Classifications

• • 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
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
  • G01N33/54326—Magnetic particles
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• • 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
• • 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
• • 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
  • G01N33/483—Physical analysis of biological material
• • 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
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
  • G01N33/54346—Nanoparticles

Definitions

• Kit and method for simultaneous electrochemical determination of at least two protein analytes Field of Art
• the present invention relates to a kit and method for simultaneous determination of two or three protein analytes, using an electrochemical technique.
• body fluids such as blood, plasma, cerebrospinal fluid, lymph, effusion or amniotic fluid during pregnancy, is always a reflection of the current state of the body.
• the presence or concentration of specific analytes, referred to in clinical practice as biomarkers is measured and evaluated as an indicator of physiological and/or pathological processes (inflammation, infection, tumor growth, tissue damage etc.) or response to therapeutic intervention.
• the so-called detection conjugate ie the secondary antibody linked with the detection tag (also called “indicator”), binds to the formed immunocomplex.
• This tag is responsible for generating the resulting signal, which is proportional to the concentration of the analyte to be determined in the analysed sample.
• the tag is typically an enzyme, a fluorescent substance or a radioisotope.
• Electrochemical biosensors represent an alternative to immunoanalytical methods.
• the electrochemical biosensors exploit the advantages of immunoassay methods, in combination with electrochemical detection.
• An electrochemical immunosensor consists of a biorecognition part and a physico-chemical converter providing a measurable signal.
• the biorecognition part contains a carrier with covalently bound specific antibody for capturing the analyte molecules.
• a secondary antibody is added to the reaction medium, which carries a tag yielding an electrochemical signal.
• a physico-chemical converter is a device that converts information from a chemical reaction to a measurable signal recorded by an electronic unit.
• Electrochemical detection offers many advantages, such as the ability to detect an analyte in nano-, pico- or even femtomolar quantities, the measurement being robust, easily miniaturizable and automatizable.
• Summary of the Invention The present invention aims to provide a system suitable for simultaneous detection of two to three protein or protein-type analytes (ie analytes of a proteinaceous nature).
• the system should be sufficiently specific and robust, with zero interference of up to three signals and achieving the required sensitivity.
• the present invention provides a kit and a method for the detection and quantification of protein analytes, the presence or concentration of which in body fluids change as a result of the development of inflammation, tumor growth or infection.
• An essential feature of the invention is the combination of two or three types of detection conjugates containing detection particles suitable for simultaneous electrochemical detection without mutual interference and for quantification of two or three different analytes in one sample in one analysis, while providing a sufficiently strong and robust signal to detect very low levels of analytes.
• the present invention therefore, provides a kit for simultaneous detection and/or quantification of two to three target protein analytes, comprising one or more types of separation conjugate(s) for each target analyte and at least two types of detection conjugates, wherein each type of the detection conjugate is destined for one analyte, wherein - each type of separation conjugate contains a primary antibody against the target analyte, said primary antibody being bound to the surface of a magnetic particle; - each detection conjugate contains a secondary antibody for binding to the target analyte, wherein each type of the detection conjugate contains a conjugation particle which is covalently bound to the secondary antibody, and wherein a plurality of detection particles of one type, selected from CdTe quantum dots, PbS quantum dots and gold nanoparticles, are bound to the surface of the conjugation particle, wherein each type of the detection conjugate comprises a different type of detection particles; wherein the said at least two types of detection conjugates are: - the detection conjugate containing CdT
• the kit may further comprise disposable (single-use) sensors for detecting the electrochemical signal from the detection particles.
• disposable sensors comprising a ceramic or plastic plate, a working electrode and a reference electrode, and optionally also an auxiliary electrode.
• the material of the electrodes and the surface treatment of the electrodes may be selected based on the reaction conditions, the type of analyte to be determined (detected and/or quantified) and the detection method used.
• the disposable sensor may be a three-electrode SPCE (C-C-Ag) sensor with a carbon working electrode, a carbon auxiliary electrode and a silver reference electrode.
• a physico-chemical converter is used to measure the electrochemical signal.
• a physico-chemical converter is a device which converts information from the chemical reaction to a measurable signal.
• An electrochemical voltammetric converter is preferably used as the physico-chemical converter.
• the detection method in this preferred embodiment is square-wave anodic / cathodic stripping voltammetry (SWASV / SWCSV).
