WO2022165576A1 - Label-free biosensor based on a zeolitic imidazolate structure, method for manufacturing said biosensor, and method for detecting protein-protein interactions - Google Patents

Label-free biosensor based on a zeolitic imidazolate structure, method for manufacturing said biosensor, and method for detecting protein-protein interactions Download PDF

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WO2022165576A1
WO2022165576A1 PCT/BR2022/050033 BR2022050033W WO2022165576A1 WO 2022165576 A1 WO2022165576 A1 WO 2022165576A1 BR 2022050033 W BR2022050033 W BR 2022050033W WO 2022165576 A1 WO2022165576 A1 WO 2022165576A1
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interdigitated electrodes
protein
biosensor
zif
trx
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PCT/BR2022/050033
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French (fr)
Portuguese (pt)
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Luciana Daniele TRINO ALBANO
Luiz Gustavo SIMÃO ALBANO
Daniela CAMPOS GRANATO
Aline GUIMARÃES SANTANA
Davi Henrique STARNINI DE CAMARGO
Cátia CRISPILHO CORRÊA
Carlos Cesar Bof Bufon
Adriana Franco PAES LEME
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Cnpem - Centro Nacional De Pesquisa Em Energia E Materiais
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/02—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds

Definitions

  • ADAM17 The protein Disintegrin and Metalloproteinase 17, called ADAM17, is considered one of the main proteases responsible for the release of the ectodomain of surface proteins, and is mainly associated with pathological processes of diseases such as cancer, cardiovascular disorders, neurological problems, and rheumatoid arthritis. More specifically, with respect to cancer, ADAM17 overexpression is involved in the initiation, progression and growth of different types of tumors. Thioredoxin-1 (Trx-1 ) is a well-known interaction partner of the ADAM17 cytoplasmic domain (ADAM17cyto) that plays a crucial role in the regulation of ADAM17 activity.
  • Detection of PPIs is commonly performed by fluorescence or chemiluminescence methods.
  • fluorescence or chemiluminescence methods require markers such as a fluorophore to detect signals, significantly altering binding to the biological target.
  • the interaction can also be limited by photodegradation and background autofluorescence interference.
  • affinity-based techniques the use of electrochemical biosensors measuring impedance by electrochemical impedance spectroscopy (EIS) has attracted attention in recent years.
  • EIS electrochemical impedance spectroscopy
  • Protein-based electrochemical biosensors have been developed for different types of disease detection, such as quantification of Tau protein for neurodegeneration diagnosis, NF-kB protein and H5 ribonuclease for HIV diagnosis and FAM134B protein for colon cancer detection.
  • a typical example is the electrochemical blood glucose biosensor for self-monitoring of diabetics. The glucometer was implemented in 1987 and is commonly used until today.
  • Electrochemical biosensors combine the sensitivity of electrochemical transducers with high specificity during biological recognition processes. These devices allow the recognition of analytes containing different biological elements (eg proteins, antibodies, enzymes, nucleic acids).
  • a transducer element is responsible for translating specific reactions into variations in the impedance spectrum at certain frequencies, in the case of EIS.
  • MOFs metal-organic structures
  • MOFs metal-organic structures
  • MOFs are porous materials based on inorganic constitution units (metal ions) connected through organic ligands.
  • This class of material has been successfully applied in strategic fields such as catalysis, drug delivery, energy harvesting, and electronics.
  • the increased interest in these structures is related to their high versatility, which can be adapted to different strategies to achieve a specific application. For example, crystallinity or pore size can be shaped by the choice of organic binder.
  • modifications can be made incorporating host molecules into the pores of MOFs during synthesis or post-synthesis.
  • MOFs have several structures, among them, we highlight the structures of zeolitic imidazolate-8 (ZIF-8) which is a type of MOF formed by bridges between tetrahedral Zn metal ions and 2-methylimidazole ligands.
  • ZIF-8 zeolitic imidazolate-8
  • the intrinsic high porosity of this structure results in a large surface area (about 1,800 m 2 /g) and makes ZIF-8 attractive for biomedical sensing, allowing the adsorption of target elements for biosensing applications.
  • the adsorption of such elements can be enhanced with functional groups included in the structure.
  • its high chemical stability up to 550 °C provides reasonable integrity under different conditions.
  • ZIF-8 The interaction between ZIF-8 and proteins depends on the electrostatic attraction between the positively charged Zn ions of ZIF-8 and the negatively charged regions of the protein structure, rich in Glu and Asp amino acid residues. This interaction can also occur between free imidazole ligands and biomolecules through hydrogen bonds.
  • ZIF-8 on the surface of the modified electrode allows for dense immobilization, long-term stability, low non-specific binding of biomolecules and adequate biomolecular orientation to allow fast and direct specific interactions.
  • Pan et al. 2018, Anal. Biochem., 546, 5-9 reveal the fabrication of ZIF-8-based electrochemical biosensors and their use for HIV-1 DNA detection, for early-stage diagnosis of HIV. illness.
  • Zhang et al (2018, J. Electroanal. Chem., 2018, 823, 40-46) present the fabrication of electrochemical biosensors based on ZIF-8 with glucose oxidase enzymes incorporated, and their use in glucose detection.
  • biosensors based on ZIF-8 are described whose detection method is related to specific interactions of antibodies, aptamers or enzymatic reactions.
  • No prior art presents a biosensor based on ZIF-8 whose detection method is related to a PPI detection mechanism.
  • PPI protein-protein interactions
  • ADAM17cyto cytoplasmic domain of ADAM17
  • ZIF zeolitic imidazolate-8
  • a biosensor without markers based on a zeolitic imidazolate structure
  • the biosensor comprising: a substrate of SiO 2 ; a pair of interdigitated electrodes deposited on the substrate, the electrodes consisting of a lower layer of 15 to 25 nm of Cr and an upper layer of 15 to 25 nm of Au, and the interdigitated electrodes having an active area of 10 to 20 mm 2 with at least 60 pairs of interdigits; a film of zeolitic-8 imidazolate structure deposited on the active area of the interdigitated electrodes, modifying the electrodes; Thioredoxin-1 protein immobilized on the zeolitic-8 imidazolate structure; and, means of performing electrochemical analysis connected to the interdigitated electrodes.
  • Said biosensor is configured to receive an ADAM17 protein cytoplasmic domain on its electrodes and conduct electrochemical impedance spectroscopy measurements.
  • the objects of the present invention are also achieved by a PPI detection method driven by electrochemical impedance spectroscopy measurements of the modified interdigitated electrodes.
  • Figure 1 is a schematic representation of a set of steps in the manufacturing process of an interdigitated electrode of a embodiment of the biosensor of the present disclosure.
  • Figure 2 is a schematic representation of a set of steps of deposition of zeolitic-8 imidazolate structures on an interdigitated electrode of a biosensor modality of the present description.
  • Figure 3 is a schematic representation of a set of protein expression and purification steps used in a biosensor embodiment of the present description.
  • Figure 4 is a schematic representation of a set of steps for immobilizing proteins in a biosensor modality of the present description.
  • Figure 5 is a schematic representation of a set of steps for detecting protein-protein interaction in a biosensor embodiment of the present description.
  • Figure 6 is a schematic representation of a set of steps for detection of protein-protein interaction by a state-of-the-art method.
  • Figure 7a is a schematic representation of a biosensor embodiment of the present description.
  • Figures 7b and 7c are photographs of an embodiment of the biosensor of the present description.
  • Figure 8 is a graph obtained by X-ray diffraction test with grazing incidence of a zeolitic-8 imidazolate structure synthesized according to an embodiment of the manufacturing process of the present description, compared with the structure diffractogram of zeolitic-8 imidazolate obtained by computer simulation.
  • Figures 9a-9c are a sequence of microscopy images, accompanied by schematic figures, showing the evolution of the crystallographic structure of the zeolitic-8 imidazolate during the growth stages according to one embodiment of the manufacturing process of the present description.
  • Figure 10 is a graph of three Raman spectroscopies, respectively, for an interdigitated electrode with deposition of structures of zeolitic imidazolate-8 of a biosensor embodiment of the present description (bottom row), for the same electrode with Thioredoxin-1 immobilization (center row), and for the same electrode with Thioredoxin-1 and ADAM17 cytoplasmic domain (top row).
  • Figures 11a-11c are schematic representations of the mechanism of interaction between Thioredoxin-1 and the ADAM17 cytoplasmic domain, in a first, second and third step, respectively.
  • Figure 12 is a graph of electrochemical impedance spectroscopy results, showing the phase angle as a function of frequency for an interdigitated electrode without zeolitic-8 imidazolate structure, an interdigitated electrode with zeolitic-8 imidazolate structure and for various concentrations of ADAM17 cytoplasmic domain over the interdigitated electrode, in accordance with an embodiment of the present disclosure.
  • Figure 13 is a Bode diagram of electrochemical impedance spectroscopy results, showing impedance as a function of frequency for an interdigitated electrode without zeolitic-8 imidazolate structure, an interdigitated electrode with zeolitic-8 imidazolate structure and for various concentrations of ADAM17 cytoplasmic domain over the interdigitated electrode, in accordance with an embodiment of the present disclosure.
  • Figures 14a-f are Nyquist diagrams of an electrochemical impedance spectroscopy, for an interdigitated electrode without zeolitic-8 imidazolate structure, an interdigitated electrode with zeolitic-8 imidazolate structure and for different concentrations of ADAM17 cytoplasmic domain on the interdigitated electrode, in accordance with an embodiment of the present description.
  • Figure 15 is a graph showing the impedances of the Bode diagram of Figure 13, at a frequency of 0.1 Hz, taken from a biosensor subjected to various concentrations of ADAM17 cytoplasmic domain, in accordance with an embodiment of the present description.
  • Figure 16 is a graph of results of a solid-phase binding assay commonly used in the prior art to assess protein-protein interaction.
  • the present description refers to a non-labeled electrochemical biosensor, based on zeolitic imidazolate-8 (ZIF-8) structure, to monitor protein-protein interactions (PPIs).
  • ZIF-8 is deposited on interdigitated electrodes, used as transducer material for the sensor, modifying the interdigitated electrodes. These modified interdigitated electrodes are exposed to the protein thioredoxin-1 (Trx-1 ), which is immobilized on them, followed by the deposition of certain concentrations of the cytoplasmic domain of disintegrin and metalloproteinase 17 (ADAM17cyto) known as the binding partner of Trx-1. , for detection of PPL
  • the ZIF-8 crystals have a rhombic dodecahedral structure with (011 ) preferentially exposed facets, an average particle size of 205 ( ⁇ 22) nm and a film thickness of ZIF-8 around 61 ( ⁇ 6) nm.
  • Trx-1 and ADAM17cyto proteins were analyzed by electrochemical impedance spectroscopy (EIS).
  • EIS electrochemical impedance spectroscopy
  • the developed biosensor provides faster analysis, label-free detection and lower cost than the technique used for comparison, namely the solid phase binding assay (SPB). While the SPB assay requires two days to provide results, the EIS measurement with the biosensor disclosed here takes less than two hours.
  • This reliable and suitable detection platform does not require antibodies, leading to lower costs per analysis and avoiding problems associated with antibody-based assays.
  • the ZIF-8-based electrochemical biosensor can be considered a suitable platform for the future of point-of-care devices, making EIS a promising tool for the reliable detection of PP Is.
  • FIG. 1 In figure 1 are presented steps of fabrication of interdigitated electrodes, common to the state of the art.
  • a photoresist layer (2) is deposited on a SiO 2 substrate (1 ).
  • the photoresist layer (2) is patterned in the form of interdigitated electrodes by photolithography.
  • Cr/Au layers (3) are deposited on the photoresist, the Au layer being on the CR layer, the latter making the adhesion of the first to the substrate.
  • Each layer (Cr/Au) is 15 to 25 nm thick.
  • the set is immersed in acetone solution to remove the photoresist, forming the pattern of interdigitated electrodes (4).
  • the surface of the interdigitated electrodes (4) is cleaned with acetone and isopropanol for 10 to 15 min, dried with a flow of N 2 , and the sensor is stored in a vacuum desiccator.
  • Figure 2 shows the stages of growth of the ZIF-8 layer on the interdigitated electrodes, resulting in modified interdigitated electrodes (5), which is an essential step for the realization of the present invention.
  • the sensor is immersed in piranha solution at 60 to 70 °C for 15 to 25 min and then washed with distilled water.
  • For deposition of the ZIF-8 film on the active area of the interdigitated electrodes (4) they are immersed in a mixture of 8 to 12 ml_ of Zn(NO 3 ) 2 to 25 mM solution in methanol with 8 to 12 ml of 50 mM solution of 2-methylimidazole in methanol for 6 to 8 hours under stirring at room temperature.
  • FIG 3 are illustrated steps of expression and purification of proteins to obtain the proteins Trx-1 and ADAM17cyto used in the sensor of the present description.
  • Figure 4 presents a set of steps for immobilization of Trx-1 proteins on the interdigitated electrodes modified by ZIF-8 (5) of the present description.
  • the steps include cleaning the surface of the modified interdigitated electrodes (5) with methanol and drying with N 2 flow; exposing the surface of the modified interdigitated electrodes (5) to a mixture containing between 0.8 and 1.2 pg of Trx-1 in 20 pL of PBS solution, for 25 to 35 min, resulting in interdigitated electrodes with immobilized Trx-1 ( 6); clean the surface of the electrodes with PBS solution and dry with N 2 flow.
  • the objects of the present invention are also achieved by a PPI detection method driven by electrochemical impedance spectroscopy measurements.
  • Figure 5 is a schematic representation of a set of steps for detecting protein-protein interaction in a biosensor embodiment of the present description, while figure 6 illustrates a set of steps for detecting protein-protein interaction by a method of the state of the art.
  • the process of detecting protein-protein interactions with the The biosensor disclosed herein comprises: exposing the surface of the interdigitated electrodes to a mixture containing between 50 nM to 8 pM of ADAM17 protein cytoplasmic domain in 20 pL of PBS solution, for 25 to 35 min; clean the surface of the interdigitated electrodes with PSB solution and dry with N2 flow; conduct electrochemical impedance spectroscopy measurements using actuation and electrical reading means electrically connected to the interdigitated electrodes.
  • Kanamycin sulfate C 18 H 38 N 4 O 15 S, 95-100%) and disodium salt of ethylenedinitrilotetraacetic acid dihydrate (EDTA, 0.5M pH 8).
  • Imidazole C 3 H 4 N 2 , 99% was purchased from Oakwood Chemical (West Columbia, SC, USA).
  • Citric acid C 6 H 8 O 7 .H 2 O, 99.5-100%
  • dibasic sodium phosphate Na 2 HPO 4 , 99%
  • potassium dihydrogen phosphate KH 2 PO 4 , 99.5-100 %) were purchased from Merck (Burlington, MA, USA).
