Immunochromatographic test strip for multiplex analysis

Abstract

The utility model is designed for the rapid (up to 20 minutes) detection of at least 10 types of analytes in environmental samples (water, air, soil, etc.) and clinical specimens. The immunochromatographic test strip for multiplex analysis includes a polymer substrate with an applied immunochromatographic membrane and a capture pad. The test and control zones of the test strip are designed as two rows of transverse microstrips occupying no more than half the width of the test strip. The test strip includes a marker for coordinating the cutting of test strips. The technical result consists in reducing the time for recording the luminescent signal from test strips in an automated system. 3 c.p. phyl., 3 fig., 2 tables.

Description

[0001] The utility model relates to devices for the immunochromatographic determination of a wide range of analytes, including pathogens of infectious diseases and toxins of bacterial and plant origin, and can be used in healthcare, biotechnology and immunology.

[0002] The claimed immunochromatographic test strip design enables multiplex analysis for the detection of viral antigens, microorganisms, toxins, and other biologically active compounds in biological fluid and water samples. The utility model is intended for use in portable immunochromatographic assay devices based on the recording of a luminescent signal from the surface of test strips during their two-dimensional movement.

[0003] The patented object is the design of a multiplex immunochromatographic test strip designed for the simultaneous detection of up to 20 types of analytes (biopathogens) in a sample. Variations in the design of the test strip's analytical reagent microstrips allow it to be used to expand the dynamic range of analytes detected.

[0004] Multiplex test strips for the immunochromatographic determination of analytes are known (Zhang X. et al. / / Journal of Agricultural and Food Chemistry. - 2017. - Vol. 65. N. 36. -P. 8063-807; Kong D. et al. / / Nanoscale. - 2016. - Vol. 8. N. 9. - P. 5245-5253) in the form of cartridges of several test strips, including a holder (a plastic frame with windows for introducing a sample and reading an optical signal). The test strip contains a substrate for laminating porous materials, on which a filter pad for applying the test sample, a pad for applying the conjugate, a porous membrane and an absorption pad are located sequentially along the length and with an overlap. The conjugate application pad is impregnated with colloidal gold particles. The number of analytes detected on a single test strip is determined by the number of analytical zones located transverse to the flow of the liquid (the sample being tested).The assay zones are biospecific binding reagents (antigens, antibodies, DNA probes, etc.) applied as a narrow strip 0.5-1 mm wide at least 2 mm apart. Typically, the number of assay zones on a single test strip does not exceed four.

[0005] There are known designs in which nanoparticles with europium ion complexones are used as a developing reagent in test strips instead of colloidal gold conjugates (Chen K. et al. / / Int J Environ Res Public Health. - 2021. - Vol. 18. N. 9. - P. 4574. doi: 10.3390 / ijerphl8094574; Valanne A. et al. / / J. Clin. Virol. - 2005. - Vol. 33. - P. 217-223.; Jaakohuhta S. et al. / / Int. J. Food Microbiol. - 2007. - Vol. 114. - P. 288-294). In this case, the signal from the test strip is recorded in the time-resolved luminescence mode using specialized devices - time-resolved fluorimeters.

[0006] The time-resolved mode provides increased sensitivity by allowing detection of lower concentrations of fluorescent marker than with photometric detection of the signal from colloidal gold particles.

[0007] In known analogs, test strips contain three to four analytical zones located transverse to the direction of the immunochromatographic flow of the sample liquid. The test strip design includes a housing, which is a plastic frame with a window for sample introduction and windows for viewing results. Inside, the housing contains a substrate, a pad for applying the liquid sample and conjugate, a filter pad, a porous chromatographic membrane, and an absorption pad.

[0008] The pad for applying the sample and conjugate is made of cellulose or glass microfiber, or polyester microfiber with a pore size of 20-25 μm and an absorption capacity of at least 55 mg / cm 2 .

[0009] The filter pad is made of porous polysulfone material with a layer thickness of 400-500 μm and a pore volume of 55-57 μl / cm 2 .

[0010] The porous membrane is made of nitrocellulose or polyethersulfone or nylon with a layer thickness of 80-150 μm.

[0011] The absorption pillow is made of porous cotton fluff material or glass cellulose fiber with a layer thickness of 700-900 microns.