• the kit of the present invention together with the physico-chemical converter forms an electrochemical immunosensor.
• the biorecognition part of the immunosensor are the separation and detection conjugates.
• the specific primary antibodies bound to the surface of the magnetic particles bind the target analytes from the aqueous medium of the sample to form immunocomplexes, and then the resulting immunocomplexes are labeled with specific secondary antibodies carrying the signal generating particles (detection conjugate).
• the kit of the present invention may further comprise a magnetic separator, e.g. a stand for reaction tubes or microtubes provided with an NdFeB magnet.
• the kit of the present invention may further contain an agent for acidic hydrolysis of the detection particles.
• the agent may be, for example, an inorganic acid such as hydrochloric acid.
• the invention also provides a process for preparing the kit according to the present invention, wherein to prepare the detection conjugates, carboxyl groups on the surface of the detection particles are activated with N-ethyl-N ⁇ -(2-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) to form detection particles with o-acylisourea ester reactive groups on the surface.
• EDAC N-ethyl-N ⁇ -(2-dimethylaminopropyl)carbodiimide hydrochloride
• the o-acylisourea ester reactive groups further react with amino groups on the surface of the conjugate particles to form signal generating particles.
• hydroxyl functional groups of monosaccharide units present in the structure of the secondary antibody are oxidized to form reactive aldehyde groups, which are then reacted with amino groups on the surface of the conjugate particles to form the detection conjugate.
• carboxyl groups on the surface of the magnetic particles are activated with N-ethyl-N ⁇ -(2-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) to form activated magnetic particles with reactive o-acylisourea ester groups on the surface and then the activated magnetic particles are reacted with amino groups present in the structure of the primary antibody molecule.
• EDAC N-ethyl-N ⁇ -(2-dimethylaminopropyl)carbodiimide hydrochloride
• the unreacted detection particles are separated from the signal generating particles by a gravity-based separation method or centrifugal separation method, for example by centrifugation.
• the unreacted activated functional groups of the detection conjugate or the signal generating particles, respectively are subjected to a blocking reaction, preferably with a mixture of 2-(N- morpholino)ethanesulfonic acid (MES, MES buffer), tris(hydroxymethyl)aminomethane (Tris buffer) and EDAC for activated carboxyl groups, or with NaCNBH 3 in NaOH solution for reactive aldehyde groups.
• Another aspect of the invention is a method for simultaneous electrochemical detection and/or quantification of two to three protein analytes in a sample, using the kit described above.
• This method includes the following steps: a) contacting the sample (in which the analytes should be detected and/or quantified) with the separation conjugates in order to form immunocomplexes by binding the target analytes to the corresponding separation conjugates; b) subjecting the sample to magnetic field separation to separate the separation conjugates and/or the immunocomplexes; c) contacting the separated separation conjugates and/or immunocomplexes with the detection conjugates to bind the detection conjugates to the immunocomplexes; d) eluting unbound detection conjugates; e) subjecting the immunocomplexes with the bound detection conjugates to acidic hydrolysis; and f) determining metal ions released from the detection particles of the detection conjugates in the acidic hydrolysis product by the SWASV / SWCSV method.
• Metal ions released from the nucleus of quantum dots and gold nanoparticles are determined electrochemically using SWASV / SWCSV.
• disposable screen-printed three-electrode sensors are used for the measurement, enabling the reduction of the analysed volume to the necessary minimum (eg 50 to 100 ⁇ l, depending on the size of the electrode).
• the resulting signal is directly proportional to the amount of metal ions released, and thus the concentration of the analyte to be determined in the sample.
• the separation conjugate contains magnetic particles with a covalently bound primary antibody.
• a primary antibody is an antibody that specifically binds to the corresponding target analyte.
• Magnetic particles are particles having a size within the range of 0.7 to 3 ⁇ m, having a magnetic core exhibiting superparamagnetic properties.
• the superparamagnetic properties of the particles enable fast and efficient separation of particles from the liquid sample and from contaminants, and the subsequent washing of the magnetic particles.
• the magnetic particles are surface-modified with functional groups via which the primary antibodies are covalently bound.
• Such magnetic particles are known in the art, and are commercially available.
• Commercially available magnetic particles usually contain functional groups on the surface, which may be first subjected to activation and then the primary antibodies are bound to the (activated) functional groups. Typically, these reactions follow the manufacturer's / supplier's instructions.