  • Sodium hydroxide NaOH, 97%) was purchased from Synth (Diadema, SP, Brazil). Isopropyl-[3-d-thiogalactopyranoside (IPTG, 99%) was purchased from Promega (Madison, Wl, USA). Sulfuric acid (H 2 SO 4 , 95-98%) was purchased from JT Baker (Phillipsburg, NJ, USA). Tris(hydroxymethyl)-aminomethane (Tris, NH 2 C(CH 2 OH) 3 , 99.8%) was purchased from Affymetrix (Santa Clara, CA, USA).
  • Anti-ADAM17 antibody (AB19027) was purchased from EMD Millipore (Burlington, MA, USA), and peroxidase-linked anti-horse rabbit secondary antibody (DC03L) from Calbiochem (San Diego, CA, USA).
  • the protease inhibitor cocktail was purchased from Roche (Basel, Switzerland).
  • the interdigitated electrodes used in the examples were manufactured in conductive silicon plates (100) with 2 pm thickness of SiO 2 .
  • an AZ5214E photoresist was standardized by conventional photolithography followed by deposition of Cr/Au (20/20 nm), both metallic layers using electron beam evaporation and deposited at a rate of 1A/S.
  • the first Cr-based metallic layer aims to improve the adhesion of the second Au-based metallic layer.
  • the lift-off process was performed to remove the photoresist, immersing the interdigitated electrodes in acetone.
  • the interdigitated electrodes have 60 electrode arrays confined to an active area of approximately 15 mm 2 . The distance between each electrode array is 10 pm (channel length).
  • the total channel width-to-length (W/L) ratio is 50,000.
  • the size of each substrate with 60 integrated interdigitated electrodes is 12 x 7 mm 2 (illustrated in Figures 7a, 7b and 7c).
  • the sample surfaces were cleaned with acetone and isopropanol for 10 minutes each, in an ultrasonic bath, and dried with a flow of N 2 . Prior to deposition of the ZIF-8, each interdigitated electrode was tested for possible leakage current.
  • the interdigitated electrodes were immersed in a piranha solution (H 2 SO 4 /H 2 O 2 , 7:3 (V/V)) at 65 °C for 20 minutes to increase the density of hydroxyl functional groups on the SiO 2 surface.
  • a piranha solution H 2 SO 4 /H 2 O 2 , 7:3 (V/V)
  • the deposition of the ZIF-8 film occurs with the immersion of the electrodes in a fresh mixture of 10 ml_ of Zn(NO 3 ) 2 (25 mM in methanol) and 10 ml of 2-methylimidazole (2-Melm, 50 mM in methanol) for 6 hours under stirring at room temperature.
  • the ZIF-8 films were washed with methanol, dried with N 2 flow and stored under vacuum.
  • ZIF-8 films were morphologically characterized by Scanning Electron Microscopy (FEG-SEM, Inspect F50 from FEI, Hillsboro, Oregon, USA).
  • the crystalline structure of the ZIF-8 films was determined by X-ray synchrotron radiation in the XRD2 beamline of the Brazilian Synchrotron Light Laboratory (LNLS/CNPEM, Campinas, Brazil), with incident wavelength ( ⁇ ) of 1 .54979 A.
  • Diffractograms were obtained in the range of 5° to 20° (20°) using a Mythen linear detector for data acquisition.
  • Atomic Force Microscopy (AFM, Park NX10, Santa Clara, CA, USA) was used to analyze the surface roughness and film thickness of the interdigitated electrodes modified by ZIF-8 using the Gwyddion software.
  • the Raman spectrum was acquired using a Raman Xplora Plus spectrometer equipped with an optical microscope (Horiba, Kyoto, Japan), operated with laser excitation at 638 nm and a 100x lens under atmospheric conditions.
  • the proteins used in the present examples were obtained by conventional expression and purification procedures, described in Arag ⁇ o et al. (2012, J. Biol. Chem., 287 (51), 43071-43082).
  • ADAM17cyto proteins were expressed in competent BL21 (DE3) cells at 37 °C for 4 hours after induction with 0.5 mM IPTG in LB broth (Luria Bertani) containing kanamycin. 1 L pellets of expressed proteins were separated by 15% SDS-PAGE under denaturing conditions to confirm protein induction.
  • Recovered cells were resuspended in buffer A (50 mM Tris pH 7.5, 2 mM CaCl 2 and 150 mM NaCl) supplemented with 1 mM PMSF and a protease inhibitor cocktail tablet as protease inhibitors. After homogenization, cells were lysed by sonication (Vibracell VCX 500; Sonics & Materials, Inc., Newtown, CT, USA). The suspensions were centrifuged at 14,000 g for 30 min at 4°C.
  • the second step of purification was carried out by gel filtration chromatography to obtain the monomeric state of each protein, eliminating the possible impurities that persisted after the affinity chromatography and separating the species with distinct oligomeric states.
  • This procedure was performed by preparing the concentrated protein solution to a volume of 0.5 ml using a 10 kDa cut-off Amicon filter (Millipore, Billerica, MA, USA). Protein suspensions (Trx-1 and ADAM17cyto) were loaded separately onto Superdex 75 10/300 GL analytical gel filtration columns (GE Healthcare, Chicago, IL, USA) using a flow rate of 0.2 mL/min of PBS with pH 7.2.
  • Proteins were eluted using 1.2 times column volumes of cooled PBS and then collected in 0.5 mL fractions. These fractions were then separated by 15% SDS-PAGE under denaturing conditions, followed by Coomassie blue staining to assess the quality of purification. Finally, the pure fractions were pooled and concentrated to a volume of 1 mL using a 10 kDa cut-off Amicon filter (Millipore, Billerica, MA, USA). The final protein concentrations obtained for Trx-1 and ADAM17cyto were 11.0 pg/pl and 7.1 pg/pl, respectively. Protein concentration was determined using the NanoDrop 2000/2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) at 280 nm.
  • Electrochemical impedance spectroscopy measurements were performed with the Solartron model SI 1260 A impedance analyzer (Solartron Analytical, Farnborough, Hampshire, UK), in the frequency range from 1 MHz to 10 mHz (10 points per decade, oscillation amplitude 100 mV and voltage offset 0 V).
  • the modified interdigitated electrodes were first exposed to Trx-1 (1 pg in 20 pL of PBS solution) for 30 minutes. Subsequently, the surface of the electrodes was washed with PBS solution and dried with N 2 flow.
  • Figure 7a is a schematic representation of a biosensor embodiment of the present description.
  • Figures 7b and 7c are photographs of the exemplary modality of the biosensor, according to the process described herein.
  • Figure 8 is a graph obtained by X-ray diffraction test with grazing incidence of a zeolitic-8 imidazolate structure synthesized according to an exemplary modality of the manufacturing process, being compared with the imidazolate structure diffractogram zeolitic-8 obtained by computer simulation.
  • the ZIF-8 film acts as an active layer where solutions containing Trx-1 and ADAM17cyto proteins are deposited.
  • the presence of high intensity peaks (011), (002), (112), (022), (013) and (222) indicates the formation of ZIF-8.
  • a preferred growth orientation of the ZIF-8 film is observed at 7.4° (angle 29), corresponding to the (011°) plane.
  • ZIF-8 film growth occurs from the nucleation of ZIF-8 crystals, which is enhanced by the presence of methanol (a practical solvent) and a higher concentration of 2-methylimidazole than Zn(NO 3 ) 2 .
  • methanol a practical solvent
  • 2-methylimidazole a higher concentration of 2-methylimidazole than Zn(NO 3 ) 2 .
  • Methanol is responsible for dissolving the precursors and dissociate the H + from the 2-methylimidazole.
  • the Zn 2+ cations combine with the deprotonated 2-methylimidazole to form individual units of ZIF-8, which then serve as the building block for the formation of ZIF-8 crystals.
  • Figures 9a-9c are a sequence of microscopy images, accompanied by schematic figures, showing the evolution of the crystallographic structure of the zeolitic-8 imidazolate during the growth stages according to one embodiment of the manufacturing process of the present description.
  • ZIF-8 begins to crystallize in a cubic form ( Figure 9a), changing to a truncated dodecahedral rhombic (TRD) structure in sequence ( Figure 9b), and gradually assuming a rhombic crystal structure.
  • TRD truncated dodecahedral rhombic
  • RD dodecahedral
  • the morphological evolution scheme of the ZIF-8 crystals is presented in these figures, starting with a cubic crystal oriented to [100], which evolves into TRD and assumes the final form of RD. According to Wulff's rule, the most stable crystal morphology occurs by the slowest growing facet. For ZIF-8, the growth direction [011] makes the RD crystal structure the most stable equilibrium morphology.
  • Figure 10 is a graph of three Raman spectroscopies, respectively, for the one interdigitated electrode with deposition of zeolitic-8 imidazolate structures of a biosensor modality of the present description (bottom line), for the same electrode with immobilization of Thioredoxin-1 (center line), and to the same electrode with Thioredoxin-1 and ADAM17 cytoplasmic domain (top line). An intense peak was observed for all samples analyzed at 519 cm -1 , being attributed to the SiO 2 of the substrate.
  • the SS elongation mode can have two contributions, the first from the disulfide bond formed in the heterodimer between Trx-1 and ADAM17cyto, and the other related to the disulfide bond present in the ADAM17cyto structure.
  • disulfide bond formation is a slow process at physiological pH (7.4), many proteins such as ADAM17cyto and Trx-1 can interact through cysteine dimerization.
  • FIGs 11 a-11 c are schematic representations of the mechanism of interaction between Thioredoxin-1 and the ADAM17 cytoplasmic domain, in a first, second and third step, respectively.
  • the formation of the disulfide bond occurs spontaneously in vitro and involves the deprotonation of two cysteine thiols (-SH) to form charged thiolates (RS-), as seen in Figure 11 a.
  • the thiolate ions react with an electron acceptor such as molecular oxygen, as seen in Figure 11b, to generate the reactive radical species (R-S') that react and form the disulfide bond, as seen in Figure 11c.
  • Figure 12 is a graph of electrochemical impedance spectroscopy results, showing the phase angle as a function of frequency for an interdigitated electrode without zeolitic-8 imidazolate structure, an interdigitated electrode with zeolitic-8 imidazolate structure and for ADAM17 cytoplasmic domain concentrations on the interdigitated electrode ranging from 50 nM to 8 pM, according to an embodiment of the present disclosure.
  • concentrations of 50 nM to 4 pM of ADAM17cyto a predominant transport of ions occurs at lower frequencies. This event may be related to the formation of thiolates from cysteines.
  • the concentration of ADAM17cyto increases from 4 pM to 8 pM, the amount of ADAM17cyto protein interacting with Trx-1 also increases - a direct effect of the higher electron transfer to form the disulfide bond observed at higher frequencies. Therefore, the charge transfer response shifts to lower frequencies as the concentration of ADAM17cyto decreases (increasing capacitive behavior).
  • Figure 13 is a Bode diagram corresponding to the test results of Figure 12.
  • the unmodified interdigitated electrode has a high impedance (
  • Trx-1 By adding the Trx-1 , followed by different At ADAM17cyto concentrations, a significant change in impedance is observed at low frequencies (from 10 mHz to 10 Hz).
  • decreased from 8.5x10 9 O to 4.8x10 6 O with an increase in ADAM17cyto concentration (see Table 1). In this low frequency region, where most ionic effects occur, the interaction of proteins on the biosensor surface led to an increase in conductivity.
  • Table 1 Impedance at 0.1 Hz and peak frequencies analyzed in interdigitated electrodes (4), interdigitated electrodes modified with ZIF-8 (5) and interdigitated electrodes with immobilized Trx-1 (6) and different concentrations of ADAM17cyto.
  • Figures 14a-f are Nyquist diagrams of an electrochemical impedance spectroscopy, for an interdigitated electrode without zeolitic-8 imidazolate structure, an interdigitated electrode with zeolitic-8 imidazolate structure and for different concentrations of ADAM17 cytoplasmic domain on the interdigitated electrode, in accordance with an embodiment of the present description.
  • Nyquist diagrams for the biosensor response under different concentrations of ADAM17cyto, show a semicircle in the high frequency range, followed by a linear tail in the lower frequencies. The semicircle is associated with charge transfer resistance, while the straight tail is associated with ionic diffusion.
  • the diameter of the semicircle decreases with increasing ADAM17cyto concentration, indicating less resistance to charge transfer.
  • ADAM17cyto 8 pM a small semicircle is observed, indicating enhanced charge transfer kinetics.
  • Figure 15 is a graph showing the impedances of the Bode diagram of Figure 12, at a frequency of 0.1 Hz, taken from a biosensor subjected to various concentrations of ADAM17 cytoplasmic domain, in accordance with an embodiment of the present description.
  • gradually decreases with increasing concentration of ADAM17cyto at 0.1 Hz (the dotted line in Figure 12) between 50 nM and 8 pM.
  • a linear relationship (correlation coefficient of 0.9902) is found between Z and ADAM17cyto concentration for the analyzed biosensor modalities.
  • Figure 16 is a graph of results of a solid phase binding assay (SPB) commonly used in the state of the art to evaluate protein-protein interaction.
  • SPB solid phase binding assay
  • the results obtained compared to a solid phase binding assay, widely used in the current technique demonstrate a better performance of the ZIF-8 based biosensor for the detection of PPIs disclosed here, especially when the sensitivity and reproducibility are evaluated.
  • the biosensor detection limit was determined to be as low as 50 nM, with a range of 1.0 to 11.4% - for the solid phase binding assay, the lower detection limit was 0.5 pM, with a variation of 5.4 to 27.5%.
  • the inventive concept presented here of detecting PPIs in markerless biosensors, based on ZIF-8 can also be extended to the recognition of other related biomolecules, showing great potential for future use as point-of-care devices. for accurate diagnosis or prognosis of clinical diseases.

Abstract

The present description relates to a label-free biosensor with interdigitated electrodes based on ZIF-8 for detecting protein-protein interactions. The method for detection using said biosensor is performed by means of electrochemical impedance spectroscopy measurements from the electrodes. Also described is the method for manufacturing said biosensors, including steps of manufacturing the interdigitated electrode (4), modifying the electrode with ZIF-8 (5), immobilizing a protein on the electrode (6), and exposing the electrode to a concentration of another protein, which is an interaction partner of the first protein, to detect interaction therebetween.

Description

BIOSSENSOR SEM MARCAÇÃO BASEADO EM ESTRUTURA DE IMIDAZOLATO ZEOLÍTICO, PROCESSO DE FABRICAÇÃO DO MESMO E PROCESSO DE DETECÇÃO DE INTERAÇÕES PROTEÍNA-PROTEÍNA CAMPO DA DESCRIÇÃO BIOSSENSOR WITHOUT MARKING BASED ON ZEOLITICAL IMDAZOLATE STRUCTURE, MANUFACTURING PROCESS AND PROCESS FOR DETECTION OF PROTEIN-PROTEIN INTERACTIONS FIELD OF DESCRIPTION
[0001] A presente descrição é do campo de análise de materiais pela investigação de variáveis eletroquímicas. [0001] The present description is from the field of materials analysis by the investigation of electrochemical variables.
