Apparatus and method for the detection of molecules in lateral flow assays

Abstract

An apparatus and a method for the detection of a biomolecule (e.g., nucleic acid, protein, hormone) in a sample by using a lateral flow assay. The apparatus comprises a holder (8) which accommodates a lateral flow assay strip (1). The strip (1) is illuminated through an aperture (10) of the holder (8) and the light transmitted through the strip (1) is detected by a detector (23). An optical slit (25), is positioned between the detector (23) and the strip (1). The apparatus further comprises a processor configured to process the readings of the detector (23).

Description

[0001] APPARATUS AND METHOD FOR THE DETECTION OF MOLECULES IN LATERAL FLOW ASSAYS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to an apparatus and a method for detecting biomolecules captured on lateral flow assay strips by using light.

[0004] BACKGROUND OF THE INVENTION

[0005] In recent years, the demand for rapid and reliable point-of-care (PoC) diagnostic tests has increased due to the need for time and cost-effective healthcare. Among the various diagnostic tools available, lateral flow assays (LFAs) have emerged as a versatile and valuable technology for PoC testing. LFAs offer a simple and user-friendly format that allows rapid detection of target analytes such as pathogens, biomarkers, or specific molecules (e.g. nucleic acids, proteins, hormones) with minimal instrument requirements (Sadeghi et al., 2021). Lateral flow assays are based on the principles of immunoassays and utilize the specific binding capabilities of antibodies or other biological recognition elements. Based on the recognition elements involved in the assay, LFAs can be categorized into different types, immunoassays (LFIA) (i.e., assays detecting antibodies, hormones, proteins etc.) and the nucleic acid lateral flow assays (NALFA) (Koczula & Gallotta, 2016). The assay is designed to produce a visible signal, usually through the appearance of colored bands indicating the presence or absence of the target analyte. This simple visual indication eliminates the need for complex instrumentation and makes LFAs particularly suitable for resource-limited environments, remote or rural areas, or situations where immediate results are important (Ochola et al., 2022).

[0006] The basic structure of an LFA comprises several components mounted on a strip. The crude sample, such as blood, urine, nasopharyngeal or vaginal swabs, is prepared with specific sampling protocols (sample collection, buffers, incubation time) varying by the type of disease, sample matrix and analyte nature (Parolo et al., 2020). The prepared sample then migrates through the porous membrane by capillary action, taking the target analyte with it. As the sample flows, it encounters a conjugate pad containing labeled molecules, such as antibodies conjugated with gold nanoparticles or other recognition molecules specific for the target analyte (Budd et al., 2023). When the target molecule is present in the sample, it forms a complex with the labeled molecule. The complex is then directed to the test area of the strip, which comprises immobilized capture molecules that bind to the complex of target analyte and labeled molecule. When the target analyte is present, a visible colored band appears on the test area indicating a positive result. For ensuring the validity of the test result and the completion of the assay, a second set of labeled molecules continue to migrate and reach the control area. This area contains immobilized molecules that capture the labeled molecules and produce a visible band regardless of the presence of the target analyte (Park, 2022).

[0007] The simplicity and rapidity of lateral flow assays have made them invaluable tools for various PoC applications. The increasing need for PoC testing that emerged during the pandemic COVID-19 has driven the need for research and innovation to expand the capabilities of LFAs to include improved sensitivity, integration of nanomaterials, purification and amplification of the samples, and detection of an increasing number of different analytes from the same sample (i.e. , multiplexing) (Liu et al., 2021). However, certain drawbacks associated with reading results significantly limit the use of various diagnostic methods and protocols using LFA technology. In particular, the interpretation of the test band and therefore the presence of the target molecule(s) can be subjective due to color changes during the testing process or misinterpretations by the user. Additionally, many commercially available LFA tests are limited to providing qualitative or semi-quantitative results, making it impossible to determine the exact amount of analyte in a sample. Finally, due to the cross-reactivity of similar molecules, false positive or false negative results may be obtained, which is particularly common in complex samples. Even if a control band shows that the test is working correctly, the user cannot determine if the target molecule is actually present in the sample, which compromises the specificity and precision of the test.

[0008] The need for accurate and quantitative analysis of lateral flow assays led to the introduction of digital technologies in various forms. Quantitative analysis readers are primarily based on optical, magnetic, photothermal, and electrochemical sensors, and recent trends aim to develop readers that are portable and inexpensive, especially for PoC applications (Park, 2022). Many LFA readers are based on the optical properties of LFAs and employ instruments that can read the detection area of the test by various means such as image sensors, spectrometers, and many others. Most of the available and proposed optical readers use complex instrumentation and methods to minimize variations in illumination conditions and optical settings. The need to fine-tune optical parameters and maximize signal-to-noise ratio (SNR) by using sophisticated lenses and instrumentation can affect compatibility with different assays and the ability to analyze multiplex assays. In addition, increasing complexity can have a significant impact on user training, platform maintenance, calibration and financial burden. Although some innovations, such as disposable electronic pregnancy tests, have attempted to avoid the above limitations, the problems of increasing electronic waste and a potential semiconductor crisis could significantly impact the manufacture and availability of these readers in the near future. Other alternatives propose the use of smartphones as quantitative LFA readers that can read the intensity of the bands using the camera, as well as applications that analyze the collected data using image analysis methods (Sena-Torralba et al., 2022). Although this approach minimizes the need for optical instrumentation and can be sustainable in low-resource applications, it requires specific light and optics settings for reproducible and accurate results, and these can be drastically affected by instrument and user variability. This can greatly affect assay accuracy and sensitivity, and can lead the user to false negative or positive results.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention provides an apparatus for the detection of a biomolecule in a sample by using a lateral flow assay.