• the magnetic particles contain carboxyl groups as functional groups on the surface.
• the primary antibodies may be monoclonal or polyclonal, preferably monoclonal antibodies.
• the primary antibodies are IgG class antibodies.
• the detection conjugate contains a secondary antibody to which a signal generating particle is covalently bound.
• the signal generating particle is a conjugation particle to which detection particles are covalently bound.
• the detection conjugate forms a specific complex with the analyte trapped/bound on the surface of the separation conjugate (ie in an immunocomplex). This step yields an amount of detection particles which is proportional to the amount of the analyte trapped/bound in the immunocomplex. This is a non-competitive arrangement, wherein an equilibrium state must be reached in the liquid environment between the individual steps.
• the secondary antibodies may be monoclonal or polyclonal antibodies, preferably polyclonal antibodies.
• the secondary antibodies are IgG class antibodies.
• the IgG antibodies used to prepare the separation and detection conjugates for a particular analyte may be of mono- or polyclonal origin. Antibodies differing in specificity as well as in the composition of the antigenic determinants against which they are directed are typically used for these two types of conjugates. However, the antigenic determinants must be sterically accessible to allow the binding of the antibody to the analyte.
• IgG antibodies to many protein analytes are commercially available or can be prepared by known methods, such as the animal immunization method or the hybridoma method.
• the conjugation particle is covalently bound to the antibody, preferably it is bound to the carbohydrate portion of the antibody by an amide bond.
• One or more conjugation particles may be bound to a single antibody. Anchoring the conjugation particle with detection quantum dots or gold nanoparticles to the carbohydrate portion of the antibody ensures that the detection portion will not interfere with the binding portions of the antibody, the so-called Fab fragments, and the antibody can thus effectively bind to the target analyte. This also ensures steric access to the detection particles (quantum dots or gold nanoparticles) during acidic hydrolysis, and thus a quantitative release of metals for the electrochemical detection.
• the conjugation particle is preferably a mesoporous nanoparticle based on SiO 2 , surface-modified with functional groups via which the detection particles are covalently bound. (In some embodiments, the conjugation particle is surface-modified with amino groups.)
• the nanoparticle has a size in the range of 10 to 900 nm, preferably 50 to 500 nm, more preferably 100 to 300 nm.
• the size of the mesopores of the particle is preferably in the range of 2 to 50 nm, more preferably up to 10 nm.
• the conjugation particles are commercially available.
• the conjugation particles in the complex with the detection particles represent a suitable tag for the preparation of the detection conjugate.
• the degree of contamination of the final product with free quantum dots, free gold nanoparticles and reactive reagents is considerable in the procedures of the prior art.
• the present invention allows to obtain a product which can be easily separated from the mixture and obtain a highly pure product. This product is then easily conjugated with the secondary antibody.
• the binding of the detection quantum dots or gold nanoparticles by means of the conjugation particle to the secondary antibody allows to significantly amplify the signal even with a small amount of the target analyte.
• the detection particles are metal ion-based particles and have a size in the range of 1 to 160 nm. They are surface-modified by functional groups through which they covalently bind to the conjugation particle.
• the detection particles are surface-modified with carboxyl groups.
• Commercially available detection particles and conjugation particles usually contain functional groups on the surface (i.e. are surface-modified with functional groups), which are first activated and then subjected to reactions leading to covalent bonding, always following the manufacturer's / supplier's instructions.
• Different detection conjugate types contain different detection particles.
• the detection particles are CdTe quantum dots, PbS quantum dots, and gold nanoparticles. In the present invention, it has been found that their electrochemical signals do not overlap, and at the same time, they can be used in the same system simultaneously.
• the CdTe quantum dots have a particle size typically ranging from 5 to 20 nm, preferably 6 to 10 nm; PbS quantum dots have a particle size typically ranging from 60 to 90 nm.
• the gold nanoparticles preferably have a size in the range of 1 nm to 100 nm, more preferably 10 to 30 nm. All three types of detection particles can thus be classified as nanoparticles. Thus, three analytes can be detected by a combination of three types of detection particles. These three types of detection particles are CdTe quantum dots for first analyte detection, PbS quantum dots for second analyte detection, and gold nanoparticles for third analyte detection.
• the analytes determined by this method may generally be substances of protein nature (protein analytes), preferably of clinical significance, whose presence or concentration in body fluids varies depending on the presence of inflammation, infection or tumor growth.
