BG110556A - Method and sensor for immune metric nano analysis - Google Patents

Abstract

The method and the sensor can be applied in medical clinical diagnosis, life sciences, control of organic production, ecological biological control, bio safety, etc. The biochemical method with calculation procedures, the micro-sensor with a high sensitivity, the equipment (laboratory and field) with specific calculation procedures and measurement method combined in a functional relation, jointly achieve measurement of the immune responses, habitual for the specialists and with accuracy preset only by the fundamental limitations arising from the very nature of the task, as well as detection of microscopic biological particles - viruses bio nano-particles, bacteria and bacterial spores, cell organelles, etc. The advantage is the individual measurement of irreversible immune reactions in their natural environment, using amounts of molecules of only one individual living cell.

Description

TECHNICAL FIELD

The invention is applicable: for clinical diagnostics in medicine; in life sciences research and development; for control in bio-technological production; for control and protection of the environment bio-environment; for biosecurity control, etc.

For the most part, life is based on a limited class of stereoconform biochemical reactions (immune) and has a single primary organization (cellular). The billions of years of evolution, however, have generated an incredible variety of biomolecules, regulatory cycles, and reactions. The analysis of immune responses for their measurement, understanding and cataloging is a major ongoing activity in this field, and the main difficulty is in the anomalous amounts of biomolecules that need to be measured / recorded in order to analyze the processes in a single living cell.

BACKGROUND OF THE INVENTION © The life sciences are at the heart of modern technological and scientific development and are being given the most resources. Each year, thousands of new biomacromolecules (antibodies, biomarkers, antigens, etc.) are cataloged by researchers, medical professionals, and biologists using immune biochemical reactions [1]. This activity (for historical reasons) is based on molecular biological methods that are no longer quite adequate. They use significant amounts of biomolecules and / or bio-particles and their collection / acquisition requires separation or multiplication, which are multi-stage, lengthy and complex procedures adding a variety of intrinsic effects, artifacts, changes and more. Separation of the results is also done by processes that are not peculiar to the immune reactions - diffusion, electrophoresis, binding to optically active molecules, gravitational / inertial separation, etc. - which further add new artifacts, selectivity, chemical changes, etc. The registration is performed with poorly sensitive methods, historically oriented eventually to human eye observation or its modern equivalents: images from CCD cameras; scanned micrographs or microplates, etc.

Bio-sensors [2,3] are the current trend of development. Currently, the most promising and sensitive sensors for detecting and measuring nanoscale bioagents (bio-macromolecules, viruses, bacterial spores, etc.) are micro-console MEMS (Micro Electro-Mechanical Systems) sensors [4]. Technologies for the functionalization of these sensors are also being developed intensively.

A number of similarly named devices have been patented, but they are substantially different from the present invention. There is a known "Piezo Immune Sensor" [9], which has a similar task for immune chemical detection of "analytes" but uses a completely different type of sensor. Also known is the "Automatic Immune Analyzer" [10], which is a complex robotic installation that has the task of automating the operation of the operator (to replace the operator) and in which orthodox molecular biological tests are applied. Also known is the Immunoassay Analyzer [11], which is computerized laboratory equipment ("smart furniture") designed to prevent improper operation and to ensure the safety of the operator.

Many chemical and biochemical reactions are known that are used to detect, analyze and measure bio molecules and their reactions in different methods and procedures for different types of micro cantilever sensors. Many methods are known for the functionalization [4, 6, 7, 8] of sensors, but none of them are intended to be performed by the operator, not the manufacturer, for the single functionalization of the sensor before each measurement for the purpose of specific study Q directly from the researcher at his or her own laboratory, nor allow the investigation of irreversible reactions.

All INDIVIDUAL elements of the sensor are known: sensors with several microconsoles (2 to 256) [7, 8], but for these sensors the microconsoles are either all the same or all different; microconsoles in the form of an elongated letter "P" [13];

active-section microconsoles [6, 7]; Built-in piezoresistors [6] and microconsoles themselves piezoresistors [13]; Sensors with reference microcontrollers [6] but not sensors with reference microcontroller pair whose ratio is the reference value; Individual actuators (vibrators, transmitters ...), as well as magnetic or electromagnetic micro-actuators, which excite the resonant oscillations of the micro-consoles [14].

A variety of support structures of micro-cantilever structures are known [6]. Methods for creating hydrophilic zones [12] on a hydrophobic surface for various purposes are known, but their use for fixing / retaining at the appropriate site the reaction volume in which the biochemical actions necessary for the operation of a sensor are performed have not been published.