FUNDAMENTOS DA DESCRIÇÃO DESCRIPTION FUNDAMENTALS
[0002] Os mecanismos básicos da vida celular dependem fortemente do funcionamento correto das interações proteína-proteína (PP Is). Várias doenças humanas podem ser rastreadas por PPIs anormais. Nesse contexto, por meio do monitoramento dessas PPIs, algumas doenças graves podem ser identificadas em seus estágios iniciais. A proteína Desintegrina e Metaloproteinase 17, chamada ADAM17, é considerada uma das principais proteases responsáveis pela liberação do ectodomínio de proteínas de superfície, e está associada principalmente a processos patológicos de doenças, como câncer, distúrbios cardiovasculares, problemas neurológicos, e artrite reumatoide. Mais especificamente, com relação ao câncer, a superexpressão de ADAM17 está envolvida na iniciação, progressão e crescimento de diferentes tipos de tumores. A Tioredoxina-1 (Trx-1 ) é um parceiro de interação bem conhecido do domínio citoplasmático ADAM17 (ADAM17cyto) que desempenha um papel crucial na regulação da atividade de ADAM17. [0002] The basic mechanisms of cellular life depend heavily on the correct functioning of protein-protein interactions (PP Is). Various human diseases can be tracked by abnormal PPIs. In this context, by monitoring these PPIs, some serious diseases can be identified in their early stages. The protein Disintegrin and Metalloproteinase 17, called ADAM17, is considered one of the main proteases responsible for the release of the ectodomain of surface proteins, and is mainly associated with pathological processes of diseases such as cancer, cardiovascular disorders, neurological problems, and rheumatoid arthritis. More specifically, with respect to cancer, ADAM17 overexpression is involved in the initiation, progression and growth of different types of tumors. Thioredoxin-1 (Trx-1 ) is a well-known interaction partner of the ADAM17 cytoplasmic domain (ADAM17cyto) that plays a crucial role in the regulation of ADAM17 activity.
[0003] A detecção de PPIs, como a interação entre ADAM-17cyto e Trx-1 , é comumente realizada por métodos de fluorescência ou quimioluminescência. No entanto, esses métodos requerem marcadores, como um fluoróforo para detectar sinais, alterando significativamente a ligação ao alvo biológico. A interação também pode ser limitada por fotodegradação e interferência de autofluorescência de fundo. Entre as técnicas baseadas em afinidade, o uso de biossensores eletroquímicos medindo a impedância por espectroscopia de impedância eletroquímica (EIS) tem atraído atenção nos últimos anos. [0004] Biossensores eletroquímicos são considerados plataformas tecnológicas versáteis e bem estabelecidas para aplicações bioeletrônicas avançadas. Biossensores eletroquímicos baseados em proteínas foram desenvolvidos para diferentes tipos de detecção de doenças, como a quantificação da proteína Tau para diagnóstico de neurodegeneração, proteína NF-kB e ribonuclease H5 para diagnóstico de HIV e proteína FAM134B para detecção de câncer de cólon. Um exemplo típico é o biossensor eletroquímico de glicose no sangue para automonitoramento de diabéticos. O glicosímetro foi implementado em 1987, sendo comumente empregado até hoje. Os biossensores eletroquímicos combinam a sensibilidade dos transdutores eletroquímicos com alta especificidade durante os processos de reconhecimento biológico. Esses dispositivos permitem o reconhecimento de analitos contendo diferentes elementos biológicos (por exemplo, proteínas, anticorpos, enzimas, ácidos nucleicos). Um elemento transdutor é responsável por traduzir reações específicas em variações do espectro de impedância em determinadas frequências, no caso da EIS. [0003] Detection of PPIs, such as the interaction between ADAM-17cyto and Trx-1, is commonly performed by fluorescence or chemiluminescence methods. However, these methods require markers such as a fluorophore to detect signals, significantly altering binding to the biological target. The interaction can also be limited by photodegradation and background autofluorescence interference. Among affinity-based techniques, the use of electrochemical biosensors measuring impedance by electrochemical impedance spectroscopy (EIS) has attracted attention in recent years. [0004] Electrochemical biosensors are considered versatile and well-established technology platforms for advanced bioelectronic applications. Protein-based electrochemical biosensors have been developed for different types of disease detection, such as quantification of Tau protein for neurodegeneration diagnosis, NF-kB protein and H5 ribonuclease for HIV diagnosis and FAM134B protein for colon cancer detection. A typical example is the electrochemical blood glucose biosensor for self-monitoring of diabetics. The glucometer was implemented in 1987 and is commonly used until today. Electrochemical biosensors combine the sensitivity of electrochemical transducers with high specificity during biological recognition processes. These devices allow the recognition of analytes containing different biological elements (eg proteins, antibodies, enzymes, nucleic acids). A transducer element is responsible for translating specific reactions into variations in the impedance spectrum at certain frequencies, in the case of EIS.
[0005] Esforços para desenvolvimento nessa área têm se voltado para fabricação de biossensores eletroquímicos baseados em materiais orgânicos e híbridos, devido às suas características de produção em escala e baixo custo, mantendo parâmetros operacionais razoáveis com boa flexibilidade mecânica. Entre os diferentes materiais, estruturas metal-orgânicas (MOFs) têm mostrado grande potencial para detecção de PPI. MOFs são materiais porosos baseados em unidades de constituição inorgânicas (íons metálicos) conectadas por meio de ligantes orgânicos. Esta classe de material tem sido aplicada com sucesso em campos estratégicos como catálise, entrega de medicamentos, captação de energia, e eletrônica. O aumento do interesse por essas estruturas está relacionado à sua alta versatilidade, a qual pode ser adequada a diferentes estratégias para alcançar uma aplicação específica. Por exemplo, a cristalinidade ou o tamanho dos poros podem ser moldados pela escolha do ligante orgânico. Além disso, modificações podem ser realizadas incorporando-se moléculas hospedeiras aos poros das MOFs durante a síntese ou pós-síntese. [0005] Efforts for development in this area have focused on the fabrication of electrochemical biosensors based on organic and hybrid materials, due to their characteristics of scale production and low cost, maintaining reasonable operational parameters with good mechanical flexibility. Among the different materials, metal-organic structures (MOFs) have shown great potential for PPI detection. MOFs are porous materials based on inorganic constitution units (metal ions) connected through organic ligands. This class of material has been successfully applied in strategic fields such as catalysis, drug delivery, energy harvesting, and electronics. The increased interest in these structures is related to their high versatility, which can be adapted to different strategies to achieve a specific application. For example, crystallinity or pore size can be shaped by the choice of organic binder. In addition, modifications can be made incorporating host molecules into the pores of MOFs during synthesis or post-synthesis.
[0006] MOFs apresentam diversas estruturas, dentre elas, destaca-se as estruturas de imidazolato zeolítico-8 (ZIF-8) que é um tipo de MOF formado por pontes entre íons metálicos de Zn tetraédricos e ligantes 2-metilimidazol. A alta porosidade intrínseca dessa estrutura resulta em uma grande área de superfície (cerca de 1.800 m2/g) e torna a ZIF-8 atraente para sensoriamento biomédico, permitindo a adsorção de elementos alvo para aplicações de biossensoriamento. A adsorção de tais elementos pode ser intensificada com grupos funcionais, incluídos na estrutura. Além disso, sua alta estabilidade química (até 550 °C) fornece integridade razoável sob diferentes condições. A interação entre ZIF-8 e proteínas depende da atração eletrostática entre os íons Zn carregados positivamente de ZIF-8 e regiões carregadas negativamente da estrutura das proteínas, rica em resíduos de aminoácidos Glu e Asp. Essa interação também pode ocorrer entre ligantes de imidazol livres e biomoléculas por meio de ligações de hidrogênio. Dessa forma, a presença de ZIF-8 na superfície do eletrodo modificado permite uma imobilização densa, estabilidade de longo prazo, baixa ligação não específica de biomoléculas e orientação biomolecular adequada para permitir interações específicas rápidas e diretas. [0006] MOFs have several structures, among them, we highlight the structures of zeolitic imidazolate-8 (ZIF-8) which is a type of MOF formed by bridges between tetrahedral Zn metal ions and 2-methylimidazole ligands. The intrinsic high porosity of this structure results in a large surface area (about 1,800 m 2 /g) and makes ZIF-8 attractive for biomedical sensing, allowing the adsorption of target elements for biosensing applications. The adsorption of such elements can be enhanced with functional groups included in the structure. In addition, its high chemical stability (up to 550 °C) provides reasonable integrity under different conditions. The interaction between ZIF-8 and proteins depends on the electrostatic attraction between the positively charged Zn ions of ZIF-8 and the negatively charged regions of the protein structure, rich in Glu and Asp amino acid residues. This interaction can also occur between free imidazole ligands and biomolecules through hydrogen bonds. Thus, the presence of ZIF-8 on the surface of the modified electrode allows for dense immobilization, long-term stability, low non-specific binding of biomolecules and adequate biomolecular orientation to allow fast and direct specific interactions.
ESTADO DA TÉCNICA STATE OF THE TECHNIQUE
[0007] Ma e colaboradores (2013, Anal. Chem., 85(15), 7550-7557) reportam a fabricação de biossensores eletroquímicos baseados em ZIFs, incluindo, mas não exclusivamente, ZIF-8, e o seu uso na medição in vivo de neuroquímicos. [0007] Ma et al (2013, Anal. Chem., 85(15), 7550-7557) report the fabrication of electrochemical biosensors based on ZIFs, including, but not limited to, ZIF-8, and their use in in-house measurement. neurochemicals alive.
[0008] Pan e colaboradores (2018, Anal. Biochem., 546, 5-9) revelam a fabricação de biossensores eletroquímicos baseados em ZIF-8 e o seu uso para detecção de DNA de HIV-1 , para diagnóstico em estágio precoce da doença. [0008] Pan et al. (2018, Anal. Biochem., 546, 5-9) reveal the fabrication of ZIF-8-based electrochemical biosensors and their use for HIV-1 DNA detection, for early-stage diagnosis of HIV. illness.
[0009] Zhang e colaboradores (2018, J. Electroanal. Chem., 2018, 823, 40-46) apresentam a fabricação de biossensores eletroquímicos baseados em ZIF-8 com enzimas glicose oxidase incorporada, e o seu uso na detecção de glucose. [0009] Zhang et al (2018, J. Electroanal. Chem., 2018, 823, 40-46) present the fabrication of electrochemical biosensors based on ZIF-8 with glucose oxidase enzymes incorporated, and their use in glucose detection.
[0010] Yang e colaboradores (2018, J. Electrochem. Soc., 2018, 165 (5), H247-H250) descrevem a fabricação de biossensores eletroquímicos baseados em ZIF-8 coberto com uma camada de nanoplatina, e o seu uso na detecção de sarcosina para diagnóstico de câncer de próstata. [0010] Yang et al (2018, J. Electrochem. Soc., 2018, 165 (5), H247-H250) describe the fabrication of electrochemical biosensors based on ZIF-8 covered with a layer of nanoplatinum, and its use in detection of sarcosine for diagnosis of prostate cancer.
[0011] Em todas as técnicas anteriores aqui apresentadas, são descritos biossensores baseados em ZIF-8 cujo método de detecção está relacionado a interações específicas de anticorpos, aptâmeros ou reações enzimáticas. Nenhuma anterioridade apresenta um biossensor baseado em ZIF-8 cujo método de detecção está relacionado a um mecanismo de detecção de PPI. BREVE DESCRIÇÃO DA INVENÇÃO [0011] In all previous techniques presented here, biosensors based on ZIF-8 are described whose detection method is related to specific interactions of antibodies, aptamers or enzymatic reactions. No prior art presents a biosensor based on ZIF-8 whose detection method is related to a PPI detection mechanism. BRIEF DESCRIPTION OF THE INVENTION
[0012] É um dos objetivos da presente invenção prover um biossensor para detecção de interações proteína-proteína (PPI) com o domínio citoplasmático de ADAM17 (ADAM17cyto), tendo como material de transdução eletrodos modificados baseados em estruturas de imidazolato zeolítico-8 (ZIF-8), em que as medições são feitas por espectroscopia de impedância eletroquímica (EIS). [0012] It is one of the objectives of the present invention to provide a biosensor for detecting protein-protein interactions (PPI) with the cytoplasmic domain of ADAM17 (ADAM17cyto), having as transduction material modified electrodes based on zeolitic imidazolate-8 (ZIF) structures -8), where measurements are made by electrochemical impedance spectroscopy (EIS).
[0013] Os objetivos da presente invenção são alcançados por um biossensor sem marcadores, baseado em estrutura de imidazolato zeolítico, o biossensor compreendendo: um substrato de SiO2; um par de eletrodos interdigitados depositados sobre o substrato, os eletrodos constituídos de uma camada inferior de 15 a 25 nm de Cr e uma camada superior de 15 a 25 nm de Au, e os eletrodos interdigitados possuindo uma área ativa de 10 a 20 mm2 com pelo menos 60 pares de interdígitos; um filme de estrutura de imidazolato zeolítico-8 depositado sobre a área ativa dos eletrodos interdigitados, modificando os eletrodos; proteína Tioredoxina-1 imobilizada sobre a estrutura de imidazolato zeolítico-8; e, meios de realizar análise eletroquímica conectados aos eletrodos interdigitados. O referido biossensor é configurado para receber um domínio citoplasmático de proteína ADAM17 em seus eletrodos e conduzir medições de espectroscopia de impedância eletroquímica. [0013] The objectives of the present invention are achieved by a biosensor without markers, based on a zeolitic imidazolate structure, the biosensor comprising: a substrate of SiO 2 ; a pair of interdigitated electrodes deposited on the substrate, the electrodes consisting of a lower layer of 15 to 25 nm of Cr and an upper layer of 15 to 25 nm of Au, and the interdigitated electrodes having an active area of 10 to 20 mm 2 with at least 60 pairs of interdigits; a film of zeolitic-8 imidazolate structure deposited on the active area of the interdigitated electrodes, modifying the electrodes; Thioredoxin-1 protein immobilized on the zeolitic-8 imidazolate structure; and, means of performing electrochemical analysis connected to the interdigitated electrodes. Said biosensor is configured to receive an ADAM17 protein cytoplasmic domain on its electrodes and conduct electrochemical impedance spectroscopy measurements.