• the protein analytes include proteins, glycoproteins, phosphoproteins, nucleoproteins or lipoproteins.
• the protein analytes are analytes for which specific antibodies can be prepared.
• an analyte is understood to mean in particular a clinically important molecule of a protein type, the levels of which in body fluids change in connection with inflammation, infection, tumor growth or other pathological conditions.
• the analytes may be determined in an aqueous environment, in body fluids such as serum, plasma, cerebrospinal fluid, cervical mucus, effusion, or amniotic fluid (in the event of pregnancy).
• the analytes are pentraxin 3 (PTX3), calreticulin (CALR) and interleukin 6 (IL-6).
• analytes play an important role in the pathophysiology of specific pregnancy complications such as inflammation and infection in the amniotic cavity (the cavity inside the pregnant uterus, filled with amniotic fluid, in which the fetus is connected to the placenta). These analytes are determined in an aqueous buffered medium and in a suitably diluted amniotic fluid sample.
• the primary and secondary antibodies used in the kit of the present invention are then anti-PTX3 antibodies, anti- CALR antibodies and anti-IL-6 antibodies, in particular IgG class antibodies.
• PTX3, CALR and IL-6 in amniotic fluid have been found to be an optimal combination of analytes to determine and predict inflammation and infection in the amniotic cavity in patients with premature amniotic fluid outflow before delivery.
• the levels of pentraxin 3 (PTX3), calreticulin (CALR) and interleukin 6 (IL-6) in the amniotic fluid taken from a patient to be diagnosed are indicative of inflammation and/or infection in the amniotic cavity. Inflammation and infection in the amniotic cavity represent a risk during childbirth and may result in a decision to speed-up the childbirth or perform a Cesarean section.
• the levels of the markers are typically compared with the levels obtained for healthy pregnant women not suffering from inflammation or infection in the amniotic cavity.
• the present invention provides the possibility of simultaneous, i.e. parallel, detection of two or three analytes.
• the ability to simultaneously detect multiple analytes in a single sample in a single analytical procedure offers a number of advantages, including lower reagent consumption, smaller sample volume, shorter time to result, less labor for laboratory workers and final lower costs as the most significant benefits.
• the development and validation of such an electrochemical immunosensor for clinical practice are challenging.
• the system must be sufficiently reliable, robust and must allow the detection and quantification of up to three analytes simultaneously, without mutual interference, even if their concentrations differ significantly.
• the quality of the method for simultaneous detection of analytes using an electrochemical sensor is fundamentally based on the choice of electrochemical indicators, which must provide a sufficiently strong signal and at the same time, there must be no overlap of detection potentials. Another condition is that the generated signals must not interfere with each other in any way. Another parameter that must be taken into account is the sensitivity of the assay. If it is necessary to detect analytes present in biological material in nano- to picogram quantities, then detection particles with appropriate properties and parameters must be used. The intensity of the generated signal per mole of test substance must meet the requirements of clinical practice for the minimum detection limit (MDL) and the limit for quantification (LOQ) for each of the analytes to be determined. The above requirements are fully met by the kit and method of the present invention.
• MDL minimum detection limit
• LOQ limit for quantification
• the present invention eliminates the limitations of immunoanalytical methods which are currently used for the simultaneous detection of analytes in body fluids or other samples.
• the invention in contrast to conventional ELISA methods, offers the possibility of simultaneous detection of up to three analytes in one assayed sample.
• the method according to the invention is robust. Due to the simultaneous detection and the small amount of reagents required (detection conjugates and separation conjugates), the cost per analysis is relatively low. Using the invention, high sensitivity of the method and a low limit of detection for analytes in nano to picogram amounts (ng-pg / mL of analysed sample) can be achieved.
• the design of the detection conjugate offers the ability to amplify a measurable signal and increase measurement sensitivity. Due to the specific surface area of the conjugation nanoparticles, a larger number of detection nanoparticles are bound within one detection conjugate, and thus the signal generated per analyte molecule is amplified. Another indisputable advantage is the possibility to use simple and inexpensive separation techniques (centrifugation, membrane filtration) in the preparation of the detection conjugates and thus achieve high yields and purity with minimal losses during the preparation. Another advantage is the gentle way of labelling the secondary antibody even under the conditions of signal amplification.
• the present invention allows to use electrochemical methods which do not require expensive equipment, automatic analysers, an equipped laboratory nor specifically educated personnel.
