BR102012003901A2 - optical device for detection of specific marker of cardiac injury - Google Patents

Abstract

The present invention relates to the manufacture of an optical device capable of detecting troponin macromolecule t which has a direct relationship with the cardiac lesion. The device is composed of a laser source that focuses a bioelectrode that becomes luminescent after recognizing the specific marker of cardiac injury. a photomultiplier coupled to a spectrometer amplifies this signal, which in turn is acquired by interfacing with a specific software whose output is expressed as luminescence in the form of a Gaussian curve where the peak is related on the abscissa axis with the characteristic wavelength of fluorophores. consisting of quantum dots and the ordinate axis with the concentration of troponin t. The making of the bioelectrode is made possible by modifying a vitreous electrode with a translucent polymeric film that covalently binds to the anti-troponin t antibody. Selective recognition of the troponin t heart injury marker by this bioelectrode is indirectly indicated by luminescence emission after additional high specificity couplings and can thus be used for diagnostic purposes.

Description

OPTICAL DEVICE FOR DETECTION OF SPECIFIC HEART INJURY MARKER

Field of the Invention The present invention relates to the manufacture of an optical device capable of diagnosing specific marker of cardiac injury. It is basically composed of a bioelectrode formed by antibodies immobilized on electroplated aminophenol-derived polymeric film on a fluorine-doped tin oxide glass slide. The device of the present invention features a versatile coupling system allowing the antigen to be recognized simultaneously by the bioelectrode and a biotinylated antibody capable of binding to conjugated streptavidin to a fluorophore that emits luminescence after laser irradiation.

State of the art Spending on cardiovascular disease in Brazil is around 1.74% of GDP and according to SUS data, annual mortality in Brazil reaches about 66,000 people, with the US being the record country, with 375,000 deaths / year, in that approximately 60% of infarction deaths occur within the first hours after the onset of symptoms. Clinical diagnosis fails in 33% of cases and electrocardiographic in 50% of cases, a fact that has led the world scientific community to conduct various researches to replace and or refine conventional techniques as in the case of UA 61930 which proposes a system differentiated electrocardiographic test to determine the extent of ischemia. JP 6114059 provides high precision images of the heart muscle and also quantifies the extent of the lesion through tomography which is usually very costly.

Macromolecules such as troponins T and I are markers that accurately indicate and quantify the extent of cardiac injury and can be detected in blood serum samples, whose biochemical diagnosis is currently centered in laboratories using specialized human resources to perform the technique called Enzyme Linked Immunosorbent Assay (ELISA). This technique is very sensitive, but it has some disadvantages, such as the large sample volume and the delayed delivery of results, whose average time in a conventional laboratory is around two days. CN 1837822 proposes a reliable and specific diagnosis by a similar technique to ELISA in 96-hole plates for antibody-antigen-labeling enzyme complex formation, however this technique has basically the same disadvantages as ELISA.

A more viable alternative for early, rapid and inexpensive diagnosis can be obtained by using biosensors in which a biological material acts as an immobilized recognition agent in a transducer, which converts the biological signal into a physical and / or chemical signal. JP 11211725 discloses troponin T antigen detections via surface plasmon resonance (SPR) by a chip made by attaching the anti-troponin T antibody to a metal surface by means of a silicon-based coupling agent. However, as magnetic resonance imaging (CT) techniques are expensive. Optical detection in biosensors can be made possible by fluorophores such as cyanine, rhodamine and alexa fluorine, however quantum dots have been superior to all of these due to their extraordinary photophysical properties. DE 102006057975 provides an arrangement for detecting microorganisms by means of the fluorescent signal of quantum dots. CN 101793899 discloses an optical quantum dot biosensor capable of detecting brain natriuretic peptide by means of a complex system of magnetic probes. KR 20100137944 proposes an optical sensor with detection of quantum dot luminescence, whose light transmission occurs by optical fiber coupled to a detector.

Sensory transducers are commonly obtained by chemically modifying electrodes with polymeric films such as polythiophene, polypyrrole and polyaniline, widely studied, produced by chemical synthesis or electropolymerization. Polyaminophenols originate from the anithin substituted with the group OH, being the study of these polymers more recent and less explored. When using optical detection in biosensors, glassy substrates containing materials such as fluorine doped tin oxide (FTO) and indium doped tin oxide (ITO) become attractive due to their characteristics such as good electrical conductivity, high transparency, chemical inertness and reproducibility. The present invention enables the detection of quantum dot troponin T by means of a bioelectrode constructed by immobilizing the anti-troponin T antibody covalently to the electropolymerized poly (3-aminophenol) on an FTO surface.