[0011] The apparatus comprises a lateral flow strip and a holder which accommodates the strip. The holder comprises an aperture which exposes both the detection side and the backing side of the strip, in particular around the test area and the control area of the strip. The apparatus further comprises means configured to move the holder along the longitudinal axis of the strip. In addition, the apparatus comprises a light source configured to illuminate the backing side or the detection side of the strip. Furthermore, the apparatus comprises a detector, configured to detect the light which is transmitted through the strip, and an optical slit, which is positioned between the detector and the strip. The apparatus further comprises a processor configured to process the signal of the detector.

[0012] The present invention provides also a method for the detection of a biomolecule in a sample with a lateral flow strip.

[0013] The method comprises injecting the sample on a lateral flow strip and placing the strip in a strip holder which comprises an aperture which exposes both the detection side and the backing side of the strip, in particular around the test area and the control area of the strip. The method further includes illuminating the detection side or the backing side of the strip, collimating and narrowing the light beam which is transmitted through the strip with an optical slit, detecting the light which is transmitted through the strip and the optical slit, moving the strip containing holder along the longitudinal axis of the strip in steps, repeating for each step the illumination of the strip and the detection of the light transmitted through the strip and the optical slit. The method further comprises processing the signal obtained by the detector to determine the presence of the biomolecule in the sample.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 shows a typical example of a lateral flow strip and its components.

[0016] Figure 2 shows a lateral flow strip and a holder of an apparatus according to the present invention.

[0017] Figure 3 shows a lateral flow strip inside a holder, a support base for a holder, a spur gear, a motor with an output shaft and a support base for a motor of an apparatus according to the present invention.

[0018] Figure 4 shows a side view of an apparatus according to the present invention with a lateral flow strip inside a holder, a support base for a holder, a support base with a motor, a light source, a detector and an optical slit.

[0019] Figure 5 shows an apparatus according to the present invention with a lateral flow strip holder, a moving mechanism for a holder, a detector, a processing unit, a user interface, and a housing with a hinged door and a removable lid.

[0020] Figure 6 shows the data obtained from an assay performed according to the present invention.

[0021] Figure 7 shows the linear correlation of the maximum signal intensity of the test band of a lateral flow strip (y axis) against the logarithmic concentration of a DNA target (x axis) obtained from an assay performed according to the present invention.

[0022] DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention provides an apparatus for the detection of a biomolecule (e.g., nucleic acid, protein, hormone) in a sample with a lateral flow assay.

[0024] The apparatus of the present invention comprises

[0025] - a housing, - a lateral flow assay strip comprising two opposite sides, a backing side and a detection side, wherein the detection side comprises a test area and a control area,

[0026] - a holder which accommodates the strip, wherein the holder comprises an aperture which exposes the test area and the control area of the detection side of the strip, and the portion of the backing side of the strip which corresponds to the test area and to the control area,

[0027] - means configured to move the holder along the longitudinal axis of the strip,

[0028] - a light source configured to illuminate the strip through the aperture of the holder,

[0029] - a detector configured to detect the light which is transmitted through the strip,

[0030] - an optical slit positioned between the strip and the detector, and

[0031] - a processor configured to process the signal of the detector.

[0032] The present invention further provides a method for the detection of a biomolecule in a sample with a lateral flow assay.

[0033] The method comprises a) injecting the sample on a lateral flow assay strip, wherein the strip comprises two opposite sides, a backing side and a detection side, wherein the detection side comprises a test area and a control area, b) placing the strip in a strip holder, wherein the holder comprises an aperture which exposes the test area and the control area of the detection side of the strip, and the portion of the backing side of the strip which corresponds to the test area and to the control area, c) illuminating with a light source the strip through the aperture of the holder, d) collimating and narrowing with an optical slit the beam of light which is transmitted through the strip, e) detecting with a detector the light which is transmitted through the strip and through the optical slit, f) moving the holder stepwise along the longitudinal axis of the strip and repeating after each moving step steps c), d, and e) until the strip is scanned g) processing the signal obtained from the detector to determine the presence of the biomolecule in the sample.

[0034] The present invention is based on the detection of a biomolecule in a sample by detecting the light which transmitted through the LFA strip.

