minutes, the polymer layer 2 is heated (as described above) up to a temperature of about 70-75°C and the membrane or heat-sensitive film 2 melts by contacting the product of the enzymatic reaction with the metal precursors present on the reacting membrane 3 which constitutes the second reaction area.

Membrane 3 can consist of a polymer material. Examples of polymers comprise, without being limited to these, nylon, cellulose such as for instance, but not limited to cellulose acetate, nitrocellulose, hydroxypropyl-cellulose, hydroxyethyl-cellulose, carboxy-methylcellulose; polycarbonate, polyether sulphones or poly-sulphones, polyvinylidenes, poly-(vinyl alcohol), poly-(vinyl acetate), polyamides, polysiloxanes such as poly-dimethyl siloxanes, polyesters, polystyrenes, polypropylenes, polyacrylates, polyvinyl compounds (for instance, poly-vinyl chloride, polyvinyl pyrrolidone, poly-(l-vinyl. pyrrolidone-co-vinyl acetate), polyvinyl acetate)), fluoropolymers, such as fluorinated ethylenes and polytetrafluoroethylenes (PTFE), nitrocellulose, cotton, polyglycolic acids (PGA), dextranes, gelatin, agarose, xanthan gum, glass, or their combinations. The polymers can be advantageously pigmented with metal oxides such as with TiC , ZrO2, SiO2 ZnO and mixtures thereof.

The reagents of the colorimetric transduction reaction are loaded on the membrane 3.

The colorimetric reaction is based on the Redox transition of a metal precursor, placed on membrane 3, whose oxidation status is reduced by the reducing species, consisting of the electron transporter (for instance NADH or NADPH), which is conveyed on the membrane 3 following the melting of membrane 2, at the end of the enzymatic reaction.

The metal precursors are preferably commercial Au salts such as chloroauric acid H[AuCk].

Advantageously, the metal precursor is conveyed on the membrane 3 as a watery solution. In the reaction environment, a surfactant is preferably added, such as a quaternary ammonium salt, for instance cetyl trimethylammonium bromide (CTAB) to accelerate the reaction kinetics. To the aqueous solution, nucleation centers can also be added, whose surface helps and accelerates the formation of metal nanostructures. The aqueous solution containing the metal precursor is then distributed on the membrane 3. A drying step follows. The membrane 3 is so ready to be positioned inside the sensor.

The nanometric metal particles that develop following the mixing of the metal precursor with the reduced cofactor present in the membrane 2, after the melting at 70-75°C, have a characteristic color due to their nanometric dimensions.

Once the reaction has occurred, this is highlighted through the underlying transparent film or reading window 4.

To view the coloring, it is possible to use any polymer that is transparent to the reading wavelength in order not to hinder the colorimetric reading.

The "normal" interval of the concentration of L-Phe in the blood is about 0.5-2 mg/dl. HPA subjects can present concentration values well above the threshold value (up to 20 mg/dl) the present invention shows a good performance in terms of sensitivity and specificity in the analytical interval from 0 to 25 mg/dl (0-1513.4 pM) of L-Phe.

The concentration of L-Phe in the blood is a critical parameter for the neonatal determination of PKU, a pre- and post-evaluation of the subjects with food restrictions, and a monitoring after the administration of therapeutic drugs.

The volume of blood used in the device, using a whole blood sample is less than 25 pl. This allows an easy use by the patient.

Two significant contributions of the present invention are the ability to reveal low concentration levels of L-Phe, high sensitivity and the ability to discriminate between various concentrations of L-Phe in the clinically significant interval.

The invention uses reagents (golden precursors) which are very stable and are deposited on the cellulose membrane, the polymer film guarantees the separation between the two reactions (enzymatic/colorimetric) allowing the achievement of good analytical performances.

The main advantages of the present invention with respect to existing solutions are:

1 ) Reduced analysis times. This invention allows a time of analysis from the withdrawal to the result of about 1 .5 hours.

2) Ease of use. The invention is characterized by a high ease of use, thanks to the integration of all the analysis steps within a single device and kit that contains all the reagents necessary for the execution of the test.

3) Low analysis costs. Thanks to the small size of the device and the possibility of integrating all the tests in a few steps, the present invention provides for an initial cost for prototype tests of about 10 euros. On the other hand, the standard method provides costs of more than 50 euros per single sample, in addition to the need to have a centralized laboratory with highly qualified staff.