OBJECTIVE OF THE INVENTION

A universal and complex solution for immunometric analysis, which exactly matches, and the well-established habitual ways of work of specialists, and the nature of biochemical processes in nature.

An essential concomitant goal is the registration of microscopic bioparticles (viruses, bacteria and bacterial spores, © bio-nanoparticles, cellular organelles, etc.) by immune responses between structural groups located on the surface of the bioparticles and antibodies specific to these groups.

The object of the invention

Achieving the complex object of the invention by means of a functional combination: High sensitivity sensor; Method with calculation procedure; Apparatus with specific measuring methods and calculation procedures; jointly to achieve a new unified effect - the "habitual" measurement of immune responses, as well as the registration of microscopic bioparticles with an accuracy set only by the fundamental constraints arising from the very nature of the task. The following elements of the task are essential to the invention, namely:

Universal, replaceable, reusable, resonant microcontroller sensor that is operationally and locally prepared (functionalized) for each measurement and / or diagnosis independently by the operator (not the manufacturer !!) immediately before use / measurement and restores immediately after this.

A method using such a sensor to individually measure irreversible immune responses that deplete prior functionalization in their natural reaction medium (solutions, sera, plasma), using nano-sized samples containing amounts of molecules corresponding to the amounts present in only one single living cell.

A small, reliable and easy-to-use instrumentation [5] operating in the field and in laboratories, employing a specific method of measurement and calculation algorithm whose accuracy is limited only by the random, irreversible natural phase noise of the sensor.

"SOLVING" PROBLEM AND DIFFICULTY

Immune reactions are highly specific stereoconform biochemical reactions - ie. reactions between two reagents whose spatial structure / shape is exactly "three-dimensional" mirror (exactly corresponding in space), with any difference and / or discrepancy preventing the reaction. For their part, the spatial structure of bio-reagents (antibodies, antigens, bio-macromolecules, etc.) is often very variable and highly dependent on the environment (temperature, dissolved ions, pH, impurities, physical conditions, other active bio- molecules, etc.).

In common with all immune responses, their result is that the two reagents merge (irreversibly) into a new common spatial structure. However, the infinite variety of immune responses gives rise to an infinite variety of resultant spatial structures, each with its own specific characteristics and chemical affinity and requiring an individual, highly specific mode of registration. The only universal result of stereo conformal (unifying) reactions is the emergence of structures with masses equal to the sum of the masses of the two reagents, and this is the only possible basis for a universal approach.

The invention measures immune responses precisely through this universal feature. The difficulty, however, is that although we have a "double" mass increase in the aggregate structure relative to the reagents, in absolute terms the mass increase remains very small. The mass of the largest biomacromolecules is also negligible compared to the usual laboratory volumes (micro liters), as well as to the mass of the sensors. О Micro sensors of the most sensitive type are required and their response must be measured with the highest possible accuracy - ie. accuracy limited only by the irreversible natural noise.

The complex nature of both purpose and decision is the second half of the "decisive" difficulty - ie. finding a balanced approach to such a diverse set of elements is difficult. Actions used, biochemical procedures, techniques, calculations, sensors, structures, etc. in spite of their differences, they need to be aligned evenly in their functional relationship, so as to ensure "coherent integration".

SUMMARY AND EFFECT OF THE INVENTION

The object of the invention is solved by a method consisting of four sequential actions of a biochemical nature, a sensor representing a micro-console structure (figure 1) on a carrier (figure 2), measuring the sensor apparatus [5] realizing the accuracy limited only by the irreversible natural phase noise of the sensor and a procedure (the fifth action of the method) to calculate the desired end result (unknown parameters of the immune response).

The biochemical protocols (sets of operations) of each of the METHODS of the METHOD, although clearly specified, are mainly characterized by their purpose / purpose and are not fixed in all their details. This is a direct consequence of the universal applicability of the invention to immune responses - i.e. to every possible “antigen-antibody” pair of infinite natural diversity. For example, for some of the applications of the invention, the binding operations can be performed with specific reagents and / or performed in two or more steps without changing their nature or purpose. There are also possible applications of the invention that may require additional, specific operations of ancillary nature to the mandatory operations. However, these two types of additions are not a modification of the method, but merely a specific detailing for a particular application, for a particular pair of biomolecules. This feature of this universal method is a major advantage of the invention - the operator has the freedom to adjust the operations of the method according to the current concrete study.