SUMMARY OF THE INVENTION In the present invention, 3-aminophenol cyclic voltammetry was electropolymerized in FTO, whose visible ultraviolet spectra showed absorption at approximately 290 nm for both 3-aminophenol (3AMF) and poly (3- aminophenol) (P3AMF) suggesting that there is preservation of aromaticity in the polymer, which besides this absorption showed two more small bands at 350 and 500 nm indicating the extent of conjugation. Ion exchange analysis of the redox iron / potassium ferricyanide and hexaminututhenium chloride (II) probes demonstrated that the material is attracted to cationic structures and repulsion to anionic structures at neutral pH. Field emission scanning electron microscopy (FEG-SEM) and atomic force (AFM) images showed almost full rough polymeric coating of the FTO surface, whose maximum laser interferometry thickness was estimated at 375 ± 75 nm. Electrical characterization and electrochemical impedance spectroscopy (EIS) demonstrated that the material has a high passivating character, being applied as a matrix in the construction of a bioelectrode to diagnose cardiac injury by forming a stable amide bond between the NH2 groups of the poly. (3-aminophenol) and the terminal carboxyls (Fc) of anti-troponin T via 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) / N-hydroxysuccinimide (NHS). Additionally this antibody was modified with biotin, its affinity being streptavidin or avidin, as well as antibody and antigen specificity explored in the design of this system. Selective detection of troponin T at a concentration of 5 x 10'7 mol.L'1 was performed by quantum dot photoluminescence.

Detailed Description of the Invention The present invention is described in FIGURE 1, where an optical arrangement has been fitted with lenses and holders to obtain the best luminescence signals. The optical detection system consists of a diode laser in the range 400 to 650 nm (1) which in turn is focused on the device comprising a bioelectrode (2) at an angle between 30 and 90 ° of the spectrometer slot coupled to photomultiplier (3) that amplifies the luminescence signal. A bandpass filter (4) was placed between the bioelectrode (2) and the spectrometric slit to suppress laser emission in the detection system, whose fluorophore-specific luminescence (5) is converted to a signal by interface to a specific software. (6) However, prior to detection, to the bioelectrode (2) is consecutively added the sample containing the disease-indicating (target) biomolecule, its biotin-labeled complement and finally the streptavidin-fluorophore conjugate. Translucent bioelectrode (2) may be obtained on transparent glassy or polymeric surfaces containing tin oxide (TO), fluorine doped tin oxide (FTO), indium doped tin oxide (ITO) or other semiconductor or conductive compounds chemically or electrochemically modified by translucent polymers, whether or not conductive, capable of physically or chemically interacting with capture biomolecules, such as antigens, antibodies, oligonucleotides, DNAs, RNAs, aptamers, enzymes, microorganisms, proteins in general and other macromolecules. natural or synthetic origin. Target biomolecules normally available in body fluids such as blood, saliva and others, and may also be antigens, antibodies, oligonucleotides, DNAs, RNAs, enzymes, microorganisms, proteins in general and other macromolecules are thus specifically recognized by the bioelectrode (2). Consecutively biotin-labeled complementary biomolecules are capable of binding simultaneously to the target and conjugated streptavidin to fluorophores which may be quantum dots, cyanine, fluorocaine, rhodamine, DyLight fluorine, alexa, bodipy, coumarins, cascade blue, lucifer yellow or others, as well as enzymes such as peroxidase or others acting on certain substrates such as diaminobenzidines and the like, providing reactions whose compounds are colored. The preparation of the plate of FTO, ITO, TO or other semiconductor or conductor compounds deposited on glass or plastic modified with polymeric film can be obtained chemically or electrochemically commonly by cyclic voltammetry. The immobilization of the biomolecules on the modified transparent electrode with thin layer of translucent polymeric material can be accomplished by physical adsorption provided that appropriate conditions such as pH, temperature, net charge of polymer and biomolecule are promoted. More effective covalent bonds can be performed using bioconjugation systems such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), EDC and N-hydroxy succinimide (NHS), EDC plus N-hydroxysulfosuccinimide (Sulfo-NHS) , 1-cyclohexyl-3- (2-morpholinethyl) carbodiimide (CMC), dicyclohexyl carbodiimide (DCC), diisopropyl carbodiimide (DIC), Woodward's Reagents K, Ν, β-carbonyldiimidazole, Schiff base formation or reduction of amines or others. Gluteraldehyde cross-linking or biomolecule entrapment during chemical or electrochemical modification may also be used.

Biotinylation of biomolecules can be successfully performed by various methodologies (G. T. Hermanson, Bioconjugate Techniques, 2nd edition, Pierce Biotechnology, Thermo Fisher Scientific, Rockford, Illinois, USA, 2008).