4) Effective monitoring of therapy: thanks to the possibility of transmitting in real time data to the doctor/reference center. In practice, the patient, in the early steps of the day, performs the reading and based on the value of L-Phe communicated to the reference center, can modulate the diet to follow.

The proposed invention has its final scope of the realization of a device which, in a growing attention to what is called "patient wellness", improves the quality of life of a subject suffering from a rare disease. The device therefore aims to facilitate the patient's follow up and goes beyond the concept of "screening tests", to which the definitions of false positive and false negative are adequate. In particular, this method allows the measurement of the levels of an analyte (L-Phe) which is normally present in the blood. The measured concentration values are used for the sole purpose of monitoring the effectiveness of patient therapy effects from PKU (follow up). The parameters that define the performance of the invention device are: Specificity :> 90%

Detection limit: 40 pM

Linearity: 50-2000 pM

Response time: 90 min

Excellent reproducibility: cv = variation coefficient ~ 4%

The following examples illustrate the invention and are not to be considered limiting the relative scope.

EXAMPLES

SENSOR PREPARATION METHODOLOGY

Preparation of membrane 1

The filtration membrane 1 (PALL Grade-GR, diameter of about 0.8 cm) is set, e.g. fixed through glue or double-sided system or other similar fixing system, on the lower end of the filtration module or storage compartment.

Preparation of the membrane 2

In the example shown, the heat-sensitive membrane 2 consists of a volume of 140 pl of agarose 0.4% p/v. The membrane 2 is prepared by solubilizing in hot deionized water (80°C) a quantity of agarose until a concentration 0.4% p/v is obtained (for instance 0.4 grams of agarose in100 ml of H2O), always hot (using a preheated dosing system) a volume equal to 140 pl is taken and placed inside the A sensor, in contact with the membrane 3. Above the membrane 2 the reagents of the enzymatic reaction are deposited: 0.335 U PDH in deionized water and 4 pl NAD+ 75mM in deionized water.

Preparation of the membrane 3

On a disc of about 0.8 cm of reacting membrane 3, a solution containing: 62.2 pl of HAuCk (5x10’²M) and 19 pl of seeds of Au-citrate nanoparticles (whose function is to provide nucleation centers of metal nanostructures), is dried for about 30-35 min in an incubator at about 70°C. Once dry, the reacting membrane 3 is inserted inside the sensor, above the membrane 4, interposed between and in contact with the membranes 2 and 4. The membrane 3 (nitrocellulose) is chosen with white color in order to make easier the identification of the final coloring.

The membrane 4 having a diameter of about 1 cm is fixed, for instance glued via glue or double -sided system on the lower end of the sensor A.

All the chemical and solvents reagents used in the sensor preparation phase were purchased by Sigma-Aldrich.

TEST EXECUTION METHODOLOGY

Testing method

An amount of a biological sample is taken, generally a drop of blood and for instance the withdrawal is carried out with a lancing device.

The biological fluid sample (blood of volume equal to 20 pL) is filtered on the filtering membrane 1 , which separates the corpuscular part of the organic fluid (in the blood: the white and red blood cells and platelets are separated from the plasma portion that passes below the filtering membrane). After 3 minutes of filtration, the sample holder or filtering module is extracted and 96 pl of phosphate buffer (10 mM pH 8.5) are added. The A sensor is then inserted in the reader which performs a first blank zero -time reading (signal to).

At this point, the enzymatic reaction to 35-37°C for about 30 min, immediately followed by the colorimetric transduction reaction, began, after heating the sensor up to about 70-75°C (the colorimetric reaction is conducted for about 50-60 min).

At the end of the colorimetric reaction, the reader performs a final reading (n measures are performed and an average value is provided) (tf signal). The differential signal S=Stf-Sto represents the signal value of the sensor that is transferred to the app and converted into concentration of L- Phe.

OPERATION OF THE READER After sampling and filtration of the biological fluid, the A sensor is placed in connection with the reader and the enzymatic reaction is completed at 35-38°C, the reader raises the sensor temperature to about 70-75°C, the polymer 3 melts and mixes with the reagents placed in the membrane 3 below, there is a variation of coloring of the membrane 3 from white to purple depending on the amount of L-Phe initially present in the biological sample. The shade of color will be the function of the reaction environment, but the color intensity will be directly correlated to the amount of L-Phe.