[0012] The laminating substrate is made of polystyrene or acrylic adhesive material with a layer thickness of 230-270 μm.

[0013] Biospecific immunoreagents are applied to the surface of the porous membrane in the form of analytical and control transverse stripes.

[0014] The choice of materials and their characteristics is not limited to the specified parameters, but is determined as a result of optimization for specific types of desired analytes.

[0015] These technical elements taken together are necessary and sufficient for the proper functioning of the immunochromatographic cartridge.

[0016] However, this design has limitations in both multiplexity and sensitivity when detecting extremely low analyte concentrations. For example, the application of analytical (test) zones as transverse stripes significantly limits the ability to accommodate a larger number of analytical zones. Furthermore, the inclusion of a developing reagent (a conjugate of nanoparticles with a biospecific component, such as antibodies) in the test strips does not completely eliminate conjugate interference outside the analytical microzones. This limits the sensitivity of the assay, as approximately 4% of the sample and conjugate mixture is distributed on the membrane surface, creating a luminescent background that limits the detection of low analyte concentrations.

[0017] Test strip designs are known that offer increased multiplexity and are not limited to 3-4 analytes. To enhance multiplexity, the assay zones are designed as microdots with a diameter of 0.5-1 mm. Colloidal gold nanoparticles, located in the starting zone of the test strip on a separate absorbent pad, are used as the developing (detecting) system. These nanoparticles are located in the starting zone of the test strip on a separate absorbent pad. Such designs, consisting of a single test strip and a pad with colloidal gold nanoparticles in the starting zone, have limitations in multiplexity and assay sensitivity.

[0018] Increasing the multiplexity of analysis on these test strips due to a more dense arrangement of microzones requires the use of developing reagents as a mixture for each analyte or as a universal multipurpose reagent at a higher concentration than required for analyzing only one analyte. This inevitably reduces the sensitivity of the analysis due to increased background from unbound reagent. The inclusion of a starting absorbent pad in the design of these test strips eliminates the need to wash the strips to completely remove developing reagents.

[0019] The closest analogue (prototype) to the claimed utility model is a cartridge test strip design with microzones of analytical reagents applied as spots 0.3-0.7 mm in diameter, without a starting pad. The chromatographic membrane at the starting end of the test strip is immersed directly in the sample. The prototype design (a positive decision on a utility model patent for application No. 2024118733 dated July 4, 2024) is intended for use in luminescence scanners with time-resolved luminescence for high-throughput analysis of multiple samples for the presence of at least 10 types of pathogens, in particular in automated immunochromatographic analysis systems (Avtomat-IKh, TU 26.51.53.141-306-05031637-2023, FSUE GosNIIBP). Test strips, in which the analytical and control zones are designed as microzones, are cost-effective and ensure low consumption of biomaterials during their manufacture.However, to scan the luminescent signal from such test strips, it is necessary to perform two-coordinate movement with a very high spatial resolution of the scanning system, which increases the cost of the design of the optical recording unit and complicates their use in simple single-coordinate scanning devices.

[0020] Furthermore, two-axis motion systems take several times longer to scan than single-axis scanners. Specifically, two-axis scanning of a cartridge of four test strips in the automated Avtomat-IKh system takes at least 3 minutes, or 45 seconds per test strip, limiting the automated analysis throughput to 20 samples per hour. By reducing the time required to scan a test strip to 15-20 seconds, throughput can be increased by 2-2.5 times, to 40-60 samples per hour, while maintaining the specified high level of multiplexity.

[0021] The prototype design in the form of microzones with a diameter of 0.3-0.7 mm cannot be used in devices in which the scanning of test strips is carried out only along one coordinate along the test strip, since the area of ​​illumination of the microzone by the scanner beam is comparable to the size of the microzone and even small geometric deviations in the installation of the test strip in its frame and in the device lead to noticeable errors in the registration of the luminescent signal.

[0022] The proposed utility model design allows for a reduction in the luminescent signal recording time from test strips in an automated system when switching to a single-axis scanning mode, without significantly reducing testing multiplexity. This utility model doubles the analysis multiplexity of test strips used in devices with single-axis longitudinal scanning that have the ability to shift the scan line, such as the IFI-03, IFI-05, and IFI-06 manufactured by IMMUNOSKRIN LLC.