[0035] LFA strips are well known in the art and their working principle is based on chromatography where a mixture of molecules in a liquid (moving phase) is separated on the surface of a solid (stationary phase) (e.g., paper) based on the individual properties of each molecule. Similarly, in LFA tests where the sample usually comprises a mixture of different analytes, in order to capture the molecule of interest, specific entities with high affinity for the target are used on the surface of the strip. The rest of the molecules that are present in the sample, move along the surface of the strip with the moving phase. An LFA strip comprises a backing made of a rigid material, such as plastic, which supports the structure of the LFA strip. On top of the backing a thin membrane, usually made from nitrocellulose, or cellulose, functions as the detection area of the LFA strip and its major role is the formation of a uniform capillary flow. Different assays, target analytes and protocols may require selection of different membrane characteristics, such as pore size, porosity, thickness, flow rate, and surface treatment, in order to maximize the sensitivity and the specificity of the assay. The sample is usually injected in a sample pad to filter any contaminants (i.e. , dust, pigments etc.), or directly in a conjugate pad, which is usually made from a highly porous material such as glass fibers. In the conjugate pad, various reagents and labeled molecules (e.g., antibodies conjugated with gold nanoparticles) are immobilized and when a sample flows, the labelled molecules conjugate with the target analyte(s) (if present) and move with the liquid phase into the detection membrane. The analyte-labeled complexes move under capillary action on the surface of the LFA strip and the target analyte(s) are captured from entities (e.g., antibodies) that are immobilized in the test area of the strip, which is located on the surface of the detection membrane. In the case where the concentration of the analyte is higher that the lower detection limit of the test, a visible band is formed in the test area. Similarly, a control area with entities complementary to the internal control labeled molecules is found on the detection membrane downstream from the test area, which is used to ensure the effectiveness of the test. The aforementioned scheme is not exclusive, because more than one test areas can be used in an assay, even for different target analytes (multiplex detection assay), or the control area may have entities (e.g., secondary antibody) that capture the same conjugates that are used for the test area and have not been immobilized with the target (competitive assay). The formed bands may have the same or smaller width than the test and control areas depending on the concentration of the target analyte(s) and the labeling molecules in use (e.g., gold nanoparticles). Finally, at the distal end of the LFA strip, an absorbent pad made from a highly absorbent material (e.g., cellulose) is used to collect redundant liquid sample, unbound target molecules and conjugate molecules. The LFA strip may have different length, width and height, and the apparatus of the present invention is not limited by any change in the orientation of the LFA components and their dimensions. Moreover, the LFA strip may be enclosed inside a protective case for better handling and additional support of the LFA structure as it is used in many prior art applications.

[0036] According to the present invention, the LFA strip backing, is made from a translucent or from a transparent material. For example, the backing can be made from a polymer, such as polyester, polycarbonate, polypropylene.

[0037] Figure 1 shows an example of typical LFA strip (1) comprising a backing (2), and a detection membrane (3). The detection membrane (3) comprises a conjugation pad (4) a test area (5), a control area (6) and an absorbance pad (7).

[0038] After the injection of the sample on the strip, light intensity measurements are taken after a specific time interval in order for the bands to form completely and avoid any degradation in specificity and sensitivity of the assay.

[0039] According to the present invention the LFA strip is placed in a holder. The holder comprises an aperture which exposes the test area and the control area of the detection side of the strip, as well as the portion of the backing side of the strip which corresponds to the test area and to the control area.

[0040] Preferably, the conjugation pad and absorbance pad of the strip are covered by the holder and are thus not illuminated.

[0041] According to the present invention, the detection side, or the backing side of the strip are illuminated with a light source. The backing side of the strip is the side in which the backing is found. The detection side of the strip, which is opposite to the backing side, is the side in which the test area and the control area of the strip are found. In other words, the detection side of the strip is the side in which the test and / or control bands are formed during the assay. Preferably, the detection side of the strip is illuminated.

[0042] The light source may be any known source. For example, the light source may be an incandescent light, a light-emitting diode (LED), or laser diode. The selection of the light source may depend on the probe molecules and on the intrinsic fluorescence of the molecules and the materials that are used in the LFA assay. Preferably, the light source is a laser diode. The intensity of the light source may be adjusted by various means known in the art and is such that the light is transmitted through the strip and reaches the detector. For example, the intensity is such that it overcompensates the absorption of the backing of the strip and the absorption of material of the detection membrane.

[0043] The light that is transmitted through the LFA strip is collimated and narrowed with the use of an optical slit. The optical slit width is smaller than the width of the test and control areas in order to increase the reading resolution. The transmitted light is captured by a light detector which is located on the side of the LFA strip which is opposite to the side of the strip which is illuminated by the light source. Then, the holder of the apparatus is moved in a stepwise manner along the longitudinal axis of the LFA strip, with a step size that is equal or smaller than the width of the optical slit. After each step, the light transmitted by the LFA strip is detected by the detector. In this manner, the apparatus performs a scanning of the LFA strip along its longitudinal axis. The number of steps required for the scanning of the strip depends on various factors, such as the length of the strip. Typically, it is not necessary to scan the whole length of the strip and scanning the test area and the control area of the strip suffices. As the holder moves in the stepwise manner, the intensity of the transmitted light changes due the presence of bands that are formed from the capturing of the labeled target molecule in the test area and the control area of the LFA strip. The captured measurements may be analyzed and compared to standards in order to find the unknown concentration of the analyte in a sample.