Each of the biochemical actions of the method consists of one or more operations, each followed by a three-step, biochemically inactive, one-step WASH to remove chemical residues and unnecessary reaction products from the surface of the work area, from the entire micro-console structure, and from the holder of the sensor. The first step is washing with a jet of reagent washing all non-chemically bound molecules (proteins, polypeptides, oligonucleotides, etc.) from the surface. This is followed by a two-step wash with a stream of double-distilled (deionized) water.

Each of the biochemical actions of the method ends with the removal of the water retained by the capillary forces DRYING, by flushing with an alcohol stream, leaving the alcohol residues self-evaporating.

After each operation of the method, the rinsed and dried sensor is MEASURED by the apparatus described in [51 by the same-type but complex and multi-step automatic measurement procedure (according to the method described in [51), using the collected results (averaged individual measurements) in the calculation algorithm [5] to obtain a corrected "summary result" relating to the completed biochemical step.

To improve accuracy, the measurements and calculations for an action can be repeated many times, averaging the resulting set of “summary results”.

All biochemical actions of the method are performed using accurate reaction quantities / volumes delivered to the sensor by (manual or automatic) micropipette. These quantities of reagents are captured / fixed / retained by capillary forces as a drop of liquid on a hydrophilic zone (9) positioned on the surface of the carrier (7) of the sensor (Figure 2). The rest of the carrier surface (7) is made hydrophobic - for example, covered with a thin but complete Teflon layer. The dimensions of the hydrophilic zone are chosen such that the working amount of fluid (aqueous solution, serum, plasma, etc.) is sufficient to wet the entire area, but not to exceed the maximum possible volume of one formed by capillary forces, a drop covering the whole hydrophilic zone (9).

Thus, the L / L reaction volumes / volumes are held securely in the correct place on the carrier (7).

The micro-console structure (10) is mounted in a suitable place on the sensor carrier (7) (Figure 2) so that all four of its micro-consoles (two pairs) enter the hydrophilic (9) zone (overhang it) and are covered by ( penetrate into the) capillary drop. Thus, the surfaces of the microconsoles become directly exposed to the biochemical reactions occurring in the volume of the capillary droplet.

Capillary drop volume is the reaction volume in which the biochemical actions of the method are performed.

All the steps of the method are performed / performed directly by the OPERATOR using the apparatus and this is an important advantage of the invention.

The method starts with the fact that the cleaned (washed) and dried sensor is measured and the result obtained is recorded as its initial, starting state (ie - the initial masses of the microcontroller). Each of the following biochemical actions increases the masses of the two microconsoles of the "measuring" microconsole pair, and the corresponding measurements record these increases. The masses of the two microconsoles of the "reference" microconsole pair remain constant.

The first biochemical action of the method aims at ACTIVATION of the surfaces of the working zones of the two microconsoles of the "measuring" microconsole pair and contains two mandatory operations:

· Activation of the inorganic (metal) surface only in the working zones of the two microcontrolls of the "measuring" pair of microcontrollers of the sensor immediately followed by;

2. Building on the surface thus activated an organic base layer (protein) in one or more steps.

This action should not affect any surface outside the work areas, nor the surfaces of the two microconsoles of the "reference" microconsole pair, or create a base layer on any of them.

The second biochemical action aims at a specific FUNCTIONALIZATION of the working zones of the two microconsoles of the "measuring" microconsole pair and also contains two mandatory operations:

1. Construction / activation of a specific intermediate layer securing the antibodies (or antigens or first reagent) to the surfaces of the working areas coated with the base layer;

2. Attachment of specific antibodies (or antigens or first reagent) to the working areas of a precise working volume of known concentration.

This action must neither affect any part of the surface of the two microconsoles of the "reference" dual microconsoles nor attach molecules of the reagent to them.

The third biochemical measuring action is carried out between antibodies (or antigens or the first reagent) affixed to the work areas by an IMMUNE REACTION with the antigens (or antibodies or the second reagent) and fixes the result thereof. The reaction proceeds in the exact working volume of the test solution, or serum or plasma, with precise control of the temperature in the working volume and accurate recording of the time during which the reaction takes place.

The reaction is terminated (i.e., the current result is fixed) by abruptly flushing with a stream of double-distilled (deionized) water at a much lower temperature than that of the working volume, the exact duration of the reaction being determined at the time of the abrupt fall of the reaction. the temperature of the microconsoles of the sensor.

This action and the immune response should not affect any part of the surface of the two microconsoles of the "reference" micro-console pair, since no molecules of the first reagent are attached to their surface.

u The fourth chemical action of the method is intended

Restoration of the sensor by removal of all biomolecules together with the bio-structures formed in the previous steps from the surfaces of the working areas, from the whole micro-console structure, as well as from the sensor holder. The action restores the sensor to its state from before the first operation of the method - preparing it for new use.