Once the color has been developed (about 50-60 min from the start of the fusion of polymer 2) the light intensity reader measures the intensity of light absorbed by the colored reaction product and, by difference between the value at the beginning and at the end of the coloring reaction, calculates the intensity of absorbed radiation, which is proportional to the coloring and therefore to the quantity of phenylalanine.

The working conditions were chosen in such a way as to perform the test in conditions of maximum selectivity.

The specificity tests were performed with some of the main analytes present in human blood (see fig. 5). The data confirm the high specificity of the proposed analytical method.

The invention device was tested with real samples (study approved by the Ethical Committee of the A.O.U. Policlinico di Catania with related informed consent no. 1327/2020) of a healthy subject S01 and subjects affected by HPA under therapy (S02 and S03). For this purpose, a volume equal to 20 pl of fresh blood was deposited on membrane 1 inside the filtration module positioned inside the sensor A. After 3 minutes of filtration the module was removed and on the membrane 2 containing the reagents for the Enzymatic reaction were added 96 pl of phosphate buffer 10 mM pH 8.5 and the A sensor was inserted into the reader. The reader is started by the software and the two consecutive reactions start automatically: enzymatic sensing at 37°C for about 30 min and colorimetric transduction for about 50 min at about 70°C. The concentration values of L-Phe obtained from the sensor are shown in Table 1 . By comparison the blood samples were analyzed within the screening center of A.O.U. Policlinico di Catania using the standard Tandem Mass method. It is clearly seen that the concentration values of L-Phe obtained with the sensor correspond well with the concentration values obtained with mass spectrometry technique, showing percentage differences of less than 5%.

Table 1

REFERENCES

1- E. Kuah, S. Toh, J. Yee, Q. Ma, Z.Gao Enzyme Mimics: Advances and Applications, Chem. Eur.J.2016, 22, 8404- 8430.

2- Y. Asano, A. Nakazawa, K.Endo. Novel phenylalanine Dehydrogenases from Sporosarcina ureae and Bacillus sphaericus. The Journal of Biological Chemistry. Vol. 262, N. 21 , pp. 10346-10354. 1987

3- Y.Asano, A. Nakazawa, K.Endo, Y. Hibino, M. Ohmori, N. Numao, K. Kondo, phenylalanine dehydrogenase of Bacillus badius. Purification, characterization and gene cloning. Eur. J. Biochem. 168, pp. 153 - 159. 1987

4- N.M.W. Brunhuber, J.B. Thoden, J.S. Blanchard, J.L. Vanhooke. Rhodococcus L-phenylalanine Dehydrogenase: Kinetics, Mechanism, and Structural Basis for Catalytic Specifity. Biochemistry. 39, pp. 9174-9187. 2000

5- 4- S.Y.K. Seah, K.L. Britton, D.W. Rice, Y. Asano, P.C. Engel. Single Amino Acid Substitution in Bacillus sphaericus phenylalanineDehydrogenase Dramatically Increases Its Discrimination between phenylalanine and Tyrosine Substrates. Biochemistry. 41 , pp. 11390-11397. 2002

Claims

1. A method for determining L-phenylalanine levels in an amount of a biological fluid of a human subject, said method being carried out on two overlapping areas and separated from each other by a heat-sensitive surface, said method comprising the following steps:

(a) in the first area, putting in contact an amount of the biological fluid, previously filtered, with an electrons transporter cofactor chosen preferably between NAD+ and NADP+ and a metabolic enzyme that is L- phenylalanine dehydrogenase or its functional fragment and letting to react enzymatically at a temperature of 35-38°C;

(b) subsequently, heating the heat-sensitive surface to its melting temperature so that said first and said second area come into communication;

(c) maintaining at a temperature up to 70-75°C to develop colored nanostructures due to the contact between the products of the enzymatic reaction of the first area and a metal precursor present in the second area;

(d) quantifying the concentration value of L-phenylalanine in the biological sample under test by measuring the intensity of the color obtained in the previous step at the wavelength of 500-1500 nm, preferably 550-800 nm.

2. The method according to the previous claim wherein the colored nanostructures are metal nanoparticles characterized by an optical absorption or plasmonic absorption of localized resonance which is within the range of 500-1500 nm, preferably 550-800 nm.

