阅读说明：本技术 表面和扩散增强的生物传感器 (Surface and diffusion enhanced biosensor ) 是由 全珍雨 黄惠珍 朴莲洙 金起范 于 2019-04-22 设计创作，主要内容包括：所公开的技术总体上涉及被配置用于免疫测定的生物传感器,并且更具体地涉及具有被配置成增强免疫测定的速度和灵敏度的结构的生物传感器,并且涉及测试试剂盒及使用该种测试试剂盒的免疫测定的方法。在一个方面,适用于酶联免疫吸附测定(ELISA)的传感器组件包括传感器条,该传感器条包括形成在该传感器条中的一个或更多个凹孔。该传感器组件附加地包括一个或更多个检测结构,该一个或更多个检测结构连接至一个或更多个凹孔中的每个凹孔的侧壁,其中该一个或更多个检测结构被配置成将生物分子直接固定化在该一个或更多个检测结构上。(The disclosed technology relates generally to biosensors configured for immunoassays, and more particularly to biosensors having structures configured to enhance the speed and sensitivity of immunoassays, and to test kits and immunoassay methods using such test kits. In one aspect, a sensor assembly suitable for use in an enzyme-linked immunosorbent assay (ELISA) comprises a sensor strip comprising one or more wells formed in the sensor strip. The sensor assembly additionally includes one or more detection structures connected to a sidewall of each of the one or more wells, wherein the one or more detection structures are configured to immobilize a biomolecule directly on the one or more detection structures.)

1. A sensor assembly suitable for use in an enzyme-linked immunosorbent assay (ELISA), the sensor assembly comprising:

a sensor strip comprising one or more wells formed in the sensor strip, each well of the one or more wells having a side wall and a bottom surface; and

one or more detection structures connected to the sidewall of each of the one or more wells,

wherein the one or more detection structures are configured to immobilize a biomolecule directly on the one or more detection structures.

2. The sensor assembly of claim 1, further comprising the biomolecule immobilized directly on the one or more detection structures.

3. The sensor assembly of claim 2, wherein the biomolecule comprises an antibody configured to specifically bind to a target analyte of the enzyme-linked immunosorbent assay.

4. The sensor assembly of claim 1, wherein each of the one or more detection structures extends laterally toward a central region of a respective one of the one or more wells.

5. The sensor assembly of claim 1, wherein the one or more detection structures comprise one or more first detection structures formed at a first vertical level in a depth direction of the one or more recessed holes.

6. The sensor assembly of claim 5, wherein the one or more detection structures include one or more second detection structures formed at a second vertical level in the depth direction that is deeper than the first depth.

7. The sensor assembly of claim 6, wherein at least a portion of the one or more first detection structures does not overlap the one or more second detection structures in the depth direction of the one or more recessed apertures.

8. The sensor assembly of claim 1, wherein the sensor strip comprises a plurality of layers each having an opening formed therethrough, wherein at least one of the layers comprises the one or more detection structures protruding from a sidewall of the opening.

9. The sensor assembly of claim 8, wherein the sensor strip comprises at least two layers including one or more detection structures vertically separated by a spacer layer.

10. The sensor assembly of claim 1, wherein the one or more detection structures comprise a wave that protrudes laterally toward a central region of a respective one of the one or more wells and is elongated vertically along a depth of the respective one of the one or more wells.

11. The sensor assembly of claim 1, wherein the one or more detection structures comprise the following surfaces: the surface is textured to have a plurality of micro-or nanostructures formed on the surface.

12. The sensor assembly of claim 1, wherein each of the one or more detection structures comprises a plate structure disposed laterally at a central region of a respective one of the one or more wells.

13. The sensor assembly of claim 1, wherein the one or more detection structures comprise at least two substantially parallel plate structures that substantially overlap each other in a depth direction of the one or more recessed wells.

14. The sensor assembly of claim 13, wherein vertically adjacent ones of the at least two plate structures are separated from each other in a depth direction of the one or more recessed holes by a distance of between 500 microns and 8 millimeters.

15. The sensor assembly of claim 1, wherein the one or more recesses are cylindrical recesses having a flat bottom surface.

16. The sensor assembly of claim 15, wherein each of the wells is configured to hold a sample having a volume of about 50 μ L to about 500 μ L.

17. The sensor assembly of claim 15, wherein the one or more recessed holes have a diameter of between about 2mm and about 9 mm.

18. The sensor assembly of claim 1, wherein, when the one or more wells are filled with a sample such that the one or more detection structures are immersed in the sample, a ratio of a combined surface area in contact with the sample to a volume of the sample is about 0.25mm²mu.L to about 8mm²/μL。

19. A sensor assembly suitable for use in an enzyme-linked immunosorbent assay (ELISA), the sensor assembly comprising:

a cuvette including a cavity;

a cover configured to enclose the cavity; and

one or more detection structures connected to the lid and configured to be at least partially immersed in a liquid sample when present in the cavity,

wherein the one or more detection structures are configured to immobilize a biomolecule directly on the one or more detection structures.

20. The sensor assembly of claim 19, further comprising the biomolecule immobilized directly on the one or more detection structures.

21. The sensor assembly of claim 19, wherein the biomolecule comprises an antibody configured to specifically bind to a target analyte of the ELISA.

22. The sensor assembly of claim 19, wherein each of the one or more detection structures comprises a plate structure having opposing major surfaces that are substantially parallel to a depth direction of the cavity of the cuvette.

23. The sensor assembly of claim 19, wherein each of the one or more detection structures comprises a plate structure having opposing major surfaces that are substantially parallel to each other.

24. The sensor assembly of claim 19, comprising two or more detection structures, wherein major surfaces of directly adjacent detection structures of the two or more detection structures that directly face each other are separated from each other by a distance of between about 500 microns and 8 millimeters.

25. The sensor assembly of claim 19, comprising two or more detection structures, wherein major surfaces of directly adjacent detection structures of the two or more detection structures that directly face each other are substantially parallel to each other.

26. The sensor assembly of claim 19, wherein each of the one or more detection structures comprises a plate structure having a straight edge extending in a depth direction of the cavity, wherein the straight edge comprises one or more recessed regions that reduce a width of the plate structure.

27. The sensor assembly of claim 19, wherein at least one of the one or more detection structures comprises the following surfaces: the surface is textured to have a plurality of micro-or nanostructures formed on the surface.

The sensor assembly of claim 19, wherein the cuvette is configured to accommodate a cuvette having a diameter of between about 50mm³And about 3000mm³The volume of liquid in between.

28. The sensor assembly of claim 19, wherein, when the cavity is filled with a sample such that the one or more detection structures are immersed in the sample, a ratio of a combined surface area in contact with the sample to a volume of the sample is about 0.1mm²mu.L to about 8mm²/μL。

Technical Field

FIELD

The disclosed technology relates generally to biosensors configured for immunoassay technology, and more particularly to biosensors having structures configured to enhance the speed and sensitivity of immunoassay technology, as well as to test kits and methods of immunoassay technology using the same.

Background

ELISA (enzyme-linked immunosorbent assay) is an assay technique for the detection and quantification of target analytes, which may include substances such as peptides, proteins, antibodies, and hormones. In an ELISA, an analyte of interest (e.g., an antigen) is immobilized on a solid surface and then complexed with an enzyme-associated reagent (e.g., an antibody). Detection is accomplished by assessing conjugated enzyme activity via incubation with substrate (substrate) to produce a detectable reaction product.

Disclosure of Invention

The disclosed embodiments are generally directed to a biosensor assembly with enhanced sensitivity and faster detection of target analytes. The biosensor assembly according to the embodiment is configured such that the concentration of immobilized reactants or antibodies is increased, thereby increasing the speed of various ELISA techniques. The biosensor assembly according to embodiments may also reduce the hook effect, which is a disadvantage of the currently known one-step ELISA technique. The disclosed embodiments are also directed to providing a biosensor assembly that is very convenient to use and can greatly reduce the complexity and time (e.g., less than 45 minutes) consumed by the analysis as compared to conventional immunoassay techniques.

In a first aspect, a sensor assembly suitable for use in an enzyme-linked immunosorbent assay (ELISA) comprises a sensor strip comprising one or more wells formed in the sensor strip. The sensor assembly also includes one or more detection structures coupled to a sidewall of each of the one or more wells, wherein the one or more detection structures are configured to immobilize a biomolecule directly on the one or more detection structures.

In a second aspect, a sensor assembly suitable for use in ELISA includes a cuvette including a cavity and a lid configured to enclose the cavity. The sensor assembly additionally includes one or more detection structures coupled to the lid and configured to be at least partially immersed in the liquid sample when present in the cuvette, wherein the one or more detection structures are configured to immobilize a biomolecule directly on the one or more detection structures.

In a third aspect, an enzyme-linked immunosorbent assay (ELISA) kit comprises one or more for ELISAReagents and sensor components suitable for use in the ELISA. The sensor assembly includes: a container, such as a transparent container, having at least one transparent surface, with one or more cavities formed therein; a plurality of active surfaces disposed in each of the one or more cavities and configured to immobilize a reagent on the plurality of active surfaces; and one or more detection structures, such as transparent detection structures, disposed in each of the one or more cavities. Each of the transparent detection structures includes one or more major surfaces that provide one or more of the active surfaces. The one or more cavities are configured to be filled with a liquid such that each of the transparent detection structures is at least partially immersed in the liquid. The ratio of the combined surface area of the transparent structure in contact with the liquid to the volume of the liquid exceeds about 0.25mm²Per microliter. Each of the active surfaces is separated from an immediately adjacent one of the active surfaces by a distance of more than about 500 microns.

In a fourth aspect, an enzyme-linked immunosorbent assay (ELISA) kit comprises one or more reagents for ELISA and a sensor component suitable for the ELISA. The sensor assembly includes: a transparent container having one or more cavities formed therein; a plurality of active surfaces disposed in each of the one or more cavities and configured to immobilize a reagent on the plurality of active surfaces; and one or more transparent detection structures disposed in each of the one or more cavities, wherein each of the transparent detection structures includes one or more major surfaces that provide one or more of the active surfaces. The one or more cavities are configured to be filled with a liquid such that each of the transparent detection structures is at least partially immersed in the liquid. Penetration in contact with liquidsThe ratio of the combined surface area of the clear structures to the volume of liquid is between about 0.25mm²Per microliter to about 8.0mm²Between each microliter.

In a fifth aspect, an enzyme-linked immunosorbent assay (ELISA) kit comprises one or more reagents for ELISA and a sensor component suitable for the ELISA. The sensor assembly includes: a transparent container having one or more cavities formed therein; a plurality of active surfaces disposed in each of the one or more cavities and configured to immobilize a reagent on the plurality of active surfaces; and one or more transparent detection structures disposed in each of the one or more cavities. Each of the transparent detection structures includes one or more major surfaces that provide one or more of the active surfaces. Each of the active surfaces is separated from an immediately adjacent one of the active surfaces by a distance of between about 500 microns to about 8 millimeters.

In a sixth aspect, an enzyme-linked immunosorbent assay (ELISA) kit comprises one or more reagents for ELISA and a sensor component suitable for the ELISA. The sensor assembly includes: a transparent container having one or more cavities formed therein; a plurality of active surfaces disposed in each of the one or more cavities and configured to immobilize a reagent on the plurality of active surfaces; and one or more transparent detection structures disposed in each of the one or more cavities. Each of the transparent detection structures includes one or more major surfaces that provide one or more of the active surfaces. At least one of the active surfaces comprises a textured polymeric surface having micro-or nano-structures.

In a seventh aspect, an enzyme-linked immunosorbent assay (ELISA) kit comprises one or more reagents for an ELISA and a sensor component suitable for the ELISA. The sensor assembly includes: a transparent container having one or more cavities formed therein; and one or more transparent detection structures disposed in each of the one or more cavities. The inner surface of the cavity and the major surface of the one or more transparent detection structures provide an active surface thereon configured for immobilizing a reagent configured to specifically bind to an analyte. The major surface of the transparent detection structure is configured such that: when performing an ELISA, the detectable optical density corresponding to the analyte specifically binding to the immobilized reagent is increased without decreasing the rate of specific binding of the analyte to the immobilized reagent relative to a sensor assembly without one or more transparent detection structures.

In an eighth aspect, a method of performing an enzyme-linked immunosorbent assay (ELISA) comprises: providing an ELISA kit according to any of the above embodiments and performing the ELISA reaction in an optically transparent container. Performing an ELISA reaction includes: providing a solution comprising a target analyte and a detection reagent labeled with a label, the detection reagent configured to specifically bind to the target analyte; immobilizing a capture reagent configured to specifically bind to the target analyte on an active surface of the sensor assembly; at least partially immersing the active surface in the solution to cause specific binding of the target analyte to the capture reagent and to the labeled detection reagent; and detecting the target analyte that specifically binds to the capture reagent and to the labeled detection reagent.

In a ninth aspect, a method of performing an enzyme-linked immunosorbent assay (ELISA) comprises: providing an ELISA well, wherein the ELISA well comprises: a transparent container and one or more enhancement layers within the optically transparent container, wherein the one or more enhancement layers are configured to allow binding of an antibody thereto, wherein the one or more enhancement layers provide a ratio of a combined surface area to a liquid volume of the one or more enhancement layers, the ratio being between about 0.25mm²Per microliter and about 8.0mm²Between each microliter. The method further comprises performing an ELISA with the optically clear container, wherein only a single wash is involved in the ELISA.

In a tenth aspect, a biosensor according to the disclosed embodiments includes a detection structure in the shape of a plate having a first surface and a second surface opposite the first surface, wherein immobilized biomolecules specifically binding to target analytes are disposed on at least one of the first surface and the second surface.

In the biosensor according to the tenth aspect, the microstructures or nanostructures in the form of protrusions are formed on at least one of the first surface and the second surface of the detection structure, and the immobilized biomolecules are attached on the outer surface of the microstructures or nanostructures.

In the biosensor of the tenth aspect, the target analyte is selected from the group consisting of: amino acids, peptides, polypeptides, proteins, glycoproteins, lipoproteins, nucleosides, nucleotides, oligonucleotides, nucleic acids, sugars, carbohydrates, oligosaccharides, polysaccharides, fatty acids, lipids, hormones, metabolites, cytokines, chemokines, receptors, neurotransmitters, antigens, allergens, antibodies, substrates, cofactors, inhibitors, drugs (drugs), pharmaceuticals (pharmaceutics), nutrients, prions, toxins, poisons, explosives, pesticides, chemical warfare agents, biohazards, bacteria, viruses, radioisotopes, vitamins, heterocyclic aromatic compounds, carcinogens, mutagens, narcotics, amphetamines, barbiturates, hallucinogens, waste products, pollutants, and mixtures thereof.

In the biosensor according to the tenth aspect, the detection structure is inserted and immersed in a cuvette containing a sample containing a target analyte, so that the immobilized biomolecules react with the target analyte.

The biosensor according to the tenth aspect, further comprising a grip member connected to one end of the detection structure and gripped by a user.

The biosensor according to the tenth aspect, further comprising a lid connecting the detection structure to the gripping member and being releasably insertable into the inlet of the cuvette.

The biosensor according to the tenth aspect, further comprising a fixing member that is disposed on an outer surface of the cap and whose shape is changed to generate elasticity when the cap is inserted into the cuvette, wherein the fixing member is in close contact with an inner circumferential surface of the cuvette by the elasticity.

In the biosensor according to the tenth aspect, the detection structure is divided into an immersion portion that is immersed in the sample and a non-immersion portion that has a narrow portion having a width smaller than that of the immersion portion.

In the biosensor according to the tenth aspect, the narrow portion is recessed from at least one of both sides of the detection structure and extends along a length direction of the detection structure.

In the biosensor according to the tenth aspect, the detection structure is provided in plurality, and the detection structures are spaced apart from and parallel to each other.

The biosensor according to the tenth aspect, further comprising a pair of guards facing each other through the detection structure to protect the detection structure.

The biosensor according to the tenth aspect, further comprising at least one sensor strip comprising a body having a predetermined length and a plurality of reaction chambers recessed from one surface of the body to accommodate a sample containing the target analyte, wherein the detection structure is disposed in each of the reaction chambers.

The biosensor according to the tenth aspect, further comprising a fixing plate having the following surfaces: the sensor strip is removably attached to the surface.