[0044] The detector may be any type of photodetector that converts light intensity into any other form of measurable signal. For example, the detector may be a photodiode, phototransistor, photoresistor, or any other known detector.

[0045] According to the present invention, an optical slit is positioned between the LFA strip and the detector. Thus, the beam of light that is propagated through the LFA strip is collimated and narrowed by the optical slit before it reaches the detector.

[0046] The present inventors have surprisingly found that the position of the optical slit affects to a great extent the quality of the signal obtained by the detector. Thus, when the optical slit is placed between the LFA strip and the detector, the signal obtained from the detector is significantly better than the signal obtained from the detector when the optical slit is positioned between the light source and the LFA strip. Preferably, the width of the optical slit is no more than 50% of the width of the control area and no more than 50% of the test area of the LFA strip. This allows the transmitted light that reaches the detector to be collimated and narrowed in order to increase the reading resolution. The width of the control area and the width of the test area is typically from 0.5 mm to 1 mm. Preferably, the width of the optical slit is 0.5 mm, more preferably, 0.25 mm.

[0047] Preferably, the thickness of the optical slit is 1 mm. More preferably, the thickness of the optical slit is 0.5 mm.

[0048] Moreover, the inventors have found that the optical slit position with respect to the LFA strip significantly affects the signal quality. Thus, preferably, the optical slit is positioned as close as possible to the LFA strip. Since the holder is needed to support and facilitate the movement of the LFA strip, the distance of LFA strip with respect to the optical slit is limited to the positioning and dimensions of the holder. The gap between the LFA strip and the optical slit may be limited by the dimensions of the holder and the slot (through which the LFA strip is inserted into the holder). More preferably, the gap between the LFA strip and the optical slit is less than 2 mm. Even more preferably, the gap between the LFA strip and the optical slit is 1.8 mm. Preferably, there is a safety clearance gap of less than 0.2 mm between the optical slit and the holder in order to avoid any accidental collision between the two parts during the readout. Moreover, the gap between the optical slit and the detector is preferably no more than 2 mm.

[0049] Preferably, the surfaces of the optical slit are covered with a material that absorbs and suppresses light reflections, in order to minimize unwanted interferences with the measured signal. In this manner, the signal-to-noise ratio (SNR) is increased and the sensitivity of the detection is also increased.

[0050] The present inventors have also found that the optical slit can be made of a polymer. It is well known in the art that optical slits are made of metal, such as stainless steel molybdenum or tungsten. An optical slit made of a polymer is easier and cheaper to manufacture compared to a slit made of metal. Thus, according to a preferred embodiment of the present invention, the optical slit is made of a polymer. The optical slit can be obtained using methods well known in the art, such as 3D printing or injection molding. Preferably, the polymer is polylactic acid (PLA), acrylonitrile butadiene styrene (ABS), or polypropylene (PP). Preferably, the surfaces of the optical slit are covered, with a material that absorbs or reflects unwanted light. More preferably, the optical slit is made of matte black PLA. The inventors also found that the optical slit can be obtained by a fused filament fabrication method. Fused filament fabrication methods are well known in the art. Preferably, the layer resolution during the fabrication of the optical slit is high enough that will not allow any unwanted light that is generated from the light source to penetrate through the surface of the optical slit that faces the light source. Preferably, the minimum layer resolution of the optical slit is 0.25 mm and the total thickness of the optical slit is 1 mm. Even more preferably, the layer resolution is 0.05 mm and the total thickness of the optical slit is 0.5 mm. With the aforementioned layer and optical slit thicknesses, the inventors have found that the optical slit is capable to block any unwanted light and obtain good quality readouts. Moreover, with this approach, the manufacturing time, cost and energy are significantly reduced compared to other manufacturing and machining approaches of other materials known in the art, such as laser cutting and computer numeric control (CNC) of steel.

[0051] The apparatus of the present invention further comprises a processor configured to process the signal of the detector. Preferably, the processor communicates with the detector with the use of a converter or directly by recording the raw measurements with the use of an integrated analog to digital convert.

[0052] The processor may be configured to perform additional tasks. For example, the processor may be configured to perform a specific routine of enabling the means for moving the holder to take one reading step and advance the LFA strip holder by a user determined distance, activate the light source and take one or more intensity measurements of a specific area of the LFA strip with the use of the detector. This routine may be performed for each reading step of the moving means and scan the whole detection area of the LFA strip. The processor may be, for example, configured to perform the aforementioned tasks according to user commands that can be performed through a user interface. In addition, the processor may be, for example, configured to record the intensity measurements over the number of reading steps, and plot them in real time on a screen, or send them to another device as time series data through a wired connection or wirelessly, for further analysis.

