Lateral flow immunoassay instrument for detecting cytokeratin-18 by using SERS (Surface Enhanced Raman Scattering) active particles

Abstract

A lateral flow immunometer 10 includes a sample accommodation portion 20 configured to accommodate a sample; a SERS active particle configured to bind to cytokeratin-18 (K18) in the sample, and a test portion 40 configured to immobilize the SERS active particle that has bound to K18.

Description

Technical Field

[0001] This invention relates to an apparatus and method for detecting and / or measuring biomarkers of drug-induced liver injury. The invention particularly (but is not limited thereto) relates to a lateral flow immunoassay analyzer and a method for detecting and / or measuring cytokeratin-18 (K18) in a sample using surface-enhanced Raman scattering (SERS). Background Technology

[0002] Paracetamol (acetaminophen, APAP) is the most widely used analgesic worldwide. [1] Paracetamol is considered to have excellent safety when taken in adults at the recommended therapeutic dose, i.e., no more than 4 g in 24 hours. [2] However, overdose of paracetamol can cause hepatotoxicity and may lead to drug-induced liver injury (DILI). This is because paracetamol is metabolized and oxidized to the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI), which saturates the detoxification pathway and binds to cellular proteins in the liver. [3] DILI can be avoided if the patient is treated with the antidote N-acetylcysteine ​​(NAC) within 8 hours of taking an overdose of paracetamol. [4] When the initiation of NAC is delayed, its efficacy is reduced, which may lead to liver failure, requiring liver transplantation or death. Although NAC is effective when delivered early enough, adverse drug reactions to NAC are common and NAC should not be administered without proper clinical risk assessment. Paracetamol overdose can be classified as acute overdose, fractional overdose, or therapeutic overdose. Patients are often asymptomatic or only exhibit mild, nonspecific gastrointestinal symptoms. [5] Therefore, clinical trials are necessary to determine the severity of damage caused by paracetamol overdose and whether NAC should be delivered.

[0003] When a patient is brought to the emergency room (A&E) after an overdose, the severity of the overdose is determined by venous blood sampling, which is then analyzed in a central laboratory within the hospital. Ideally, samples are collected 4 hours or more after the overdose to indicate drug absorption and tissue distribution. [6] However, since patients often arrive at the A&E several hours after an overdose, the time required for sampling can be much longer. In the hospital laboratory, a series of liver function tests (LFT) are performed. LFT examines the levels of certain enzymes and proteins in the blood, and elevated levels may indicate liver damage. Typically, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), serum bilirubin, total protein, and albumin are measured. The concentration of acetaminophen in the serum is also determined. The results and interpretation of these tests can take a considerable amount of time. Late arrival and long testing times mean that the critical 8-hour NAC treatment window is approaching, and effective treatment may be missed. These tests also lack sensitivity and specificity, which may lead to missed diagnoses of DILI. [7] This is well illustrated by a 2014 case study that followed patients who had taken a single overdose of compound codeine (60-70 tablets, totaling 30-35 grams of paracetamol) and had blood drawn 4.5 hours later. [8] The paracetamol concentration was 107 mg / L, below the "200-line" on the UK paracetamol treatment NAK table, and ALT was 34 U / L, also below the upper limit of normal (ULN) – 50 U / L. Therefore, the risk of DILI was considered extremely low, and the patient was discharged without NAC treatment. However, 43 hours later, the patient returned to the hospital with vomiting. A repeat test revealed a significant increase in ALT (333-fold). In fact, the paracetamol overdose caused DILI, but this was missed due to the low sensitivity of ALT. The UK National Poison Information Service (an organization responsible for developing clinical guidelines for paracetamol overdose in the UK) stated that there is an urgent need to develop new DILI biomarker tests to detect and assess patients who have overdosed on paracetamol, thereby more effectively improving current standards of care.

[0004] Recently, a novel protein biomarker, cytokeratin-18 (K18), has emerged as a candidate for accurate and early detection of DILI. [9, 10] K18 is an intermediate filament protein abundant in hepatocytes. During acute and chronic hepatocellular injury, such as DILI, necrotic cells passively release full-length K18 into circulation due to loss of cell membrane integrity.

[11] Studies have shown that K18 levels can accurately distinguish patients with or without DILI at an earlier time point compared to ALT.

[12] In a case study, K18 concentrations in samples were retrospectively measured at 4.5 hours and found to be higher than the ULN (30 ng / mL in serum), indicating that K18 could have correctly detected DILI earlier. K18 has received regulatory support from the European Medicines Agency and the U.S. Food and Drug Administration for its role in predicting DILI and assessing prognostic outcomes, further highlighting its potential as a powerful biomarker for DILI. [7, 13] The gold standard method for K18 quantification is enzyme-linked immunosorbent assay (ELISA). When K18 binds, the associated reaction produces a detectable signal, typically a color change. Quantification is performed by measuring the absorbance of the solution using an ELISA reader.