In addition, a CONTROL is performed, and if, after cleaning the sensor, the measurement result differs significantly from the result of its initial measurement ^w from before the first action, it is an indication of either irreversible change / damage to the sensor or a significant error of the sensor. the operator when performing the method actions. In this case, the results from the previous three actions are rejected, the sensor is replaced if it is damaged and the method actions are repeated from the beginning.

The fifth action of the method is CALCULATION of the desired end result (unknown parameters of the immune response) by a calculation procedure. This procedure uses the already collected five “summarized results”, which are 3 “measurement” results characterizing the first three biochemical actions, and 2 “control” results (before the first and after the fourth action) obtained from the apparatus [5].

The relation between the period (P) of the fundamental resonance of a micro-console and its effective mass (M) is: Μ = α P ² , where "a" is a collecting factor specific to the sensor construction and simultaneously reflecting (as corrections) the momentary influence of the environment the environment and production variations of the particular copy. Since all four sensor microcontrollers are created simultaneously in the same production process and measured simultaneously in a common environment, they have the same coefficient a (but their effective masses are substantially different). Each of the first three biochemical actions increases (or does not increase) the mass of each of the microconsoles to a different degree, and the fourth "cleansing" action restores their original meaning.

For each sensor measurement, the exact ratio of the squares of the resonance frequency periods of all 4 microconsoles is determined. These relationships directly correspond to the instantaneous relationships of the masses, so that after the first 3 biochemical actions, the 12 "measured" periods give rise to a system of 12 equations (quite voluminous and not completely independent, but obvious equations). In this system of equations, the experimental meanings of periods are related to masses by 15 quantities of different types:

a) 5 pcs. values characterizing the immune response investigated - the molecular weight of the antibodies "mb"; the molecular weight of the "ml" antigens; number of antibodies immobilized on active sites "N"; the total number of antigen molecules in the reaction volume T tested; and the reaction constant antibody-antigen "C".

b) 3 pcs. their coefficients, a ₂ and a _3, reflecting the environmental impact at the time of the three measurements, each of which participates equally and in the four equations derived from the respective measurement;

c) 3 pcs. Constants specific to the specific construction reported by the sensor manufacturer and 2 pcs. Manufacturer-measurable individual constants reflecting possible small (<1%) individual variations of each sensor instance;

d) 2 pcs. very small quantities («0.1%) of random nature and zero mean (statistical fluctuations of the experiment), which can be made arbitrarily small by repeating it repeatedly.

The three coefficients of point “b” are self-exclusive when using relationships. The five constants of point “c” are known and are entered into the computer of the apparatus [5] (but can also be determined by the operator in a series of measurements without biochemical action). The fluctuation values of point “d” have little influence or can be neglected at all. Thus, the system of 12 non-independent_ equations is reduced to 9 equations linking the five essential quantities of point “a” and both of point “d” with the experimental meanings of the periods, with at least one of the quantities of point “a” known in advance. As a result, the system becomes overridden and easily solved (by the computer in [5]), which gives unknown parameters of the immune response along with the error with which they were determined.

The micro-console structure of the SENSOR (Figure 1) consists of two identical pairs of micro-consoles (1 and 2) created simultaneously in the same production process. Each of the four microconsoles can be made up of one or more parallelepiped-shaped segments, e.g. in the form of an elongated letter "P". Each pair of microconsoles (1 and 2) consists of two separate microconsoles differing only in length. The resonance frequency of the microcontrollers decreases as their length and / or mass increases. The difference in the lengths of the two separate microconsoles from the pairs of microconsoles (1 and 2) must be such as to provide the necessary difference in the resonance frequencies to eliminate the acoustic interference / interconnection between them. The two identical pairs of micro-brackets (1 and 2) are spaced apart on a common base (6). Only one of the micro-console pairs (2-measuring), at an appropriate location on its two separate micro-consoles, creates two exactly identical active regions (3) whose surface allows (or is suitable for) the attachment of biomolecules or microscopic bio -particles of interest. The rest of the surface of these two micro-consoles, as well as the entire surface of the two micro-consoles of the other (1 reference) micro-console pair, does not allow (not suitable for) the attachment of bio-molecules or microscopic bio-particles of interest.