[0053] According to the present invention, the data generated by the detector may be obtained in raw form, represented as arbitrary units of signal intensity (as shown in Figure 6A, y- axis) at each individual reading step (as shown in Figure 6A, x-axis).

[0054] Preferably, the data undergoes preprocessing steps to enhance its suitability for subsequent analysis. The preprocessing steps may, for example, include: (i) Filtering out non-specific signals caused by the sample pad / conjugation pad (left of line 34) and the absorbent pad (right of line 35). (ii) Signal intensity normalization and baseline correction (36). The primary objective of preprocessing is to enhance data quality and remove undesired artifacts, from further analysis.

[0055] Preferably, after preprocessing, the data is utilized to extract features associated with the control and test absorption peaks. These features typically include: peak absolute height of the control band (37), peak absolute height of the test band (38), scan position at the peak height of the control band (39), scan position at the peak height of the test band (40), full width at half maximum (FWHM) of the control band (41), full width at half maximum (FWHM) of the test band (42). Additionally, other parameters such as peak area may also be taken into consideration.

[0056] Preferably, the next step of the analysis is a quality control (QC) validation. The QC may confirm that the control band parameters (37, 39, 41) and the distance between the scan position of the control band (39) and the scan position of the test band(s) (40) are within an expected range. For example: (i) absence of the control band (37, 39, 41) may indicate a non-proper function of the lateral flow strip, (ii) absence of a symmetrical pick and / or a deviation from the expected scan positions of the control band (39) and / or the test band(s) (40) may indicate the improper insertion of the lateral flow strip in the apparatus.

[0057] If the QC criteria are met, the extracted data can be utilized for further analysis. For assays utilizing a lateral flow strip with a control band and a single test band, a simple regression analysis may be performed; the band intensity or the FWHM of the control band may be correlated with the concentration of the sample input. Furthermore, in the case of multiplex assays with one control line and two or more test bands, multiple regression analysis may be conducted.

[0058] In certain setups, one or more spike bands of known intensity may be added to the strip. These spike bands may serve as a signal intensity reference, improving the quantitative analysis, particularly in cases where peak characteristics correlate with one another (e.g., biochemical antagonistic assays or multiplex nucleic acid amplification methods), as well as serving as a saturation threshold.

[0059] By employing this approach, the present invention provides an efficient solution for preprocessing and analyzing raw instrument data of any form. It ensures accurate and reliable qualitative, semi-quantitative, and / or quantitative results, addressing the specific needs of various applications.

[0060] The inventors have found that the presented apparatus and method is also effective in qualifying the intensity of formed bands in commercially available LFA strips that have a transparent cover foil on the upper side of the detection membrane. This cover foil is used for the protection of detection membrane from physical damage, such as scratches or dents. The inventors compared the reading results from LFA strips with and without the transparent cover foil and concluded that it does not have an effect on the signal quality and intensity. Thus, the LFA strips may include a transparent cover foil, minimizing the possibility of false or low-quality readings due to physical damage of the LFA strip.

[0061] The advantage of the apparatus and method of the present invention is the lack of sophisticated and complicated optical arrangements and instruments that are used in prior art approaches. Therefore, the present invention, is a more user friendly, easy to fabricate and a low-cost approach that can report to any available medium, such as a smartphone or a personal computer, the results of an assay precisely and in a short amount of time.

[0062] The apparatus and methods of the present invention can be used as a portable or bench-top platform. Preferably, the platform is portable, lightweight and has low-energy consumption for use in any environment and application.

[0063] The assay used and the biomolecule to be detected depend on the need of the user / facility / laboratory and it can be either a protein, for example an antibody, a nucleic acid target, for example a DNA or RNA sequence, a living microorganism, or any known molecule in existence, such as chemical compounds, toxins etc. Preferably, the biomolecule is selected from the group consisting of a peptide, a hormone, a protein, a nucleotide, an oligonucleotide, a polynucleotide, a saccharide, an oligosaccharide, and a lipid.

[0064] Preferably, the labelling of the probe molecules that capture the target analyte is done with any organic or inorganic molecule / particle / compound that has or have optical properties. The labels that are used in the LFA strip are typically detected in the visible, ultra violet, and infrared spectrum. For example, the labels can be quantum dots (QDs), upconverting nanoparticles (UCNPs), gold / silver / carbon nanoparticles (AuNPs, AgNPs, CNPs, respectively), carbon nanotubes (CNTs), or latex nanoparticles, with different colors based on the target analyte, the desired stability and sensitivity, and the need for multiple detection assays.

[0065] Preferably, the light source and the light detector can be adjusted depending on the labeling of the receptor molecule, the intrinsic fluorescence or absorbance of the target molecule, the sensitivity of the light detector in the corresponding wavelengths and the intrinsic fluorescence characteristics of the LFA strip.

[0066] Figure 1 shows an embodiment in which the detection side of the LFA strip (1) is illuminated (direction of arrows). In this embodiment, the detection area (3) with the formed bands (5, 6) is located on the same side with a light source.