In the biosensor according to the tenth aspect, the detection structure is provided in plurality, and the detection structures are vertically spaced apart from each other, for example, in the depth direction.

The biosensor according to the tenth aspect, further comprising a sample injection hole recessed from one surface of the body to communicate with the reaction chamber.

The biosensor according to the tenth aspect, further comprising an insertion protrusion protruding from one surface of the fixing plate, wherein the body is recessed or perforated to form an insertion recess into which the insertion protrusion is inserted so that the sensor strip is adhered to the fixing plate.

The biosensor according to the tenth aspect further comprises a fixing protrusion spaced apart from the insertion protrusion and protruding from one surface of the fixing plate such that the insertion protrusion contacts the inwardly recessed corner of the one end of the body when inserted into the insertion hole.

Features and advantages in accordance with the disclosed embodiments will become apparent from the following description with reference to the accompanying drawings.

The biosensor according to the disclosed embodiments is configured such that the concentration of the reacted receptor or antibody per unit volume is increased. Due to this configuration, the biosensor according to the disclosed embodiments facilitates one-step assay, significantly reduces the time required for analysis, and achieves further improved sensitivity.

Drawings

These and/or other aspects and advantages of the present invention will become apparent and more readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings.

FIG. 1A is a schematic flow diagram showing the sequence of steps in an enzyme-linked immunosorbent assay (ELISA) based on sequential reactions;

FIG. 1B is a schematic flow chart showing the sequence of steps in a one-step ELISA based on simultaneous reactions;

fig. 2A to 2C are sectional side views schematically showing a detection structure of a biosensor according to an exemplary embodiment;

fig. 3A to 3E are perspective views schematically illustrating microstructures or nanostructures formed on a biosensor according to an exemplary embodiment;

fig. 3F is a scanning electron microscope image of a nanopillar formed on a biosensor according to an exemplary embodiment;

FIG. 4A is a schematic cross-sectional view of a cuvette-type biosensor assembly;

FIG. 4B is a schematic cross-sectional view of a cuvette-type biosensor assembly including a detection structure according to an exemplary embodiment;

fig. 4C is a perspective view of a cuvette-type biosensor according to an embodiment;

FIG. 5 illustrates a front view of a biosensor assembly including the biosensor shown in FIG. 4C and a cuvette to be inserted into the biosensor, according to an embodiment;

fig. 6 is a side view illustrating a state in which the biosensor shown in fig. 4C is inserted into a cuvette according to an embodiment;

FIG. 7 is a perspective view of a cuvette-type biosensor according to further embodiments;

FIG. 8 is a perspective view of a strip type biosensor component according to an embodiment;

FIG. 9 is a perspective view of a detection structure of a strip type biosensor assembly according to an embodiment;

fig. 10 is a perspective view of a sensor strip of a strip-type biosensor component according to an embodiment;

FIG. 11 is a perspective view of a strip type biosensor assembly according to further embodiments;

fig. 12A and 12B are perspective views of a portion of a sensor strip of a strip-type biosensor assembly according to an embodiment;

FIG. 12C is a perspective view of a component layer of a portion of the sensor strip shown in FIGS. 12A and 12B;

FIG. 12D shows a perspective view of a partially overlapped portion of the sensor strip shown in FIGS. 12A and 12B;

12E-12G illustrate different portions of a sensor strip of a strip-type biosensor assembly having different stacking configurations, according to an embodiment;

fig. 12H is an experimental absorbance plot obtained using a strip-type biosensor assembly according to an embodiment;

fig. 13A schematically illustrates a top view of a component layer of a sensor strip of a strip-type biosensor assembly according to an embodiment;

13B-13D show perspective and top views of the sensor strip shown in FIG. 13A;

fig. 14A schematically illustrates a top view of a component layer of a sensor strip of a strip-type biosensor assembly according to an embodiment;

14B-14D show photographs of a perspective view and a top view of the sensor strip shown in FIG. 14A;

fig. 15 illustrates a perspective view of a sensor strip of a strip-type biosensor assembly according to an embodiment;

fig. 16A and 16B are upper and lower perspective views of a component layer of a sensor strip of a strip-type biosensor package according to an embodiment, respectively;

FIGS. 16C and 16D are detailed upper and lower perspective views, respectively, of the component layers of the sensor strip shown in FIGS. 16A and 16B;

fig. 17A and 17B are experimental measurements of absorbance and assay time and absorbance and concentration, respectively, for an analyte (HE4) measured using a biosensor assembly according to an embodiment.

Fig. 18A illustrates a perspective view of a portion of a sensor strip of a strip-type biosensor assembly, in accordance with an embodiment;

18B-18E illustrate top views of portions of a sensor strip of different strip-type biosensor assemblies, according to embodiments;

fig. 19A illustrates a perspective view of a portion of a sensor strip of a strip-type biosensor assembly, in accordance with an embodiment;

FIGS. 19B-19F show top views of portions of a sensor strip of different strip-type biosensor assemblies, according to embodiments;

FIGS. 20A-20D show perspective views of a reaction chamber of a sensor strip of different strip-type biosensor assemblies according to embodiments;

FIGS. 21A and 21B are calculated graphs of surface area in contact with a sample and diameter of a reaction chamber for a strip type biosensor assembly according to an embodiment;

fig. 22 is a flowchart illustrating a method of performing ELISA using a cuvette-type biosensor assembly according to an embodiment;

fig. 23 is a flowchart illustrating a method of performing ELISA using a strip type biosensor assembly according to an embodiment;

fig. 24A to 24F are experimental measurements of the absorbance of reaction products with various analytes at different concentrations, measured using a biosensor assembly according to an embodiment; and

fig. 25A and 25B are experimental measurements of absorbance of reaction products with various analytes at different concentrations, measured using biosensor components with different numbers of detection structures, according to embodiments.

Detailed Description

Other objects, advantages and novel features of the invention will become apparent from the following detailed description of the preferred embodiment when considered in conjunction with the drawings. In the drawings, the same elements are denoted by the same reference numerals even though they are shown in different drawings. In the description of the disclosed technology, a detailed description of the related art will be omitted when it is considered that it may unnecessarily obscure the essence of the disclosed technology.

Various ELISA techniques using conventional sensor assemblies include multiple steps including washing and/or incubation (incubate) and take a long time to complete, e.g., >120 minutes. Therefore, there is a need for a biosensor cartridge that can implement a simpler and faster ELISA technique.

Regardless of the types of ELISA techniques described above, a general challenge facing faster and more sensitive ELISAs involves increasing the signal of the target analyte detected while having a relatively high reaction rate. These and other challenges are addressed by various embodiments of the disclosed technology, which will now be described in detail with reference to the accompanying drawings.

The reactivity in an immunoassay is determined by a number of factors. In particular, the concentration of a protein in a sample participating in a reaction and the concentration of a receptor or antibody reacting with the protein are considered to be the most important factors.

For general ELISA, the reaction area of a microtiter plate used for immunoassay is limited to the area including the sample, thereby limiting the concentration of receptor or antibody participating in the reaction per volume of the same sample. However, the inventors have recognized that some techniques that attempt to increase the surface to volume ratio to increase sensitivity may reduce the diffusion of various reagents and/or analytes in an ELISA, resulting in longer reaction times. Various embodiments described herein address these and other competing needs, thereby increasing overall sensitivity while reducing reaction time.

Fig. 1A and 1B show the trade-off between speed and accuracy observed in ELISA techniques. For illustrative purposes only, this trade-off is described in the context of a sandwich ELISA. However, similar trade-offs may exist in the context of other ELISA techniques. Referring to fig. 1A, a general sandwich ELISA procedure 1A is shown. In the ELISA process 1A, a sample containing a target analyte 2 (e.g., a protein or antigen) is reacted with a receptor or capture antibody C immobilized on a substrate 10. For example, the substrate 10 may be a detection structure or an ELISA well plate. Subsequently, the reaction mixture is washed, and the reaction product 4 is reacted with the detection antibody 6 labeled with a label. In the embodiment shown, the detection antibody 6 is conjugated directly or indirectly to an enzyme, such as horseradish peroxidase (HRP). Due to the complexity of the sequential reactions, ELISA techniques (such as the sandwich ELISA technique shown) are typically performed in the laboratory by skilled users and can last for up to 4 more hours to complete.

Referring to fig. 1B, a modified sandwich ELISA procedure 1B is shown. In ELISA procedure 1B, to reduce the time and complexity of the sequential sandwich ELISA shown in fig. 1A, a labeled detection antibody 6 is first mixed with a sample containing the analyte of interest 2 (e.g., a protein or antigen) and the mixture is allowed to react. The reaction mixture is reacted with the receptor or capture antibody C immobilized on the substrate 10. When the receptor or capture antibody C is immobilized on the substrate 10 before the reaction, immunoassay can be performed by one-time injection of the sample without involving a complicated analysis process, which may make the technique more convenient and shorten the analysis time to about one eighth or less compared with that of some conventional techniques. However, these benefits are often not realized for various reasons. For example, when a large amount of a target protein is present, a part of the target protein is not reacted with a detection antibody labeled with a label, and may be reacted with an antibody immobilized on a substrate. This phenomenon is called the "hook effect". Furthermore, the presence of unlabeled target protein makes it impossible to accurately determine the concentration of the target protein. These problems can be alleviated or solved when the concentration of the receptor or capture antibody involved in the reaction can be made higher than the concentration of the target protein. Therefore, there is a need for sensor assemblies suitable for ELISA techniques (e.g., sandwich ELISA techniques) that are faster, more sensitive, and/or less complex.

Sensor assembly with micro-or nanostructured active surface for enhanced sensitivity

As used herein, a biomolecule refers to any organic compound or reagent, including antibodies and antigens, that can participate directly or indirectly in an immunoassay (e.g., ELISA). For example, a biomolecule may include a receptor or capture antibody, a detection antibody, an enzyme, a substrate, a label, and/or an analyte, to name a few, or any combination or complex formed from these molecules. It will be appreciated that the analyte may be an organic compound or an inorganic compound. When the analyte is an inorganic compound, the compound that forms the analyte and another biomolecule (e.g., a capture antibody or a detection antibody) may be collectively referred to as a biomolecule.

As described above, the inventors have recognized that increasing the active surface area of the detection structure of a biosensor component, according to embodiments disclosed herein, can increase the speed and sensitivity of immunoassay techniques. The active region is configured for immobilizing a biomolecule directly or indirectly on the active region. Increasing the active area can increase the density of immobilized biomolecules, which in turn can increase the sensitivity of an immunoassay (e.g., ELISA). In addition, according to embodiments of the biosensor assembly disclosed herein, the active surface area may be increased while reducing some of the possible negative effects on the diffusive transport of various biomolecules, reagents, and/or analytes. To meet these and other needs, according to various embodiments, a biosensor component according to some embodiments has an active surface area that includes a textured or modified surface (e.g., a textured or modified polymer surface) that may have microstructures or nanostructures according to various embodiments. As used herein, microstructures have one or more physical dimensions, such as length, width, height, diameter, and the like, from about 100nm to about 500 μm. The nanostructures have one or more physical dimensions of less than about 100 nm.

Fig. 2A to 2C are sectional views or side views schematically showing a detection structure 10 of a biosensor having one or more main surfaces on which biomolecules are attached. It is to be understood that although this embodiment depicts the receptor or capture antibody C attached to one or more major surfaces for illustrative purposes, the embodiments are not so limited. In various embodiments, the biomolecule attached to one or more major surfaces can be any biomolecule described herein that can be attached directly or indirectly to one or more major surfaces. According to various exemplary embodiments, the biomolecule may be, for example, an antibody, such as a capture antibody, a target analyte and/or a detection antibody. The detection structure 10 shown in fig. 2B and 2C may also have one or more major surfaces that are textured or include micro-or nano-structures 11. Fig. 3A to 3E are perspective views schematically illustrating various examples of micro-or nanostructures 11 formed on one or more major surfaces of a biosensor according to some other exemplary embodiments.

Fig. 3F is a scanning electron microscope image of nano-pillars or nano-fibers formed on a biosensor according to an exemplary embodiment. The nano-column or nano-fiber is prepared by reacting Ar and CF₄Etching in plasma.

As shown in fig. 2A to 2C, the biosensor according to some embodiments includes a detection structure 10 in a plate shape having one or more major surfaces, e.g., a first surface and a second surface opposite to the first surface. In the illustrated embodiment, each of the first and second major surfaces opposite each other includes an active surface. However, embodiments are not limited thereto, and in some other embodiments, one but not the other of the first and second major surfaces opposite to each other includes an active surface. In some embodiments, one or both of the first surface and the second surface comprises an active surface configured to immobilize a biomolecule thereon, either directly or indirectly. In some embodiments, for example, in the embodiments shown with respect to fig. 2B and 2C, one or more major surfaces, such as first and second major surfaces opposite one another, may include a textured polymeric surface having microstructures or nanostructures 11. In some embodiments, the microstructures or nanostructures 11 may be formed of a polymer material, for example, the same or different polymer material as the bulk (bulk) of the detection structure 10.

As described herein, an active surface refers to a surface on which one or more biomolecules and/or analytes can be immobilized for performing an immunoassay. The active surface may be chemically treated or functionalized so that biomolecules and/or analytes (e.g., as antibodies or antigens) may be specifically bound relative to the inactive surface. For example, when exposed to the same solution under the same conditions, the specificity of the active surface for a particular antibody or analyte is higher than that of the inactive surface by a factor of more than, for example, 2, 4, 6, 8, 10-fold or higher.

Referring to fig. 2A-2C, immobilized biomolecules C or reagents (e.g., antibodies) are disposed on at least one of the major surfaces (e.g., the planar first and second surfaces) of the planar detection structure 10. Here, for illustrative purposes, immobilized biomolecule C is a biomolecule (e.g., a capture antibody) that is configured to be specifically bound or bind specifically to a target analyte. The target analyte may be provided separately or may be present in the sample. The sample may further comprise a detection biomolecule or reagent (e.g. an antibody) labelled with a label.

The target analyte may be an organic compound or an inorganic compound. Non-limiting examples of target analytes include: amino acids, peptides, polypeptides, proteins, glycoproteins, lipoproteins, nucleosides, nucleotides, oligonucleotides, nucleic acids, sugars, carbohydrates, oligosaccharides, polysaccharides, fatty acids, lipids, hormones, metabolites, cytokines, chemokines, receptors, neurotransmitters, antigens, allergens, antibodies, substrates, cofactors, inhibitors, drugs, nutrients, prions, toxins, poisons, explosives, pesticides, chemical warfare agents, biohazards, bacteria, viruses, radioisotopes, vitamins, heterocyclic aromatic compounds, carcinogens, mutagens, narcotics, amphetamines, barbiturates, hallucinogens, waste products, pollutants, and mixtures thereof.

It will be appreciated that the immobilized biomolecule C or reagent, e.g. an antibody (such as a capture antibody or a detection antibody), configured to specifically bind or bind with the target analyte is determined in dependence of the target analyte. Non-limiting examples of immobilized biomolecule C include: low molecular weight compounds, antigens, antibodies, proteins, peptides, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), Peptide Nucleic Acid (PNA), enzymes, enzyme substrates, hormones, hormone receptors, and synthetic reagents having functional substrates, mimetics thereof, and combinations thereof.

Non-limiting examples of labels for labeling antibodies include: horseradish peroxidase (HRP), alkaline phosphatase, and fluorescein, to name a few.

Non-limiting examples of substrate solutions include: 2,2 ' -diazanyl-bis [ 3-ethylbenzothiazoline-6-sulfonic acid ] diammonium salt (ABTS) or 3,3 ', 5 ' -Tetramethylbenzidine (TMB) as a reagent reacting with the label (to name a few). In some embodiments, any enzymatic substrate can be used in the methods, kits, and devices provided herein. In some embodiments, the substrate is at least one or more of: OPD (o-phenylenediamine dihydrochloride) and/or pNPP (p-nitrophenyl phosphate disodium salt).

It is to be understood that the disclosed examples of target analytes, immobilized biomolecules C, labels, and reagents are provided as non-limiting examples and are not necessarily limited thereto.