The creation of the active sites (3) is carried out simultaneously in one production process equally for the two sites (by selectively adding one or more additional layers of suitable materials / substances) in a way that substantially increases the mass of the microconsoles of this (measuring) double- so that the resonant frequency of both its microcontrolls is lowered enough to provide the necessary difference in the resonant frequencies to eliminate the acoustic interaction with the microcontroller of the other a (reference) double-micro-console.

Figure 2 shows how the micro-console structure (10) is mounted on the carrier (7) so that the two identical two-micro-consoles (1 and 2) are located directly above the hydrophilic zone (9), which is of suitable shape and size.

The resonance oscillations of each of the four microconsoles must be recorded by "sensing devices" that produce a signal proportional to their bending. They can be implemented as separate devices (optical / laser displacement meters, capacitive distance / distance sensors, etc.), and can be created / positioned directly on or inside the microcontroller. The micro-console structure (10) has four "sensor devices" in the form of built-in "four" individual piezoresisters (4), or the structural elements of the microconsoles themselves, can perform the functions of piezoresistors. Piezoresistors may have separate electrical terminals (8 pins) or their pins may be partially interconnected to form a circuit with a reduced number of pins (5-pin star, 4-bridge bridge, etc.). The electrical terminals of the piezo resistors (4) reach the contact pads (5) and from there they are transferred to the carrier (7) and connected to its electrical terminals (8) - the gold-plated contact feathers at its end.

The transfer of electrical terminals from the piezo resistors (4) to contact pads on the lower surface of the common base (6) can be done by means of insulated (eg p-n transitions) vertical conductive "columns" or "openings with conductive walls" located therein. . These lower contact pads, not shown in the figures, can be soldered directly to the respective contact pads on the surface of the carrier (7) and from there to be connected to its electrical terminals (8). In this way, both electrical connection and mechanical attachment of the micro-console structure to the carrier can be carried out simultaneously.

As a replaceable element, the carrier (7) is inserted into a holder which is simultaneously an electrical connector connecting the contacts (8) to the measuring apparatus [5].

The exciter (not shown in the figures) generates controlled-frequency acoustic waves that excite mechanical vibrations in each of the microcontroller pairs (1 and 2), including the oscillations of the resonant frequency of the individual microcircuits required for the sensor to operate. The exciter can be a separate device in acoustic connection with the micro-console structure (10) or with the carrier (7) - for example: piezo radiator, loudspeaker, electromagnetic vibrator, electrostatic vibrator, etc., or placed / created directly on the micro-console structure - for example: magnetic micro-actuator, electromagnetic micro-actuator, bimorphic thermo-actuator, electrostatic or piezomechanical micro-actuator, etc.

The carrier (7), together with the micro-console structure (10), is placed in a reactor connected to the computer equipment [5], which automatically measures the sensor repeatedly with accuracy limited only by its irreversible natural phase noise and additionally performs the calculation of the unknown immune response parameters from the collected intermediate results by the calculation procedure of the fifth action of the method of this invention.

SUMMARY OF THE INVENTION

The invention is undoubtedly industrially applicable and has already been realized.

A step forward is to develop a method for one-off individual functionalization of sensors, directly from the researcher, in his own laboratory and for his specific research.

A versatile, replaceable and reusable sensor incorporating a "difference between two differences" type standard and related apparatus [5] limited only by the indistinguishable natural phase noise, jointly reaching a sensitivity of several NUMBERS of the molecules of the reagents used (eg <10 antibodies) is a significant step.

Separately, each of the elements of the sensor structure is known. Microconsoles, P-shaped microcontrollers, piezoresistors (common from microsegments), additional sections and layers incl. contact pads, “vertical” joints, various micro-actuators and carriers, hydrophilic and hydrophobic regions, etc. - they can all be found in known structures. Many of the individual elements (methods) of the method are also known: chemical reactions cleaning, surface polymerisations, activating, functionalizing; computing procedures; etc.

The novelty of the invention is in the balanced combination of known elements and (methodical) techniques combined in a functional connection and corresponding to the specific set of requirements and features specific to this task only. The combined application of the biochemical method, the sensor, the measuring apparatus [5] and the calculating procedures leads to a new unified effect - the measurement of stereo-conformal reactions between quantities of molecules contained in a single living cell.

What is new is the formulation of the task itself - a "personal" universal device for all molecular biological tasks.

ADVANTAGES OF THE INVENTION

The invention is universal - i.e. The METHOD is applicable to any stereo conform reactions in their natural reaction environment, and the SENSOR is suitable for any variant of the biochemical actions (protocols) of the method.

The invention can also be applied to the registration of microscopic bioparticles - viruses, bacteria and bacterial spores, bio nanoparticles, cellular organelles, etc.