[0014] Os objetivos da presente invenção também são alcançados por um método de fabricação do referido biossensor com etapas de fabricação de eletrodos interdigitados conhecidas do estado da técnica, seguidas de: [0014] The objectives of the present invention are also achieved by a method of manufacturing said biosensor with steps of manufacturing interdigitated electrodes known in the prior art, followed by:
[0015] depositar um filme de estrutura de imidazolato zeolítico-8 sobre a área ativa dos eletrodos interdigitados, imergindo o substrato em uma mistura de 8 a 12 mL de solução de Zn(NO3)2 25mM em metanol com de 8 a 12 mL de solução de 2-metilimidazol 50 mM em metanol, por 6 a 8 horas sob agitação, em temperatura ambiente; [0015] deposit a film of zeolitic-8 imidazolate structure on the active area of the interdigitated electrodes, immersing the substrate in a mixture of 8 to 12 mL of 25mM Zn(NO 3 ) 2 solution in methanol with 8 to 12 mL of 50 mM 2-methylimidazole solution in methanol for 6 to 8 hours under stirring at room temperature;
[0016] limpar a superfície dos eletrodos interdigitados com metanol e secar com fluxo de N2; [0016] clean the surface of the interdigitated electrodes with methanol and dry with N 2 flow;
[0017] expor a superfície dos eletrodos interdigitados a uma mistura contendo entre 0,8 e 1 ,2 pg de Tioredoxina-1 em 20 pL de solução tampão de fosfato salino (PBS), por 25 a 35 min; [0017] expose the surface of the interdigitated electrodes to a mixture containing between 0.8 and 1.2 pg of Thioredoxin-1 in 20 pL of phosphate buffer saline solution (PBS), for 25 to 35 min;
[0018] limpar a superfície dos eletrodos interdigitados com solução PSB e secar com fluxo de N2; [0018] clean the surface of the interdigitated electrodes with PSB solution and dry with N 2 flow;
[0019] expor a superfície dos eletrodos interdigitados a uma mistura contendo entre 50 nM a 8 pM de domínio citoplasmático de proteína ADAM17 em 20 pL de solução PBS, por 25 a 35 min; [0019] expose the surface of the interdigitated electrodes to a mixture containing between 50 nM to 8 pM of ADAM17 protein cytoplasmic domain in 20 pL of PBS solution, for 25 to 35 min;
[0020] limpar a superfície dos eletrodos interdigitados com solução PBS e secar com fluxo de N2. [0020] Clean the surface of the interdigitated electrodes with PBS solution and dry with N 2 flow.
[0021] Os objetivos da presente invenção também são alcançados por um método de detecção de PPI conduzido por medições de espectroscopia de impedância eletroquímica dos eletrodos interdigitados modificados. [0021] The objects of the present invention are also achieved by a PPI detection method driven by electrochemical impedance spectroscopy measurements of the modified interdigitated electrodes.
BREVE DESCRIÇÃO DAS FIGURAS BRIEF DESCRIPTION OF THE FIGURES
[0022] A presente invenção é ilustrada em uma modalidade representada nos desenhos. [0022] The present invention is illustrated in an embodiment shown in the drawings.
[0023] A figura 1 é uma representação esquemática de um conjunto de etapas do processo de fabricação de um eletrodo interdigitado de uma modalidade do biossensor da presente descrição. [0023] Figure 1 is a schematic representation of a set of steps in the manufacturing process of an interdigitated electrode of a embodiment of the biosensor of the present disclosure.
[0024] A figura 2 é uma representação esquemática de um conjunto de etapas de deposição de estruturas de imidazolato zeolítico-8 em um eletrodo interdigitado de uma modalidade do biossensor da presente descrição. [0024] Figure 2 is a schematic representation of a set of steps of deposition of zeolitic-8 imidazolate structures on an interdigitated electrode of a biosensor modality of the present description.
[0025] A figura 3 é uma representação esquemática de um conjunto de etapas de expressão e purificação das proteínas utilizadas em uma modalidade do biossensor da presente descrição. [0025] Figure 3 is a schematic representation of a set of protein expression and purification steps used in a biosensor embodiment of the present description.
[0026] A figura 4 é uma representação esquemática de um conjunto de etapas para imobilização de proteínas em uma modalidade do biossensor da presente descrição. [0026] Figure 4 is a schematic representation of a set of steps for immobilizing proteins in a biosensor modality of the present description.
[0027] A figura 5 é uma representação esquemática de um conjunto de etapas para detecção de interação proteína-proteína em uma modalidade do biossensor da presente descrição. [0027] Figure 5 is a schematic representation of a set of steps for detecting protein-protein interaction in a biosensor embodiment of the present description.
[0028] A figura 6 é uma representação esquemática de um conjunto de etapas para detecção de interação proteína-proteína por um método do estado da técnica. [0028] Figure 6 is a schematic representation of a set of steps for detection of protein-protein interaction by a state-of-the-art method.
[0029] A figura 7a é uma representação esquemática de uma modalidade do biossensor da presente descrição. As figuras 7b e 7c são fotografias de uma modalidade do biossensor da presente descrição. [0029] Figure 7a is a schematic representation of a biosensor embodiment of the present description. Figures 7b and 7c are photographs of an embodiment of the biosensor of the present description.
[0030] A figura 8 é um gráfico obtido por ensaio de difração de raios-X com incidência rasante de uma estrutura de imidazolato zeolítico-8 sintetizada de acordo com uma modalidade do processo de fabricação da presente descrição, comparado com o difratograma de estrutura de imidazolato zeolítico-8 obtido por simulação computacional. [0030] Figure 8 is a graph obtained by X-ray diffraction test with grazing incidence of a zeolitic-8 imidazolate structure synthesized according to an embodiment of the manufacturing process of the present description, compared with the structure diffractogram of zeolitic-8 imidazolate obtained by computer simulation.
[0031] As figuras 9a-9c são uma sequência de imagens de microscopia, acompanhadas de figuras esquemáticas, apresentando a evolução da estrutura cristalográfica do imidazolato zeolítico-8 durante as etapas de crescimento de acordo com uma modalidade do processo de fabricação da presente descrição. [0031] Figures 9a-9c are a sequence of microscopy images, accompanied by schematic figures, showing the evolution of the crystallographic structure of the zeolitic-8 imidazolate during the growth stages according to one embodiment of the manufacturing process of the present description.
[0032] A figura 10 é um gráfico de três espectroscopias Raman, sendo, respectivamente, para um eletrodo interdigitado com deposição de estruturas de imidazolato zeolitico-8 de uma modalidade do biossensor da presente descrição (linha inferior), para o mesmo eletrodo com imobiliização de Tioredoxina-1 (linha central), e para mesmo eletrodo com Tioredoxina-1 e domínio citoplasmático ADAM17 (linha superior). [0032] Figure 10 is a graph of three Raman spectroscopies, respectively, for an interdigitated electrode with deposition of structures of zeolitic imidazolate-8 of a biosensor embodiment of the present description (bottom row), for the same electrode with Thioredoxin-1 immobilization (center row), and for the same electrode with Thioredoxin-1 and ADAM17 cytoplasmic domain (top row).
[0033] As figuras 11a-11c são representações esquemáticas do mecanismo de interação entre Tioredoxina-1 e o domínio citoplasmático ADAM17, em uma primeira, segunda e terceira etapa, respectivamente. [0033] Figures 11a-11c are schematic representations of the mechanism of interaction between Thioredoxin-1 and the ADAM17 cytoplasmic domain, in a first, second and third step, respectively.
[0034] A figura 12 é um gráfico de resultados de uma espectroscopia de impedância eletroquímica, mostrando o ângulo de fase em função da frequência para um eletrodo interdigitado sem estrutura de imidazolato zeolítico-8, um eletrodo interdigitado com estrutura de imidazolato zeolítico-8 e para diversas concentrações de domínio citoplasmático ADAM17 sobre o eletrodo interdigitado, de acordo com uma modalidade da presente descrição. [0034] Figure 12 is a graph of electrochemical impedance spectroscopy results, showing the phase angle as a function of frequency for an interdigitated electrode without zeolitic-8 imidazolate structure, an interdigitated electrode with zeolitic-8 imidazolate structure and for various concentrations of ADAM17 cytoplasmic domain over the interdigitated electrode, in accordance with an embodiment of the present disclosure.
[0035] A figura 13 é um diagrama de Bode de resultados de uma espectroscopia de impedância eletroquímica, mostrando a impedância em função da frequência para um eletrodo interdigitado sem estrutura de imidazolato zeolítico-8, um eletrodo interdigitado com estrutura de imidazolato zeolítico-8 e para diversas concentrações de domínio citoplasmático ADAM17 sobre o eletrodo interdigitado, de acordo com uma modalidade da presente descrição. [0035] Figure 13 is a Bode diagram of electrochemical impedance spectroscopy results, showing impedance as a function of frequency for an interdigitated electrode without zeolitic-8 imidazolate structure, an interdigitated electrode with zeolitic-8 imidazolate structure and for various concentrations of ADAM17 cytoplasmic domain over the interdigitated electrode, in accordance with an embodiment of the present disclosure.
[0036] As figuras 14a-f são diagramas de Nyquist de uma espectroscopia de impedância eletroquímica, para um eletrodo interdigitado sem estrutura de imidazolato zeolítico-8, um eletrodo interdigitado com estrutura de imidazolato zeolítico-8 e para diversas concentrações de domínio citoplasmático ADAM17 sobre o eletrodo interdigitado, de acordo com uma modalidade da presente descrição. [0036] Figures 14a-f are Nyquist diagrams of an electrochemical impedance spectroscopy, for an interdigitated electrode without zeolitic-8 imidazolate structure, an interdigitated electrode with zeolitic-8 imidazolate structure and for different concentrations of ADAM17 cytoplasmic domain on the interdigitated electrode, in accordance with an embodiment of the present description.
[0037] A figura 15 é um gráfico mostrando as impedâncias do diagrama de Bode da figura 13, na frequência de 0,1 Hz, tomadas de um biossensor submetido a diversas concentrações de domínio citoplasmático ADAM17, de acordo com uma modalidade da presente descrição. [0038] A figura 16 é um gráfico de resultados de um ensaio de ligação em fase sólida comumente usado no estado da técnica para avaliação da interação proteína-proteína. [0037] Figure 15 is a graph showing the impedances of the Bode diagram of Figure 13, at a frequency of 0.1 Hz, taken from a biosensor subjected to various concentrations of ADAM17 cytoplasmic domain, in accordance with an embodiment of the present description. [0038] Figure 16 is a graph of results of a solid-phase binding assay commonly used in the prior art to assess protein-protein interaction.
DESCRIÇÃO DETALHADA DA INVENÇÃO DETAILED DESCRIPTION OF THE INVENTION
[0039] A presente descrição se refere a um biossensor eletroquímico sem marcação, baseado em estrutura de imidazolato zeolítico-8 (ZIF-8), para monitorar interações proteína-proteína (PPIs). O ZIF-8 é depositado em eletrodos interdigitados, empregados como material transdutor do sensor, modificando os eletrodos interdigitados. Esses eletrodos interdigitados modificados são expostos a proteína tiorredoxina-1 (Trx-1 ), sendo essa proteína imobilizada nos mesmos, seguida pela deposição de certas concentrações de domínio citoplasmático de desintegrina e metaloproteinase 17 (ADAM17cyto) conhecido como parceiro de ligação da Trx-1 , para detecção da PPL [0039] The present description refers to a non-labeled electrochemical biosensor, based on zeolitic imidazolate-8 (ZIF-8) structure, to monitor protein-protein interactions (PPIs). ZIF-8 is deposited on interdigitated electrodes, used as transducer material for the sensor, modifying the interdigitated electrodes. These modified interdigitated electrodes are exposed to the protein thioredoxin-1 (Trx-1 ), which is immobilized on them, followed by the deposition of certain concentrations of the cytoplasmic domain of disintegrin and metalloproteinase 17 (ADAM17cyto) known as the binding partner of Trx-1. , for detection of PPL
[0040] Caracterizações estruturais e morfológicas foram utilizadas para validar e verificar a formação do ZIF-8. Os cristais ZIF-8 apresentam uma estrutura rômbica dodecaédrica com facetas (011 ) preferencialmente expostas, um tamanho médio de partícula de 205 (± 22) nm e uma espessura de filme de ZIF-8 em tomo de 61 (± 6) nm. [0040] Structural and morphological characterizations were used to validate and verify the formation of ZIF-8. The ZIF-8 crystals have a rhombic dodecahedral structure with (011 ) preferentially exposed facets, an average particle size of 205 (± 22) nm and a film thickness of ZIF-8 around 61 (± 6) nm.
[0041] A interação entre as proteínas Trx-1 e ADAM17cyto foi analisada por espectroscopia de impedância eletroquímica (EIS). Os resultados indicam uma relação linear e inversa entre as respostas de impedância em 0,1 Hz para concentrações de ADAM17cyto de 50 nM a 8 pM, com um coeficiente de variação de 1 ,0 a 11 ,4%. [0041] The interaction between Trx-1 and ADAM17cyto proteins was analyzed by electrochemical impedance spectroscopy (EIS). The results indicate a linear and inverse relationship between impedance responses at 0.1 Hz for ADAM17cyto concentrations from 50 nM to 8 pM, with a coefficient of variation of 1.0 to 11.4%.
[0042] Como prova do conceito inventivo, os resultados foram comparados com um tipo de ensaio PPI amplamente usado no estado da técnica, baseado em anticorpos, que é o ensaio de ligação em fase sólida usando as mesmas proteínas. O ensaio de ligação em fase sólida foi capaz de detectar uma ligação significativa apenas em concentrações de ADAM17cyto acima de 0,5 pM, com um coeficiente de variação variando de 5,4 a 27,5%. Os resultados demonstram que o biossensor desenvolvido foi 10x mais sensível e reprodutível do que o ensaio de ligação de fase sólida convencional. Além disso, o biossensor eletroquímico desenvolvido baseado em ZIF-8 fornece uma análise de detecção mais rápida, sem marcação e de baixo custo, representando vantagens na detecção de PP Is frente ao estado da técnica. [0042] As an inventive proof of concept, the results were compared with a type of PPI assay widely used in the prior art, antibody-based, which is the solid phase binding assay using the same proteins. The solid phase binding assay was able to detect significant binding only at ADAM17cyto concentrations above 0.5 pM, with a coefficient of variation ranging from 5.4 to 27.5%. You Results demonstrate that the developed biosensor was 10x more sensitive and reproducible than the conventional solid phase binding assay. In addition, the electrochemical biosensor developed based on ZIF-8 provides a faster detection analysis, without labeling and at low cost, representing advantages in the detection of PP Is compared to the state of the art.
[0043] O biossensor desenvolvido fornece uma análise mais rápida, detecção sem marcação (label-free) e custo mais baixo que a técnica usada para comparação, qual seja, o ensaio de ligação em fase sólida (SPB). Enquanto o ensaio SPB requer dois dias para fornecer os resultados, a medição por EIS com o biossensor aqui revelado leva menos de duas horas. Essa plataforma de detecção confiável e adequada não requer anticorpos, levando a custos mais baixos por análise e evitando problemas associados a ensaios baseados em anticorpos. O biossensor eletroquímico baseado em ZIF-8 pode ser considerado uma plataforma adequada para o futuro de dispositivos point-of-care, tornando o EIS uma ferramenta promissora para a detecção confiável de PP Is. [0043] The developed biosensor provides faster analysis, label-free detection and lower cost than the technique used for comparison, namely the solid phase binding assay (SPB). While the SPB assay requires two days to provide results, the EIS measurement with the biosensor disclosed here takes less than two hours. This reliable and suitable detection platform does not require antibodies, leading to lower costs per analysis and avoiding problems associated with antibody-based assays. The ZIF-8-based electrochemical biosensor can be considered a suitable platform for the future of point-of-care devices, making EIS a promising tool for the reliable detection of PP Is.