[0023] The test strip is designed as a test strip (an immunochromatographic membrane on a polymer backing) 4-6 mm wide with an absorbent pad 10-20 mm long. The test strip contains test (analytical) and control zones made of biological material in the form of transverse microbands 0.3-0.8 mm thick and 1.5-2.5 mm long, arranged one below the other in two rows with a distance of 2.5-3 mm between the strips. The distance between adjacent analytical microbands should be greater than 1.5-2 times the diameter of the scanning light beam, ensuring reliable separation of signals from individual microbands. The design of the test strips includes an additional element - a microzone with a diameter of 0.1-0.2 mm, which is located between the control micro strips and the capture pad and consists of a luminescent reagent or a perforated hole in the membrane of the same diameter.The microzone is designed for precise cutting of test strips from commercial source material, manufactured as immunochromatographic strips 300 mm long and 50-80 mm wide. The type of luminescent dye is not critical. Cutting accuracy (the precise positioning of the microbands on the test strip), which is ensured by referencing the microzone coordinate, is crucial for precise cutting of the test strips and subsequent scanning of the luminescent signal of the test and control zones along the center of the microbands containing the analytical reagents. The luminescent microzone or opening in the membrane serves as a reference for optical sensors, ensuring precise cutting of the test strips.

[0024] To expand the dynamic range of analyte detection in samples with widely varying concentrations, the design utilizes microbands of the same antibodies arranged one above the other. In this case, the analyte concentration in the sample is estimated by the decrease in the luminescence signal in the microbands arranged one above the other.

[0025] Fig. 1 shows the appearance of the test strip. The test strip includes a laminated substrate - 1 with a coated chromatographic membrane composite of nitrocellulose - 2, a catching "pad" - 3, as well as a microzone - 4 (the location of the luminescent marker or microholes, control (pos. 5, 6) and analytical microbands (pos. 7-16), located along line A (left row of microbands) and line B (right row of microbands).

[0026] The luminescent microzone (mark) or perforated hole is used only in the production (coordination of cutting) of test strips.

[0027] Scanning the fluorescent signal from the test strip is carried out along line A and line B.

[0028] The test strip can be placed in a plastic frame, which must have a window for scanning the micro strip area and the starting end of the test strip must protrude at least 5-10 mm from the frame for incubation with the sample.

[0029] Fig. 2 graphically shows the results of scanning the luminescent signal from the surface of the membrane composite of the proposed test strip.

[0030] Fig. 2 illustrates the possibility of detecting 10 analytes (microorganism antigens and their toxins) using the example of detecting two biopathogen imitators (brucellosis) and botulinum toxin type B in a sample.

[0031] The abscissa axis shows the locations of the microbands on the test strip along line A and line B. The control microband zones include sheep anti-mouse (pos. 17) and sheep anti-rabbit (pos. 18) antibodies. Along line A are analytical microbands for detecting antigens of the microorganisms that cause plague - 19, anthrax - 20, tularemia - 21, glanders and melioidosis - 22, and brucellosis - 23.

[0032] Along line B there are analytical micro strips for detecting bacterial toxins botulinum type A - 24, botulinum type B - 25, botulinum type E - 26, cholera - 27, staphylococcal enterotoxin type B - 28.

[0033] The luminescent signal (relative units) when scanning the test strip along line A is shown as curve 29, when scanning along line B is shown as curve 30.

[0034] Fig. 3 graphically shows the results of scanning the luminescent signal from the surface of the membrane composite test strip designed to detect 10 low-molecular-weight narcotic drugs in a competitive immunoassay.

[0035] Fig. 3 illustrates the results of analysis on the proposed design of test strips of a urine sample containing low-molecular compounds in the form of two narcotic drugs: amphetamine and fentanyl.

[0036] The abscissa axis shows the locations of the micro strips on the test strip along line A and line B. The areas of the control micro strips K1, K2 include anti-mouse sheep antibodies - 31 along lines A and B. Along line A are analytical micro strips of conjugates of bovine serum albumin with narcotic compounds for the detection of morphine (MOR) - 32, benzoylecgonine (BZE) - 33, amphetamine (AMP) - 34, methamphetamine (mAMP) - 35, methadone (MTD) - 36; Along line B there are analytical micro strips of conjugates of bovine serum albumin with narcotic compounds for the detection of benzodiazepine (BZD) - 37, barbiturates (BAR) - 38, Δ9-tetrahydrocannabinol (THC) - 39, fentanyl - 40, cocaine - 41.