Fig. 3A to 3E are perspective views schematically illustrating various examples of micro-or nano-structures 11 formed on one or more major surfaces of a detection structure 10 of a biosensor according to an exemplary embodiment. As shown in fig. 3A to 3E, the microstructures or nanostructures 11 may be formed on at least one of the major surfaces, for example, on the first surface and/or the second surface of the detection structure 10. In some embodiments, the first and second surfaces may be opposing major surfaces. However, embodiments are not limited thereto, and in some other embodiments, the first and second surfaces may be abutting surfaces. The microstructures or nanostructures 11 may be formed over the entire or substantially the entire area of the first surface and/or the second surface of the detection structure 10. Alternatively, the microstructures or nanostructures 11 may be formed in some sections or portions of the first surface and/or the second surface of the detection structure 10. The immobilized biomolecules C may be disposed on the outer surface of at least some or substantially all of the microstructures or nanostructures 11. With this arrangement, a relatively larger amount of immobilized biomolecules C may be formed on the surface of the detection structure 10 compared to a detection structure not formed with the microstructures or nanostructures 11, because the microstructures or nanostructures 11 provide a relatively larger surface area compared to a planar detection structure not formed with the microstructures or nanostructures 11 (e.g., fig. 2A).

The microstructures or nanostructures 11 may take the form of protrusions or protuberances. In various embodiments, the microstructures or nanostructures 11 comprise protrusions having a cross-sectional area that decreases away from the base (i.e., closer to the solid substrate in which they are formed). Advantageously, these structures can significantly increase the surface area available for immobilization of biomolecule C and/or analytes while reducing the possible negative impact on diffusive transport of various reagents and/or analytes. As described herein, the microstructures or nanostructures 11 may have any suitable shape that can increase the active surface area for immobilizing biomolecules thereon. The suitable shape may have at least one surface that at least partially approximates a polygon, circle or ellipse. For example, the shape of the microstructures or nanostructures 11 may include at least a portion of: spheres, ovoids, pyramids (e.g., rectangular, triangular), prisms (e.g., rectangular, triangular), cones, cubes, cylinders, plates, discs, wires, rods, sheets, and fractals, to name a few. The microstructures or nanostructures 11 may comprise any truncated or distorted form of these various shapes.

In some embodiments, as shown in fig. 3A, the microstructures or nanostructures 11 comprise protrusions having the shape of truncated spheres (e.g., hemispheres). The hemispherical protrusions may be arranged regularly, for example in a regular array. In the illustrated embodiment, the protrusions are in a zig-zag configuration. This configuration allows adjacent protrusions to contact each other in a two-dimensional close-packed configuration. However, embodiments are not so limited, and in some other embodiments, the protrusions may be arranged in any suitable configuration, such as in a rectangular array or randomly. As used herein, microstructures have one or more physical dimensions, such as length, width, height, diameter, and the like, from about 100nm to about 500 μm. The nanostructures have one or more physical dimensions of less than about 100 nm.

In other embodiments, the protrusions may be prismatic (see fig. 3B) or semi-cylindrical (see fig. 3C).

In still other embodiments, the protrusions may be pyramidal or conical. For example, the protrusions may have the shape of a pyramidal frustum (see fig. 3D) or a conical frustum (see fig. 3E), the cross-sectional area of which tapers in a direction perpendicular to the main surface of the detection structure 10.

The use of micro-or nanostructures 11 having a large surface area may increase the amount and/or concentration of receptors or antibodies involved in the reaction, thereby achieving improved sensitivity compared to conventional ELISA techniques. In particular, the use of micro-or nanostructures 11 may be effective in controlling the hook effect when the protein is present at a higher concentration than the receptor or antibody.

In various embodiments, the microstructures or nanostructures 11 have an average value of the lateral base dimension (e.g., a hemisphere diameter in FIG. 3A, a prism width in FIG. 3B, a half cylinder width in FIG. 3C, a pyramid frustum base width in FIG. 3D, a cone frustum base diameter in FIG. 3E, or a fiber diameter in FIG. 3F) that is about 1-10nm, 10-100nm, 100-1000nm, 0.1-1 μm, 1-10mm, 10-100 μm, 100-500 μm, or a value within a range defined by any of these values.

In some embodiments, the microstructures or nanostructures 10 are arranged regularly, e.g., periodically. However, embodiments are not limited thereto, and in other embodiments, the microstructures or nanostructures may be randomly arranged or pseudo-randomly arranged, e.g., regularly arranged in one direction and randomly arranged in another direction.

Cuvette-type sensor assembly configured for enhanced sensitivity and reagent diffusion

The biosensor according to some embodiments may be a cuvette type or a strip type, which will be explained in detail separately. As mentioned above, it would be advantageous to have a biosensor assembly with an increased active surface area of the detection structure, while also reducing the possible negative effects on the diffusive transport of various biomolecules, reagents and/or analytes. To meet these and other needs, according to various embodiments, a biosensor assembly according to various embodiments has a partially or fully transparent container with one or more cavities formed therein. The one or more cavities are configured to hold a liquid sample. The container may include a plurality of active surfaces disposed in each of the one or more cavities configured to immobilize biomolecules or reagents thereon. The biosensor further comprises one or more detection structures, which may be partially or fully transparent or may be opaque, configured to be at least partially arranged in each of the one or more cavities such that when the one or more cavities are filled with the liquid sample, the one or more transparent detection structures are configured to be at least partially immersed in the liquid sample. Each of the detection structures includes one or more major surfaces that provide one or more of the active surfaces. As configured, the plurality of active surfaces increases the available active surface area for immobilization of biomolecules and/or analytes thereon. In addition, each of the active surfaces is separated from an immediately adjacent one of the active surfaces by an appropriate distance to facilitate diffusion transport or to reduce retardation of diffusion transport of various biomolecules, reagents and/or analytes involved in the ELISA process.

Fig. 4A is a schematic cross-sectional view of a cuvette-type biosensor assembly 400A according to some embodiments. The biosensor assembly shown in fig. 4A includes a cuvette 1 in which a cavity 130 for containing a liquid sample is formed. The active region 11 covers at least a portion of the inner surface of the cavity 130 of the cuvette 1. As described herein, the active region 11 is configured to immobilize the biomolecule C with higher specificity than the inactive region 14.

Fig. 4B is a schematic cross-sectional view of a cuvette-type biosensor assembly 400B according to some other embodiments. The biosensor assembly 400B shown in fig. 4B includes a cuvette 1, which may be arranged similarly to the embodiment shown in fig. 4A, that includes a cavity 130, which may or may not have an active region 11 formed on a sidewall. Unlike the cuvette-type biosensor described above with respect to fig. 4A, in addition to having an active region 11 covering at least a portion of the inner surface of the cavity 130 of the cuvette 1, the biosensor assembly 400B additionally includes one or more transparent detection structures 10 disposed in the cavity 130, wherein each of the transparent detection structures 11 includes one or more major surfaces that provide one or more of the active surfaces 11. As configured, the plurality of active surfaces increases the available active surface area for immobilization of biomolecules and/or analytes thereon. In addition, each of the active surfaces 11 is separated from an immediately adjacent one of the active surfaces 11 or an inactive surface by a suitable distance to facilitate or reduce retardation of diffusive transport of various biomolecules, reagents and/or analytes involved in the ELISA process.

The inventors have found that suitable distances or gaps between immediately adjacent surfaces for unimpeded diffusive transport of various biomolecules, reagents and/or analytes used in an ELISA are distances of about 500 microns, 1000 microns, 2000 microns, 3000 microns, 4000 microns, 5000 microns, 6000 microns, 7000 microns, 8000 microns, 9000 microns or within a range defined by any of these values, depending on the particular configuration of the biosensor component and the target analyte to be detected or quantified. For a given configuration of the biosensor assembly, in order to obtain various advantageous experimental results described herein, including high sensitivity and fast reaction times, the inventors have found that it may be critical to have a separation distance between immediately adjacent surfaces, wherein at least one of the surfaces is an active surface that is greater than or equal to one or more of these values.

The inventors have also found that a suitable combined active area per volume of cavity for immobilizing various biomolecules and/or analytes for use in an ELISA exceeds about 0.1mm²/μl、1.0mm²/μl、1.5mm²/μl、2.0mm²/μl、2.5mm²/μl、3.0mm²/μl、3.5mm²/μl、4.0mm²/μl、4.5mm²/μl、5.0mm²/μl、5.5mm²/μl、6.0mm²/μl、6.5mm²/μl、7.0mm²/μl、7.5mm²/μl、8.0mm²μ l, or have a value within a range defined by any of these values, depending on the particular configuration of the biosensor component and the target analyte to be detected or quantified. For a given configuration of biosensor components, in order to obtain the various advantageous experimental results described herein, including high sensitivity and rapid reaction times, the inventors have found that it is critical to have a combined active surface area that is greater than or equal to one or more of these values.

The inventors have also discovered that when the active region of the sensor assembly is configured as described herein, the detectable concentration of analyte and/or the resulting detectable optical density that specifically binds to the immobilized biomolecule or reagent is increased by at least a factor of 1.1, 2, 5, 10, 15, 20, or a factor within a range defined by any of these values, without substantially reducing the rate at which the analyte (e.g., antigen) specifically binds to the biomolecule (e.g., antibody), relative to a comparable sensor assembly that does not have one or more transparent detection structures. For example, sensor assemblies 400B and 400A with and without the detection structure 10 shown in fig. 4B and 4A, respectively, can have these ratios of specifically bound analyte.

Figure 4C is a perspective view of a cuvette-type biosensor 400C in accordance with one embodiment of the disclosed technology. Fig. 5 shows a front view of the biosensor 400C and the cuvette 1 shown in fig. 4C, the biosensor 400C is configured to be inserted into the cuvette 1, and fig. 6 is a side view 600 showing a state in which the biosensor 400C shown in fig. 5 is inserted into the cuvette 1.

As shown in fig. 4B to 6, the cuvette-type biosensor assembly according to various embodiments has a structure in which the detection structure 10 is inserted into the cuvette 1. Due to this structure, the immobilized biomolecules disposed on the detection structure 10 react with the target analyte contained in the cuvette 1. A target analyte may be present in the sample 3 contained in the cuvette 1. In this case, the immobilized biomolecules react with the target analyte while the detection structure 10 is immersed in the sample 3. As described above, immobilized biomolecules are arranged on the detection structure 10. In some embodiments, microstructures or nanostructures 11 may be formed on the detection structure 10, as described above, for example, with respect to fig. 2B-3E. The microstructures or nanostructures 11 may be formed on at least a portion of the active area of each of the major surfaces of the detection structure 10. In some embodiments, the entire area of the first and/or second surface (which may be the opposite major surfaces of each of the detection structures 10) is configured as an active area such that they are covered by the microstructures or nanostructures 11. Alternatively, a portion of the first surface and/or the second surface of the detection structure 10 is configured as an active region, such that the microstructures or nanostructures 11 are selectively formed on some portions of the first surface and/or the second surface, but not on other portions. The immobilized biomolecules may be disposed on an outer surface of the detection structure 10. The absorbance (absorbance) is measured in a state where the detection structure 10 is inserted into the cuvette 1. Accordingly, the detection structure 10 and cuvette 1 may have a predetermined absorbance, for example, to provide a reference absorbance curve.

Advantageously, one or more of the detection structures 10 and the cuvette 1 as shown in fig. 4A-6 may be formed from a solid polymer material. For example, the detection structure 10 and the cuvette 1 may be made of a polymer material such as polycarbonate, polyethylene terephthalate, polymethyl methacrylate, triacetyl cellulose, cycloolefin, polyarylate, polyacrylate, polyethylene naphthalate, polybutylene terephthalate, or polyamide. However, these polymer materials are merely exemplary, and other suitable light-transmitting polymer materials may be used without particular limitation. It will be appreciated that, in addition to providing optical transparency for the immunoassay, the selection of appropriate materials for the detection structure 10 and/or cuvette 1 may also advantageously enable efficient formation of an active surface by surface chemical modification or functionalization. In addition, the selection of appropriate materials for the detection structure 10 and/or cuvette 1 may advantageously enable the formation of appropriate surface topographies, including microstructures or nanostructures 11 (fig. 3A-3E), by directly modifying the surface thereof. For example, any of the microstructures or nanostructures 11 described herein for increasing surface area may be formed as an integral part of a solid substrate (e.g., a solid polymer substrate). The microstructures or nanostructures 11 may be formed directly on the surface of the detection structure 10 and/or cuvette 1 by a suitable method. For example, the microstructures or nanostructures 11 may be molded (mold) as part of a solid polymer substrate, or may be formed by a suitable post-processing method, such as by etching or plasma treatment. Thus, in some embodiments, each of the active surfaces comprises a solid polymer surface that directly serves as an active surface for immobilizing biomolecules and/or analytes without forming a separate layer with topology (topology). In these embodiments, the active surface may not include an additional material formed thereon that serves as an active surface. That is, the surface on which the biomolecules are immobilized may be the same material as the solid substrate. When the solid substrate is formed of a polymeric material, the active surface may not comprise another material, for example a metal, a semiconductor, an inorganic glass, or a polymeric material different from the polymeric material of the solid substrate may not be present between the solid substrate and the biomolecules attached thereto. However, embodiments are not limited thereto, and in other embodiments, additional materials may be formed on the active surface.

Referring to fig. 4C-6, the cuvette-type biosensor may further include a grip member or handle 20 connected to one end of each of the detection structures 10. The gripping member 20 forms a base from which the detection structure 10 extends and is configured to be gripped by a user. The user can insert the detection structure 10 into the cuvette 1 while holding the grip member 20. At this point, the free end of the detection structure 10 first enters the cuvette 1. The inserted test structure 10 is immersed in the sample 3. The free end of the detection structure 10 refers to the end opposite to the end of the detection structure 10 connected to the gripping member 20.

Each of the detection structures 10 is fixedly connected to the gripping member 20. The detection structures 10 are spaced apart from and parallel to each other such that the major surfaces of adjacent detection structures 10 face each other. The separation distance between immediately adjacent active surfaces may be any of the distances described herein, and may be critical to achieving various advantages associated with increased optical density and/or reduced reaction time. The formation of the plurality of detection structures 10 increases the density (or concentration) of immobilized biomolecules per unit volume of the sample 3 containing the analyte by increasing the surface area available for immobilization of the biomolecules. As a result, an improved sensitivity of the sensor and control for the hook effect may be achieved.

Still referring to fig. 4C-6, the cuvette-type biosensor may further include a cap 30. The lid 30 is configured to be releasably inserted into the open inlet or opening of the cuvette 1 to close the inlet of the open cuvette 1. The inlet of the cuvette 1 may be substantially or completely sealed or closed by a lid 30. Alternatively, only a portion of the inlet of the cuvette 1 may be closed by the lid 30. A cover 30 is provided under the grip member 20 to connect the grip member 20 to the detection structure 10. The lid 30 is held in contact with the inner surface of the cuvette 1 to prevent movement of the detection structure 10 within the cuvette 1.

The inventors have realised that depending on the size of the inlet of the cuvette 1, there may be a gap between the outer surface of the lid 30 and the inner surface of the inlet of the cuvette 1. Thus, the lid 30 may remain unsecured to the cuvette 1, making it difficult to accurately analyze the sample 3 without, for example, leaking the sample 3. To avoid this problem, the biosensor 400C (fig. 4C, 5, 6) may further include a fixing member or fastening member 40 for fixing or fastening the detection structure 10 regardless of the close matching between the relative sizes of the inlet of the cuvette 1 and the cover 30.

The fixing member 40 is disposed on an outer surface of the cover 30. With this arrangement, when the cover 30 is inserted into the cuvette 1, the original position or shape of the fixing member 40 is changed to generate elasticity. The fixing member 40 is in close contact with the inner circumferential surface of the cuvette 1 by this elasticity.

For example, referring to fig. 5 and 6, when the cover 30 is inserted into the cuvette 1, the fixing member 40 may be pressed and deformed, such as elastically bent or deformed, by the inner surface of the cuvette 1. Accordingly, the fixing member 40 may be made of an elastic material such as rubber. The elasticity of the elastic material allows the fixing member 40 to contact the inner surface of the inlet of the cuvette 1. The securing member 40 may utilize the inherent elasticity of the elastic material to fixedly hold the cover 30 in place with the sensing structure 10. Alternatively, the fixing member 40 may include an elastic member such as a spring. In this case, the fixing member 40 may fixedly hold the cover 30 and the sensing structure 10 in place using the elastic force of the spring. However, the embodiment is not limited thereto, and the fixing member 40 may not necessarily rely on the elasticity of the elastic material or member. For example, the fixing member 40 may be structurally modified such that the detection structure 10 is fixed, which will be described in detail below.