[0044] A presente descrição também trata do processo de fabricação do referido biossensor, que tem algumas de suas etapas ilustradas nas representações esquemáticas das figuras 1 , 2 e 4. [0044] This description also deals with the manufacturing process of said biosensor, which has some of its steps illustrated in the schematic representations of figures 1, 2 and 4.
[0045] Na figura 1 são apresentadas etapas de fabricação de eletrodos interdigitados, comuns ao estado da técnica. Inicialmente, é depositada uma camada de fotoresiste (2) sobre um substrato de SiO2 (1 ). A camada fotoresiste (2) é padronizada no formato de eletrodos interdigitados por fotolitografia. São depositadas sobre o fotoresiste camadas de Cr/Au (3), sendo a camada de Au sobre a camada de CR, essa segunda fazendo a adesão da primeira no substrato. Cada uma das camadas (Cr/Au) apresenta de 15 a 25 nm de espessura. Finalmente, o conjunto é imerso em solução de acetona, para remoção do fotoresiste, formando o padrão dos eletrodos interdigitados (4). A superfície dos eletrodos interdigitados (4) é limpa com acetona e isopropanol por 10 a 15 min, seca com fluxo de N2, sendo o sensor armazenado em um dessecador a vácuo. [0045] In figure 1 are presented steps of fabrication of interdigitated electrodes, common to the state of the art. Initially, a photoresist layer (2) is deposited on a SiO 2 substrate (1 ). The photoresist layer (2) is patterned in the form of interdigitated electrodes by photolithography. Cr/Au layers (3) are deposited on the photoresist, the Au layer being on the CR layer, the latter making the adhesion of the first to the substrate. Each layer (Cr/Au) is 15 to 25 nm thick. Finally, the set is immersed in acetone solution to remove the photoresist, forming the pattern of interdigitated electrodes (4). The surface of the interdigitated electrodes (4) is cleaned with acetone and isopropanol for 10 to 15 min, dried with a flow of N 2 , and the sensor is stored in a vacuum desiccator.
[0046] Na figura 2 são apresentadas as etapas de crescimento da camada de ZIF-8 sobre os eletrodos interdigitados, resultando em eletrodos interdigitados modificados (5), sendo essa uma etapa essencial a realização da presente invenção. O sensor é imerso em solução piranha da 60 a 70 °C, por 15 a 25 min, e lavado com água destilada em seguida. Para deposição do filme de ZIF-8 sobre a área ativa dos eletrodos interdigitados (4), os mesmos são imersos em uma mistura de 8 a 12 ml_ de solução de Zn(NO3)2 a 25mM em metanol com 8 a 12 mL de solução de 2-metilimidazol a 50 mM em metanol, por 6 a 8 horas, sob agitação, em temperatura ambiente. [0046] Figure 2 shows the stages of growth of the ZIF-8 layer on the interdigitated electrodes, resulting in modified interdigitated electrodes (5), which is an essential step for the realization of the present invention. The sensor is immersed in piranha solution at 60 to 70 °C for 15 to 25 min and then washed with distilled water. For deposition of the ZIF-8 film on the active area of the interdigitated electrodes (4), they are immersed in a mixture of 8 to 12 ml_ of Zn(NO 3 ) 2 to 25 mM solution in methanol with 8 to 12 ml of 50 mM solution of 2-methylimidazole in methanol for 6 to 8 hours under stirring at room temperature.
[0047] Na figura 3 são ilustradas etapas de expressão e purificação de proteínas para obtenção das proteínas Trx-1 e ADAM17cyto utilizadas no sensor da presente descrição. [0047] In figure 3 are illustrated steps of expression and purification of proteins to obtain the proteins Trx-1 and ADAM17cyto used in the sensor of the present description.
[0048] A figura 4 apresenta um conjunto de etapas para imobilização de proteínas Trx-1 nos eletrodos interdigitados modificados por ZIF-8 (5) da presente descrição. As etapas incluem limpar a superfície dos eletrodos interdigitados modificados (5) com metanol e secar com fluxo de N2; expor a superfície dos eletrodos interdigitados modificados (5) a uma mistura contendo entre 0,8 e 1 ,2 pg de Trx-1 em 20 pL de solução PBS, por 25 a 35 min, resultando em eletrodos interdigitados com Trx-1 imobilizada (6); limpar a superfície dos eletrodos com solução PBS e secar com fluxo de N2. [0048] Figure 4 presents a set of steps for immobilization of Trx-1 proteins on the interdigitated electrodes modified by ZIF-8 (5) of the present description. The steps include cleaning the surface of the modified interdigitated electrodes (5) with methanol and drying with N 2 flow; exposing the surface of the modified interdigitated electrodes (5) to a mixture containing between 0.8 and 1.2 pg of Trx-1 in 20 pL of PBS solution, for 25 to 35 min, resulting in interdigitated electrodes with immobilized Trx-1 ( 6); clean the surface of the electrodes with PBS solution and dry with N 2 flow.
[0049] Os objetivos da presente invenção também são alcançados por um método de detecção de PPI conduzido por medições de espectroscopia de impedância eletroquímica. [0049] The objects of the present invention are also achieved by a PPI detection method driven by electrochemical impedance spectroscopy measurements.
[0050] A figura 5 é uma representação esquemática de um conjunto de etapas para detecção de interação proteína-proteína em uma modalidade do biossensor da presente descrição, enquanto a figura 6 ilustra um conjunto de etapas para detecção de interação proteína-proteína por um método do estado da técnica. [0050] Figure 5 is a schematic representation of a set of steps for detecting protein-protein interaction in a biosensor embodiment of the present description, while figure 6 illustrates a set of steps for detecting protein-protein interaction by a method of the state of the art.
[0051 ] O processo de detecção de interações proteína-proteína com o biossensor aqui revelado compreende: expor a superfície dos eletrodos interdigitados a uma mistura contendo entre 50 nM a 8 pM de domínio citoplasmático de proteína ADAM17 em 20 pL de solução PBS, por 25 a 35 min; limpar a superfície dos eletrodos interdigitados com solução PSB e secar com fluxo de N2; conduzir medições de espectroscopia de impedância eletroquímica utilizando meios de atuação e leitura elétrica ligados eletricamente aos eletrodos interdigitados. [0051] The process of detecting protein-protein interactions with the The biosensor disclosed herein comprises: exposing the surface of the interdigitated electrodes to a mixture containing between 50 nM to 8 pM of ADAM17 protein cytoplasmic domain in 20 pL of PBS solution, for 25 to 35 min; clean the surface of the interdigitated electrodes with PSB solution and dry with N2 flow; conduct electrochemical impedance spectroscopy measurements using actuation and electrical reading means electrically connected to the interdigitated electrodes.
EXEMPLOS DE CONCRETIZAÇÃO DAS MODALIDADES DESCRITAS EXAMPLES OF IMPLEMENTATION OF THE MODALITIES DESCRIBED
[0052] No que segue, algumas modalidades exemplares do objeto descrito, bem como modalidades do estado da técnica para fins de comparação, serão apresentadas aqui em concretizações não restritivas do escopo da invenção ora revelada. [0052] In what follows, some exemplary embodiments of the object described, as well as embodiments of the state of the art for comparison purposes, will be presented here in non-restrictive embodiments of the scope of the invention disclosed herein.
[0053] Todos os reagentes químicos utilizados nos exemplos foram obtidos de fornecedores comerciais. Foram adquiridos da Sigma-Aldrich (San Luis, MO, EUA): Nitrato de zinco hexa-hidratado (Zn(NO3)2 6H2O, a 98%), 2-metilimidazol (C4H6N2, 99%), caldo de lisogenia, cloreto de sódio (NaCI, 99%), cloreto de cálcio di-hidratado (CaCI2.2H2O, 99%), albumina de soro bovino (BSA, 98,5%), solução de peróxido de hidrogênio (H2O2, 30%), Tween-20 (C58H114O26), 2,2'-Azino-bis (ácido 3-etilbenzotiazolina-6-ácido sulfônico) sal de diamônio (ABTS, C18H24N6O6S4, 98%), Triton X-100 (f-Oct-C6H4-(OCH2CH2)xOH, x = 9-10), fluoreto de fenilmetilsulfonil (PMSF, 98%), ácido clorídrico (HCI, 37%) e cloreto de potássio (KCI, 99%). Foram adquiridos de Gibco (Waltham, MA, EUA): Sulfato de canamicina (C18H38N4O15S, 95-100%) e sal dissódico de ácido etilenodinitrilotetraacético di-hidratado (EDTA, 0,5M pH 8). O imidazol (C3H4N2, 99%) foi adquirido na Oakwood Chemical (West Columbia, SC, EUA). Ácido cítrico (C6H8O7.H2O, 99,5-100%), fosfato de sódio dibásico (Na2HPO4, 99%) e dihidrogenofosfato de potássio (KH2PO4, 99,5-100%) foram adquiridos da Merck (Burlington, MA, EUA). Hidróxido de sódio (NaOH, 97%) foi adquirido da Synth (Diadema, SP, Brasil). O isopropil-[3-d-tiogalactopiranosídeo (IPTG, 99%) foi adquirido na Promega (Madison, Wl, EUA). O ácido sulfúrico (H2SO4, 95-98%) foi adquirido a J. T. Baker (Phillipsburg, NJ, EUA). Tris (hidroximetil)-aminometano (Tris, NH2C(CH2OH)3, 99,8%) foi adquirido na Affymetrix (Santa Clara, CA, EUA). 0 anticorpo anti-ADAM17 (AB19027) foi adquirido da EMD Millipore (Burlington, MA, EUA), e o anticorpo secundário anti-coelho de cavalo ligado à peroxidase (DC03L) da Calbiochem (San Diego, CA, EUA). O coquetel inibidor de protease foi adquirido pela Roche (Basel, Suíça). [0053] All chemical reagents used in the examples were obtained from commercial suppliers. Purchased from Sigma-Aldrich (San Luis, MO, USA): Zinc nitrate hexahydrate (Zn(NO 3 ) 2 6H 2 O, 98%), 2-methylimidazole (C 4 H 6 N 2 , 99% ), lysogen broth, sodium chloride (NaCI, 99%), calcium chloride dihydrate (CaCl 2 .2H 2 O, 99%), bovine serum albumin (BSA, 98.5%), peroxide solution of hydrogen (H 2 O 2 , 30%), Tween-20 (C 58 H 114 O 26 ), 2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS, C 18 H 24 N 6 O 6 S 4 , 98%), Triton X-100 (f-Oct-C 6 H 4 -(OCH 2 CH 2 ) x OH, x = 9-10), phenylmethylsulfonyl fluoride (PMSF, 98%), hydrochloric acid (HCl, 37%) and potassium chloride (KCI, 99%). The following were purchased from Gibco (Waltham, MA, USA): Kanamycin sulfate (C 18 H 38 N 4 O 15 S, 95-100%) and disodium salt of ethylenedinitrilotetraacetic acid dihydrate (EDTA, 0.5M pH 8). Imidazole (C 3 H 4 N 2 , 99%) was purchased from Oakwood Chemical (West Columbia, SC, USA). Citric acid (C 6 H 8 O 7 .H 2 O, 99.5-100%), dibasic sodium phosphate (Na 2 HPO 4 , 99%) and potassium dihydrogen phosphate (KH 2 PO 4 , 99.5-100 %) were purchased from Merck (Burlington, MA, USA). Sodium hydroxide (NaOH, 97%) was purchased from Synth (Diadema, SP, Brazil). Isopropyl-[3-d-thiogalactopyranoside (IPTG, 99%) was purchased from Promega (Madison, Wl, USA). Sulfuric acid (H 2 SO 4 , 95-98%) was purchased from JT Baker (Phillipsburg, NJ, USA). Tris(hydroxymethyl)-aminomethane (Tris, NH 2 C(CH 2 OH) 3 , 99.8%) was purchased from Affymetrix (Santa Clara, CA, USA). Anti-ADAM17 antibody (AB19027) was purchased from EMD Millipore (Burlington, MA, USA), and peroxidase-linked anti-horse rabbit secondary antibody (DC03L) from Calbiochem (San Diego, CA, USA). The protease inhibitor cocktail was purchased from Roche (Basel, Switzerland).
[0054] Os eletrodos interdigitados utilizados nos exemplos foram fabricados em placas de silício condutor (100) com 2 pm de espessura de SiO2. Primeiro, um fotoresiste AZ5214E foi padronizado por fotolitografia convencional seguido pela deposição de Cr/Au (20/20 nm), ambas camadas metálicas usando evaporação de feixe de elétrons e depositadas a velocidade de 1A/S. A primeira camada metálica à base de Cr visa melhorar a adesão da segunda camada metálica à base de Au. Em seguida, foi realizado o processo de lift-off para remoção do fotoresiste, imergindo os eletrodos interdigitados em acetona. Os eletrodos interdigitados têm 60 matrizes de eletrodos confinados a uma área ativa de aproximadamente 15 mm2. A distância entre cada matriz de eletrodos é de 10 pm (comprimento do canal). A relação largura-comprimento total do canal (W/L) é 50.000. O tamanho de cada substrato com 60 eletrodos interdigitados integrados é 12 x 7 mm2 (ilustrado nas Figuras 7a, 7b e 7c). As superfícies da amostra foram limpas com acetona e isopropanol por 10 minutos cada, em banho ultrassónico, e secas com fluxo de N2. Antes da deposição do ZIF-8, cada eletrodo interdigitado foi testado para verificar uma possível corrente de fuga. [0054] The interdigitated electrodes used in the examples were manufactured in conductive silicon plates (100) with 2 pm thickness of SiO 2 . First, an AZ5214E photoresist was standardized by conventional photolithography followed by deposition of Cr/Au (20/20 nm), both metallic layers using electron beam evaporation and deposited at a rate of 1A/S. The first Cr-based metallic layer aims to improve the adhesion of the second Au-based metallic layer. Then, the lift-off process was performed to remove the photoresist, immersing the interdigitated electrodes in acetone. The interdigitated electrodes have 60 electrode arrays confined to an active area of approximately 15 mm 2 . The distance between each electrode array is 10 pm (channel length). The total channel width-to-length (W/L) ratio is 50,000. The size of each substrate with 60 integrated interdigitated electrodes is 12 x 7 mm 2 (illustrated in Figures 7a, 7b and 7c). The sample surfaces were cleaned with acetone and isopropanol for 10 minutes each, in an ultrasonic bath, and dried with a flow of N 2 . Prior to deposition of the ZIF-8, each interdigitated electrode was tested for possible leakage current.