[0067] Figures 2 to 5 show embodiments and aspects of an apparatus according to the present invention.

[0068] According to a preferred embodiment, the apparatus of the present invention comprises a holder (8), such as that shown in Figure 2. In the holder (8), the LFA strip (1) is inserted from the slot (9), leaving only a small part of the LFA strip (1) exposed outside the borders of the holder (8) for easiness of handling and removal. The slot (9) may have dimensions similar to that of the LFA strip (1). On the top and the bottom side of the holder (8), an aperture (10) is located in order to expose the detection area of the LFA strip and the corresponding portion of the backing side. In the embodiment shown in Figure 2, the LFA strip (1) in inserted in the holder (8) in such way that the backing side of the strip (1) faces the light source. Nevertheless, preferably the LFA strip (1) is inserted in the holder (8) in such a way that the detection side of the strip (1) faces the light source.

[0069] The conjugation pad (4) and the absorbent pad (7) are located inside the holder (8), and outside the borders of the aperture (10).

[0070] In this embodiment, the LFA strip (1) is inserted inside the holder (8) from the side that the absorbent pad (7) is located.

[0071] Figure 3 shows a moving mechanism of an apparatus according to the present invention, such as the apparatus of Figure 5. The mechanism comprises a holder (8) that encloses an LFA strip (1). The side walls of the holder (8) have channels (12) that fit with the guiding rails (13) of a support structure (11). The channels (12) and guiding rails (13) are used to facilitate the movement of the holder (8) and a LFA strip (1) in one axis. The holder (8) may move freely, with respect to the support structure (11) without any considerable amount of friction. The channels (12) and guiding rails (13) are similar to linear motion guides that are known in the art, in order to minimize friction, vibrations and any form of instability during movement.

[0072] The holder (8) can move in relation to a stable support structure (11), with the use of any form of engaging mechanisms, parts or means. A wall of the holder (8) has one or more gear racks (14) that engage with a moving gear (15). The gear (15) is connected with a shaft (16) that rotates with the use of an actuator (17). The actuator (17) is a mechanical or electrical motor that rotates with constant speed. The actuator (17) can be an electrical motor known in the art that can be controlled and interfaced via a connector (18).

[0073] The motion from the actuator (17) is transferred directly to a spur gear (15) with the use of a shaft (16), or through a gearbox (not depicted) that transforms the torque and the speed accordingly.

[0074] The actuator (17), and the support structure (11) are mounted on a support base (19) capable of stabilizing the aforementioned structure and absorb any vibrations.

[0075] The holder (8) and support structure (11), can be made from any material known in art, while, the channels (12), guiding rails (13), gear racks (14) and spur gear (15), can be made from any type of durable and low friction material, such as metals (e.g., aluminum) or polymers (e.g., Teflon).

[0076] Figure 4 shows a side view of a moving mechanism, a light source and a detection unit of an apparatus according to the present invention, such as the apparatus of Figure 5. The LFA strip (1) which is located inside the holder (8), moves along the horizontal axis of Figure 4, as it is indicated by the arrow, with the means of an actuator (17) and a support structure (11). On top of a support base (19), a mounting frame (20) facilitates the adjustment of the light source (21) position with respect to the aperture (10) (Figure 2). In the present embodiment, the light source is a laser light with an adjustable beam by means of a focus lens or lenses (22). Furthermore, the orientation of the LFA strip (1) is such (not shown) that the detection side of the strip faces the light source. The light from the light source (21) travels through the aperture (10) (Figures 2, 3) directly on the detection side of the LFA strip (1), propagates through the LFA strip (1), and reaches the optical slit (25), which collimates and narrows the beam of light transmitted through the LFA strip (1). The optical slit (25) is positioned near the surface of the holder (8), leaving a safety clearance gap. The light transmitted through the optical slit (25) finally reaches a detector (23), which is mounted on a support case (24). The support case (24) is used for the alignment of the detector (23) with respect to the LFA strip (1), the aperture (10) in the holder (8), and the light source (21). The support case (24) also protects the detector from any other source of light rather than that of the present apparatus (e.g., sun light), in order to avoid any interference with the measurements. The surfaces of the support case (24) that surround the detector (23) and are illuminated from the light source (21) may be made from, or covered with materials that absorb and suppress light reflections, in order to minimize unwanted interferences with the measured signal.

[0077] Figure 5 shows a moving mechanism, a light source and a detection unit of an apparatus according to the present invention. The LFA strip (1) is inserted into a holder (8) through an opening (29) with a hinged door (30) that is located on the body of a housing (31), and may be closed during a test procedure in order to reduce / block any light that is not used by the apparatus. The moving mechanism moves the LFA strip (1) in its longitudinal axis and illuminates the LFA strip (1).