In some embodiments, the fixing member 40 may extend from an outer surface of the cover 30 and may be bent in a predetermined direction. For example, the fixing member 40 may extend outward from the outer surface of the cover 30, and may be bent in parallel with the outer surface of the cover 30 to form an inverted L-shape. The outwardly protruding protrusion formed at one end of the fixing member 40 is pressed against the inner surface of the cuvette 1, and as a result, the fixing member 40 can be brought into close contact with the inner surface of the cuvette 1 by tension. At this time, since the fixing member 40 moves toward the cover 30 when being pressed, a portion of the outer surface of the cover 30 opposite to the fixing member 40 may be recessed.

Alternatively, the fixing member 40 may extend to a recessed portion of the cover 30, and may protrude outward from the outer surface of the cover 30 to form an "L" shape.

In summary, the fixing member 40 may be freely modified as long as it can be brought into close contact with the inner surface of the cuvette 1 when the cover 30 is inserted into the cuvette 1.

Referring to fig. 5 and 6, each of the detection structures 10, when immersed in the sample 3, may be divided into an immersed portion immersed in the sample 3 and a non-immersed portion above the liquid sample, the non-immersed portion including a narrow portion 12 having a width smaller than that of the entrance portion.

When the detection structures 10 are inserted into the cuvette 1 for analyzing the sample 3, capillary forces are generated in the gap between one or both of the detection structures 10 and the respective inner surface(s) of the cuvette 1, and/or in the gap(s) between the detection structures 10 arranged in parallel. This capillary force may increase the level of the sample 3, requiring a large amount of sample 3 to be analyzed, which may seriously degrade the analytical reliability of the sensor. The inventors have recognized that this problem can be alleviated or solved by forming the narrowed portion 12. The level of the sample 3 rises along the detection structure 10 by the attraction between the sample 3 and the surface of the detection structure 10. Accordingly, forming the narrow portion 12 having a smaller width reduces the contact area between the detecting structure 10 and the sample 3, thereby reducing or preventing the level of the sample 3 from rising.

Still referring to fig. 5 and 6, the narrowed portion 12 may have one or more recesses, notches, or indentations 17 recessed from one or both sides of the detection structure 10, the anti-lifting recesses 17 being formed to a predetermined depth in a direction from one side of the detection structure 10 to the other opposite side. Accordingly, at the position where the rise-preventing recessed portion 17 is formed, the width of the detecting structure 10 (i.e., the distance between the opposite sides of the detecting structure 10) is reduced to be smaller than the width of the detecting structure at the dipping portion below the lowest recessed portion 17. The rising-prevention recess 17 may be formed on one side or both sides of the sensing structure 10. When formed on both sides, the rising-prevention recesses 17 located at both sides of the detecting structure 10 may be arranged at the same vertical level. However, embodiments are not necessarily limited to this arrangement. For example, the rising-prevention recesses 17 may be alternately arranged in a zigzag pattern. The rising-prevention recesses 17 may be formed at predetermined intervals in a length direction along the side of the sensing structure 10.

The anti-lifting recess 17 may be circular as shown, but is not necessarily limited to this shape. The rising-prevention recess 17 may have any shape as long as the width of the detection structure 10 is narrowed.

Fig. 7 is a perspective view of a cuvette-type biosensor 700 in accordance with yet another embodiment of the disclosed technology. Biosensor 700 has various features similar to those described above with respect to fig. 4C-6. For the sake of brevity, a detailed description thereof will not be repeated here. In the embodiment shown in fig. 7, biosensor 700 further includes one or more guards 50 configured to protect detection structure 10. In the illustrated embodiment, a pair of guard members 50 face each other and are inserted into and spaced apart from the test structure 10 by the test structure 10. The number of detection structures 10 interposed between a pair of guard members 50 is not limited to any particular number, but may be defined by the internal dimensions of the cuvette 1, the thickness of the detection structures 10, and the spacing between adjacent detection structures 10. By being configured as the outermost structure, the guard 50 prevents the detection structure 10 from contacting the inner surface of the cuvette 1 and protects the detection structure 10 from external factors such as impact. The guard 50 may be plate-shaped as shown, but is not necessarily limited to this shape. When the guard 50 is provided in a plate shape, the sample 3 may rise in the gap between the detection structures 10 arranged in parallel or in the gap between the detection structures 10 and the inner surface of the cuvette 1. Accordingly, in a similar manner to the detection structure described above with respect to fig. 5 and 6, the guard 50 may include a narrow portion 12a having a width smaller than that of the immersed portion, the narrow portion being formed at a predetermined height in the guard 50. The narrow portion 12a may have a recess 17a recessed from the side of the guard 50. However, it is not essential to form the narrow portion 12a in the protection member 50 or to form the rise-preventing recess 17a in the narrow portion 12 a.

In some embodiments, none of the opposing major surfaces of one or both of the guards 50 may be configured for immobilizing biomolecules and/or forming microstructures or nanostructures thereon. In some embodiments, one of the opposing major surfaces of one or both of the guards 50 (e.g., the major surface facing the detection structure 10) may be configured for immobilizing biomolecules and/or forming microstructures or nanostructures thereon. In still other embodiments, the immobilized biomolecules may be attached to one or both surfaces of one or both of the guards 50. As a result, the density of immobilized biomolecules per unit volume can be increased.

In each of the embodiments described herein, e.g., with respect to fig. 2A-7, the detection structure 10 can have a thickness of about 100 to 5000 microns, 100 to 500 microns, 500 to 1000 microns, 1000 to 1500 microns, 1500 to 2000 microns, 2000 to 2500 microns, 2500 to 3000 microns, 3000 to 3500 microns, 3500 to 4000 microns, 4000 microns to 4500 microns, 4500 to 5000 microns, or within a range defined by any of these values.

In each of the embodiments described herein, e.g., with respect to fig. 2A-7, the detection structure 10 can have one or more major surfaces, e.g., a first surface and a second surface, each major surface having an area of about 10 to 100mm²10 to 20mm²20 to 30mm²30 to 40mm²40 to 50mm²50 to 60mm²60 to 70mm²70 to 80mm²80 to 90mm²90 to 100mm²Or within a range defined by any of these values, e.g., about 51mm². Furthermore, in each of the embodiments described herein, e.g., with reference to fig. 2A-7, a suitable portion of each of the major surfaces, e.g., 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, or any percentage within the range defined by these values, may be an active surface.

In each of the embodiments described herein, e.g., with respect to fig. 2A-7, the cuvette 1 is configured to contain a liquid having a volume as follows: about 50mm³-3000mm³、50mm³-300mm³、300mm³-600mm³、600mm³-900mm³、900mm³-1200mm³、1200mm³-1500mm³、1500mm³-1800mm³、1800mm³-2100mm³、2100mm³-2400mm³、2400mm³-2700mm³、2700mm³-3000mm³Or a value within a range defined by any of these values.

In each of the embodiments described herein, e.g., with respect to fig. 2A-7, the cuvette 1 and the detection structures 10 are of suitable dimensions, including suitable separation distances between adjacent surfaces (e.g., between active surfaces), and suitable combined active areas for immobilizing various biomolecules and/or analytes, such that the cuvette is configured to receive 1 to 20, 1 to 5,5 to 10, 10 to 15, 15 to 20, or any number of detection structures within a range defined by any of these values.

Bar-type sensor assembly configured for enhanced sensitivity and reagent diffusion

Hereinafter, a bar type biosensor assembly according to an embodiment is described. In these embodiments, an optically transparent container configured to receive one or more detection structures comprises a strip container comprising a plurality of cavities formed therein. Unlike the cuvette-type biosensor assembly described above, in the cuvette-type biosensor assembly, one or more detection structures may have the following plate structures: the plate structure has opposing major surfaces that may be substantially parallel to a depth direction of the cavity; in the strip-type biosensor disclosed herein, the one or more transparent detection structures comprise the following plate structures: the plate structure has opposing major surfaces substantially perpendicular to a depth direction of the cavity. In some embodiments, each of the one or more transparent detection structures comprises the following plate structure: the plate structure has opposing major surfaces that are substantially parallel to each other. A major surface of one of the one or more transparent detection structures that directly faces the bottom surface of a respective one of the cavities may be substantially parallel to the bottom surface of the respective one of the cavities and may be separated from the bottom surface of the respective one of the cavities by a distance of more than about 500 microns.

In some embodiments, each of the one or more transparent detection structures comprises a plate structure disposed laterally at a central region of a respective one of the cavities, as exemplified by the various embodiments described herein, including with respect to fig. 8-11. In some embodiments, one or more of the major surfaces of the one or more transparent detection structures may substantially overlap each other in a vertical direction substantially parallel to a depth direction of the cavity. In some embodiments, the sensor assembly comprises two or more transparent detection structures, wherein major surfaces of directly adjacent transparent detection structures of the two or more transparent detection structures that directly face each other are separated from each other by a distance of more than 500 micrometers. FIG. 8 is a perspective view of a strip-type biosensor in accordance with one embodiment of the disclosed technology. Fig. 9 is a perspective view schematically illustrating a detection structure of a bar-type biosensor according to some embodiments, and fig. 10 is a perspective view illustrating a sensor bar of the bar-type biosensor according to some embodiments.

As shown in fig. 8 to 10, the strip-type biosensor assembly according to the embodiment includes one or more sensor strips 100, each of which includes a body 150 having a predetermined length and a plurality of reaction chambers, recesses, or cavities 130 recessed from one surface of the body 150 to accommodate a sample containing a target analyte. One or more detection structures 10 are disposed in each of the reaction chambers 130.

Specifically, a strip-type biosensor assembly according to some embodiments includes sensor strips 100, each of the sensor strips 100 having the following structure: in the structure in which the reaction chamber 130 is formed in the body 150 in the shape of an elongated bar having a predetermined length and width. The body 150 comprises one or more chambers 130 in the form of recessed wells or cavities, and at least one detection structure 10 is arranged in each of the reaction chambers 130.

The reaction chamber 130 is recessed from one of the outer surfaces of the body 150 to receive a sample therein, and is arranged in the length direction of the body 150. The immobilized biomolecules may be disposed at least on the bottom surface of the reaction chamber 130 (not shown). In some embodiments, the microstructures or nanostructures 11 in the form of protrusions similar to the microstructures or nanostructures 11 described in the previous embodiments may be formed at the bottom surface of the reaction chamber 130, and the immobilized biomolecules are arranged in the form of protrusions on the outer surface of the microstructures or nanostructures. The formation of the microstructures or nanostructures 11 increases the density of immobilized biomolecules per unit volume in a manner similar to that described above. However, embodiments are not limited thereto, and in some other embodiments, the microstructures or nanostructures 11 may be omitted from the bottom surface of the reaction chamber 130.

The detection structure 10 is arranged in each of the plurality of reaction chambers 130 such that the detection structure is configured to be immersed in a sample contained in the reaction chamber 130. In some embodiments, the detection structure 10 has a plate structure with opposing major planar surfaces. Referring to fig. 9, in some embodiments, microstructures or nanostructures 11 are formed on one or both of the opposing planar surfaces in a manner similar to that described above with respect to the cuvette-type sensor assembly. In some embodiments, the microstructures or nanostructures 11 are formed on substantially the entire area of a first surface and/or a second surface opposite the first surface of the detection structure 10. Alternatively, the microstructures or nanostructures 11 may be formed in some but not other portions of the first and/or second surface. The immobilized biomolecules may be disposed on an outer surface of the detection structure 10. However, embodiments are not limited thereto, and in some other embodiments, the microstructures or nanostructures 11 may be omitted from the surface of the detection structure 10.

Forming multiple reaction chambers 130 and detection structures 10 in each of the sensor strips 100 enables analysis of multiple reactions in parallel or simultaneously. In certain analytical techniques, the same sample may be used to analyze different reactions. For example, when different capture antibodies are immobilized on the detection structure 10 in different reaction chambers 130, the same sample can be used to analyze different reactions. In some other analytical techniques, different samples may be used to analyze different reactions. For example, different samples introduced into different reaction chambers may be used to analyze different reactions. Still referring to fig. 9, a surface opposite to the surface of the sensor strip 100 where the opening of the reaction chamber 130 is formed may be disposed on the fixing plate 200. The fixing plate 200 may be configured to fix a plurality of sensor bars 100. As arranged, n × m reactions may be analyzed, where n may represent the number of reaction chambers per sensor strip 100, and m may represent the number of sensor strips 100 that may be secured on the fixation plate 200.

The fixing plate 200 has a predetermined width and thickness. The sensor strip 100 is detachably attached to one surface of the fixing plate 200. The sensor strip 100 may be attached to and detached from the fixing plate 200 through the insertion protrusion 400 and the insertion hole 120. The insertion protrusion 400 is inserted into and fixed to the insertion hole 120. The insertion hole 120 may be recessed or perforated to have a shape corresponding to the outer shape of the insertion protrusion 400. Due to their corresponding shapes, the insertion protrusions 400 are releasably withdrawn from the insertion holes 120. The insertion protrusion 400 may protrude from one surface of the fixing plate 200, and the insertion hole 120 may be formed on the opposite surface of the body 150 of the sensor strip 100, so that the sensor strip 100 may be attached to and detached from the fixing plate 200. Alternatively, the insertion protrusion 400 may be formed on the sensor strip 100, and the insertion hole 120 may be formed in the fixing plate 200.

The biosensor component according to various embodiments is configured for analyzing a target analyte based on absorbance measurements. Therefore, the detection structure 10 is configured to be irradiated with external light. Accordingly, the fixing plate 200 may be perforated in a thickness direction thereof to form the hole 210 to allow unimpeded light to pass therethrough. The reaction chamber 130 is configured to be disposed over a hole 210 formed in the fixing plate 200. With this arrangement, the reaction chambers 130 and the holes 210 may be provided in the same number at their corresponding positions. However, embodiments are not limited thereto, and in some other embodiments, the fixing plate 200 may include a frame configured to fix or support one or more edges of one or more sensor bars 100 thereon, with the remaining portion removed. For example, in some configurations, the plurality of holes 210 corresponding to a sensor strip 100 may be replaced by an elongated slot that extends along the length of the sensor strip 100 to overlap a plurality of reaction chambers 130 in the same sensor strip 100. In some other configurations, some or substantially all of the portion of the fixing plate that overlaps sensor strip 100 may be removed, except for a frame that includes the portion of the fixing plate on which insertion protrusion 400 is formed. In this configuration, the removed portion of the fixing plate may overlap with the plurality of reaction chambers 130 in different sensor bars 100. As configured, light may be transmitted through the detection structure 10 and the bottom of the reaction chamber 130. Accordingly, the detection structure 10 and the bottom of the reaction chamber 130 are made of a light-transmitting material such as polycarbonate, polyethylene terephthalate, polymethyl methacrylate, triacetyl cellulose, cycloolefin, polyarylate, polyacrylate, polyethylene naphthalate, polybutylene terephthalate, or polyamide. However, these polymer materials are merely exemplary, and the present invention is not necessarily limited thereto.

Referring to fig. 10, each of the sensor bars 100 may include a plurality of sensing structures 10, 10a, 10b, and 10 c. The plurality of detection structures 10, 10a, 10b, and 10c may be vertically spaced apart from each other along the depth direction of the reaction chamber 130. The detection structure 10a may be arranged to face at least one other detection structure, e.g. detection structure 10b or 10 c. The arrangement of the plurality of detection structures 10 increases the density (or concentration) of immobilized biomolecules per unit volume of the sample. In some embodiments, the microstructures or nanostructures 11 may be formed on the surface of one or more of the detection structures 10, and the immobilized biomolecules may in turn be disposed on the outer surface of the microstructures or nanostructures 11.

Referring to fig. 10, the sensor strip 100 of the strip type biosensor assembly according to the embodiment further includes a sample injection hole 300. The sample injection hole 300 may be recessed from one surface of the body 150 so as to communicate with the reaction chamber 130. The sample is injected into the reaction chamber 130 through the sample injection hole 300, and the sensing structure 10 is immersed in the sample.