[0055] Para preparação da superfície e posterior deposição do filme de ZIF-8, os eletrodos interdigitados foram imersos em uma solução piranha (H2SO4/H2O2, 7:3 (V/V)) a 65 °C por 20 minutos para aumentar a densidade de grupos funcionais hidroxila na superfície de SiO2. Depois de ter sua superfície lavada com água destilada, a deposição do filme ZIF-8 ocorre com a imersão dos eletrodos em uma mistura fresca de 10 ml_ de Zn(NO3)2 (25 mM em metanol) e 10 mL de 2-metilimidazol (2-Melm, 50 mM em metanol) durante 6 horas, sob agitação, à temperatura ambiente. Após a deposição, os filmes de ZIF-8 foram lavados com metanol, secos com fluxo de N2 e armazenados sob vácuo. [0055] For surface preparation and subsequent deposition of the ZIF-8 film, the interdigitated electrodes were immersed in a piranha solution (H 2 SO 4 /H 2 O 2 , 7:3 (V/V)) at 65 °C for 20 minutes to increase the density of hydroxyl functional groups on the SiO 2 surface. After having its surface washed with distilled water, the deposition of the ZIF-8 film occurs with the immersion of the electrodes in a fresh mixture of 10 ml_ of Zn(NO 3 ) 2 (25 mM in methanol) and 10 ml of 2-methylimidazole (2-Melm, 50 mM in methanol) for 6 hours under stirring at room temperature. After deposition, the ZIF-8 films were washed with methanol, dried with N 2 flow and stored under vacuum.
[0056] Os filmes ZIF-8 foram caracterizados morfologicamente por Microscopia Eletrônica de Varredura (FEG-SEM, Inspect F50 da FEI, Hillsboro, Oregon, EUA). A estrutura cristalina dos filmes de ZIF-8 foi determinada por radiação síncrotron de raios X na linha de luz XRD2 do Laboratório Brasileiro de Luz Síncrotron (LNLS/CNPEM, Campinas, Brasil), com comprimento de onda incidente (À) de 1 ,54979 A. Os difratogramas foram obtidos na faixa de 5o a 20° (20) usando um detector linear Mythen para aquisição de dados. Microscopia de Força Atômica (AFM, Park NX10, Santa Clara, CA, EUA) foi usada para analisar a rugosidade da superfície e espessura do filme dos eletrodos interdigitados modificados por ZIF-8 através do software Gwyddion. O espectro Raman foi adquirido usando o espectrômetro Raman Xplora Plus equipado com um microscópio óptico (Horiba, Quioto, Japão), operado com excitação a laser a 638 nm e lente 100x em condições atmosféricas. [0056] ZIF-8 films were morphologically characterized by Scanning Electron Microscopy (FEG-SEM, Inspect F50 from FEI, Hillsboro, Oregon, USA). The crystalline structure of the ZIF-8 films was determined by X-ray synchrotron radiation in the XRD2 beamline of the Brazilian Synchrotron Light Laboratory (LNLS/CNPEM, Campinas, Brazil), with incident wavelength (À) of 1 .54979 A. Diffractograms were obtained in the range of 5° to 20° (20°) using a Mythen linear detector for data acquisition. Atomic Force Microscopy (AFM, Park NX10, Santa Clara, CA, USA) was used to analyze the surface roughness and film thickness of the interdigitated electrodes modified by ZIF-8 using the Gwyddion software. The Raman spectrum was acquired using a Raman Xplora Plus spectrometer equipped with an optical microscope (Horiba, Kyoto, Japan), operated with laser excitation at 638 nm and a 100x lens under atmospheric conditions.
[0057] As proteínas utilizadas nos presentes exemplos foram obtidas por processos convencionais de expressão e purificação, descritos em Aragão e colaboradores (2012, J. Biol. Chem., 287 (51 ), 43071-43082). As proteínas ADAM17cyto foram expressas em células competentes BL21 (DE3), a 37 °C por 4 horas após a indução com IPTG 0,5 mM em caldo LB (Luria Bertani) contendo canamicina. Os pellets de 1 L das proteínas expressas foram separados por SDS-PAGE a 15% em condições desnaturantes para confirmar a indução da proteína. As células recuperadas foram ressuspensas em tampão A (Tris 50 mM pH 7,5, CaCI2 2 mM e NaCI 150 mM) suplementado com PMSF 1 mM e um comprimido de coquetel de inibidor de protease, como inibidores de protease. Após a homogeneização, as células foram lisadas por sonicação (Vibracell VCX 500; Sonics & Materials, Inc., Newtown, CT, EUA). As suspensões foram centrifugadas a 14.000 g, durante 30 min, a 4 °C. O sobrenadante foi carregado em colunas quelantes HisTrap (GE Healthcare, Chicago, IL, EUA) de 1 ml_ carregadas com níquel e previamente equilibradas com tampão A suplementado com imidazol 20 mM, a uma taxa de fluxo de 1 mL/min. As proteínas foram eluídas usando um gradiente linear de imidazol (0,02-1 M). A solução tampão de fosfato de sódio salino (PBS) foi preparada com NaCI (137 mM), KCI (2,7 mM), Na2HPO4 (10 mM) e KH2PO4 (1 ,8 mM), o pH foi ajustado para 7,2 com HCI para 1 L de uma solução aquosa . Todas as frações purificadas para cada proteína foram combinadas em um filtro Am icon de corte de 10 kDa (Millipore, Billerica, MA, EUA), e o tampão foi alterado para PBS pH 7,2. As frações purificadas foram separadas por SDS-PAGE 15% em condições desnaturantes para avaliar a qualidade da purificação. [0057] The proteins used in the present examples were obtained by conventional expression and purification procedures, described in Aragão et al. (2012, J. Biol. Chem., 287 (51), 43071-43082). ADAM17cyto proteins were expressed in competent BL21 (DE3) cells at 37 °C for 4 hours after induction with 0.5 mM IPTG in LB broth (Luria Bertani) containing kanamycin. 1 L pellets of expressed proteins were separated by 15% SDS-PAGE under denaturing conditions to confirm protein induction. Recovered cells were resuspended in buffer A (50 mM Tris pH 7.5, 2 mM CaCl 2 and 150 mM NaCl) supplemented with 1 mM PMSF and a protease inhibitor cocktail tablet as protease inhibitors. After homogenization, cells were lysed by sonication (Vibracell VCX 500; Sonics & Materials, Inc., Newtown, CT, USA). The suspensions were centrifuged at 14,000 g for 30 min at 4°C. O supernatant was loaded onto 1 ml HisTrap chelating columns (GE Healthcare, Chicago, IL, USA) loaded with nickel and pre-equilibrated with buffer A supplemented with 20 mM imidazole at a flow rate of 1 ml/min. Proteins were eluted using a linear gradient of imidazole (0.02-1 M). Sodium phosphate buffer saline (PBS) was prepared with NaCl (137 mM), KCI (2.7 mM), Na 2 HPO 4 (10 mM) and KH 2 PO 4 (1.8 mM), the pH was adjusted to 7.2 with HCl to 1 L of an aqueous solution. All purified fractions for each protein were combined on a 10 kDa cut-off Am icon filter (Millipore, Billerica, MA, USA), and the buffer changed to PBS pH 7.2. Purified fractions were separated by 15% SDS-PAGE under denaturing conditions to assess the quality of purification.
[0058] Procedeu-se com a segunda etapa de purificação por cromatografia de filtração em gel para obter o estado monomérico de cada proteína, eliminando as possíveis impurezas que persistiram após a cromatografia de afinidade e separando as espécies com estados oligoméricos distintos. Este procedimento foi realizado preparando a solução de proteína concentrada a um volume de 0,5 ml_ usando um filtro Amicon de corte de 10 kDa (Millipore, Billerica, MA, EUA). As suspensões de proteína (Trx-1 e ADAM17cyto) foram carregadas separadamente em colunas analíticas de gel de filtração Superdex 75 10/300 GL (GE Healthcare, Chicago, IL, EUA) usando uma taxa de fluxo de 0,2 mL/min de PBS com pH 7,2. As proteínas foram eluídas usando 1 ,2 vezes os volumes de coluna de PBS resfriado e, em seguida, coletadas em frações de 0,5 mL. Essas frações foram então separadas por SDS-PAGE 15% em condições de desnaturação, seguido por coloração com azul de Coomassie, para avaliar a qualidade da purificação. Finalmente, as frações puras foram reunidas e concentradas até um volume de 1 mL usando um filtro Amicon de corte de 10 kDa (Millipore, Billerica, MA, EUA). As concentrações finais de proteína obtidas para Trx-1 e ADAM17cyto foram 11 ,0 pg/pl e 7,1 pg/pl, respectivamente. A concentração de proteínas foi determinada pelo espectrofotômetro NanoDrop 2000/2000c (Thermo Fisher Scientific, Waltham, MA, EUA) a 280 nm. [0058] The second step of purification was carried out by gel filtration chromatography to obtain the monomeric state of each protein, eliminating the possible impurities that persisted after the affinity chromatography and separating the species with distinct oligomeric states. This procedure was performed by preparing the concentrated protein solution to a volume of 0.5 ml using a 10 kDa cut-off Amicon filter (Millipore, Billerica, MA, USA). Protein suspensions (Trx-1 and ADAM17cyto) were loaded separately onto Superdex 75 10/300 GL analytical gel filtration columns (GE Healthcare, Chicago, IL, USA) using a flow rate of 0.2 mL/min of PBS with pH 7.2. Proteins were eluted using 1.2 times column volumes of cooled PBS and then collected in 0.5 mL fractions. These fractions were then separated by 15% SDS-PAGE under denaturing conditions, followed by Coomassie blue staining to assess the quality of purification. Finally, the pure fractions were pooled and concentrated to a volume of 1 mL using a 10 kDa cut-off Amicon filter (Millipore, Billerica, MA, USA). The final protein concentrations obtained for Trx-1 and ADAM17cyto were 11.0 pg/pl and 7.1 pg/pl, respectively. Protein concentration was determined using the NanoDrop 2000/2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) at 280 nm.
[0059] As medições de espectroscopia de impedância eletroquímica foram realizadas com o analisador de impedância Solartron modelo SI 1260 A (Solartron Analytical, Farnborough, Hampshire, Reino Unido), na faixa de frequência de 1 MHz a 10 mHz (10 pontos por década, amplitude de oscilação de 100 mV e deslocamento de voltagem 0 V). Os eletrodos interdigitados modificados foram primeiro expostos a Trx-1 (1 pg em 20 pL de solução de PBS) por 30 minutos. Posteriormente, a superfície dos eletrodos foi lavada com solução de PBS e seca com fluxo de N2. Em seguida, 20 pL de soluções com domínio citoplasmático ADAM17 (de 50 nM a 8 pM em solução de PBS) foram deixados nos eletrodos funcionalizados por 30 minutos, seguido de uma etapa de lavagem em solução de PBS. Finalmente, a superfície foi seca com fluxo de N2. [0059] Electrochemical impedance spectroscopy measurements were performed with the Solartron model SI 1260 A impedance analyzer (Solartron Analytical, Farnborough, Hampshire, UK), in the frequency range from 1 MHz to 10 mHz (10 points per decade, oscillation amplitude 100 mV and voltage offset 0 V). The modified interdigitated electrodes were first exposed to Trx-1 (1 pg in 20 pL of PBS solution) for 30 minutes. Subsequently, the surface of the electrodes was washed with PBS solution and dried with N 2 flow. Then, 20 pL of solutions with ADAM17 cytoplasmic domain (from 50 nM to 8 pM in PBS solution) were left on the functionalized electrodes for 30 minutes, followed by a washing step in PBS solution. Finally, the surface was dried with N 2 flux.
[0060] A figura 7a é uma representação esquemática de uma modalidade do biossensor da presente descrição. As figuras 7b e 7c são fotografias da modalidade exemplar do biossensor, conforme processo ora descrito. [0060] Figure 7a is a schematic representation of a biosensor embodiment of the present description. Figures 7b and 7c are photographs of the exemplary modality of the biosensor, according to the process described herein.
[0061 ] A figura 8 é um gráfico obtido por ensaio de difração de raios-X com incidência rasante de uma estrutura de imidazolato zeolítico-8 sintetizada de acordo com uma modalidade exemplar do processo de fabricação, sendo comparado com o difratograma de estrutura de imidazolato zeolítico-8 obtido por simulação computacional. O filme ZIF-8 atua como uma camada ativa onde as soluções contendo as proteínas Trx-1 e ADAM17cyto são depositadas. A presença de picos de alta intensidade (011 ), (002), (112), (022), (013) e (222) indica a formação de ZIF-8. Uma orientação de crescimento preferencial do filme ZIF-8 é observada em 7,4° (ângulo 29), correspondendo ao plano (011 ). O crescimento do filme ZIF-8 ocorre a partir da nucleação dos cristais de ZIF-8, que é melhorado pela presença de metanol (um solvente prático) e uma concentração maior de 2-metilimidazol do que Zn(NO3)2. O excesso de ligante orgânico é crucial para melhorar o crescimento da nucleação e estabilizar os colóides iniciais. O metanol é responsável por dissolver os precursores e dissociar o H+ do 2-metilimidazol. Subsequentemente, os cátions Zn2+ se combinam com o 2-metilimidazol desprotonado para formar unidades individuais de ZIF-8, que então servem como bloco de construção para a formação de cristais de ZIF-8. [0061] Figure 8 is a graph obtained by X-ray diffraction test with grazing incidence of a zeolitic-8 imidazolate structure synthesized according to an exemplary modality of the manufacturing process, being compared with the imidazolate structure diffractogram zeolitic-8 obtained by computer simulation. The ZIF-8 film acts as an active layer where solutions containing Trx-1 and ADAM17cyto proteins are deposited. The presence of high intensity peaks (011), (002), (112), (022), (013) and (222) indicates the formation of ZIF-8. A preferred growth orientation of the ZIF-8 film is observed at 7.4° (angle 29), corresponding to the (011°) plane. ZIF-8 film growth occurs from the nucleation of ZIF-8 crystals, which is enhanced by the presence of methanol (a practical solvent) and a higher concentration of 2-methylimidazole than Zn(NO 3 ) 2 . Excess organic ligand is crucial to improve nucleation growth and stabilize initial colloids. Methanol is responsible for dissolving the precursors and dissociate the H + from the 2-methylimidazole. Subsequently, the Zn 2+ cations combine with the deprotonated 2-methylimidazole to form individual units of ZIF-8, which then serve as the building block for the formation of ZIF-8 crystals.