[0037] The luminescent signal (relative units) when scanning the test strip along line A is shown as curve 42, when scanning along line B is shown as curve 43.

[0038] The performance of the proposed test strip design is confirmed using the example of a test strip for determining protein antigens of biopathogens (bacterial group pathogens and their toxins) and a test strip for determining narcotic drugs.

[0039] The reagents used to prepare test strips and conduct the analysis to detect protein antigens of biopathogens (bacterial agents and their toxins) in samples and their working concentrations are presented in Table 1.

[0040] The reagents used to prepare test strips and conduct the analysis to determine narcotics in urine samples and their working concentrations are presented in Table 2.

[0041] The procedure for setting up an analysis for the detection of biopathogen imitators using the proposed test strip design included adding a mixture of conjugates of luminescent nanoparticles with antibodies to the desired pathogens in a volume of 50 μl, containing 10, to a well with a sample volume of 100 μl. 8 Nanoparticles in the sample were detected, the free end of the test strip was immersed in a well containing a 150-µl sample, incubated for 5 minutes, the test strip was transferred to a well containing assay buffer for 10 minutes, and then transferred to the luminescence signal recording unit. All operations were performed in an automated immunochromatographic analysis system (Avtomat-IKh).

[0042] The sample contained the F1 fraction of the plague microbe at a concentration of 10 ng / ml of water and 10 ng / ml of staphylococcal enterotoxin type B.

[0043] The results of testing bacterial biopathogens are shown in Fig. 2.

[0044] When scanning the test strip along line A-29, luminescence signals are detected at the locations of the brucellosis microstrip (item 23), and when scanning along line B-30, in the botulinum toxin type B microstrip (item 25), indicating their presence in the sample. High signal levels in control zones K1-17 and K2-18 indicate the passage of the reaction mixture across the membrane and the correctness of the analysis.

[0045] The procedure for setting up an analysis for the detection of narcotic drugs using the proposed design of the test strip included adding a mixture of specific mouse monoclonal antibodies to narcotic drugs in a volume of 50 μl to a well with a urine sample of 100 μl, immersing the free end of the test strip in the sample and incubating for 5 minutes, then transferring the test strip to a well containing luminescent nanoparticles coated with anti-mouse sheep antibodies in a volume of 100 μl in an amount of 10 9Nanoparticles. The test strip was incubated for 5 minutes and transferred to a well containing assay buffer, followed by another 5 minutes of incubation. The test strip was then transferred to the luminescence signal recording unit. All operations were performed automatically using an automated immunochromatographic assay system (Avtomat-IKh). The urine sample contained 80 ng / ml amphetamine and 50 ng / ml fentanyl.

[0046] Competitive analysis involves the binding of specific antibodies to a microband and their subsequent detection in this microband using luminescent nanoparticles coated with anti-mouse antibodies. If drugs are present in the sample, specific antibodies compete for binding between the sample drugs and the drug-analog material in the microband, resulting in a decrease in their binding level in the microband and, consequently, a decrease in the luminescent signal.

[0047] The results of testing for the presence of 2 drugs in a urine sample are shown in Figure 3.

[0048] Figure 3 shows the scanning curves along line A - 42 and line B - 43 when recording the luminescent signal from a test strip with a sample containing two types of narcotics: amphetamine (item 34) and fentanyl (item 40). When scanning the test strip, the luminescence signals decrease at the locations of the microstrips with conjugates to amphetamine and fentanyl, indicating the presence of these narcotics in the urine sample.

[0049] High signal levels in the control zones - 31 indicate that the reaction mixture has passed through the membrane and that the analysis is set up correctly.

[0050] Compared with the prototype, the presented test strip design can reduce the signal reading and processing time.