[0005] Although the assay yields accurate and quantitative results, the process takes several hours and must be performed by trained personnel using specialized equipment and expensive materials.

[14] In clinical practice, the long wait for results means that NAC treatment will miss the critical 8-hour window.

[0006] One object of the present invention is to solve and / or mitigate one or more problems associated with the prior art.

[0007] The purpose of this invention is to provide a rapid quantitative test for detecting K18 in subject samples.

[0008] One object of the present invention is to provide a test that can be used at the point of care (POC) to quickly provide results for determining the DILI status, which is easy to use, requires minimal training, and / or has low production and consumable costs. Summary of the Invention

[0009] According to the first aspect, an immunoassay is provided, the immunoassay comprising: The sample holding section is configured to hold samples. SERS active particles, configured to bind to cytokeratin-18 (K18) in the sample; and In the testing section, the configuration was set to immobilize the SERS active particles that had been combined with K18.

[0010] Advantageously, this method allows for the combination of SERS quantitative measurement technology with convenient immunoassay methods. This can help reduce testing time typically associated with K18 testing, improve the sensitivity of K18 measurements, and / or provide a convenient point-of-care (POC) approach requiring minimal training or clinical expertise.

[0011] The immunoassay instrument may include or may be an ELISA instrument.

[0012] The immunoassay instrument may include, or may be based on, a bead-based instrument.

[0013] Preferably, the immunoassay device may include or may be a lateral flow immunoassay (LFIA). This provides a convenient and efficient point-of-care (POC) device that requires minimal training or clinical expertise.

[0014] An immunoassay instrument, such as an LFIA, can extend between a first end and a second end.

[0015] An LFIA typically includes a backing disposed in the lower portion (usually the bottommost portion) of the LFIA. This backing can be made of any suitable material such as polyvinyl chloride.

[0016] The LFIA may include capillary elements, such as capillary membranes. The capillary membrane may be configured to allow fluid wicking, for example, from a first end to a second end. The capillary element typically includes or may be composed of a cellulose material, such as a cellulose nitrate membrane. The capillary element may typically be disposed on the upper surface of a backing.

[0017] The terms “upper” and “lower” should not be understood as absolute terms in this document, but rather as terms representing the sample side. In other words, the “upper” side may face the side configured to accommodate the sample, while the “lower” side may face the side opposite to the sample side, and / or may face the support surface.

[0018] The LFIA may include a wicking or absorption element, typically disposed at or near its second end. The wicking or absorption element may be configured to draw a sample through the capillary element via capillary action, for example, from a first end of the capillary element to a second end.

[0019] Typically, the sample container can be configured to hold a liquid sample. The liquid sample may include the subject's bodily fluids. The liquid sample may include blood, serum, plasma, etc., or may be blood, serum, plasma, etc. Conveniently, the sample may include or may be blood, such as whole blood, for example, blood drawn from a fingertip. This improves convenience and efficiency at the point of care by avoiding the need for venous blood collection and / or sample processing or preparation prior to application.

[0020] The sample receiving portion may include at least one sample receiving element, such as at least one sample pad or sample membrane.

[0021] The sample container may be located at or near the first end of an immunoassay instrument such as an LFIA.

[0022] The at least one sample accommodating element may be made of fibrous materials such as cellulose fibers or glass fibers.

[0023] The at least one sample receiving element may serve as a filter element, such as a blood filter element. The at least one sample receiving element may be configured to remove and / or block one or more blood components, such as cellular material.

[0024] The sample containment portion may include a first sample containment element, such as a first sample filter membrane. The first sample containment element may be treated with a first treatment agent. The first treatment agent may include sodium chloride. The first sample containment element may be treated with an aqueous solution of NaCl, for example, at a concentration of about 0.25% to 2.5%, such as about 1% (w / v). Advantageously, using a NaCl-treated membrane causes aggregation of cellular blood components. This effectively converts blood into plasma within the sample containment portion. This allows the use of whole blood as a sample for the subject, thus avoiding the need for time-consuming serum or plasma extraction. It has been found that excessively low NaCl concentrations can lead to suboptimal aggregation, while excessively high concentrations can lead to instability of nanoparticles in the conjugate pad.