[0078] According to the embodiment presented in Figure 5, the illuminated detection area (3) (Figures 1 , 2) is captured by the detector (23). The formation of bands (5, 6) on the detection area (3) (Figures 1 , 2) results in the absorbance of the light, therefore the intensity of the light over the bands is reduced proportionally to the concentration of the conjugate (i.e., target biomolecule conjugated with labeled molecules) in the test area. The detection area (3) is scanned in a number of finite points (steps) and a recording from the detector (23) is captured. Preferably, the number of the points may is large enough to capture any formed bands in the detection side of the strip (1), which may be visible or non-visible to the human eye. When the width of the optical slit (25) is no more than 50% of the width of the control area and no more than 50% of the width of the test area, a specific reaction site (e.g., test band (5)), is scanned in more than one points, resulting in higher resolution and offering better signal-to-noise ratio (SNR) in cases that the background noise is higher.

[0079] The apparatus of Figure 5 also comprises a microcontroller or a microprocessor unit that may be configured to perform multiple tasks, such as, to control the number of reading steps of the actuator (17) (Figures 3, 4), adjusting the speed, adjust the light source (21) intensity and focus.

[0080] The apparatus of Figure 5 also comprises a removable lid (32) for accessing the moving mechanism, the light apparatus and the microcontroller. Preferably, the housing (31) and the removable lid (32) are made from lightweight materials, such as plastics, in order to reduce the overall weight of the apparatus. Preferably, the inside walls of the housing (31) and the lid (32) are colored or covered with light absorbent materials or colors (e.g., black), in order to minimize the scattering and reflections of the light that is produced from the light source (21).

[0081] Example

[0082] Quantitative detection of a nucleic acid target using lateral flow strips

[0083] As an example, the detection of RNA or DNA targets involves isolating the target from a biological sample and amplifying it using an amplification method ((RT-) PCR and / or (RT-) RPA or LAMP). The amplified products, when appropriately labeled, can bind to the test area on the lateral flow strips to form a distinct band that can be further analyzed with the device of the present invention. A common labeling approach is to biotinylate the 5’ end of a primer and insert a 5’-labeled FAM probe into the reaction. The biotin allows the amplified product to bind to the lateral flow strip, while the FAM probes are specifically recognized by anti FAM gold nanoparticles. The conjugation of the amplified products with the gold nanoparticles form a distinct colored band whose intensity corresponds to the concentration of the bound amplified product on the test line of the strip. To visually illustrate this process, Figure 7 presents the correlation between the maximum values of signal intensity (x-axis) of the test band and the log of various concentrations of an isothermally amplified DNA product (y-axis). The graph demonstrates a strong correlation between the template concentration and the increase in the maximum peak observed at the test line, as evidenced by the eguation: Signal intensity = 0.3 * loglO(Copies) - 0.68, R2= 0.99, p < 2.2e-16.

[0084] REFERENCES

[0085] Budd, J., Miller, B. S., Weckman, N. E. et al. (2023). Lateral Flow Test Engineering and lessons learned from COVID-19. Nature Reviews Bioengineering, 7(1), 13-31.

[0086] Koczula, K. M., & Gallotta, A. (2016). Lateral flow assays. Essays in Biochemistry, 60(1), 111-120. Liu, Y., Zhan, L., Qin, Z., Sackrison, J., & Bischof, J. C. (2021). Ultrasensitive and highly specific lateral flow assays for point-of-care diagnosis. ACS Nano, 75(3), 3593-3611.

[0087] Ochola, L., Ogongo, P., Mungai, S., Gitaka, J., & Suliman, S. (2022). Performance evaluation of lateral flow assays for coronavirus disease-19 serology. Clinics in Laboratory Medicine, 42(1), 31-56.

[0088] Park, J. (2022). Lateral flow immunoassay reader technologies for quantitative point-of- care testing. Sensors, 22(19), 7398.

[0089] Parolo, C., Sena-Torralba, A., Bergua, J. F., Calucho, E., Fuentes-Chust, C., Hu, L., Rivas, L., Alvarez-Diduk, R., Nguyen, E. P., Cinti, S., Quesada-Gonzalez, D., & Merkogi, A. (2020). Tutorial: Design and fabrication of nanoparticle-based lateral-flow immunoassays. Nature Protocols, 15(12), 3788-3816.

[0090] Sadeghi, P., Sohrabi, H., Hejazi, M., Jahanban-Esfahlan, A., Baradaran, B., Tohidast, M., Majidi, M. R., Mokhtarzadeh, A., Tavangar, S. M., & de la Guardia, M. (2021). Lateral flow assays (LFA) as an alternative medical diagnosis method for detection of virus species: The intertwine of nanotechnology with sensing strategies. TrAC Trends in Analytical Chemistry, 145, 116460.

[0091] Sena-Torralba, A., Alvarez-Diduk, R., Parolo, C., Piper, A., & Merkogi, A. (2022). Toward next generation lateral flow assays: Integration of nanomaterials. Chemical Reviews, 722(18), 14881-14910.