• the method and the kit according to the invention are versatile. Adaptation for individual analytes is ensured by antibody selection, and antibodies to various analytes are often commercially available or can be prepared by generally known methods.
• the method of simultaneous detection of analytes according to the invention provides a more sensitive diagnosis or prediction of a given pathological condition (so-called receiver operating characteristic curve; ROC, sensitivity and specificity of the determination and the so-called separation criterion).
• the method in this arrangement meets the requirements of point of care testing (POCT), ie it allows the determination of selected analytes outside standard diagnostic laboratories.
• POCT point of care testing
• the only instrumentation required is an easily transportable potentiostat for measuring the electrochemical signal on the surface of the screen-printed three-electrode sensor.
• the present invention provides the possibility to simultaneously detect and quantify two or three analytes related to the pathophysiology of inflammation and infection in the amniotic cavity, namely PTX3, CALR and IL-6. Reliable and timely acquisition of information about the presence of inflammation and infection in the amniotic cavity, by assaying a sample of amniotic fluid, is very important to determine the optimal therapeutic management in patients with premature amniotic fluid outflow before delivery.
• Optimal therapeutic management is the only way to minimize short- and long-term neonatal morbidity and mortality, but also maternal morbidity.
• the timeliness of obtaining information about the condition of the amniotic cavity is crucial from a clinical point of view.
• There is currently no suitable POCT method allowing the simultaneous detection/quantification of more than one analyte in amniotic fluid to determine and predict inflammation and infection in amniotic fluid.
• the test For optimal use directly at the patient's bedside or in the delivery room, it is absolutely necessary that the test must be in the form of a POCT which is a form that can be performed anywhere, with minimal and cheap instrumental equipment, and with low demands on the technical education of the staff.
• the use of the kit according to the present invention meets these conditions.
• FIG. 1 Schematic representation of the principle of the signal generating particles preparation. Reaction between amino groups (-NH 2 ) of SiO 2 -based conjugation particles and carboxyl groups (- COOH) of detection quantum dots or gold nanoparticles activated by EDAC.
• Figure 2. Schematic representation of the principle of preparation of the detection conjugate. The signal generating particles (1) react with specific antibodies oxidized by NaIO 4 (2) to form a detection conjugate (3). Residual aldehyde groups are blocked by NaCNBH 3 (4).
• Figure 8. Calibration curves of detection conjugates using signal generating particles composed of conjugation particles and detection gold nanoparticles AuNPs without blocking (top) and with blocking (bottom) of residual reactive groups measured separately (curve 1) and simultaneously (curve 2).
• Voltammogram (top) and calibration curve (bottom) for IL-6 protein analyte determination 1 - IL-6 concentration 0 ⁇ g mL -1 ; 2 - IL-6 concentration 0.015 ⁇ g mL -1 ; 3 - IL-6 concentration 0.03 ⁇ g mL -1 .
• Figure 12. Voltammogram (top) and calibration curve (bottom) for determination of protein analyte PTX 3.1 - PTX 3 concentration 0 ⁇ g mL -1 ; 2 - PTX 3 concentration 0.01 ⁇ g mL -1 ; 3 - PTX concentration 30.02 ⁇ g mL -1 .
• Voltammogram (top) and calibration curve (bottom) for CALR protein analyte determination 1 - CALR concentration 0 ⁇ g mL -1 ; 2 - CALR concentration 0.075 ⁇ g mL -1 ; 3 - CALR concentration 0.15 ⁇ g mL -1 .
• Figure 14 Voltammogram for determination of IL-6 protein analyte to verify the effect of a real sample matrix - amniotic fluid.
• 1 - IL-6 at a concentration of 0 ⁇ g mL -1 in 0.2 M Tris-HCl buffer pH 8.0 with 1% BSA and 0.1% Tween 20; 2 - IL-6 at a concentration of 0 ⁇ g mL -1 in a sample of amniotic fluid diluted 1:1; 3 - IL-6 at a concentration of 0.015 ⁇ g mL -1 in a sample of amniotic fluid diluted 1:1; 4 - IL-6 at a concentration of 0.015 ⁇ g mL -1 in 0.2 M Tris-HCl buffer pH 8.0 with 1% BSA and 0.1% Tween 20.
• Figure 15
• Figure 19 Calibration curves of simultaneous detection of three biomarkers.