FIG. 11 is a perspective view of a strip-type biosensor assembly, according to another embodiment of the disclosed technology. The illustrated embodiment is similar in some respects to the strip-type biosensor assembly described above with respect to fig. 8-10, and similar detailed descriptions are not repeated here. Referring to fig. 11, the strip type biosensor may further include a fixing protrusion 500 to more firmly fix the sensor strip 100 to the fixing plate 200. The fixing protrusion 500 protrudes from one surface of the fixing plate 200, and is disposed at a predetermined interval from the insertion protrusion 400. The distance between the fixing protrusions 500 and the insertion protrusion 400 is determined such that each of the fixing protrusions 500 is in contact with the outer surface of one end of the body 150 of the sensor strip 100 when the insertion protrusion 400 is inserted into the insertion hole 120. A corner of one end of the body 150 of the sensor strip 100 may be recessed inward. When the insertion protrusion 400 is inserted into the insertion hole 120, the corner of the depression comes into close contact with the fixing protrusion 500, and as a result, the sensor strip 100 is firmly fixed to the fixing plate 200.

In the strip-type biosensor assembly described above with respect to fig. 10, the detection structures 10 are substantially similar in shape and are substantially identically positioned with respect to a lateral position within the reaction chamber 130. Thus, the detection structures 10 substantially overlap each other when viewed in a top-down direction. Various other embodiments are also possible, as described below.

Fig. 12A-12D, 13A-13D, and 14A-14D illustrate embodiments of a strip-type biosensor according to some other embodiments, wherein each of the one or more transparent detection structures includes a protrusion extending from an inner surface of a respective one of the cavities.

Fig. 12A and 12B show perspective views at different angles of a portion of a sensor strip stack 1200 as part of a strip-type biosensor assembly, according to an embodiment. For illustrative purposes, the portion includes a reaction chamber or cavity 130 in which the first detection structure 10A and the second detection structure 10B are formed. Sensor strip stack 1200 is formed by stacking multiple component layers. Sensor strip stack 1200 includes a backplane 1200-4, one or more detection structure layers 1200-1, 1200-2, and one or more spacer (spacer) layers 1200-3. The one or more detection structure layers 1200-1, 1200-2 and the one or more spacer layers 1200-3 may be stacked in any suitable order. In the embodiment shown, sensor strip stack 1200 includes, in a bottom-up direction: a backplane 1200-4, a first spacer layer 1200-3, a first test structure layer 1200-1 comprising first test structures 10A, a second spacer layer 1200-3, a second test structure layer 1200-2 comprising second test structures 10B, and third and fourth spacer layers 1200-3. In addition to the backplane 1200-4, each of the detection structure layers 1200-1 and 1200-2 and each of the spacer layers 1200-3 includes an opening formed therethrough. Unlike the spacer layer 1200-3, each of the detection structure layers 1200-1 and 1200-2 includes three detection structures formed at intervals of approximately 120 ° along the circumference of the respective opening formed therethrough. For illustrative purposes, FIG. 12C shows disassembled component layers that may be stacked to form sensor strip stack 1200 shown in FIGS. 12A and 12B. As assembled, the portion of the sensor strip shown in fig. 12A and 12B has a reaction chamber or cavity 130 in which the first and second transparent detection structures 10A and 10B are formed. Each of the first and second transparent detection structures 10A, 10B comprises a plate structure extending laterally towards a central region of a respective one of the cavities. The first detection structure 10A of the detection structure layer 1200-1 and the second detection structure 10B of the detection structure layer 1200-2 are angularly rotated about 60 deg. with respect to each other.

To more clearly illustrate the arrangement of the first and second sensing structures 10A and 10B, FIG. 12D illustrates a portion of a partial sensor strip stack 1200' without the top two spacer layers 1200-3. As shown, the one or more transparent detection structures include: one or more first detection structures 10A formed as lateral protrusions at a first vertical level in the depth direction (z direction) of the reaction chamber, well, or cavity 130 by a pattern of the detection structure layer 1200-1; and one or more second detection structures 10B formed as lateral protrusions at a second vertical level in the depth direction of the reaction chamber or cavity 130 by the pattern of the detection structure layer 1200-2. Unlike the bar type sensor assemblies described above with respect to fig. 8-11, the sensor assembly shown in fig. 12A-12D is configured such that the first and second detection structures 10A and 10B, which are vertically separated by one or more spacer layers 1200-3, are laterally rotated relative to each other. As a result, at least a portion of each of the first detection structures 10A does not overlap any of the vertically adjacent second detection structures 10B in a lateral direction (x, y direction) perpendicular to the depth direction (z direction) of the reaction chamber or cavity 130, such that non-overlapping portions of the first and second detection structures 10A, 10B that are rotationally offset with respect to each other are visible to a user when viewed in the z direction. However, the embodiment is not limited thereto, and in other embodiments, the first detection structure 10A and the second detection structure 10B substantially overlap in the lateral direction. In still other embodiments, the first detection structure 10A and the second detection structure 10B substantially overlap each other in the lateral direction.

Still referring to fig. 12D, in the illustrated embodiment, the first and second detection structures 10A and 10B are regularly (e.g., periodically) arranged around the inner surface of the reaction chamber or cavity 130. For example, in the illustrated embodiment, adjacent ones of the first and second detection structures 10A and 10B are arranged about 120 ° apart around the perimeter of the reaction chamber or cavity 130. However, embodiments are not so limited, and there may be any suitable number of first and second detection structures 10A, 10B, such that the first and second protrusions 10A, 10B are arranged to be angularly separated around the perimeter of the reaction chamber 130 by about 30 °, 45 °, 60 °, 90 °, 120 °, or 180 ° (to name a few examples), or an angle defined by 360 °/n, where n is an integer. Additionally, in the illustrated embodiment, the first detection structure 10A and the second detection structure 10B are rotationally offset from each other by any fraction of θ. That is, when the angular interval between circumferentially adjacent ones of first sensing structure 10A and/or second sensing structure 10B is θ, the angular offset between vertically adjacent ones of first sensing structure 10A and second sensing structure 10B may be represented as θ/m, where m is any value greater than 1.

Advantageously, in order to provide an additional volume of liquid adjacent to the active surface for enhancing diffusion and entry of the active surface by biomolecules, reagents and/or analytes in the sample to facilitate the ELISA process, the one or more first detection structures 10 and the one or more second detection structures 10B are separated in the depth direction of the cavity by a spacer region formed by a spacer layer 1200-3 having a target thickness. Alternatively, one or more spacer layers 1200-3 may be formed between vertically adjacent detection structure layers 1200-1, 1200-2. Similarly, one or more spacer layers 1200-3 of suitable thickness may be disposed above the detection structure layer 1200-2 and/or below the detection structure layer 1200-1. An appropriate number of spacer layers and/or their thicknesses may be tailored to enhance the ability to diffuse into the active surface by biomolecules and/or reagents in the sample that come into contact with the sample.

In addition, in a manner similar to that described above with respect to various embodiments, one or more of the interior surfaces of the respective ones of the reaction chambers or cavities 130 may be used as active surfaces in addition to one or more of the major surfaces of the detection structures 10A, 10B.

The stacked layer configuration of the bar sensor assembly shown in fig. 12A-12D may provide a number of advantages. For example, by customizing the number of detection layers and structures, the overall reaction surface area can be customized. Furthermore, by tailoring the thickness or number of spacer layers between vertically adjacent detection structures, the volume of sample available for immediate diffusion of biomolecules or reagents into the active surface can be tailored.

In addition, since first detection structure 10A and second detection structure 10B are laterally offset with respect to each other, the vertical distance therebetween can be reduced without adversely affecting the diffusive entry of vertically adjacent detection structures. As a result, a greater number of detection structures can be formed per unit depth of the reaction chamber or cavity 130 than in an arrangement where vertically adjacent detection structures overlap significantly in the lateral direction. In addition, a central region of each of the reaction chambers or cavities 130 not occupied by the one or more transparent detection structures 10A, 10B is configured to readily receive a sample therein, e.g., using the tip of a pipette (pipette).

The customizability of the various layers is further illustrated with respect to fig. 12E-12G. FIG. 12E shows sensor strip stack 1200C, which includes two structural layers of detection, sensor I and sensor II. The sensors I may be arranged in a similar manner as the detection structure layer 1200-1, and the sensors II may be arranged in a similar manner as the detection structure layer 1200-2 described above with respect to FIGS. 12A-12D. Each of the detection structure layers includes three detection structures formed at intervals of about 120 ° along the perimeter of the opening formed therethrough, in a manner similar to the arrangement shown in fig. 12A-12B. In addition, the detection structures of vertically adjacent ones of the detection structure layers are angularly rotated about 60 ° relative to each other, and adjacent ones of the detection structure layers are vertically separated by a spacer layer. Fig. 12F and 12G show sensor strip stacks 1200B and 1200C, respectively, comprising three and four detection structure layers, respectively. Each of the detection structure layers includes three detection structures formed at intervals of about 120 ° along a circumference of an opening formed therethrough, and vertically adjacent detection structure layers are separated by a spacer layer. Sensor strip stack 1200B includes two layers of sensor I (1200-1) interposed by sensor II (1200-2). Sensor strip stack 1200C includes two layers of sensors I (1200-1) and two layers of sensors II (1200-2) arranged alternately.

12E-12G may have the same total number of layers, and when the different layers have the same thickness, the sensor stacks may have substantially the same overall thickness (and depth of the cavity) while having varying or customizable sensitivity. This customizability is shown in fig. 12H, which shows a graph showing the absorbance intensity at 655nm for the same nominal concentration of mouse IgG (immunoglobulin G). By increasing the number of detection structure layers and thus the number of detection structures, the absorbance is increased. For example, as shown, when the mouse IgG concentration is about 25ng/mL, with three and four detection structure layers, each of which contains three detection structures, the absorbance increases by 16% and 23%, respectively, relative to having only two detection structure layers. Thus, as shown in fig. 12E-12G, the stacked layer configuration of the sensor strip may enable tailoring of the sensitivity of the strip-type sensor assembly comprised in the ELISA kit.

Fig. 13A shows a top view of a component layer of a sensor strip that is part of a strip-type biosensor assembly similar to that described above with respect to fig. 12A-12G, including: a bottom plate 1200-4; a detection structure layer 1200-1 comprising a first detection structure 10A or a second detection structure 10B; and one or more spacer layers 1200-1, which may be assembled into a sensor strip stack 1300. Figures 13B-13D show photographic images of a perspective view, a top view, and a close-up top view, respectively, of the assembled sensor strip stack 1300 shown in figure 13A.

Fig. 14A is a top view of a component layer of a sensor strip, except as part of a strip-type biosensor assembly similar to that described above with respect to fig. 12A-12G, comprising: a bottom plate 1200-4; a detection structure layer 1200-1 comprising a first detection structure 10A; a test structure layer 1200-2 comprising a second test structure 10B; and one or more spacer layers 1200-1 that may be assembled into sensor strip stack 1400. Figures 14B-14D show photographic images of a perspective view, a top view, and a close-up top view, respectively, of the assembled sensor strip stack 1400 shown in figure 14A.

FIG. 15 is a perspective view of a sensor strip 1500 as part of a strip-type biosensor assembly, in accordance with yet another embodiment of the disclosed technology. The sensor strip 1500 is similar in some respects to the strip-type biosensor assembly described above with respect to fig. 8-14D, and for the sake of brevity, similar detailed descriptions are not repeated here. Sensor strip 1500 includes a plurality of reaction chambers, cavities, or wells 130 in a manner similar to those described above with respect to fig. 8-14D. However, unlike the sensor strip described above with respect to fig. 8-14D, the reaction chamber 130 has a contoured sidewall with a plurality of undulations or detection structures 1504. In the illustrated embodiment, the undulations 1504 are regularly, e.g., periodically, arranged around the inner surface of each of the reaction chambers or cavities 130. For example, in the illustrated embodiment, adjacent ones of the undulations 1504 are arranged about 60 ° apart around the perimeter of the reaction chamber or cavity 130. However, embodiments are not so limited, and there may be any suitable number of undulations 1504 arranged angularly spaced at any angle θ defined by 360 °/n, where n is an integer. Although in the illustrated embodiment, the undulations 1504 are circular undulations, embodiments are not so limited, and the undulations 1504 can have any suitable shape including spheres, ovoids, pyramids (e.g., rectangular, triangular), prisms (e.g., rectangular, triangular), cones, cubes, cylinders, plates, discs, wires, rods, plates, and portions of fractal, to name a few. In addition, although in the illustrated embodiment, the undulations 1504 are regularly spaced around the perimeter of the wall of the reaction chamber 130, embodiments are not limited thereto, and in some other embodiments, the undulations 1504 may be irregularly spaced around the perimeter of the wall of the reaction chamber 130. In the illustrated embodiment, sensor strip 1500 is formed as a single piece article. However, embodiments are not so limited, and in some other embodiments, sensor strip 1500 may be formed from multiple component layers, as described below.

Figures 16A-16D are perspective views of a sensor strip stack 1600 as part of a strip type biosensor assembly, in accordance with yet another embodiment of the disclosed technology. Sensor strip stack 1600 is similar in some respects to the strip-type biosensor assembly described above with respect to fig. 8-15, and for the sake of brevity, similar detailed descriptions are not repeated here. In a manner similar to that described above with respect to figures 12A-14D, sensor strip stack 1600 includes a plurality of detection structure layers 1604. Fig. 16A and 16B show perspective views showing the upper and lower surfaces, respectively, of detection structure layer 1604. Fig. 16C and 16D show detailed perspective views showing the upper and lower surfaces, respectively, of a portion of the detection structure layer 1604. When assembled, sensor strip stack 1600 additionally includes a base plate similar to base plates 1300-4, which are not shown in figures 16A-16D for the sake of brevity. When fully assembled, sensor strip stack 1600 includes a plurality of reaction chambers, cavities, or wells 130 in a manner similar to those described above with respect to fig. 8-15.

Referring to the detailed bottom view of fig. 16D, each of detection structure layers 1600 has a first thickness portion 1600A and a second thickness portion 1600B. First thickness portion 1600A includes a detection layer portion having a plurality of undulations 1604 regularly arranged (e.g., periodically arranged) around an inner surface of an opening formed therethrough. The shape and location of the undulations 1604 may be similar to the undulations described above with respect to fig. 15. And a detailed description will not be repeated herein for the sake of brevity. Unlike the first thickness portion 1600A, the second thickness portion 1600B includes a spacer layer portion that does not have undulations and is arranged in a manner similar to the spacer layer 1300-3 described above with respect to fig. 12A-12G. Thus, as a whole, first thickness portion 1600A and second thickness portion 1600B are similar in structure to the combination of detection structure layers 1300-1/1300-2 and spacer layers 1300-3 described above with respect to fig. 12A-12G. As arranged, the first thickness portion 1600A with undulations 1604 may be arranged similarly to the test structure layer 1300-1/1300-2, provide similar advantages to test structure layer 1300-1/1300-2, and serve similar functions to test structure layer 1300-1/1300-2, and the second thickness portion 1600B may be arranged similarly to spacer layer 1300-3 described above with respect to fig. 12A-12G, provide similar advantages to spacer layer 1300-3 described above with respect to fig. 12A-12G, and serve similar functions to spacer layer 1300-3 described above with respect to fig. 12A-12G.

Fig. 17A is an experimental measurement of absorbance versus assay time for HE4 with a concentration of 62.5pM (picomoles per liter) using a TMB substrate solution, measured using a biosensor assembly, according to an embodiment. This measurement shows the saturation point reached at an assay time of about 30 minutes. Fig. 17B is an experimental measurement of absorbance versus concentration for HE4 with a concentration of 62.5pM using a TMB substrate solution, measured using a biosensor assembly, according to an embodiment. The results are summarized in table 1 below.

TABLE 1

FIGS. 18A-18E illustrate portions of sensor bars 1800A-1800E, respectively, as part of a bar type biosensor assembly, according to further embodiments of the disclosed technology. In particular, the portions of sensor bars 1800A-1000E illustrate alternative arrangements of detection structures extending from the side walls of the reaction chamber. Fig. 18A shows a perspective view, and fig. 18B to 18E are plan views. Portions of sensor bars 1800A-1800E are arranged in some respects similarly to the sensor bars described above with respect to FIG. 15, and for the sake of brevity, similar detailed descriptions are not repeated here. As shown, portions of sensor bars 1800A-1800E include undulations or sensing structures 1804A-1804E, respectively, having various shapes and arrangements. In particular, the undulations 1804A-1804E have various shapes including at least partial prisms (1804A, 1804B, 1804D), partial cylinders, wires, or rods (1804D), and sharp edges (1804C), to provide some examples.