An advantage of the invention is the ability to conduct all standard (standardized) clinical trials with easily comparable "absolute" results.

The invention also creates the possibility of autonomy in its application for research and / or measurements, since it is not necessary for biochemical reagents (antigens, antibodies ....) to be produced and sold anymore, but may be created for the purposes of a particular study directly in the process of work (collect, isolate, multiply ...) directly by the researcher, and in his own laboratory.

An advantage of the invention is that the operator, rather than the manufacturer, performs a single functionalization of the sensor prior to each measurement, which provides the opportunity to investigate irreversible reactions, reactions that would otherwise immediately wear out a permanently functionalized sensor.

An important advantage of the invention, applied in conjunction with the measuring apparatus [5], is the achievable sensitivity of UNITS of the molecules of the reagents used - a sensor specially designed for maximum sensitivity can provide ± 1 antibody. Such high sensitivity makes it possible to study nano-sized samples containing amounts of molecules corresponding to the quantities present in only one living cell.

The invention also provides significant operational advantages: 1) the sensor is versatile, replaceable, reusable and self-calibrating; 2) the operation of the method is familiar to the specialists, working with standard reaction volumes, no special training or training of the operator is needed, ordinary laboratory devices are used; 3) the reactor in which the sensor and the measuring equipment [5] are placed are automatic, small and economical, suitable for both laboratory and field (expedition) conditions, and the computer system of the measuring equipment [5] allows for the rapid addition of new ones. methodologies and refinement of measurement actions and / or procedures.

EXAMPLE FOR THE IMPLEMENTATION OF THE INVENTION

The object of this "not optimized" embodiment is to perform and measure the immune response between sensor-immobilized antibodies (Rabbit IgG) and human serum serum albumin (HSA).

Each operation in the biochemical actions of the exemplary embodiment is followed by a three-stage wash - first wash with a 1% solution of sodium dodecyl sulphate (SDS) and two wash steps with double-distilled and deionized water spray. Immediately followed by methyl alcohol drying (CH ₃ OH) and Measurement of changes in masses of the sensor micro-consoles by the method and apparatus according to G51.

A biochemical protocol was used in which the first operation of the Activation Action is carried out with a first step flowing in a reaction volume of 25μΙ of an activation solution containing 1% of beta-mercaptoethanol (β-mercaptoethanol - BME) followed by a second stage flowing in a reaction volume of 25µΙ reducing solution of Tris (2-carboxy-ethyl) phosphine hydrochloride (TCEP) at 5mM concentration. The second operation (the construction of the basic protein layer) is carried out / is carried out in a reaction volume of 30μΙ (15μΙ + 15μΙ) by polymerization surface reaction between two proteins: 1) monomeric outer membrane purine multiplied by plasmid pOR-LA18 E.coli BL21 (DE3); 2) Z-domain of Staphylococcus aureus protein A (SpA), at a combined concentration of 0.2 and 0.5 mg / ml, respectively. Sufficient polymerization time at 37 ° C is 20 minutes.

The first operation of the Functionalizing Action (creating an activated bonding layer) was carried out for 10 minutes at 37 ° C in a reaction volume of 25μΙ of a 1% solution of n-Octylglucoside to which 0.25mg / ml HS- (CH ₂ ) was added. _ir (OCH ₂ CH ₂ ) ₃ -OH (trioPEG) which solution is activated during its preparation by raising the temperature to 55 ° C and subsequent natural cooling to 37 ° C. The second operation (antibody fixation) was performed for 20 minutes at 37 ° C in a reaction volume of 20μΙ containing phosphate buffer diluted to 1: 100 antibody preparation in this exemplary embodiment of Rabbit IgG against HSA antibodies.

The Immune Reaction Action is carried out in a reaction volume of 10μΙ of test serum containing human albumin (HSA) prepared / processed according to its origin. The reaction temperature and time are experimental variables that are selected each time according to the objectives of the study. The reaction was terminated by a stream of cooled deionized water at a temperature below 10 ° C.

The restorative action of the sensor is accomplished by washing with a gentle jet of 2.5M citric acid solution (pH 3.2) for 2-3 minutes.

A MEMS sensor with a common base size of 4900x3200x250 pm is used, and microcontrolls with 310 and 325 pm lengths, 76 pm widths and 3.5 pm thicknesses. The area of each of the active sites is 50x50 pm with a deposited layer of gold (Au) with a thickness of ~ 0.4 pm. The elasticity of the microconsoles is about ~ 3.5 N / m. The resonance frequencies of the four microconsoles are in the 50-80 kHz range (average -65kHz). Piezo resistors are on

1.5 kOhm ± 10% with a maximum signal level of ~ 1mV / V / pm. The temperature change of the resistance of the piezo resistors, measured after 24-bit accuracy after calibration, is used to determine the individual microcontroller temperatures and the flow temperature of the biochemical reactions, achieving an accuracy better than ± 0.1 ° C.