3. The method according to anyone of claims 1 -2 wherein the biological sample is a body fluid, preferably whole blood or serum or plasma or urine.

4. The method according to anyone of claims 1 -3 wherein the functional fragment of L-Phenylalanine dehydrogenase is a polypeptide portion of this enzyme of sufficient length to preserve at least a partial biological function similar or substantially similar to the function of said enzyme.

5. The method according to anyone of claims 1 -4 wherein the temperature up to 70-75°C is maintained for 40-90 minutes, preferably 50- 70 minutes.

6. The method according to anyone of claims 1 -5 wherein the heatsensitive surface is made of a polymer preferably chosen among, but not limited to, agarose, carbopol, chitosan.

7. The method according to anyone of claims 1 -6 wherein colorimetric transduction is conducted on a layer of white material, such as nitrocellulose on which metal precursors are immobilized, preferably chosen among, but not limited to, gold, silver, palladium, platinum salts, copper, cobalt and alloys such as nickel/cobalt, gold/silver and gold/platinum.

8. The method according to anyone of claims 1 -7 wherein the layer on which the colorimetric transduction reaction takes place is a polymer layer containing white pigments chosen preferably among, but not limited to, TiO2, ZrO2, SiO2 ZnO or a mixture thereof.

9. The method according to the previous claim wherein the layer on which the colorimetric transduction reaction takes place is chosen between: nylon, carbopol, agarose, hydroxypropyl cellulose, hydroxyethyl cellulose, poly-(vinyl alcohol), dextran, gelatin, agarose, sodium carboxymethylcellulose, xanthan gum, poly-vinylpyrrolidone, or poly-(l- vinylpyrrolidone-co-vinylacetate), poly-(vinylacetate), nitrocellulose, cellulose, cellulose acetate, and mixtures thereof.

10. A sensor for determining the level of L-phenylalanine in the biological fluid of a human subject, said sensor comprising two areas: a first area wherein an enzymatic reaction takes place at a temperature of 35-38°C and a second area wherein a colorimetric transduction reaction takes place at a temperature up to 70-75°C, said two areas being separated by a heat-sensitive surface; said heat-sensitive surface being made of or comprising a polymer having a melting temperature above 35-38°C and below 70-75°C and comprising at least one electron transporting cofactor chosen between NAD+ and NADP+ and a metabolic enzyme which is L-phenylalanine dehydrogenase or its functional fragment; said sensor further comprising: a reacting membrane placed below the heat-sensitive surface, said reacting membrane comprising metal precursors chosen from salts of gold, silver, palladium, platinum, copper, cobalt and alloys such as nickel/cobalt, gold/silver and gold/platinum.

11 . A sensor according to the previous claim which further comprises:

- a removable sample compartment closed on the bottom by a filtering membrane which is placed above and in contact with the heatsensitive surface which in turn is placed above the reacting membrane;

- a reading window, placed below the reacting membrane.

12. An equipment for the determination of the level of L- phenylalanine in a biological fluid of a human subject, said equipment comprising:

- a sensor according to anyone of claims 10-1 1 ;

- a reader comprising a light source connected to a light detector;

- heating means;

- means for colorimetric detection; and

- computerized means for the analysis of the data provided by the reader comprising at least one processor, a storage memory and means for viewing data.

13. The equipment according to the previous claim wherein the reader comprises a light source and a light detector, capable of correlating the variation of light radiation with an electrical signal, corresponding to a concentration value of L-Phenylalanine in the sample, said electric signal being converted into numerical value and sent to a system aimed at data displaying.

14. The equipment according to anyone of claims 12-13 wherein the means for viewing data are local, such as a display or remote such as a computer, smartphone or other electronic device.

15. A computer program which, when performed, instructs the equipment of claims 12-14 to perform a method according to claims 1 -11.

16. A computer readable memorization support comprising the program according to claim 15.

17. A computer system comprising the processor program according to claim 15, wherein the computer system can optionally be a tablet or a smartphone.

18. A kit for the determination of L-Phenylalanine level in an amount of a biological fluid of a human subject comprising: the sensor of claims 10-11 and the reader of claim 12; optionally: a lancing device and related lancets, reagents, a buffer, one or more dosing and sampling means, one or more micropipettes, a set of instructions, optionally accessible remotely through an electronic device.