According to various embodiments described herein having detection structures or undulations extending inwardly toward a central region of a reaction chamber or cavity, the detection structures or undulations have a peak distance from a sidewall of the reaction chamber that is configured to provide an enhanced degree of variation in surface area available for reaction. The peak distance may be, for example, 0.5mm to 1mm, 1mm to 1.5mm, 1.5mm to 2.0mm, 2.0mm to 2.5mm, 2.5mm to 3.0mm, 3.0mm to 3.5mm, or a value within a range defined by any of these values.

FIGS. 19A-19F illustrate portions of sensor bars 1900A-1900F, respectively, as part of a bar-type biosensor assembly, according to further embodiments of the disclosed technology. In particular, portions of sensor bars 1900A-1900F illustrate alternative arrangements of sensing structures extending from a base plate. Fig. 19A shows a perspective view, and fig. 19B to 19F are plan views. Portions of sensor bars 1900A-1900F are arranged in some respects similarly to the sensor bars described above with respect to FIGS. 15 and 18A-18E. And for the sake of brevity, similar detailed descriptions are not repeated herein. In fig. 15 and 18A-18E the undulations or detection structures extend radially inward from the side walls of the reaction chamber or cavity 130, unlike the embodiment shown with respect to fig. 15 and 18A-18E, in which the detection structures 1904A-1904F are attached to and extend from a bottom plate. As shown, portions of sensor bars 1900A-1900F include sensing structures 1904A-1904F having various shapes and arrangements, respectively. In particular, detection structures 1904A-1904F have various shapes including vertical flat strips (1904A, 1904D, 1904F), vertical curved strips (1904B), cylinders, wires or rods (1804F), and tie strips (1904C) to provide some examples. The detection structures 1904A-1904F may be detachable from or attachable to the side walls of the reaction chamber or cavity 130.

Fig. 20A-20D illustrate portions of sensor bars 2000A-2000D, respectively, as part of a bar-type biosensor assembly, in accordance with further embodiments of the disclosed technology. Fig. 20A shows a perspective view, and fig. 20B to 20D are plan views. In particular, portions of sensor strips 2000A-2000D illustrate alternative shapes for reaction chambers, cavities, or wells 130. Portions of sensor bars 2000A-2000D are arranged in some respects similarly to the sensor bars described above with respect to fig. 8-19F, and similar detailed descriptions are not repeated here for brevity. As shown, at least a portion of the reaction chamber 130 may have various shapes including a cylindrical shape (2000A, 2000D), a polygonal prism (2000B), or a cone (2000C) of circular or oval shape to provide some examples. According to different implementations, the reaction chamber 130 may have various dimensions, including height, width, and volume. According to various implementations, the reaction chambers 130 may be radially symmetric or asymmetric, and may have the same or different top and bottom dimensions.

Surface to volume ratio of reaction chamber

In each of the embodiments of the strip-type sensor assemblies described herein, e.g., with respect to fig. 8-20D, the various surface modifications described above for the cuvette-type sensor assembly described above, e.g., with respect to fig. 3A-3E. In addition, various parameters, including: for example, the number, thickness, surface area and active surface area of the test structures; volume capacity of the reaction chamber, cavity or recess; the distance between adjacent surfaces; and the ratio of the combined active surface area to the volume of liquid held by the cavity, may have one or more of the values described herein. The inventors have realized that by controlling these and various other parameters, the reaction surface area involved in the ELISA reaction can be controlled and optimized. Table 2 below is an example calculation for a reaction chamber having an arrangement similar to that described above with respect to fig. 12F-12G.

TABLE 2

The calculations in table 2 are for a cylindrical reaction chamber with a diameter of 5mm and a detection structure as a disc-shaped structure with a radius of 1.1 mm. The minimum surface area in contact with the sample and the minimum volume of the sample correspond to the minimum amount of sample required to completely immerse the detection structure. By way of comparison, for a commercially available cylindrical reaction chamber having a height of 10.75mm and a diameter of 6.66mm, and for a sample volume of 100. mu.L, the contact surface area is about 95.93mm²Corresponding to a ratio of contact surface to sample volume of less than 1. In contrast, according to embodiments, the reaction chamber with the detection structure provides much higher values for the ratio of contact surface area to sample volume, as shown in table 2.

Table 3 below is an example calculation of the reaction area that can be impregnated in a sample as a function of the diameter of a cylindrical reaction chamber for a sample volume of 100. mu.L.

TABLE 3

Diameter (mm)	Area of penetration (mm)2)
		3	140.40
4	112.57
		5	99.63
6	94.94
		7	95.63
8	100.27
		9	108.06

The calculated relationship between the saturation area and the reaction chamber diameter is graphically shown in fig. 21A. Based on this result, the inventors determined that, for a given sample volume, there is a diameter of the reaction chamber that corresponds to the minimum saturation area. In the example shown, for a sample volume of 100 μ L, a diameter of about 6mm results in about the lowest saturation area, and the saturation area increases in non-linear inverse proportion to the diameter below 6 mm. Accordingly, when the diameter is less than about 6mm, the ELISA reaction rate is increased accordingly.

Figure 21B shows the calculated relationship between the saturation area and the reaction chamber diameter for various sample volumes. As shown, for sample volumes between about 50 μ L and 200 μ L, the diameter of the reaction chamber (below which the active surface area or saturation area of the reaction chamber rapidly increases) is between about 7mm and about 5 mm. For various sample volumes and corresponding diameters (below which the saturation area increases rapidly), the saturation area may have a value of between about 50mm²And about 400mm²Any value in between. According to these observations, the reaction chamber according to an embodiment has a ratio of the saturation surface area to the volume of about 0.25mm²mu.L to about 8mm²mu.L. For example, the sample in the reaction chamber or the reaction chamber itself may have a soaked surface area to volume ratio of between about 0.25mm²mu.L to 1mm²/μL、1mm²mu.L to 2mm²/μL、2mm²mu.L to 3mm²/μL、3mm²mu.L to 4mm²/μL，4mm²mu.L to 5mm²/μL、5mm²mu.L to 6mm²/μL、6mm²mu.L to 7mm²/μL、7mm²mu.L to 8mm²μ L, or a value within a range defined by any of these values.

Immunoassay method using sensor assembly with enhanced sensitivity and diffusion

According to various embodiments, a sensor assembly according to various embodiments described herein may be advantageously used to perform one or more of a variety of immunoassays, such as an ELISA process. According to various embodiments, the method of performing an ELISA comprises providing an ELISA kit according to any one of the embodiments described herein. The ELISA kit comprises one or more reagents, biomolecules and/or analytes for ELISA and a sensor component suitable for ELISA according to the various embodiments described above. As described above, the method additionally includes performing an ELISA reaction in an optically transparent container (e.g., one or more cavities of a cuvette or strip sensor). The method additionally includes: the following solutions were provided: the solution comprises a target analyte and a labeled detection reagent configured to specifically bind to the target analyte; immobilizing a capture reagent configured to specifically bind to the target analyte on an active surface of the sensor assembly; at least partially immersing an active surface in the solution to cause specific binding of target analytes to capture reagents and to labeled detection reagents; and detecting the target analyte that specifically binds to the capture reagent and to the labeled detection reagent.

According to various embodiments, the method of performing ELISA comprises providing an ELISA well, wherein the ELISA well comprises: a transparent container, and one or more detection structures within the optically transparent container, wherein the one or more detection structures have an active surface configured to allow binding of an antibody thereto, wherein the one or more detection structures provide a ratio of a combined surface area of the one or more detection structures to a liquid volume that is between about 0.25mm²Per microliter and about 8.0mm²Between each microliter, or any value disclosed herein. The method additionally comprises performing the ELISA with an optically clear container, wherein only a single wash is involved in the ELISA.

Different types of ELISA used in the art include direct ELISA, indirect ELISA, sandwich ELISA and competitive ELISA to name a few.

In direct ELISA, an antigen is immobilized on the surface of a multiwell plate and detected with an antibody configured to specifically bind to the antigen and directly conjugated to a detection molecule such as horseradish peroxidase (HRP).

In indirect ELISA, similar to direct ELISA, the antigen is immobilized on the surface of a multiwell plate. However, the detection is performed using a two-step process whereby a primary antibody specific for the antigen binds to the target and a labeled secondary antibody directed against the host species of the primary antibody binds to the primary antibody for detection. The method can also be used to detect specific antibodies in a serum sample by replacing the primary antibodies with serum.

In a sandwich ELISA (or sandwich immunoassay), two antibodies specific for an antigen are used, sometimes referred to as a matched antibody pair. One of the antibodies is coated (coated) on the surface of the multiwell plate and serves as a capture antibody to facilitate immobilization of the antigen. Another antibody is conjugated and facilitates detection of the antigen.

In competitive ELISA (also known as inhibition ELISA or competitive immunoassay), the concentration of antigen is measured by signal interference. The sample antigen competes with the reference antigen for binding to a specific amount of labeled antibody. Reference antigens were pre-coated on multiwell plates. The sample is pre-incubated with the labeled antibody and then added to the wells. Depending on the amount of antigen in the sample, more or less free antibody will be available for binding to the reference antigen. This means that the more antigen present in the sample, the less reference antigen will be detected and the weaker the signal. The labeled antigen and the sample antigen (unlabeled) compete for binding to the primary antibody. The lower the amount of antigen in the sample, the stronger the signal due to the more labeled antigen in the wells.

According to various embodiments, the ELISA protocols and/or components described herein can be used in an indirect ELISA, a direct ELISA with streptavidin detection, a sandwich ELISA, a competitive ELISA, and/or a sandwich ELISA with strep-biotin detection.

In some embodiments, the kit may include one or more of the following: coating buffer, blocking buffer (such as PBS, optionally with 1% BSA) and washing buffer (such as PBS with 0.05% v/v Tween-20). In some embodiments, the kit may further compriseIncluding substrate solutions (such as TMB Core + (BHU062) or pNPP (BUF044)) and stop solutions (such as 0.2M H)₂SO₄Or 1M NaOH).

In some embodiments, the method of performing ELISA may comprise one or more of: coating the surface of the detection structure and/or the surface of the concave hole with an antigen solution; optionally washing the plate in water; add blocking solution and wash plate; adding unconjugated detection antibody and washing the plate; adding an enzyme-conjugated secondary antibody and washing the plate; the substrate solution is added and the reaction allowed to occur, and then the absorbance is read from a cuvette or well. This can be used for indirect ELISA.

In some embodiments, the method of performing ELISA may comprise one or more of: coating the concave hole with an antigen solution; optionally washing the plate in water; adding a blocking solution and washing the plate; adding the sample to the well; adding biotin-conjugated detection antibody to each well (optionally washed); adding enzyme-conjugated streptavidin to the wells (optionally washed); adding the substrate solution to the well (or cuvette); the absorbance was then read. This can be used for direct ELISA.

In some embodiments, the direct ELISA comprises the steps of: (i) coating the solid phase with an antigen dissolved in a coating buffer; (ii) (ii) incubating the solid phase from step (i) with a blocking reagent for 1 hour to block non-specific binding sites on the solid phase; (iii) (iii) optionally washing the solid phase from step (ii) three times with PBS or PBST for 1 minute each time; (iv) (iv) incubating the solid phase from step (iii) with a primary detection reagent that binds to the antigen; (v) (iv) optionally washing the solid support (support) from step (iii) five times for 1 minute each in PBS or PBST to remove non-specifically bound primary detection reagent; and (vi) detecting the bound primary detection reagent using a detection system, such as UV, fluorescence, chemiluminescence, or other detection methods. The primary detection reagent may be, but is not limited to: detection reagents coupled to a fluorescent dye or reporter enzyme (reporter enzyme) such as Alkaline Phosphatase (AP) or horseradish peroxidase (HRP) can convert a colorless substrate to a colored product whose optical density can be measured at a target wavelength on an ELISA plate reader.

In some embodiments, the indirect ELISA comprises the steps of: (i) coating the solid phase with an antigen dissolved in a coating buffer; (ii) (ii) incubating the solid phase from step (i) with a blocking agent for 1 hour to block non-specific binding sites on the solid phase; (iii) (iii) optionally washing the solid phase from step (ii) three times with PBS or PBST for 1 minute each time; (iv) (iv) incubating the solid phase from step (iii) with a primary detection reagent diluted in solution for 1 hour; (v) (iii) optionally washing the solid support from step (iv) five times for 1 minute in PBS or PBST to remove non-specifically bound primary detection reagent; (vi) (vi) incubating the solid support from step (v) with a secondary detection reagent diluted in solution for 1 hour; (vii) (vii) optionally washing the solid support from step (vi) five times for 1 minute in PBS or PBST to remove non-specifically bound secondary detection reagent; (viii) the bound secondary detection reagent is detected using a detection system, such as UV, fluorescence, chemiluminescence, or other methods. The secondary detection reagent is combined with the primary detection reagent. The secondary detection reagent may be, but is not limited to: a detection reagent coupled to a reporter enzyme such as Alkaline Phosphatase (AP) or horseradish peroxidase (HRP) that can convert a colorless substrate to a colored product whose optical density can be measured at a target wavelength on an ELISA plate reader.

In some embodiments, the direct ELISA procedure involves at least three incubation steps: the first is an incubation between the solid support and the antigen; the second is an incubation between the solid support and the blocking reagent; the third is an incubation between the solid support and the primary detection reagent. The incubation step can be a two-stage reaction and involves a binding reaction between the antigen on the solid support and the detection reagent.

In some embodiments, the indirect ELISA procedure involves at least four incubation steps: the first is an incubation between the solid support and the antigen; the second is an incubation between the solid support and the blocking reagent; the third is an incubation between the solid support and the primary detection reagent; the fourth is incubation between the solid support and the secondary detection reagent. The incubation step can be a two-stage reaction and involves a binding reaction between the antigen on the solid support and the detection reagent.

In some embodiments, for direct ELISA, the first incubation step, antigen coating, takes at least 2 hours, and each other incubation step takes about 1 hour.

In some embodiments, for indirect ELISA, the first incubation step, antigen coated, takes at least 2 hours, and each other incubation step takes about 1 hour.

In some embodiments, the cell-based ELISA (C-ELISA) is a medium-throughput format for the detection and quantification of the following cellular proteins: the cellular proteins include post-translational modifications associated with cellular activation (e.g., phosphorylation and degradation). The cells were plated, treated according to experimental requirements, directly fixed in a dent hole, and then permeabilized. After permeabilization, the fixed cells are subjected to a treatment similar to conventional immunoblotting, including blocking, incubation with primary antibodies, washing, incubation with secondary antibodies, addition of chemiluminescent substrate, and development.

In some embodiments, ELISA is performed by: the activated wells were coated with antigen overnight at 4 ℃, the wells were blocked at 37 ℃ for 2 hours, followed by antibody and conjugate binding at 37 ℃ for 2 hours respectively and color development, which is an enzyme-substrate reaction at room temperature for 5 minutes, followed by reading the absorbance.

In some embodiments, the ELISA kit may be one or more of: acetylcholine ELISA kit, AGE ELISA kit, CXCL13 ELISA kit, FGF23 ELISA kit, HMGB1 ELISA kit, iNOS ELISA kit, LPS ELISA kit, malondialdehyde ELISA kit, melatonin ELISA kit, NAG ELISA kit, OVA ELISA kit, oxytocin ELISA kit, PGE2 ELISA kit, PTHrP ELISA kit, S100B ELISA kit, tenascin C ELISA kit, VEGF-B ELISA kit and/or pluripotent proteoglycan ELISA kit.

In some embodiments, the ELISA may be performed under a first set of conditions that allow selective and/or specific binding of an antibody or other binding molecule to a target or antigen. The ELISA can then be continued under the same set of conditions, or those conditions can be altered during the enzymatic step of the technique. This may allow for more variation of the enzymatic process in the process parameters. In some embodiments, an antibody (as shown in fig. 1A) is used to immobilize the antigen. In some embodiments, other proteins or structures (binding molecules, such as receptor molecules or enzymes, etc.) may be immobilized as long as they are still capable of binding to the target molecule. Thus, as will be understood by those of skill in the art, although the term ELISA is used throughout, the methods and devices provided herein are not limited to "immunological" assays, but may instead employ other binding molecules in place of immunological (e.g., antibody) components for any of the embodiments provided herein. This applies to all suitable embodiments provided herein. In some embodiments, the ELISA employed is one or more of a direct ELISA, an indirect ELISA, a sandwich ELISA, a competitive ELISA, and/or a reverse ELISA.