• Example 1 Preparation of a detection conjugate Chemical reactions between the functional groups (in this example -NH 2 , -COOH, -CHO groups) present on the individual components of the detection conjugate are used to prepare the detection conjugate.
• Commercially available starting materials - conjugation particles: SiO 2 -based nanoparticles (SiNPs): commercially available (Sigma-Aldrich, St. Louis, MO, USA) - surface modification - NH 2 , mesoporous, particle size 200 nm, pore size 4 nm.
• - CdTe quantum dots (CdTe QDs) commercially available (Sigma-Aldrich, St.
• Preparation of signal generating particles the formation of covalent bonds between detection quantum dots and SiO 2 -based nanoparticles is carried out by one-step carbodiimide method using N-ethyl-N'-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDAC) as a crosslinking agent.
• EDAC N-ethyl-N'-(3- dimethylaminopropyl)carbodiimide hydrochloride
• the carboxyl groups of the detection quantum dots or gold nanoparticles are activated by EDAC to form an active intermediate (QDs/AuNPs-o-acylisourea), which immediately reacts with the amino groups on the surface of the SiO 2 -based nanoparticles to form an amide bond (see Fig.1).
• the pore size (4 nm) of the mesoporous SiO 2 -based nanoparticles sterically prevents quantum dots from penetrating the porous surface and negatively affecting their release necessary for the final electrochemical detection. Due to the high density of the signal generating particles, they are easily separated from the solution containing the reagents and free unconjugated detection quantum dots or gold nanoparticles. Unconjugated detection quantum dots or gold nanoparticles can be easily separated by centrifugation, thus avoiding the lengthy dialysis step that would reduce preparation yield. More specifically, the procedure for preparing signal-generating particles is as follows: Conjugation particles (SiNPs, 3 mg) are washed 5 times with 0.1M phosphate buffer pH 7.3 (1 mL).
• the particles are separated from the solution by centrifugation for 5 minutes at 5000 rpm. Subsequently, 0.5 mg mL- 1 EDAC solution, CdTe QDs solution (50 ⁇ L, 1 mg mL -1 ), PbS QDs (4 ⁇ L stock solution) or AuNPs (10 ⁇ L, 1 mg mL -1 ) are added. After making up the reaction volume to 1 mL, incubation for 1 hour at room temperature in the dark with gentle rotation followed. After incubation, the detection particles are washed 3x with 0.1M phosphate buffer pH 7.3 with 1M NaCl and 2x with 0.1M phosphate buffer pH 7.3 (1 mL).
• SiNPs and QDs/AuNPs is set with respect to an excess of SiO 2 -based nanoparticles with free amino functional groups. These are then used to bind specific antibodies to form a detection conjugate. Antibodies are conjugated by the site-specific covalent binding method.
• the hydroxyl functional groups of the monosaccharides present in the structure of antibody molecule and localized at the end of the glycosidic chain are first oxidized with sodium periodate to form reactive aldehydes that react spontaneously with the amino groups of the signal generating particles. The reaction leads to a formation of a stable amide bond (see Fig. 2).
• the final stabilization step is the blocking of the remaining reactive functional groups.
• Blocking substantially reduces the non-specific sorption of the detection conjugate to the surface of the separation conjugate.
• the detection conjugate was prepared as follows: Detection antibodies (20 ⁇ g) were oxidized with a solution of NaIO 4 (0.02 M) in a reaction volume of 250 ⁇ L for 30 minutes at room temperature in the dark with gentle rotation. After stopping the oxidation by adding 1.5 ⁇ L of ethylene glycol and removing the NaIO 4 by desalting using silica gel centrifugal columns, the oxidized antibodies were added to the prepared detection particles. After making up the reaction volume to 1 mL with 0.1M phosphate buffer pH 7.3, incubation followed overnight at 2-8 °C in the dark with gentle rotation.
• the particles were separated from the solution by means of a magnetic separator. Subsequently, a 7.5 mg/0.5 mL EDAC solution was added. After making up the reaction volume to 1 mL with 0.1M MES solution (MES buffer pH 5.0), incubation followed for 1 hour at room temperature with gentle rotation. After incubation, the magnetic microparticles were washed twice with 0.1M MES pH 5.0 (1 mL). Subsequently, specific IgG antibodies were added at a concentration of 50 ⁇ g mL -1 of 0.1M MES pH 5.0. This was followed by incubation for 16 hours at 4 °C with gentle rotation.