Indirect detection of biomolecules by optical systems by fluorophores luminescence or colored products by enzymatic reactions may be performed by spectroscopic techniques such as ultraviolet, fluorescence, photoluminescence, etc., capable of qualitatively discriminating the presence or absence of the target molecule and also quantitatively by relating the emission or absorption of light to their respective concentration in the sample. It is worth noting a peculiar situation where detection technology can be minimized or completely suppressed as the amount of fluorophores or enzyme substrates can be maximized to the point where visualized with the naked eye after irradiation or after the formation of colored products.

Examples Example 1: Detection of cardiac injury specific marker (troponin T) by indirect method by quantum dot luminescence. FIGURE 2 shows in detail the production of a bioelectrode (2) and its target coupling step by step until the addition of the fluorophore.

Preparation of poly (3-aminophenol) modified FTO plate (7) (8): 3-aminophenol (3AMF) 2.5 x 10 "1 to 2.5 x 1CT3 mol.L'1 diluted in acid support electrolyte 0.5 to 2.5 mol.L'1 were deaerated in ultra-dry N2 prior to the electropolymerization which was performed by cyclic voltammetry on a 25-mL three-compartment cell fed by a Model 420A potentiostat (CH Instruments). The fluoride-doped tin oxide 1x2 cm was used and the auxiliary electrode used was platinum with a geometric area of 2 cm2.The potentials were referenced to an Ag / AgCI electrode saturated with 3M KCI. potentials ranging from -0.2 to 2.3 V at a speed of 5 to 100 mV.s'1 All chemical reagents used were analytical grade The solutions were prepared with deionized water by a Millipore Milli-Q (18.2 Mqcrrf1).

Preparation of bioelectrode (2): Three solutions were added to the poly (3AMF) modified FTO electrodes (7) (8), namely: anti-troponin T antibody (600 ± 400 ng / cm2) (9), 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS), both in the range of 15 ± 10 mmol.L'1, 1: 1 (v / v) and incubated 5 ± 3 ° C overnight. In order to remove unbound antibodies, a 250 ± 150 rpm spin wash was performed for 5 ± 3 s in phosphate buffered saline (PBS). Then 15 ± 10 mmo.L "1 glycine was added for 40 ± 20 minutes to deactivate the still active succinimide groups in the antibodies. Subsequently, PBS was washed again and bovine serum albumin (BSA) solution was added. 3 ± 2% for 40 ± 20 minutes for blocking nonspecific sites thus configuring the bioelectrode (2) formed by covalent immobilization of anti-troponin T (9) to the poly (3-aminophenot) modified FTO electrode (7) (8) ) whose efficiency of this immobilization was proven by electrochemical impedance spectroscopy.

Biotinylation of the complementary anti-troponin T antibody; Conjugation of anti-troponin T with biotin was made possible based on the protocol (E. Harlow, D. Lane, Labeling Antibodies with Biotin, Coid Spring Harb Protoc, doi: 10.1101 / pdb.prot4286, 2006), where a solution was prepared. of 10 mg.mL -1 N-hydroxysuccimide-biotin in dimethyl sulfoxide (DMSO). An antibody solution of 1 to 3 mg.mL -1 in Na2B4O7.10H2O 0.1 mol.L "1, pH 8.8 was then prepared. 25 to 250 pg of biotin ester was added to the antibody, The mixture was incubated for 4 hours under slow stirring at room temperature and then 20 µl of 1 mol.L'1 NH4Cl solution was added and incubated for 10 minutes at room temperature using an Amicon ultrafiltration system. Millipore (mean diameter 3 KDa) was removed from unbound biotin Approximately 500 pL of biotin-labeled antibody solution against 4 mL of PBS buffer centrifuged 7500.g for 30 minutes with at least (3 buffer changes) Biotin-modified antibody (10) was aliquoted and kept at 4 ° C when in use or at -20 ° C when stored.The efficiency of biotin conjugation to anti-troponin T antibody was demonstrated by dot blot. ”Detection of the troponin T antigen (900 ng / cm2) (11) by photolu minescence of quantum dots after the addition of 60 ± 40 ng of the streptavidin-quantum dot CdSe / ZnS (Qdot-525®) conjugate (12).

Dynamic washes, 250 ± 150 rpm for 5 ± 3 s of bioelectrode (2) were performed in phosphate buffer (PBS) between all stages and at the end of the process for removal of nonspecific species, as follows: Addition of troponin T antigen (11 ), biotinylated anti-troponin T antibody (10) and quantum dot-streptavidin conjugate (12).