[0062] As figuras 9a-9c são uma sequência de imagens de microscopia, acompanhadas de figuras esquemáticas, apresentando a evolução da estrutura cristalográfica do imidazolato zeolítico-8 durante as etapas de crescimento de acordo com uma modalidade do processo de fabricação da presente descrição. Inicialmente, depois que as soluções precursoras foram misturadas, o ZIF-8 começa a cristalizar em uma forma cúbica (Figura 9a), mudando para uma estrutura rômbica dodecaédrica truncada (TRD) em sequência (Figura 9b), e gradualmente assumindo uma estrutura cristalina rômbica dodecaédrica (RD) no final (Figura 9c). O esquema de evolução morfológica dos cristais de ZIF-8 é apresentado nessas figuras, começando com um cristal cúbico orientado para [100], que evolui para TRD e assume a forma final de RD. De acordo com a regra de Wulff, a morfologia de cristal mais estável ocorre pela faceta de crescimento mais lento. Para o ZIF-8, a direção de crescimento [011] torna a estrutura de cristal RD a morfologia de equilíbrio mais estável. [0062] Figures 9a-9c are a sequence of microscopy images, accompanied by schematic figures, showing the evolution of the crystallographic structure of the zeolitic-8 imidazolate during the growth stages according to one embodiment of the manufacturing process of the present description. Initially, after the precursor solutions have been mixed, ZIF-8 begins to crystallize in a cubic form (Figure 9a), changing to a truncated dodecahedral rhombic (TRD) structure in sequence (Figure 9b), and gradually assuming a rhombic crystal structure. dodecahedral (RD) at the end (Figure 9c). The morphological evolution scheme of the ZIF-8 crystals is presented in these figures, starting with a cubic crystal oriented to [100], which evolves into TRD and assumes the final form of RD. According to Wulff's rule, the most stable crystal morphology occurs by the slowest growing facet. For ZIF-8, the growth direction [011] makes the RD crystal structure the most stable equilibrium morphology.
[0063] A figura 10 é um gráfico de três espectroscopias Raman, sendo, respectivamente, para o um eletrodo interdigitado com deposição de estruturas de imidazolato zeolítico-8 de uma modalidade do biossensor da presente descrição (linha inferior), para o mesmo eletrodo com imobiliização de Tioredoxina-1 (linha central), e para mesmo eletrodo com Tioredoxina-1 e domínio citoplasmático ADAM17 (linha superior). Um pico intenso foi observado para todas as amostras analisadas em 519 cm-1, sendo atribuído ao SiO2 do substrato. Para ZIF-8, picos característicos foram observados em: 683 cm-1 (contração do anel imidazólico), 832 cm-1 (C-H dobrando fora do plano), 953 cm-1 (C-H dobrando fora do plano), 1026 cm-1 (C-H dobrando fora do plano), 1143 cm-1 (C-N alongando), 1183 cm-1 ( C-N alongando + balanço N-H), 1384 cm-1 (flexão de metil), 1460 cm-1 (flexão C-H), 1510 cm-1 (alongamento C=C), 2929 cm-1 (C-H, alongamento de metil) e 3128 cm-1 (alongamento de CH do anel imidazólico). [0063] Figure 10 is a graph of three Raman spectroscopies, respectively, for the one interdigitated electrode with deposition of zeolitic-8 imidazolate structures of a biosensor modality of the present description (bottom line), for the same electrode with immobilization of Thioredoxin-1 (center line), and to the same electrode with Thioredoxin-1 and ADAM17 cytoplasmic domain (top line). An intense peak was observed for all samples analyzed at 519 cm -1 , being attributed to the SiO 2 of the substrate. For ZIF-8, characteristic peaks were observed at: 683 cm -1 (imidazole ring contraction), 832 cm -1 (CH bending out of plane), 953 cm -1 (CH bending out of plane), 1026 cm -1 (CH bending out of plane), 1143 cm -1 (CN stretching), 1183 cm -1 (CN stretching + NH balance), 1384 cm -1 (methyl flexion), 1460 cm -1 (CH flexion), 1510 cm -1 (C=C stretching), 2929 cm -1 (CH, methyl elongation) and 3128 cm -1 (CH elongation of the imidazole ring).
[0064] Observa-se que o sinal Raman para o ZIF-8 é suprimido quando a Trx-1 é imobilizada em baixas concentrações. A presença de tiol (modo de alongamento SH) de Trx-1 foi observada em 2545 cm-1. Outras vibrações envolvendo enxofre foram observadas, incluindo um alongamento C-S centrado em 657 cm-1, e um alongamento S-S em 440 cm-1. A banda de vibração em 1003 cm-1 foi atribuída ao resíduo de aminoácido fenilalanina (Phe). Além disso, é possível resolver em 1103 cm-1 (alongamento C-C), 1248 cm-1 (amida III), 1327 cm-1 e 1449 cm-1 (dobramento C-H), 1580 cm-1 (amida II), 1665 cm-1 (amida I), 2878 cm-1 e 2930 cm-1 (alongamento C-H alifático). [0064] It is observed that the Raman signal for ZIF-8 is suppressed when Trx-1 is immobilized at low concentrations. The presence of thiol (SH elongation mode) of Trx-1 was observed at 2545 cm -1 . Other vibrations involving sulfur were observed, including a CS elongation centered at 657 cm -1 , and an SS elongation at 440 cm -1 . The vibration band at 1003 cm -1 was assigned to the amino acid residue phenylalanine (Phe). Furthermore, it is possible to resolve at 1103 cm -1 (CC elongation), 1248 cm -1 (amide III), 1327 cm -1 and 1449 cm -1 (CH bending), 1580 cm -1 (amide II), 1665 cm -1 (amide I), 2878 cm -1 and 2930 cm -1 (aliphatic CH elongation).
[0065] Todos os picos relacionados a ZIF-8 desaparecem após a imobilização de Trx-1 e ADAM17cyto, confirmando a interação entre a estrutura e as proteínas. As bandas Raman observadas no espectro com ADAM17cyto em tomo de 855 cm-1, 883 cm-1 e 1004 cm-1 foram atribuídas a resíduos de aminoácidos de tirosina (Tyr), triptofano (Trp) e fenilalanina (Phe), respectivamente. Um pico adicional em 1612 cm-1 também pode ser atribuído à contribuição desses três resíduos de aminoácidos. Outros modos de vibração foram observados em 1091 cm-1 (alongamento simétrico C-N), 1175 cm-1 (curvatura C-H no plano) , 1275 cm-1 (amida III), 1449 cm-1 (curvatura C-H), 1714 cm-1 (alongamento C=O), 2930 cm-1 (alongamento C-H alifático) e 3070 cm-1 (alongamento C-H aromático). Picos de alta intensidade em 440 cm-1 e 611 cm-1 para ADAM17cyto indicam a presença de alongamento S-S e C-S, respectivamente. O modo de alongamento S-S pode ter duas contribuições, a primeira da ligação dissulfeto formada no heterodímero entre Trx-1 e ADAM17cyto, e outra relacionada à ligação dissulfeto presente na estrutura ADAM17cyto. Embora a formação da ligação dissulfeto seja um processo lento em pH fisiológico (7,4), muitas proteínas como ADAM17cyto e Trx-1 podem interagir por meio da dimerização da cisteína. [0065] All peaks related to ZIF-8 disappear after immobilization of Trx-1 and ADAM17cyto, confirming the interaction between structure and proteins. The Raman bands observed in the ADAM17cyto spectrum around 855 cm -1 , 883 cm -1 and 1004 cm -1 were assigned to amino acid residues of tyrosine (Tyr), tryptophan (Trp) and phenylalanine (Phe), respectively. An additional peak at 1612 cm -1 can also be attributed to the contribution of these three amino acid residues. Other vibration modes were observed at 1091 cm -1 (CN symmetrical elongation), 1175 cm -1 (CH curvature in the plane), 1275 cm -1 (amide III), 1449 cm -1 (CH curvature), 1714 cm -1 (C=O elongation), 2930 cm -1 (aliphatic CH elongation) and 3070 cm -1 (aromatic CH elongation). High intensity peaks at 440 cm -1 and 611 cm -1 for ADAM17cyto indicate the presence of SS and CS elongation, respectively. The SS elongation mode can have two contributions, the first from the disulfide bond formed in the heterodimer between Trx-1 and ADAM17cyto, and the other related to the disulfide bond present in the ADAM17cyto structure. Although disulfide bond formation is a slow process at physiological pH (7.4), many proteins such as ADAM17cyto and Trx-1 can interact through cysteine dimerization.
[0066] As figuras 11 a-11 c são representações esquemáticas do mecanismo de interação entre Tioredoxina-1 e o domínio citoplasmático ADAM17, em uma primeira, segunda e terceira etapa, respectivamente. A formação da ligação dissulfeto ocorre espontaneamente in vitro e envolve a desprotonação de dois tióis de cisteínas (-SH) para formar tiolatos carregados (R-S-), como visto na Figura 11 a. Os íons tiolatos reagem com um receptor de elétrons, como o oxigênio molecular, como visto na Figura 11 b, para gerar as espécies radicais reativas (R-S’) que reagem e formam a ligação dissulfeto, como visto na Figura 11c. [0066] Figures 11 a-11 c are schematic representations of the mechanism of interaction between Thioredoxin-1 and the ADAM17 cytoplasmic domain, in a first, second and third step, respectively. The formation of the disulfide bond occurs spontaneously in vitro and involves the deprotonation of two cysteine thiols (-SH) to form charged thiolates (RS-), as seen in Figure 11 a. The thiolate ions react with an electron acceptor such as molecular oxygen, as seen in Figure 11b, to generate the reactive radical species (R-S') that react and form the disulfide bond, as seen in Figure 11c.
[0067] A figura 12 é um gráfico de resultados da espectroscopia de impedância eletroquímica, mostrando o ângulo de fase em função da frequência para um eletrodo interdigitado sem estrutura de imidazolato zeolítico-8, um eletrodo interdigitado com estrutura de imidazolato zeolítico-8 e para concentrações de domínio citoplasmático ADAM17 sobre o eletrodo interdigitado variando entre 50 nM 8 pM, de acordo com uma modalidade da presente descrição. Conforme mostrado na figura, um comportamento totalmente capacitivo (9 = -90°) muda ligeiramente em frequências mais baixas após a deposição de ZIF-8. Em concentrações de 50 nM a 4 pM de ADAM17cyto, ocorre um transporte predominante de íons em frequências mais baixas. Esse evento pode estar relacionado à formação dos tiolatos a partir das cisteínas. À medida que a concentração de ADAM17cyto aumenta para de 4 pM para 8 pM, a quantidade de proteína ADAM17cyto interagindo com Trx-1 também aumenta - um efeito direto da transferência de elétrons mais alta para formar a ligação dissulfeto observada em frequências mais altas. Portanto, a resposta de transferência de carga muda para frequências mais baixas à medida que a concentração de ADAM17cyto diminui (aumentando o comportamento capacitivo). [0067] Figure 12 is a graph of electrochemical impedance spectroscopy results, showing the phase angle as a function of frequency for an interdigitated electrode without zeolitic-8 imidazolate structure, an interdigitated electrode with zeolitic-8 imidazolate structure and for ADAM17 cytoplasmic domain concentrations on the interdigitated electrode ranging from 50 nM to 8 pM, according to an embodiment of the present disclosure. As shown in the figure, a fully capacitive behavior (9 = -90°) changes slightly at lower frequencies after deposition of ZIF-8. At concentrations of 50 nM to 4 pM of ADAM17cyto, a predominant transport of ions occurs at lower frequencies. This event may be related to the formation of thiolates from cysteines. As the concentration of ADAM17cyto increases from 4 pM to 8 pM, the amount of ADAM17cyto protein interacting with Trx-1 also increases - a direct effect of the higher electron transfer to form the disulfide bond observed at higher frequencies. Therefore, the charge transfer response shifts to lower frequencies as the concentration of ADAM17cyto decreases (increasing capacitive behavior).
[0068] A figura 13 é um diagrama de Bode correspondente aos resultados de ensaios da figura 12. O eletrodo interdigitado não-modificado tem uma alta impedância (|Z|> 1011 Q a 10 mHz) que diminui ligeiramente após a modificação por deposição de ZIF-8. Ao adicionar a Trx-1 , seguido de diferentes concentrações de ADAM17cyto, uma mudança significativa na impedância é observada em baixas frequências (de 10 mHz a 10 Hz). Em 0,1 Hz, o valor da impedância |Z| diminuiu de 8,5x109 O para 4,8x106 O com um aumento na concentração de ADAM17cyto (ver Tabela 1 ). Nesta região de baixa frequência, onde ocorre a maioria dos efeitos iônicos, a interação das proteínas na superfície do biossensor levou a um aumento na condutividade. Embora as proteínas estivessem em solução de PBS antes da secagem com N2 para medições de espectroscopia de impedância, a presença de íons remanescentes não interferiu na detecção de ADAM17cyto. Na faixa de altas frequências, de 103 Hz a 106 Hz, uma relação linear pode ser observada entre a impedância |Z| e frequência. Esta região de frequência corresponde principalmente às contribuições eletrônicas das diferentes interfaces envolvidas. [0068] Figure 13 is a Bode diagram corresponding to the test results of Figure 12. The unmodified interdigitated electrode has a high impedance (|Z|> 10 11 Q at 10 mHz) that decreases slightly after deposition modification of ZIF-8. By adding the Trx-1 , followed by different At ADAM17cyto concentrations, a significant change in impedance is observed at low frequencies (from 10 mHz to 10 Hz). At 0.1 Hz, the impedance value |Z| decreased from 8.5x10 9 O to 4.8x10 6 O with an increase in ADAM17cyto concentration (see Table 1). In this low frequency region, where most ionic effects occur, the interaction of proteins on the biosensor surface led to an increase in conductivity. Although the proteins were in PBS solution before drying with N 2 for impedance spectroscopy measurements, the presence of remaining ions did not interfere with the detection of ADAM17cyto. In the high frequency range, from 10 3 Hz to 10 6 Hz, a linear relationship can be observed between the impedance |Z| and frequency. This frequency region mainly corresponds to the electronic contributions of the different interfaces involved.
Tabela 1 - Impedância a 0,1 Hz e frequências de pico analisadas nos eletrodos interdigitados (4), eletrodos interdigitados modificados com ZIF-8 (5) e eletrodos interdigitados com Trx-1 imobilizada (6) e diferentes concentrações de ADAM17cyto.
Figure imgf000021_0001
Figure imgf000022_0001
Table 1 - Impedance at 0.1 Hz and peak frequencies analyzed in interdigitated electrodes (4), interdigitated electrodes modified with ZIF-8 (5) and interdigitated electrodes with immobilized Trx-1 (6) and different concentrations of ADAM17cyto.