The carrier is a Teflon (PTFE) board measuring 25x10x0.8 mm, with nine (1 base and 8 piezo resistors) electrical outlets - gold-plated contact points at the rear. The micro-console structure of the sensor is soldered to its underside, through unleaded SMD solder, to a gilded pad (smaller than the underside) at its front end. The gold-plated pads on the upper side of the micro-console structure (piezo resistor terminals) are bonded with gold (Au) wires to the gold plated bonding pins connected to the gold plated pins at the rear end of the carrier. The naked metal resulting from this bonding (Au gold) is covered with a chemically inactive insulating layer (silicone solder mask - “240 SB”).

The carrier is inserted into a 9-pin, gas-tight plug, mounted inclined at both X and Y, at angles of 30 °, so that the jet flows from the front left corner when flushed. On the hydrophobic surface of the carrier, a hydrophilic zone with a droplet shape and an area of 20mm ² was created, by means of local electrochemical treatment [12], which ensures the use / fixation of convenient working volumes of 10 - 30μΙ.

The excitation of the oscillations of the micro-consoles is performed by an ultrasonic capacitive piezo radiator (USP-1nF), tightly pressed against the carrier for good acoustic coupling, which emits simultaneously, but with different amplitude, all resonances and frequencies.

The instrumentation [5] measures the resonant frequencies of the sensor to an accuracy of 0.56 ppm, which is determined only by the unbroken natural phase noise (-114dBc / Hz @ ~ 65kHz) of the microcontroller oscillations. With automatic repetitions, the accuracy can be increased to -0.1 ppm (~ 10 ' ⁷ or ~ 7mHz @ ~ 65 kHz). The sensor microcontrollers change the resonant frequency by about 3 Hz by increasing their mass by 10 ^'15 grams - ie. have a sensitivity of - 3 Hz / fg (- 3.3 10 ^'16 g for 1Hz), which means that a sensitivity of 2.3 10' ¹⁸ g (7mHz at 3 Hz / fg) is achievable.

The molecules of the antibodies used - the monomeric immunoglobulin “Rabbit IgG against HSA” have an approximately triangular shape with sides of -15 nm and a thickness of -4 nm. They attach at an angle of -45 ° to the surface of the active regions, covering an area of -140 nm ² , but are spaced apart. Only about 1.8 10 ⁶ pieces are attached to the area of 50x50 pm on each of the active sections, directly covering only about 10% of their area.

The Rabbit IgG against HSA monomeric immunoglobulin mass was -150 kDa (-2.5 10 ^'19 g) and the human albumin antigen HSA mass was 67 kDa (1.1 10' ¹⁹ g). The IgG + HSA immune response investigated created a new molecular structure with a mass of -217 kDa (-3.6 10 ^'19 g) - i. increases the mass by - 44%.

For the molecules of the exemplary embodiment, a maximum sensitivity of 2.3 10 ^'18 g means an accuracy of ± 9 antibodies and ± 20 protein molecules.

Random statistical fluctuations in the number of antibodies attached (in the second method) are only ± 0.075% or ± 3 10 ^'16 g (with a full coverage of-1.8 10 ⁶ antibodies), which is 130 times the resolution of the exemplary embodiment (2.3 10 ^'18 d) - i.e. the limitations of the invention are fundamental natural boundaries.

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Claims (8)