In some embodiments, the method involves: a first binding solution for allowing a desired binding selectivity; and a second solution for the enzymatic component of the assay. In some embodiments, the buffers in the solution are the same. In some embodiments, the buffer is changed throughout the process (and may change the salt concentration or the type of monovalent or divalent salt or other components present). In some embodiments, the temperature during the binding phase is designed for binding, and the temperature during the enzymatic phase is designed for enzymatic activity. In some embodiments, the temperatures are the same or substantially the same. In some embodiments, the temperature is different, but still allows for continued binding during the enzymatic phase. In some embodiments, the temperature and/or solution composition is altered between the binding and enzymatic stages to the extent that some or many or even all of the target can be dissociated from the binding molecule. However, this can be solved by: during the binding phase and through any elution phase (if present), the target is kept bound to the binding molecule, and then the solution used for the enzymatic phase is retained in the well or in the same volume of liquid. In other embodiments, the target remains bound to the antigen binding molecule throughout the process.

In the following, a description of an example homogeneous immunoassay method (referred to herein as a one-step immunoassay method) is provided for antigen detection using a biosensor according to some embodiments.

Fig. 22 is a flowchart illustrating a method of analyzing a sample using a cuvette-type biosensor according to some embodiments, and fig. 23 is a flowchart illustrating a method of analyzing a sample using a strip-type biosensor according to some embodiments.

First, as shown in fig. 22, the one-step immunoassay method for antigen detection using a cuvette-type biosensor according to some embodiments includes: (a) preparing a sample comprising an analyte of interest (e.g., an antigen) and a detection biomolecule complex solution (e.g., a detection antibody complex solution) comprising a detection biomolecule labeled with a label (e.g., a detection antibody); (b) sequentially immersing a detection structure surface capable of binding to the target analyte, binding to the immobilized biomolecule (e.g., capture antibody), in any order, in the sample and detection biomolecule complex solution, or immersing a detection structure surface bound to the immobilized biomolecule in a mixed solution of the sample and detection biomolecule complex solution, and (c) measuring the absorbance of the reaction product.

In step (b), the immobilized biomolecule, the target analyte and the detection biomolecule labeled with the label react with each other. Approximately 15-30 minutes after the reaction begins, the test construct is immersed in a cuvette containing the enzyme substrate and the absorbance of the cuvette is measured. After step (b) is complete, the detection structure can be washed to remove unreacted target analyte and/or the labeled detection biomolecule, and the detection structure can be immersed in a cuvette containing an enzyme substrate.

When the strip type biosensor is used, in step (b), the sample and the detection biomolecule complex solution are sequentially injected through the sample injection hole in any order, or a mixture of the sample and the detection biomolecule complex solution is injected through the sample injection hole. In step (c), an enzyme substrate is injected and the absorbance of the reaction product is measured.

Aflatoxin B1, streptomycin, human epididymis protein 4(HE4), carcinoembryonic antigen (CEA), mouse IgG and cortisol (cortisol) were used as target analytes and the absorbance of the reaction products with target analytes at different concentrations was measured. The results are shown in fig. 24A to 24F.

Aflatoxin B1 was detected at concentrations ranging from 19.53 to 312.5pg/mL within 45 minutes (see figure 24A). Streptomycin was detected at concentrations ranging from 0.39 to 50ng/mL within 30 minutes (see fig. 24B).

To determine whether the sensitivity of the biosensor of the present invention was improved, HE4, CEA, mouse IgG, and cortisol were analyzed using currently commercially available biosensors (ELISA kits, R & D Systems). The absorbance measured from the biosensor of the present invention and the commercial biosensor is represented by a solid line and a dotted line in fig. 24C to 24F.

The biosensors of the present invention successfully detected HE4 at concentrations ranging from 6.1 to 390pg/mL in 30 minutes, whereas the commercial biosensors detected the same analyte of interest at concentrations ranging from 78 to 5,000pg/mL in 4h (see fig. 24C).

The biosensors of the present invention successfully detected CEA at concentrations ranging from 0.2 to 390ng/mL in about 15-30 minutes, whereas commercial biosensors detected the same analyte of interest at concentrations ranging from 1 to 65ng/mL in 90 minutes (see FIG. 24D).

The biosensor of the present invention successfully detected mouse IgG at concentrations ranging from 0.05 to 25ng/mL in 30 minutes, whereas the commercial biosensor detected the same analyte of interest at concentrations ranging from 7.8 to 500ng/mL in 120 minutes (see fig. 24E).

The biosensor of the present invention successfully detected cortisol at a concentration ranging from 0.15 to 5ng/mL within 45 minutes. Whereas commercial biosensors detect the same target analyte in concentrations ranging from 0.15 to 10ng/mL in 180 minutes. (see FIG. 24F).

Fig. 25A and 25B are experimental measurements of absorbance of reaction products with various analyte antibodies at different concentrations, measured using a biosensor assembly according to an embodiment. As described above in various embodiments, the active or reaction area of various configurations of biosensor assemblies according to embodiments is much larger than that of conventional ELISA plates for comparable sample volumes. Compared to conventional ELISA plates, embodiments have a larger amount of immobilized substance (capture molecule or receptor) that can react with the target. In this regard, biosensors enable assays with reduced hook effects (enhanced assay sensitivity) and increased reaction rates (reduced assay time). This is illustrated with respect to fig. 25A and 25B, where the concentration of a receptor or antibody reactive with a protein (e.g., antigen) is greatly increased by stacking a plurality of detection structures. For example, as shown in fig. 25B, a biosensor having six detection structures detects IgG with higher sensitivity than a biosensor having two detection structures. As described above, the higher sensitivity for biosensors having a large number of detection structures can be attributed to the higher density of immobilized biomolecules and/or analytes (e.g., capture molecules or receptors) available in the defined volume, as well as their enhanced diffusion.

Taken together, these results indicate that the biosensor according to some embodiments has significantly improved sensitivity and can detect a target analyte in a short time.

Example embodiments

1. An enzyme-linked immunosorbent assay (ELISA) kit comprising:

one or more reagents for an ELISA and a sensor assembly suitable for use in the ELISA, wherein the sensor assembly comprises:

a transparent container having one or more cavities formed therein;

a plurality of active surfaces disposed in each of the one or more cavities and configured to immobilize a reagent on the plurality of active surfaces; and

one or more transparent detection structures disposed in each of the one or more cavities, wherein each of the transparent detection structures comprises one or more major surfaces that provide one or more of the active surfaces,

wherein the one or more cavities are configured to be filled with a liquid such that each of the transparent detection structures is at least partially immersed in the liquid, wherein a ratio of a combined surface area of the transparent structures in contact with the liquid to a volume of the liquid exceeds about 0.25mm²Per microliter, and

wherein each of the active surfaces is separated from an immediately adjacent one of the active surfaces by a distance of more than about 500 microns.

2. An enzyme-linked immunosorbent assay (ELISA) kit comprising:

one or more reagents for an ELISA and a sensor assembly suitable for use in the ELISA, wherein the sensor assembly comprises:

a transparent container having one or more cavities formed therein;

a plurality of active surfaces disposed in each of the one or more cavities and configured to immobilize a reagent on the plurality of active surfaces; and

one or more transparent detection structures disposed in each of the one or more cavities, wherein each of the transparent detection structures comprises one or more major surfaces that provide one or more of the active surfaces,

wherein the one or more cavities are configured to be filled with a liquid such that each of the transparent detection structures is at least partially immersed in the liquid,

wherein the ratio of the combined surface area of the transparent structure in contact with the liquid to the volume of the liquid is between about 0.25mm²Per microliter and about 8.0mm²Between each microliter.

3. An enzyme-linked immunosorbent assay (ELISA) kit comprising:

one or more reagents for an ELISA and a sensor assembly suitable for use in the ELISA, wherein the sensor assembly comprises:

a transparent container having one or more cavities formed therein;

a plurality of active surfaces disposed in each of the one or more cavities and configured to immobilize a reagent on the plurality of active surfaces; and

one or more transparent detection structures disposed in each of the one or more cavities, wherein each of the transparent detection structures comprises one or more major surfaces that provide one or more of the active surfaces,

wherein each of the active surfaces is separated from an immediately adjacent one of the active surfaces by a distance of between about 500 microns and about 8 millimeters.

4. An enzyme-linked immunosorbent assay (ELISA) kit comprising:

one or more reagents for an ELISA and a sensor assembly suitable for use in the ELISA, wherein the sensor assembly comprises:

a transparent container having one or more cavities formed therein;

a plurality of active surfaces disposed in each of the one or more cavities and configured to immobilize a reagent on the plurality of active surfaces; and

one or more transparent detection structures disposed in each of the one or more cavities, wherein each of the transparent detection structures comprises one or more major surfaces that provide one or more of the active surfaces,

wherein at least one of the active surfaces comprises a textured polymeric surface having micro-or nano-structures.

5. An enzyme-linked immunosorbent assay (ELISA) kit comprising:

one or more reagents for an ELISA and a sensor assembly suitable for use in the ELISA, wherein the sensor assembly comprises:

a transparent container having one or more cavities formed therein; and

one or more transparent detection structures disposed in each of the one or more cavities,

wherein the inner surface of the cavity and the major surface of the one or more transparent detection structures provide an active surface thereon configured for immobilizing a reagent configured to specifically bind to an analyte, and

wherein a major surface of the transparent detection structure is configured such that: when performing an ELISA, the detectable optical density corresponding to the analyte specifically binding to the immobilized reagent is increased without decreasing the rate of specific binding of the analyte to the immobilized reagent relative to a sensor assembly without one or more transparent detection structures.

6. The ELISA kit of any one of embodiments 1 to 5, wherein each of the transparent detection structures comprises a transparent solid polymer structure.

7. The ELISA kit of any one of embodiments 1 to 6, wherein each of the active surfaces comprises a solid polymer surface.

8. The ELISA kit of any one of embodiments 1 to 7, wherein the active surface does not comprise a metal formed thereon.

9. The ELISA kit of any one of embodiments 1 to 8, wherein the optically transparent container comprises a cuvette.

10. The ELISA kit of embodiment 9, wherein each of the one or more transparent detection structures comprises a plate structure having opposing major surfaces substantially parallel to a depth direction of the cavity of the cuvette.

11. The ELISA kit of embodiment 9 or embodiment 10, wherein each of the one or more transparent detection structures comprises a plate structure having opposing major surfaces that are substantially parallel to each other.

12. The ELISA kit of any one of embodiments 9 to 11, wherein a major surface of one of the one or more transparent detection structures that directly faces an interior sidewall of the cuvette is separated from the interior sidewall of the cuvette by a distance of more than about 500 micrometers.

13. The ELISA kit of any one of embodiments 9 to 12, wherein a major surface of the one or more transparent detection structures that directly faces an interior sidewall of the cuvette is substantially parallel to the interior sidewall of the cuvette.

14. The ELISA kit according to any of embodiments 9 to 13, comprising two or more transparent detection structures, wherein major surfaces of directly adjacent transparent detection structures of the two or more transparent detection structures that directly face each other are separated from each other by a distance of more than 500 micrometers.

15. The ELISA kit according to any one of embodiments 9 to 14, comprising two or more transparent detection structures, wherein the major surfaces of directly adjacent transparent detection structures of the two or more transparent detection structures that directly face each other are substantially parallel to each other.

16. The ELISA kit according to any one of embodiments 9 to 15, wherein one or more of the major surfaces of the one or more transparent detection structures and one or more of the inner side walls of the cuvette serve as active surfaces.

17. The ELISA kit of any one of embodiments 9 to 16, wherein one or more of the major surfaces of the one or more transparent detection structures substantially overlap the side wall of the cuvette in a lateral direction orthogonal to the depth direction of the cavity of the cuvette.

18. The ELISA kit of any one of embodiments 9 to 17, wherein the one or more transparent detection structures have a thickness between about 100 micrometers and about 53000 micrometers.

19. The ELISA kit of any one of embodiments 9 to 18, wherein each of the major surfaces has an area of between about 10mm²And about 100mm²In the meantime.

20. The ELISA kit of any one of embodiments 9 to 19, wherein the cuvette is configured to hold a volume of between about 50mm³And about 3000mm³In between.

21. The ELISA kit according to any one of embodiments 1 to 8, wherein the optically transparent container comprises a strip container comprising a plurality of cavities formed therein.

22. The ELISA kit of embodiment 21, wherein each of the one or more transparent detection structures comprises a plate structure having opposing major surfaces substantially perpendicular to the depth direction of the cavity.

23. The ELISA kit of embodiment 21 or embodiment 22, wherein each of the one or more transparent detection structures comprises a plate structure having opposing major surfaces that are substantially parallel to each other.

24. The ELISA kit of any one of embodiments 21 to 23, wherein a major surface of one of the one or more transparent detection structures that directly faces a bottom surface of a respective one of the cavities can be substantially separated from the bottom surface of the respective one of the cavities by a distance of more than about 500 micrometers.

25. The ELISA kit of any one of embodiments 21 to 24, wherein a major surface of one of the one or more transparent detection structures that directly faces a bottom surface of a respective one of the cavities can be substantially parallel to the bottom surface of the respective one of the cavities.

26. The ELISA kit of any one of embodiments 21 to 25, wherein each of the one or more transparent detection structures comprises a plate structure disposed laterally at a central region of a respective one of the cavities.

27. The ELISA kit of any one of embodiments 21 to 26, wherein one or more of the major surfaces of the one or more transparent detection structures substantially overlap in a vertical direction substantially parallel to the depth direction of the cavity.

28. The ELISA kit of any one of embodiments 21 to 27, comprising two or more transparent detection structures, wherein major surfaces of directly adjacent transparent detection structures of the two or more transparent detection structures that directly face each other are separated from each other by a distance of more than 500 micrometers.

29. The ELISA kit of any one of embodiments 21 to 28, comprising two or more transparent detection structures, wherein major surfaces of directly adjacent transparent detection structures of the two or more transparent detection structures that directly face each other are substantially parallel to each other.

30. The ELISA kit of any one of embodiments 21 to 29, wherein each of the transparent detection structures has a substantially circular shape.

31. The ELISA kit of any one of embodiments 21 to 25, wherein each of the one or more transparent detection structures comprises a protrusion extending from an inner surface of a respective one of the cavities.

32. The ELISA kit of embodiment 31, wherein each of the one or more transparent detection structures comprises a plate structure extending laterally towards a central region of a respective one of the cavities.

33. The ELISA kit of embodiment 31 or embodiment 32, wherein the one or more transparent detection structures comprise one or more first protrusions formed at a first vertical level in the depth direction of the cavity.

34. The ELISA kit of embodiment 33, wherein the one or more transparent detection structures comprise one or more second protrusions formed at a second vertical level in the depth direction of the cavity.

35. The ELISA kit of embodiment 34, wherein at least one of the one or more first protrusions does not overlap any of the one or more second protrusions in any lateral direction perpendicular to the depth direction of the cavity.

36. The ELISA kit of embodiment 34, wherein at least one of the one or more first protrusions at least partially overlaps one or more second protrusions in a lateral direction perpendicular to a depth direction of the cavity.

37. The ELISA kit of any one of embodiments 34 to 36, wherein the one or more transparent detection structures comprise two or more first protrusions and/or two or more second protrusions arranged periodically around the inner surface of a respective one of the cavities.

38. The ELISA kit of any one of embodiments 34 to 37, wherein the one or more first protrusions and the one or more second protrusions are separated in the depth direction of the cavity by a spacer region without protrusions.

39. The ELISA kit of any of embodiments 31 to 38, wherein one or more of the major surfaces of the one or more transparent detection structures and one or more of the inner surfaces of the respective ones of the cavities serve as active surfaces.

40. The ELISA kit of any one of embodiments 31 to 39, wherein a central region of each of the cavities not occupied by one or more transparent detection structures is configured to receive a tip of a pipette therein.

41. The ELISA kit of any of embodiments 21 to 38, wherein each of the one or more transparent detection structures has a shape comprising a portion of a circle.