• 0.1M MES solution MES buffer pH 5.0
• the separation conjugate was washed 3x with 0.1M MES pH 5.0, 2x 0.1M PBS buffer pH 7.4 and 3x 0.1M MES pH 5.0 (1 mL each).
• the separation conjugates were stored at 4 °C.
• Example 3 Procedure for analyte determination (immunocomplex formation and subsequent electrochemical detection) The individual steps take place in a total reaction volume of 100 ⁇ l. In general, these conditions are suitable for various analytes. 1. Binding of the target analyte with the separation conjugate: the binding is proceeded in a reaction solution of 0.2M Tris-HCl buffer pH 8.0 with 1% BSA and 0.1% Tween 20.
• the action of hydrochloric acid produces the intermetallic compound Au(III), the signal of which is electrochemically measured.
• the electrochemical determination is carried out in the solution after magnetic separation under the conditions listed in Table 1.
• Metal ions released from the nucleus of the quantum dots and the gold nanoparticles, which are components of the detection conjugates, are measured electrochemically using square-wave stripping voltammetry in the negative and positive potential area (SWASV / SWCSV).
• Disposable screen-printed three-electrode sensors can be used for measurement, enabling the reduction of the analysed volume to the necessary minimum (eg 50 to 100 ⁇ L, depending on the size of the electrode).
• the resulting signal is directly proportional to the amount of the metal ions released, and thus the concentration of the assayed protein analyte in the sample.
• the electrochemical detection in the examples was performed using a portable MultiEmStat 3 electrochemical device with four independent potentiostats controlled by MultiTrace 4 software (PalmSens, The Netherlands), and printed three-electrode sensors containing a carbon working electrode, carbon auxiliary electrode and silver reference electrode (C-110; W/A-C, R-Ag; Metrohm, Switzerland). Table 1.
• Example 4 Verification of the signal increase due to the construction of the detection conjugates Using the detection conjugates prepared in Example 1, the effect of SiO 2 -based conjugation particles on increasing the resulting signal was confirmed. Two types of detection conjugates were compared, without and with the conjugation particles. Detection conjugates with CdTe QDs and PbS QDs were tested. Both types of detection conjugates were analysed electrochemically under the experimental conditions listed in Table 1. The result of the analysis is shown in Fig. 3.
• Example 5 Effect of a blocking step in the preparation of the detection conjugate on the signal intensity
• the key parameter is the low signal level of negative controls. Any sorption of the detection conjugate to the surface of the separation conjugate without the participation of the analyte (so-called non-specific sorption rate) is undesirable.
• Appropriate experimental conditions for the preparation of the detection conjugate and the binding of the detection conjugate to the antigen must ensure zero non- specific sorption. The degree of non-specific sorption affects the sensitivity of the assay. In the case of detection conjugates, the remaining reactive groups of all reagents used for their preparation are mainly responsible for the non-specific sorption.
• a blocking step is preferably included in the detection conjugate preparation protocol.
• the procedure is described in Example 1.
• the signals of detection conjugates prepared without blocking the remaining reactive groups (i.e., omitting the blocking step) and with blocking (i.e., all steps of the procedure of Example 1) were compared. They were also compared from the view point of the detection method, where the individual detection conjugates were measured electrochemically as well as their mixture.
• the measuring of the mixture involved simultaneous measurement of the signals generated by the three detection conjugates in parallel.
• the electrochemical measurement was performed under the conditions shown in Table 1. The results are shown in Figures 4 to 8. In Figures 4 and 5, the results of the analysis of the individual detection conjugates are shown separately.
• Figure 4 shows the result of measuring detection conjugates with CdTe QDs detection quantum dots (curve 3) and PbS QDs detection quantum dots (curve 2) without blocking residual reactive groups.
• the step of blocking reactive groups is always used, because without the blocking step they did not provide any measurable signal.
• Figure 5 shows the result of measuring detection conjugates with CdTe QDs detection quantum dots (curve 3) and PbS QDs detection quantum dots (curve 2) with blocking of the residual reactive groups.
• the blocking of the residual reactive groups led to a decrease in non-specific sorption, ie. in the signal corresponding to the negative control, but at the same time, it led to an increase in the resulting signal.
• FIG. 6 to 8 show a comparison of calibration curves for individual and simultaneous analyses for the detection conjugates as well as a comparison of the preparation method, ie without blocking (top) and with blocking (bottom).