This example illustrates the indirect detection of T troponin by quantum dot luminescence as a function of the construction engineering of the device of the present invention which provides a coupling design set forth in FIGURE 2 wherein the bioelectrode (2) composed of (7, 8 and 9) selectively recognizes the specific troponin T target (11) indirectly indicated by biotin-modified (10) complementary anti-troponin T complementary antibody bound to the core CdSe Shell ZnS (Qdot-525®) conjugate streptavidia-quantum dots (12) green luminescence (525 nm) after laser irradiation between 400 nm and 650 nm. This bioelectrode (2) is applied to the optical detection of target biomolecules present in biological samples by coupling the respective constituent species using specific interactions, whose affinity constants range from 104 to 1012 mol.L "1 and 1015 mol Between the corresponding antigen-antibodies and biotin-streptavidin, respectively.The absence of the specific target in the sample does not cause the coupling of the system, because the species are removed during the dynamic wash steps, where the absence Emission spectra proving the efficiency of selective detection of troponin T against an irrelevant protein (anti-troponin antibody I) are available in FIGURE 3. The device of the present invention may be used for “screen” qualitative diagnosis whose positive test for cardiac injury results in a green color on the bioelectrode o (2), and the absence of color or a small sign would indicate negative test for this condition. In addition, positive diagnoses can be quantified, with the extent of cardiac ihesion proportional to the concentration of Troponin T, which in turn is proportional to the intensity of luminescence, and can also be used to verify the stage of cardiac necrosis. The device of the present invention was used for detection of troponin T at a concentration of 5 x 10 7 mol.L "1 in approximately 2 hours and the entire scheme described in FIGURE 1 can be adapted for portable on-site measurement molds. in real samples at medical clinics, health clinics, emergency rooms and hospitals by unskilled professionals. ELISA Troponin T detections have a sensitivity of 10'13 mol.L "1 in approximately 48 hours in centralized laboratories requiring qualified professionals.

Claims (9)

Optical device for detecting a specific marker for cardiac injury, comprising a diode laser (1) incident on a bioelectrode (2) at an angle ranging from 30 to 90 ° from the slot of a spectrometer coupled to a photomultiplier (3) and a bandpass filter (4) whose luminescence (5) is converted to signal via interface to a specific software (6). Device according to Claim 1, characterized in that the irradiation can be performed by lamps including in the ultraviolet region, whose photoluminescent signal from the bioelectrode can be converted into a photoelectric signal by means of avalanche diodes or photodiodes coupled to preamplifiers. and data acquisition boards. Device according to Claim 1, characterized in that the bioelectrode (2) has an electrochemically modified FTO (7) glass plate with a poly (3-aminophenol) polymer (8) in which the anti-troponin T antibody (1) has been covalently immobilized. 9) which recognizes the Troponin T target (11) which binds to the biotin-modified anti-troponin T antibody (10) which couples to the streptavidin-quantum dot conjugate CdSe / ZnS (Qdot-525®) (12). Device according to Claim 3, characterized in that the streptavidin of the streptavidin-quantum dot conjugate CdSe / ZnS (Qdot-525®) (12) may be replaced by avidin or any other bioconjugation element. Device according to Claim 3, characterized in that the streptavidin-quantum dot conjugate CdSe / ZnS (Qdot-525®) (12) can be replaced by quantum dots of the same composition that emit luminescence at other wavelengths as well as dots. consisting of other semiconductors or various fluorophores such as cyanine, fluorescein, rhodamine, DyLight fluorine, Alexa fluorine, bodipy, coumarins, cascade blue, lucifer yellow, as well as substrates capable of producing colored compounds in the presence of enzymes such as peroxidase diaminobenzidines which in turn may be conjugated to streptavidin as well as other bioconjugation elements. Device according to Claim 3, characterized in that the fluorine-doped tin oxide (FTO) electrode (7) can be replaced by tin oxide (TO), indium doped tin oxide (ITO) or other semi-compounds. -conductors or conductors deposited on transparent surfaces such as glass or plastics. Device according to Claim 3, characterized in that the poly (3-aminophenol) polymer can be replaced by another polymeric material (derived from amines (aromatic or aliphatic) and non-synthetic or naturally occurring (aromatic or aliphatic) lamines. chemically, electrochemically, biologically or physically at the electrode. Device according to Claim 3, characterized in that it can be constructed with other biomolecules, such as other antibodies or antigens, oligonucleotides, DNAs, RNAs, aptamers, enzymes, microorganisms, proteins in general and other natural or synthetic linked macromolecules. by carbodiimide covalent bonds such as EDC, EDC / NHS, EDC plus Sulfo-NHS, CMC, DCC, DIC, Woodward's Reagents K, Ν, β-carbonidiimidazole, Schiff base formation or amine reduction, or others immobilization techniques such as physical adsorption, crosslinking or entrapment. Device as defined in any one of the preceding claims, characterized in that it is used for the diagnosis of other diseases, the markers of which may also be antibodies or antigens, or other target biomolecules such as oligonucleotides, DNAs, RNAs, enzymes, microorganisms, proteins. in general and other macromolecules.