Figure imgf000021_0001
Figure imgf000022_0001
[0069] As figuras 14a-f são diagramas de Nyquist de uma espectroscopia de impedância eletroquímica, para um eletrodo interdigitado sem estrutura de imidazolato zeolitico-8, um eletrodo interdigitado com estrutura de imidazolato zeolitico-8 e para diversas concentrações de domínio citoplasmático ADAM17 sobre o eletrodo interdigitado, de acordo com uma modalidade da presente descrição. Os diagramas de Nyquist para a resposta do biossensor, sob diferentes concentrações de ADAM17cyto, mostram um semicírculo na faixa de alta frequência, seguido por uma cauda linear nas frequências mais baixas. O semicírculo está associado à resistência à transferência de carga, enquanto a cauda reta está associada à difusão iônica. Além disso, no diagrama de Nyquist, é possível observar que o diâmetro do semicírculo diminui com o aumento da concentração de ADAM17cyto, indicando menor resistência à transferência de carga. Para ADAM17cyto 8 pM, um pequeno semicírculo é observado, indicando uma cinética de transferência de carga aprimorada. Ao diminuir a concentração de ADAM17cyto até 50 nM, há um aumento na resistência à transferência de carga causada pela baixa concentração de proteína. No gráfico de Nyquist, o máximo do semicírculo é dado por CÜPT = 1 , onde cüp = 2irfp é a frequência de pico. Ao analisar os valores de frequências de pico (Tabela 1 ), o semicírculo muda para frequências mais altas à medida que a concentração de ADAM17cyto aumenta. [0069] Figures 14a-f are Nyquist diagrams of an electrochemical impedance spectroscopy, for an interdigitated electrode without zeolitic-8 imidazolate structure, an interdigitated electrode with zeolitic-8 imidazolate structure and for different concentrations of ADAM17 cytoplasmic domain on the interdigitated electrode, in accordance with an embodiment of the present description. Nyquist diagrams for the biosensor response, under different concentrations of ADAM17cyto, show a semicircle in the high frequency range, followed by a linear tail in the lower frequencies. The semicircle is associated with charge transfer resistance, while the straight tail is associated with ionic diffusion. Furthermore, in the Nyquist diagram, it is possible to observe that the diameter of the semicircle decreases with increasing ADAM17cyto concentration, indicating less resistance to charge transfer. For ADAM17cyto 8 pM, a small semicircle is observed, indicating enhanced charge transfer kinetics. By decreasing the concentration of ADAM17cyto down to 50 nM, there is an increase in the resistance to charge transfer caused by the low protein concentration. In the Nyquist plot, the maximum of the semicircle is given by CÜ P T = 1 , where cü p = 2irf p is the peak frequency. When analyzing peak frequency values (Table 1), the semicircle shifts to higher frequencies as the ADAM17cyto concentration increases.
[0070] A figura 15 é um gráfico mostrando as impedâncias do diagrama de Bode da figura 12, na frequência de 0,1 Hz, tomadas de um biossensor submetido a diversas concentrações de domínio citoplasmático ADAM17, de acordo com uma modalidade da presente descrição. Conforme mostrado na figura, a impedância |Z| diminui gradualmente com o aumento da concentração de ADAM17cyto em 0,1 Hz (a linha pontilhada na Figura 12) entre 50 nM e 8 pM. Uma relação linear (coeficiente de correlação de 0,9902) é encontrada entre Z e a concentração de ADAM17cyto para as modalidades do biossensor analisadas. [0070] Figure 15 is a graph showing the impedances of the Bode diagram of Figure 12, at a frequency of 0.1 Hz, taken from a biosensor subjected to various concentrations of ADAM17 cytoplasmic domain, in accordance with an embodiment of the present description. As shown in the figure, the impedance |Z| gradually decreases with increasing concentration of ADAM17cyto at 0.1 Hz (the dotted line in Figure 12) between 50 nM and 8 pM. A linear relationship (correlation coefficient of 0.9902) is found between Z and ADAM17cyto concentration for the analyzed biosensor modalities.
[0071] A figura 16 é um gráfico de resultados de um ensaio de ligação em fase sólida (SPB) comumente usado no estado da técnica para avaliação da interação proteína-proteína. A interação direta de Trx-1 com ADAM17cyto avaliada pelo ensaio SPB demonstrou que a detecção é menos sensível e menos reprodutível em comparação com a obtida pelo ZIF-8 baseado em biossensor. [0071] Figure 16 is a graph of results of a solid phase binding assay (SPB) commonly used in the state of the art to evaluate protein-protein interaction. The direct interaction of Trx-1 with ADAM17cyto evaluated by the SPB assay demonstrated that the detection is less sensitive and less reproducible compared to that obtained by the biosensor-based ZIF-8.
[0072] Em resumo, os resultados obtidos comparados com um ensaio de ligação em fase sólida, amplamente utilizados na técnica atual, demonstram um melhor desempenho do biossensor baseado em ZIF-8 para a detecção de PPIs aqui revelado, especialmente quando a sensibilidade e reprodutibilidade são avaliadas. O limite de detecção do biossensor foi determinado tão baixo quanto 50 nM, com uma variação de 1 ,0 a 11 ,4% - para o ensaio de ligação em fase sólida, o limite de detecção mais baixo foi de 0,5 pM, com uma variação de 5,4 a 27,5%. [0072] In summary, the results obtained compared to a solid phase binding assay, widely used in the current technique, demonstrate a better performance of the ZIF-8 based biosensor for the detection of PPIs disclosed here, especially when the sensitivity and reproducibility are evaluated. The biosensor detection limit was determined to be as low as 50 nM, with a range of 1.0 to 11.4% - for the solid phase binding assay, the lower detection limit was 0.5 pM, with a variation of 5.4 to 27.5%.
[0073] Além disso, o conceito inventivo aqui apresentado de detecção de PPIs em biossensor sem marcadores, baseados em ZIF-8 também pode ser estendido para o reconhecimento de outras biomoléculas relacionadas, mostrando grande potencial para uso futuro como dispositivos point-of-care para diagnóstico ou prognóstico preciso de doenças clínicas. [0073] Furthermore, the inventive concept presented here of detecting PPIs in markerless biosensors, based on ZIF-8, can also be extended to the recognition of other related biomolecules, showing great potential for future use as point-of-care devices. for accurate diagnosis or prognosis of clinical diseases.
[0074] Logo, embora modalidades exemplares dos processos e dispositivos descritos tenham sido apresentadas neste relatório, não se pretende que o escopo de proteção seja limitado à literalidade das mesmas. Portanto, a descrição deve ser interpretada não como limitativa, mas meramente como exemplificações de modalidades particulares que guardam o conceito inventivo aqui apresentado. Um técnico poderá prontamente aplicar ensinamentos aqui apresentados em soluções análogas, decorrentes dos mesmos, limitadas apenas pelo escopo das reivindicações deste pedido.[0074] Therefore, although exemplary modalities of the processes and provisions described have been presented in this report, the scope of protection is not intended to be limited to their literalness. Therefore, the description should be interpreted not as limiting, but merely as exemplifications of particular modalities that keep the inventive concept presented here. A technician can readily apply the teachings presented here in analogous solutions, arising from the themselves, limited only by the scope of the claims in this application.
LISTA DE NÚMEROS DE REFERÊNCIA DAS FIGURAS LIST OF REFERENCE NUMBERS OF FIGURES
1 - substrato 1 - substrate
2 - camada de fotoresiste 2 - photoresist layer
3 - camada de Cr/Au 3 - Cr/Au layer
4 - eletrodos interdigitados 4 - interdigitated electrodes
5 - eletrodos interdigitados modificados com ZIF-8; 5 - interdigitated electrodes modified with ZIF-8;
6 - eletrodos interdigitados com Trx-1 imobilizada; 6 - interdigitated electrodes with immobilized Trx-1;

Claims

23 REIVINDICAÇÕES 23 CLAIMS
1. Biossensor sem marcação baseado em estrutura de imidazolato zeolítico, caracterizado por compreender: um substrato de SiO2 (1); um par de eletrodos interdigitados (4) depositados sobre o substrato (1 ), os eletrodos interdigitados (4) constituídos de uma camada inferior de 15 a 25 nm de Cr e uma camada superior de 15 a 25 nm de Au, e os eletrodos interdigitados possuindo uma área ativa de 10 a 20 mm2 com pelo menos 60 pares de interdígitos; um filme de estrutura de imidazolato zeolítico-8 depositado sobre a área ativa dos eletrodos interdigitados (4), resultando em eletrodos interdigitados modificados com ZIF-8 (5); proteína Tioredoxina-1 imobilizada sobre os eletrodos interdigitados modificados com ZIF-8 (5), resultando em eletrodos interdigitados com Trx-1 imobilizada (6); e meios de realizar análise eletroquímica conectado aos eletrodos interdigitados com Trx-1 imobilizada (6). 1. Unlabeled biosensor based on a zeolitic imidazolate structure, characterized in that it comprises: a SiO 2 substrate (1); a pair of interdigitated electrodes (4) deposited on the substrate (1), the interdigitated electrodes (4) consisting of a lower layer of 15 to 25 nm of Cr and an upper layer of 15 to 25 nm of Au, and the interdigitated electrodes having an active area of 10 to 20 mm 2 with at least 60 pairs of interdigits; a film of zeolitic-8 imidazolate structure deposited over the active area of the interdigitated electrodes (4), resulting in interdigitated electrodes modified with ZIF-8 (5); Thioredoxin-1 protein immobilized on interdigitated electrodes modified with ZIF-8 (5), resulting in interdigitated electrodes with immobilized Trx-1 (6); and means of performing electrochemical analysis connected to interdigitated electrodes with immobilized Trx-1 (6).
2. Biossensor, de acordo com a reivindicação 1 , caracterizado pelo fato de que os eletrodos interdigitados são configurados para receber um domínio citoplasmático de proteína ADAM 17. 2. Biosensor, according to claim 1, characterized in that the interdigitated electrodes are configured to receive a cytoplasmic domain of ADAM 17 protein.
3. Biossensor, de acordo com a reivindicação 2, caracterizado pelo fato de que os meios de atuação e leitura elétrica são configurados para conduzir medições de espectroscopia de impedância eletroquímica. 3. Biosensor, according to claim 2, characterized in that the electrical actuation and reading means are configured to conduct electrochemical impedance spectroscopy measurements.
4. Processo de fabricação do biossensor sem marcação baseado em estrutura de imidazolato zeolítico das reivindicações 1 a 3, que compreende depositar uma camada de fotoresiste sobre um substrato de SiO2 e padronizar o fotoresiste por fotolitografia no formato de eletrodos interdigitados; depositar uma camada de 15 a 25 nm de Cr sobre o fotoresiste e, em seguida, depositar uma camada de 15 a 25 nm de Au sobre a camada de Cr; imergir o substrato em solução de acetona, para remoção do fotoresiste, formando eletrodos interdigitados (4); limpar a superfície dos eletrodos interdigitados (4) com acetona e isopropanol por 10 a 15 min e secar com fluxo de N2; o processo sendo caracterizado por: imergir os eletrodos interdigitados (4) em solução piranha da 60 a 70 °C, por 15 a 25 min; lavar os eletrodos interdigitados (4) com água destilada; depositar um filme de estrutura de imidazolato zeolítico-8 sobre a área ativa dos eletrodos interdigitados (4), imergindo o substrato em uma mistura de 8 a 12 ml_ de solução de Zn(NO3)2 a 25mM em metanol com de 8 a 12 ml_ de solução de 2-metilimidazol a 50 mM em metanol, por 6 a 8 horas, sob agitação, em temperatura ambiente, resultando em eletrodos interdigitados modificados com ZIF-8 (5); limpar a superfície dos eletrodos interdigitados modificados com ZIF-8 (5) com metanol e secar com fluxo de N2; expor a superfície dos eletrodos interdigitados a uma mistura contendo entre 0,8 e 1 ,2 pg de Tioredoxina-1 em 20 pL de solução PSB, por 25 a 35 min, resultando em eletrodos interdigitados com Trx-1 imobilizada (6); limpar a superfície dos eletrodos interdigitados com Trx-1 imobilizada (6) com solução PSB e secar com fluxo de N2. 4. Process of manufacturing the unmarked biosensor based on the zeolitic imidazolate structure of claims 1 to 3, which comprises depositing a layer of photoresist on a SiO 2 substrate and standardizing the photoresist by photolithography in the format of interdigitated electrodes; depositing a 15 to 25 nm layer of Cr over the photoresist and then depositing a 15 to 25 nm layer of Au over the Cr layer; immerse the substrate in acetone solution to remove the photoresist, forming interdigitated electrodes (4); clean the surface of the interdigitated electrodes (4) with acetone and isopropanol for 10 to 15 min and dry with a flow of N 2 ; the process being characterized by: immersing the interdigitated electrodes (4) in piranha solution at 60 to 70 °C, for 15 to 25 min; wash the interdigitated electrodes (4) with distilled water; deposit a film of zeolitic-8 imidazolate structure on the active area of the interdigitated electrodes (4), immersing the substrate in a mixture of 8 to 12 ml_ of Zn(NO 3 ) 2 25 mM solution in methanol with 8 to 12 ml_ of 2-methylimidazole solution at 50 mM in methanol, for 6 to 8 hours, under stirring, at room temperature, resulting in interdigitated electrodes modified with ZIF-8 (5); clean the surface of the interdigitated electrodes modified with ZIF-8 (5) with methanol and dry with N 2 flow; exposing the surface of interdigitated electrodes to a mixture containing between 0.8 and 1.2 pg of Thioredoxin-1 in 20 pL of PSB solution, for 25 to 35 min, resulting in interdigitated electrodes with immobilized Trx-1 (6); clean the surface of the interdigitated electrodes with immobilized Trx-1 (6) with PSB solution and dry with N 2 flow.
5. Processo de detecção de interações proteína-proteína com o biossensor das reivindicações 1 a 3, caracterizado por ainda compreender: expor a superfície dos eletrodos interdigitados com Trx-1 imobilizada (6) a uma mistura contendo entre 50 nM a 8 pM de domínio citoplasmático de proteína ADAM17 em 20 pL de solução PSB, por 25 a 35 min; limpar a superfície dos eletrodos interdigitados com Trx-1 imobilizada (6) com solução PSB e secar com fluxo de N2; conduzir medições de espectroscopia de impedância eletroquímica utilizando meios de atuação e leitura elétrica ligados eletricamente aos eletrodos interdigitados com Trx-1 imobilizada (6). 5. Process for detecting protein-protein interactions with the biosensor of claims 1 to 3, further comprising: exposing the surface of interdigitated electrodes with immobilized Trx-1 (6) to a mixture containing between 50 nM to 8 pM of domain cytoplasm of ADAM17 protein in 20 pL of PSB solution, for 25 to 35 min; clean the surface of the interdigitated electrodes with immobilized Trx-1 (6) with PSB solution and dry with N 2 flow; conduct electrochemical impedance spectroscopy measurements using actuation and electrical reading means electrically connected to interdigitated electrodes with immobilized Trx-1 (6).
PCT/BR2022/050033 2021-02-04 2022-02-03 Label-free biosensor based on a zeolitic imidazolate structure, method for manufacturing said biosensor, and method for detecting protein-protein interactions WO2022165576A1 (en)

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