• the current values for all three analysed detection conjugates also increase.
• there is a noticeable difference in the measured current values especially for the detection conjugates with the detection quantum dots CdTe QDs, where the resulting signal is suppressed during simultaneous analysis.
• the results clearly show that it is very advantageous to use detection conjugates after blocking for simultaneous analysis.
• FIG. 9 is a record of a measurement comparing the current response intensities of the detection conjugates measured separately (top) and simultaneously (bottom). In simultaneous measurements, the signal for PTX3 was significantly suppressed, when the detection conjugate was labelled with cadmium- based quantum dots (CdTe QDs).
• Example 6 Effect of the antibody type on the preparation of a separation conjugate The effect of the antibody type was tested using an anti-IL-6 antibody. The method of preparation of the separation conjugate and the type of specific antibodies used from the point of view of clonality (monoclonal vs.
• polyclonal are also relevant for the resulting functionality and sensitivity of the system.
• the effect of the type of antibodies against the IL-6 analyte is given as an example. Both monoclonal and polyclonal antibodies were used and tested to prepare the separation conjugate. Polyclonal antibodies were always used to prepare the detection conjugate (wider variability in polyclonal antibody specificity leads to a higher reactivity with the analyte). It was experimentally confirmed that the use of monoclonal antibodies is more advantageous for the preparation of the separation conjugate (see Figure 10). The procedure for preparing the separation conjugate is described in Example 2. Electrochemical detection was performed under the conditions listed in Table 1.
• Example 7 Analysis of analytes Standard commercially available proteins were used for separate analysis of analytes and preparation of calibration curves.
• PTX 3, IL-6 and CALR protein analyses are provided in this example.
• Monoclonal antibodies were used for PTX 3, CALR and IL-6for the preparation of the separation conjugate (preparation procedure according to Example 2).
• Monoclonal antibodies were used for PTX3 and CALR and polyclonal antibodies were used for IL-6 to prepare detection conjugates (preparation procedure according to Example 1).
• the analysis procedure is described in Example 3.
• the electrochemical detection was performed under the conditions shown in Table 1.
• the resulting voltammograms and calibration curves are shown in Figures 11-13 and 18-19.
• the resulting voltammogram ( Figure 14) demonstrates the usability of the system for real samples.
• the measured current response value for the amniotic fluid sample (diluted 1:1) with standard IL-6 analyte addition (curve 3) is comparable to the current response pattern of the sample containing the analyte (IL-6) in a buffer with defined molarity and pH (curve 4).
• the current response curve is partially different for negative controls in amniotic fluid (curve 2) and buffer (curve 1), but the absolute difference is the same for both environments and the measured analyte concentrations in the amniotic fluid sample fully correspond to the expected IL-6 levels.
• Example 9 Simultaneous analysis of three analytes
• Commercially available protein standards were used for simultaneous analysis of the analytes.
• Examples for PTX 3, IL-6 and CALR are provided.
• the immunocomplex formation procedure is described in Example 3.
• Electrochemical detection was performed under the conditions shown in Table 1.
• the resulting voltammogram is shown in Figure 15.
• Example 10 Verification of the reactivity of the detection conjugates Magnetic particles with immobilized target analytes (PTX3, IL-6, CALR) were used to confirm the reactivity of detection conjugates corresponding to the amount of the analyte.
• Example 11 Verification of non-specific sorption of prepared detection bioconjugates
• inert protein represented by bovine serum albumin (BSA) was used.
• BSA bovine serum albumin
• An increasing amount of protein was used for reaction with the constant amount of detection conjugates (125 ⁇ L corresponding to 2.5 ⁇ g of antibodies for each). Reaction was proceeded in a solution of 0.2M Tris-HCl buffer, pH 8.0 with 1% BSA and 0.1% Tween 20 for 1 hour at room temperature upon gentle mixing.
• Microbial invasion of the amniotic cavity was defined as the presence of microorganisms and/or microbial nucleic acids in the amniotic fluid .
• Intra-amniotic inflammation was defined as a concentration of IL-6 in amniotic fluid ⁇ 3,000 pg/mL using an automated electrochemiluminescence immunoassay method (described in: Musilova I, Andrys C, Holeckova M, Kolarova V, Pliskova L, Drahosova M, et al. J Matern Fetal Neonatal Med.2020;33(11):1919-26).

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