COPYRIGHT CLAIMS An immunometric analysis method using a universal, replaceable, reusable and self-calibrating sensor according to claim 2, characterized in that it consists of five actions, the first four of them being biochemical in nature and carried out in a precise reaction micro volume fixed as capillary drop at the exact location (9) on the sensor carrier (7), with the first action chemically activating the surfaces of the active sections (3) of the measuring pair of microconsoles (1), the second action creating an active point on the active sections (3) a sensible, one-off for each implementation of the method, functionalization of the sensor, in the third action in the reaction micro volume, the analyzed immune reaction is carried out with accurate keeping of temperature and with accurate reading of the elapsed reaction time, and the fourth action removes the residues from the first three actions and restores the sensor to its original state, each of the biochemical actions being followed by a single measurement [5] of the sensor, the measurement results of which relate to that biochemical action, such as p the results for the fourth action serve as a control, and the results for the first three biochemical actions are used in the fifth action in a calculation procedure for unknown parameters of the immune response, which uses as a standard the constant ratio of the resonance frequencies of the two microconsoles from the reference pair of microconsoles (2) , whose masses are not altered by biochemical actions because their surface is not suitable for attachment of biomolecules and / or microscopic bio particles. A sensor used in the method of claim 1, representing a micro-console structure (10) on a carrier (7), characterized in that it consists of four micro-consoles grouped in two identical pairs of micro-consoles (1 and 2) created simultaneously in the same production process, each of the pairs of micro-consoles (1 and 2) consists of two different micro-consoles differing only in their length, which difference in length is sufficient to provide a difference in the resonant frequencies excluding the acoustic interplay between the micro-consoles, such as all identical pairs of micro-brackets (1 and 2) are symmetrically arranged at a suitable distance on a common base (6), with only one of the pairs of micro-brackets (2) being produced at the same place on the surfaces of both its micro-brackets. , by selectively adding one or more additional layers of suitable materials, two completely identical active regions (3), the surface of which regions (3) are suitable for attaching biomolecules and / or microscopic bioparticles of interest, while the rest of the surface this two The microconsoles (2) and the entire surface of the two microconsoles of the other pair of microconsoles (1) are manufactured in such a way that they are not suitable for attaching biomolecules and / or microscopic bioparticles of interest, with each of the four microconsoles having a built-in piezoresistor ( 4) a manufacturing signal proportional to its bending, which piezoresistors (4) have two electrical terminals connected to isolated contact pads (5) on the upper surface of the common base (6), and the electrical output of the conducting common base (6) is erected on an insulated contact surface on the lower surface of the common base (6), the micro-console structure (10) being disposed on a support (7) which is a flat replaceable element of suitable shape, a hydrophobic surface and contact feathers (8) being inserted into electrical a clutch contacting the feathers (8) having contact pads electrically connected to the contact feathers (8), to which the contact surfaces are soldered and the common base (6) is mechanically attached and the terminals (5 are connected to the other contact surfaces) ) by piezoresist (4), whereby a hydrophilic zone (9) of a suitable shape and size above which the pairs of microconsoles (1 and 2) are located and on which Q is a hydrophilic zone (9) is created on the hydrophobic surface of the carrier (7) in an appropriate place. to retain by the capillary forces the working volume of the fluid in which the chemical reactions of the process of claim 1 proceed. A sensor according to claim 2, comprising a micro-console structure (10), characterized in that each of the four micro-consoles of its two micro-console pairs (1 and 2) is made up of three conductive structural elements forming an elongated letter "P" with two insulated electrical terminals (5) at the two ends of the letters and active sections (3) arranged on the transverse members of one pair of microcontroller (2), producing a signal proportional to their bending as current flows through the three structural elements; therefore n and the bending of the entire micro-console, with which the structural elements also function as piezoresistors. A sensor according to claim 2, characterized in that a layer of magnetic material and a subsequent insulating layer, such as the layer, are additionally applied to all four microconsoles of the pairs of microconsoles (1 and 2), to their entire surface or only part of it. of magnetic material, in an alternating magnetic field at an appropriate frequency, creates a variable force by which the resonant oscillations of the microconsoles are excited. A sensor according to claim 2, characterized in that one isolated conductive auxiliary path is arranged along the surface of all four microconsoles of the pairs of microconsoles (1 and 2) in such a way that an alternating current flowing therefrom at an appropriate frequency into one suitable constant magnetic field, creates a variable force with which to excite the resonant oscillations of the microconsoles. A sensor according to claim 2, characterized in that the excitation of the resonance oscillations of its four microconsoles is carried out by an acoustic wave emitting a separate device in acoustic connection with the carrier (7) and through it in acoustic connection with the microconsole structure (10). A sensor according to claim 2, comprising a micro-console structure (10) on a support (7), characterized in that the electrical terminals of the piezo-resistors (4) are connected to contact pads on the lower surface of the common base (6) by isolated vertical electrical connections by contacting the pads on the lower surface directly with the corresponding pads located on the upper surface of the carrier (7), thereby electrically connecting the piezo resistors (4) to the electrical terminals from the carrier (8) and mechanical attachment of the common base (6) to the carrier (7). A sensor according to claim 2, comprising a micro-console structure (10), characterized in that the electrical terminals of the piezoresistors (4) are partially connected to each other to form a "star" circuit with one common terminal and one individual terminal from each piezoresistor ( 4).