[0051] The test strip design allows for insertion into the wells of a microplate and immersion of the free end of the test strip into the sample volume in the well, followed by incubation of the test strips with the sample and reagents in the wells of a 96-well microplate. The test strips have only one capture pad and do not contain pads for the sample or conjugate. This test strip design allows for sample incubation in the wells of the microplate, followed by a pre-wash step (incubation of the test strips with assay buffer) to remove unbound luminescent nanoparticles in the microplate wells. The pre-wash step is only possible after the test strip has been transferred from the wells of the microplate containing the samples and developing reagents to the wells containing the assay buffer.The absence of a sample and conjugate pad in the test strip allows for the complete removal of luminescent nanoparticles from the free volume of the membrane composite within 5 minutes, thereby increasing the signal-to-background ratio by more than 10 times and raising the threshold level for detecting biopathogens several times over compared to a traditional design containing a conjugate pad and sample introduction.

[0052] For multiplex detection of one or more biopathogens using a test strip, various versions of the developing reagent can be used:

[0053] - in the form of a mixture of monospecific luminescent nanoparticles;

[0054] - in the form of luminescent nanoparticles conjugated with antibodies to all pathogens detected using a test strip;

[0055] - in the form of mixtures of biotinylated antibodies and one universal detection reagent—luminescent nanoparticles conjugated with streptovidin. All of these specific immunoreagents enable the detection of at least 10 types of biopathogens in a sample using the proposed test strip design.

[0056] In the proposed test strip design, immunoreagents are applied to test strips using a contactless nanoplotter manufactured by IMMUNOSKRIN LLC. The antibody concentration is 2 mg / ml, the amount per microstrip is 100 nl, the strip width is 1.6 mm, and the strip thickness is 0.5-0.6 mm. The distance between the microstrips along the test strip is 3 mm.

[0057] The number of analytical micro strips on the test strip is not limited to 10 and can be increased to 18 by increasing the longitudinal scanning zone to 30 mm, since in order to resolve the luminescent signals from each micro strip, it is necessary that the density of micro strips does not exceed one micro strip for every 2.5-3 mm of the test strip when using the commercially available automated immunochromatographic analyzer "Avtomat-IKh" (utility model RU No. 230227) as a recorder.

[0058] The utility model significantly simplifies the design requirements for scanning systems by forming analytical and control zones in the form of transverse microstrips, since scanning is performed only along the test strip. Moreover, the devices (IFI-03, IFI-05, IFI-06, "Avtomat-IKh") have an option for scanning test strips along lines A and B, as shown in Fig. 2 and Fig. 3. The transition from microdots to microlines eliminates scanning along the second coordinate, which reduces the signal recording time from the test strip several times, maintains the level of analysis multiplexity, but at the same time simplifies the optical system of the device, in particular, reduces the requirements for the level of light focusing on the test strip, simplifies signal processing, allows for a reduction in the weight and dimensions of the recording devices and a decrease in their cost. The proposed test strip design expands the functionality of the immunochromatographic analysis and meets the criterion of novelty.

[0059]

[0060]

[0061] Note to Tables 1 and 2:

[0062] - antibodies were immunoglobulins of class G, isolated by affinity chromatography on protein - PrG Sepharose or protein - G Sepharose.

[0063] The utility model can be used in medical clinical diagnostics, in laboratory practice for sanitary and epidemiological services, and in law enforcement agencies to ensure biological protection of troops and the population. Therefore, the utility model meets the criteria for industrial applicability.

[0064] The utility model (test system) is a consumable and is used in the operation of an automated immunochromatographic analysis system (patent for utility model RU No. 230227 “Automated immunochromatographic analyzer”).

Claims

1. An immunochromatographic test strip for multiplex analysis, comprising a polymer substrate with an applied immunochromatographic membrane and a capture pad, characterized in that the test and control zones of the test strip are made in the form of two rows of transverse micro strips occupying no more than half the width of the test strip, and includes a marker for coordinating the cutting of the test strips.

2. An immunochromatographic test strip according to paragraph 1, characterized in that the test and control zones of the test strips are made of biological materials in the form of transverse strips 1.5-2.5 mm wide and 0.3-0.8 mm thick, placed one under the other in two rows with a distance in the row between the strips of 2.5-3 mm.

3. An immunochromatographic test strip according to paragraph 1, characterized in that the marker for cutting test strips is made in the form of a perforation hole on the test strip in the area between the control micro strips and the catching pad with a diameter of 0.1-0.2 mm.

4. Immunochromatographic test strip p. 1, characterized in that the marker for cutting test strips is made in the form of a microdot with a diameter of 0.1-0.2 mm made of luminescent material in the area between the control microstrips and the catching pad.

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