42. The ELISA kit of any one of embodiments 21 to 41, wherein the one or more transparent detection structures have a thickness between about 100 and about 2000 microns.

43. According to any one of embodiment 21 to embodiment 42The ELISA kit of embodiment, wherein each of the major surfaces of the one or more transparent detection structures has between about 10mm²And about 40mm²The area in between.

44. The ELISA kit of any one of embodiments 21-43, wherein each of the cavities is configured to accommodate a volume of between about 50mm³And about 500mm³In between.

45. The ELISA kit of embodiment 31, wherein the one or more transparent detection structures comprise protrusions extending from an inner surface of a respective one of the cavities, wherein the protrusions form undulations on the inner surface of the cavity.

46. The ELISA kit of embodiment 45, wherein the undulations have a length that extends through at least a partial depth of the cavity.

47. The ELISA kit of any one of embodiments 1 to 46, wherein at least one of the active surfaces comprises a plurality of microstructures or nanostructures formed on the at least one active surface.

48. The ELISA kit of embodiment 47, wherein the microstructures or nanostructures comprise polymeric microstructures or nanostructures.

49. The ELISA kit of embodiment 47 or embodiment 48, wherein the microstructures or nanostructures comprise regularly arranged nanostructures.

50. The ELISA kit of any one of embodiments 47-49, wherein each of the microstructures or nanostructures comprises a protrusion having a cross-sectional area that decreases away from a base.

51. The ELISA kit of any one of embodiments 47 to 50, wherein the microstructures or nanostructures have the shape of truncated spheres or polygons.

52. The ELISA kit of any one of embodiments 47 to 50, wherein the microstructures or nanostructures have a prismatic shape.

53. The ELISA kit of any one of embodiments 47 to 50, wherein the microstructures or nanostructures have the shape of a truncated cylinder.

54. The ELISA kit of any one of embodiments 47 to 50, wherein the microstructures or nanostructures have the shape of a conical frustum or a pyramidal frustum.

55. The ELISA kit of embodiment 47 or embodiment 48, wherein the microstructures or nanostructures are randomly or pseudo-randomly arranged in one or more lateral directions.

56. The ELISA kit of any one of embodiment 47, embodiment 48, or embodiment 55, wherein the microstructures or nanostructures comprise microwires, microposts, microfibers, nanowires, nanopillars, or nanofibers.

57. The ELISA kit of any one of embodiments 47-56, wherein the microstructures or nanostructures form an integral extension comprising the same material as the solid substrate.

58. The ELISA kit of any one of embodiments 1 to 57, wherein the active surface of the sensor assembly has immobilized thereon a capture reagent configured to specifically bind to a target analyte.

59. The ELISA kit of any one of embodiments 1 to 58, wherein the one or more cavities have a solution comprising a target analyte and a labeled detection reagent configured to specifically bind to the target analyte.

60. The ELISA kit of any one of embodiments 1 to 59, wherein the one or more cavities have a solution comprising an analyte of interest bound to a labeled detection reagent and further comprising an enzyme substrate.

61. The ELISA kit of any one of embodiments 1 to 60, wherein the one or more cavities have ELISA products produced from an enzyme substrate in an ELISA reaction.

62. A method of performing an enzyme-linked immunosorbent assay (ELISA), the method comprising:

providing an ELISA kit according to any one of embodiments 1 to 61, and

the ELISA reaction was performed in an optically clear container.

63. The method of embodiment 62, wherein performing the ELISA reaction comprises:

providing a solution comprising a target analyte and a labeled detection reagent configured to specifically bind to the target analyte;

immobilizing a capture reagent configured to specifically bind to the target analyte on an active surface of the sensor assembly;

at least partially immersing the active surface in the solution to cause specific binding of the target analyte to the capture reagent and to the labeled detection reagent; and

detecting the target analyte that specifically binds to the capture reagent and to the labeled detection reagent.

64. The method of embodiment 66, wherein the target analyte specifically binds to the capture reagent and to the labeled detection reagent in a single reaction step in 30 minutes or less prior to detecting the target analyte specifically bound to the capture reagent and to the labeled detection reagent.

65. The method of embodiment 63 or embodiment 64, wherein the sensor assembly is washed after causing the analyte to specifically bind to the capture reagent and to the labeled detection reagent, wherein the method does not include an additional washing step.

66. The method according to any one of embodiments 62 to 65, wherein the ELISA is selected from the group comprising: direct ELISA, indirect ELISA, sandwich ELISA, competitive ELISA and Enzyme Linked Immunospot (ELISPOT) assays.

67. A method of performing an ELISA, the method comprising:

providing an ELISA well, wherein the ELISA well comprises:

1) a transparent container, and

2) one or more enhancement layers within an optically transparent container, wherein the one or more enhancement layers are configured to allow binding of an antibody to the enhancement layer, wherein the one or more enhancement layers provide a ratio of the combined surface area to liquid volume of the one or more enhancement layers, said ratio being between about 0.25mm²Per microliter and about 8.0mm²Between each microliter; and

performing an ELISA with the optically clear container, wherein only a single wash is involved in the ELISA.

68. The method of embodiment 67, wherein the ELISA comprises: the detection antibody labeled with the label is incubated with a sample containing the protein of interest to form a first reaction mixture.

69. The method of embodiment 68, wherein the ELISA further comprises: the first reaction mixture is taken up and allowed to react with the substrate on which the immobilized antibody is immobilized.

70. The method of embodiment 69, wherein said ELISA can be performed by adding said sample at once.

71. The method of any one of the embodiments provided herein, wherein the ELISA aspect comprises incubating HRP and adding a substrate solution comprising a substrate, wherein the substrate is converted to a detectable form by HRP.

72. The method of embodiment 70, wherein the detectable form comprises a color signal.

73. A biosensor comprising a detection structure in the shape of a plate having a first surface and a second surface opposite the first surface, wherein an immobilized species that specifically binds to a target analyte is disposed on at least one of the first and second surfaces.

74. The biosensor of embodiment 73, wherein microstructures or nanostructures in the form of protrusions are formed on at least one of the first surface and the second surface of the detection structure and an immobilized species is attached to an outer surface of the microstructures or nanostructures in the form of protrusions.

75. The biosensor of embodiment 73, wherein the target analyte is selected from the group consisting of: amino acids, peptides, polypeptides, proteins, glycoproteins, lipoproteins, nucleosides, nucleotides, oligonucleotides, nucleic acids, sugars, carbohydrates, oligosaccharides, polysaccharides, fatty acids, lipids, hormones, metabolites, cytokines, chemokines, receptors, neurotransmitters, antigens, allergens, antibodies, substrates, cofactors, inhibitors, drugs, nutrients, prions, toxins, poisons, explosives, pesticides, chemical warfare agents, biohazards, bacteria, viruses, radioisotopes, vitamins, heterocyclic aromatic compounds, carcinogens, mutagens, narcotics, amphetamines, barbiturates, hallucinogens, waste products, pollutants, and mixtures thereof.

76. The biosensor of embodiment 73, wherein the detection structure is inserted and immersed in a cuvette containing a sample containing the target analyte such that the immobilized species reacts with the target analyte.

77. The biosensor of embodiment 76, further comprising a grip member connected to one end of the detection structure and gripped by a user.

78. The biosensor of embodiment 77, further comprising a lid connecting the detection structure to the grasping member and releasably inserted into the inlet of the cuvette.

79. The biosensor of embodiment 78, further comprising a fixing member disposed on an outer surface of the cap and having a shape that is changed to generate elasticity when the cap is inserted into the cuvette, wherein the fixing member is in close contact with an inner circumferential surface of the cuvette by the elasticity.

80. The biosensor of embodiment 76, wherein the detection structure is divided into an immersed portion immersed in the sample and a non-immersed portion having a narrow portion with a width smaller than a width of the immersed portion.

81. The biosensor of embodiment 80, wherein the narrow portion is recessed from at least one of two sides of the detection structure and extends along a length direction of the detection structure.

82. The biosensor of embodiment 76, wherein the detection structures are provided as a plurality of detection structures, and the detection structures are spaced apart from and parallel to each other.

83. The biosensor of embodiment 73, further comprising at least one sensor strip comprising a body having a predetermined length and a plurality of reaction chambers recessed from one surface of the body to accommodate a sample containing the target analyte, wherein the detection structure is disposed in each of the reaction chambers.

84. The biosensor of embodiment 83, further comprising a fixation plate having a lower surface: the sensor strip is removably attached to the surface.

85. The biosensor of embodiment 83, wherein the detection structures are provided as a plurality of detection structures and the detection structures are vertically spaced apart from each other.

86. The biosensor of embodiment 83, further comprising a sample injection hole recessed from one surface of the body to communicate with the reaction chamber.

87. The biosensor of embodiment 84, further comprising an insertion protrusion protruding from one surface of the fixation plate, wherein the body is recessed or perforated to form an insertion recess into which the insertion protrusion is inserted such that the sensor strip is adhered to the fixation plate.

88. The biosensor of embodiment 87, further comprising a fixation protrusion spaced apart from the insertion-out portion and protruding from one surface of the fixation plate such that the insertion protrusion contacts an inwardly recessed corner of one end of the body when inserted into an insertion hole.

89. A sensor assembly suitable for use in an enzyme-linked immunosorbent assay (ELISA), the sensor assembly comprising:

a sensor strip comprising one or more wells formed in the sensor strip, each well of the one or more wells having a side wall and a bottom surface;

and

one or more detection structures connected to the sidewall of each of the one or more wells,

wherein the one or more detection structures are configured to immobilize a biomolecule directly on the one or more detection structures.

90. The sensor assembly of embodiment 89, further comprising the biomolecule immobilized directly on the one or more detection structures.

91. The sensor assembly of embodiment 89 or embodiment 90, wherein the biomolecule comprises an antibody configured to specifically bind to a target analyte of the ELISA.

92. The sensor assembly of any one of embodiments 89-91, wherein each of the one or more detection structures extends laterally toward a central region of a respective one of the one or more wells.

93. The sensor assembly of any one of embodiments 89-92, wherein the one or more detection structures comprise one or more first detection structures formed at a first vertical level in a depth direction of the one or more recessed holes.

94. The sensor assembly of any one of embodiments 89-93, wherein the one or more detection structures comprise one or more second detection structures formed at a second vertical level in the depth direction that is deeper than the first depth.

95. The sensor assembly of any one of embodiments 89-94, wherein at least a portion of the one or more first detection structures does not overlap the one or more second detection structures in a depth direction of the one or more recessed apertures.

96. The sensor assembly of any one of embodiments 89-95, wherein the sensor strip comprises a plurality of layers each having an opening formed therethrough, wherein at least one of the layers comprises one or more detection structures protruding from a sidewall of the opening.

97. The sensor assembly of any one of embodiments 89-96, wherein the sensor strip comprises at least two layers comprising one or more detection structures vertically separated by a spacer layer.

98. The sensor assembly of any one of embodiments 89-97, wherein the one or more detection structures comprise a wave that protrudes laterally toward a central region of a respective one of the one or more wells and is elongated vertically along a depth of the respective one of the one or more wells.

99. The sensor assembly of any one of embodiments 89-99, wherein the one or more detection structures comprise surfaces that: the surface is textured to have a plurality of micro-or nanostructures formed on the surface.

100. The sensor assembly of any one of embodiments 89-99, wherein each of the one or more detection structures comprises a plate structure disposed laterally at a central region of a respective one of the one or more wells.

101. The sensor assembly of any one of embodiments 89-100, wherein the one or more detection structures comprise at least two substantially parallel plate structures that substantially overlap one another in a depth direction of the one or more recessed wells.

102. The sensor assembly of any one of embodiments 89-101, wherein vertically adjacent ones of the at least two plate structures are separated from each other in a depth direction of the one or more recessed holes by a distance of between 500 microns and 8 mm.

103. The sensor assembly of any one of embodiments 89-102, wherein the one or more recesses are cylindrical recesses having a flat bottom surface.

104. The sensor assembly of any one of embodiments 89-103, wherein each of the wells is configured to hold a sample having a volume of about 50 μ Ι _ to about 500 μ Ι _.

105. The sensor assembly of any one of embodiments 89-104, wherein the one or more recessed holes have a diameter of between about 2mm and about 9 mm.

106. The sensor assembly of any one of embodiments 89-105, wherein, when one or more wells are filled with sample, the ratio of the combined surface area in contact with the sample to the volume of the sample exceeds about 0.25mm²Per microliter.

107. A sensor assembly suitable for use in an enzyme-linked immunosorbent assay (ELISA), the sensor assembly comprising:

a cuvette including a cavity;

a cover configured to enclose the cavity; and

one or more detection structures connected to the lid and configured to be at least partially immersed in a liquid sample when present in the cavity,

wherein the one or more detection structures are configured to immobilize a biomolecule directly on the one or more detection structures.

108. The sensor assembly of embodiment 107, further comprising the biomolecule immobilized directly on the one or more detection structures.

109. The sensor assembly of embodiment 107 or embodiment 108, wherein the biomolecule comprises an antibody configured to specifically bind to a target analyte of the ELISA.

110. The sensor assembly of any one of embodiments 107-109, wherein each of the one or more detection structures comprises a plate structure having opposing major surfaces that are substantially parallel to a depth direction of the cavity of the cuvette.

111. The sensor assembly of any one of embodiments 107-110, wherein each of the one or more detection structures comprises a plate structure having opposing major surfaces that are substantially parallel to each other.

112. The sensor assembly of any one of embodiments 107-111, comprising two or more detection structures, wherein major surfaces of directly adjacent detection structures of the two or more detection structures that directly face each other are separated from each other by a distance of between about 500 microns and 9 mm.

113. The sensor assembly of any one of embodiments 107-detection structures 112, comprising two or more detection structures, wherein major surfaces of directly adjacent detection structures of the two or more detection structures that directly face each other are substantially parallel to each other.

114. The sensor assembly of any one of embodiments 107-sensing structures 113, wherein each of the one or more sensing structures comprises a plate structure having a straight edge extending in a depth direction of the cavity, wherein the straight edge comprises one or more recessed regions that reduce a width of the plate structure.

115. The sensor assembly of any one of embodiments 107-sensing structures 114, wherein at least one of the one or more sensing structures comprises a surface comprising: the surface is textured to have a plurality of micro-or nanostructures formed on the surface.

116. The sensor assembly of any one of embodiments 107-detection structures 115, wherein, when the cavity is filled with a sample, the ratio of the combined surface area in contact with the sample to the volume of the sample is about 0.1-8mm²Per microliter.

Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise," "comprising," "contain," and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is, in the sense of "including, but not limited to". As generally used herein, the term "coupled" applies to two or more elements that may be connected directly or by way of one or more intervening elements. Also, as generally used herein, the word "connected" applies to two or more elements that may be connected directly or through one or more intermediate elements. Additionally, the words "herein," "above," "below," and words of similar import, when used in this application, shall apply to this application as a whole and not to any particular portions of this application. Where the context permits, words in the above detailed description using the singular or plural number may also include the plural or singular number respectively. The word "or" refers to a list of two or more items that encompasses all of the following interpretations of the word: any of the items in the list, all of the items in the list, and any combination of the items in the list.

Furthermore, conditional language such as "can," might, "" can, "" such as, "" e.g., "such as," and the like, as used herein, are generally intended to convey that certain embodiments include, but not certain features, elements, and/or states unless specifically stated otherwise or otherwise understood in the context of use. Thus, such conditional language is not generally intended to imply that features, elements, and/or states are in any way required for one or more embodiments or that these features, elements, and/or states are included in or are to be performed in any particular embodiment.

While certain embodiments have been described, these embodiments have been presented by way of example only, and are not intended to limit the scope of the present disclosure. Indeed, the novel apparatus, methods, and systems described herein may be embodied in a variety of other forms; furthermore, various omissions, substitutions and changes in the form of the methods and systems described herein may be made without departing from the spirit of the disclosure. For example, while features are presented in a given arrangement, alternative embodiments may perform similar functions with different components and/or sensor topologies, and some features may be deleted, moved, added, subdivided, combined, and/or modified. Each of these features may be implemented in a variety of different ways. Any suitable combination of the elements and acts of the various embodiments described above can be combined to provide further embodiments. The various features and processes described above may be implemented independently of one another or may be combined in various ways. All possible combinations and sub-combinations of the features of the present disclosure are intended to fall within the scope of the present disclosure.