Claims

CLAIMS1 . An apparatus for the detection of a biomolecule in a sample with a lateral flow assay, wherein the apparatus comprises- a housing (31),- a lateral flow assay strip (1) comprising two opposite sides, a backing side and a detection side, wherein the detection side comprises a test area (5) and a control area (6),- a holder (8) which accommodates the strip (1), wherein the holder comprises an aperture (10) which exposes the test area (5) and the control area (6) of the detection side of the strip (1), and the portion of the backing side of the strip (1) which corresponds to the test area (5) and to the control area (6),- means configured to move the holder (8) along the longitudinal axis of the strip (1),- a light source (21) configured to illuminate the strip through the aperture (10) of the holder (8),- a detector (23) configured to detect the light which is transmitted through the strip (1),- an optical slit (25) positioned between the strip (1) and the detector (23), and- a processor configured to process the signal of the detector (23).

2. The apparatus according to claim 1 , wherein the width of the optical slit (25) is no more than 50% of the width of the test area (5) and no more than 50% of the width the control area (6) of the strip (1).

3. The apparatus according to claim 1 or 2, wherein the width of the optical slit (25) 0.5 mm, preferably 0.25 mm.

4. The apparatus according to any one of the preceding claims, wherein the thickness of the optical slit (25) is 1 mm, preferably 0.5 mm.

5. The apparatus according to any one of the preceding claims, wherein the optical slit (25) is made of a polymer.

6. The apparatus according to claim 5, wherein the polymer is selected from polylactic acid, acrylonitrile butadiene styrene, or polypropylene.

7. The apparatus according to any one of the preceding claims, wherein the optical slit (25) is made of matte black polylactic acid.

8. The apparatus according to any one of the preceding claims, wherein the optical slit (25) is obtainable by 3D printing.

9. The apparatus according to any one of the preceding claims, wherein the optical slit (25) is obtainable by a fused filament fabrication method.

10. The apparatus according to claim 9, wherein the minimum layer resolution of the optical slit is 0.25 mm and the total thickness of the optical slit is 1 mm.

11. The apparatus according to any one of the preceding claims, wherein the gap between the strip (1) and the optical slit (25) is less than 2 mm.

12. The apparatus according to any one of the preceding claims, wherein the gap between the strip (1) and the optical slit (25) is 1.8 mm.

13. The apparatus according to any one of the preceding claims, wherein the gap between the optical slit (25) and the detector (23) is no more than 2 mm.

14. The apparatus according to any one of the preceding claims, wherein the light source (21) is configured to illuminate the detection side of the strip (1).

15. The apparatus according to any one of the preceding claims, wherein the light source (21) is selected from an incandescent light source, a light-emitting diode, or a laser diode.

16. The apparatus according to any one of the preceding claims, wherein the light source (21) is a laser diode.

17. A method for the detection of a biomolecule in a sample with a lateral flow assay, wherein the method comprises a) injecting the sample on a lateral flow assay strip (1), wherein the strip (1) comprises two opposite sides, a backing side and a detection side, wherein the detection side comprises a test area (5) and a control area (6),b) placing the strip (1) in a strip holder (8), wherein the holder comprises an aperture (10) which exposes the test area (5) and the control area (6) of the detection side of the strip (1) and the portion of the backing side of the strip (1) which corresponds to the test area (5) and to the control area (6), c) illuminating with a light source (21) the strip through the aperture (10) of the holder (8), d) collimating and narrowing with an optical slit (25) the beam of light which is transmitted through the strip, d) detecting with a detector (23) the light which is transmitted through the strip (1) and through the optical slit (25), e) moving the holder (8) stepwise along the longitudinal axis of the strip (1) and repeating after each moving step steps c), d, and e) until the strip is scanned, h) processing the signal obtained from the detector (23) to determine the presence of the biomolecule in the sample.

18. The method according to claim 17, wherein the light source (21) is configured to illuminate the detection side of the strip (1).

19. The method according to claim 17 or 18, wherein the light source is selected from an incandescent light source, a light-emitting diode, or a laser diode.

20. The method according to any one of claims 17 to 19, wherein the light source is a laser diode.

21. The method according to any one of claims 17 to 20, wherein the width of the optical slit (25) is no more than 50% of the width of the test area (5) and no more than 50% of the width the control area (6) of the strip (1).

22. The method according to any one of claims 17 to 21 , wherein the size of every moving step is equal or smaller than the width of the optical slit (25).

23. The method according to any one of claims 17 to 22, wherein the detection side of the strip (1) faces the light source (21).

24. The method according to any one of claims 17 to 23, wherein the processing of the signal obtained by the detector (23) comprises- performing baseline correction,- determining the absolute height of the peak obtained from the test area (5),- determining the absolute height of the peak obtained from the control area (6),- determining the scan position at the height of the peak obtained from the control area (6),- determining the scan position at the height of the peak obtained from the test area (5),- determining the full width at half maximum of the peak obtained from the test area (5), - determining the full width at half maximum of the peak obtained from test area(6).

25. The method according to any one of claims 17 to 24, wherein the biomolecule is selected from the group consisting of a peptide, a hormone, a protein, a nucleotide, an oligonucleotide, a polynucleotide, a saccharide, an oligosaccharide, and a lipid

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