[0025] The sample containment portion may include a second sample containment element, such as a second sample filter membrane. The second sample containment element may be treated with a second treatment agent. The second treatment agent may include a surfactant, such as a nonionic surfactant like polysorbate, for example, polysorbate 20 (Tween® 20). The second sample containment element may be treated with an aqueous solution of the surfactant, for example, at a concentration of about 1% to 10%, such as about 5% (w / v). Advantageously, it has been found that treating the second sample containment element with a surfactant such as Tween® 20 reduces surface tension, which helps increase the diffusion of samples such as blood or plasma and the wetting of the fiber network, thereby enabling faster assays. It is not desirable to be bound by theory, but it is also thought that this reduces protein adhesion to the fibers, thereby improving the sensitivity of the assay.

[0026] The LFIA may include a conduit. Typically, the conduit may be positioned adjacent to a sample receiving portion, for example, adjacent to at least one sample receiving element. The conduit may overlap with at least one sample receiving element.

[0027] The conjugate pad can be configured to contain SERS-active particles configured to bind to cytokeratin-18 (K18) in the sample. The conjugate pad may be provided with SERS-active particles configured to bind to K18 in the sample.

[0028] The bonding pad can be made of fibrous materials, such as cellulose fibers or glass fibers, usually glass fibers.

[0029] Advantageously, the binding pad can be a pre-treated binding pad. The binding pad may have been treated before the deposition of SERS active particles.

[0030] The conjugation pad may have been treated with a sucrose solution, for example, an aqueous sucrose solution with a concentration of about 0.1% to 5%, such as about 1% (w / v). The sucrose solution may further include a surfactant, such as a nonionic surfactant, like polysorbate, such as polysorbate 20 (Tween® 20). The concentration of the surfactant may be about 0.1% to 2%, such as about 0.5% (w / v).

[0031] Observations have shown that once SERS active particles deposit on the conjugate pad, they aggregate, limiting their ability to flow through the immunoassay apparatus during use, for example, to their second end. Pre-treatment of the conjugate pad with a sucrose solution was found to alleviate this problem. Without being bound by theory, it is believed that after sucrose treatment, when the SERS active particles deposit and dry, sucrose molecules can form a layer around the SERS active particles, thereby stabilizing their biological structure. Furthermore, when the sample enters the conjugate pad, the sugar molecules dissolve rapidly, carrying the particles into the flow.

[0032] In one embodiment, the binding pad was treated with a sucrose solution at a concentration of about 1% and polysorbate 20 at a concentration of about 0.5% (w / v).

[0033] The capillary element may extend at least between the conduit and the wicking or absorbent element. The conduit may overlap with the capillary element. The wicking or absorbent element may overlap with the capillary element.

[0034] As described above, the SERS active particles are configured to bind to cytokeratin-18 (K18) in the sample.

[0035] SERS-active particles may include nanoparticles (“NP”) capable of generating a SERS signal, for example, upon excitation. Typically, SERS-active particles may include metallic nanoparticles, such as noble metal nanoparticles like silver or gold nanoparticles. Alternatively, SERS-active particles may include any SERS-generating nanoparticles, such as semiconductors, alloys, core-shell particles like silver-coated gold, and / or asymmetric particles like nanostars, nanorods, nanoshells, nanoprisms, nanocubes, etc. Nanoparticles, such as individual nanoparticles, typically have a diameter of about 10-100 nm, for example, about 40-70 nm, for example, about 50-60 nm, for example, about 55 nm.

[0036] SERS active particles may include a Raman reporter molecule. Typically, the Raman reporter molecule can be a compound capable of enhancing the SERS signal, for example, upon excitation. Those skilled in the art can select from known Raman reporter molecules. In embodiments, the Raman reporter molecule may include 4,4-bipyridine (DIPY).

[0037] SERS active particles may include metal nanoparticles functionalized with Raman reporter molecules such as DIPY, such as gold or silver nanoparticles.

[0038] Raman reporter molecule-functionalized nanoparticles can comprise single nanoparticles and / or clusters of nanoparticles, such as clusters of two (dimers), three (trimers), and / or four (tetramers). Without being bound by theory, it is considered that functionalization of metallic nanoparticles, such as gold or silver nanoparticles, via Raman reporter molecules such as DIPY can promote and / or induce aggregation between nanoparticles. When functionalization occurs via symmetric and / or multifunctional Raman reporter molecules such as DIPY, functionalization can promote and / or induce aggregation between nanoparticles. This can lead to an enhancement of the electromagnetic field between NPs, commonly referred to as a “hot spot.” If a Raman reporter molecule is present at the hot spot, the resulting SERS signal can be enhanced. Therefore, advantageously, Raman reporter molecule-functionalized nanoparticles can comprise one or more clusters of nanoparticles, such as one or more clusters of two (dimers), three (trimers), or four (tetramers).

[0039] SERS-active particles, such as Raman reporter molecule-functionalized nanoparticles, can be encapsulated. These particles can be encapsulated with a protective shell. The protective shell can include an inorganic shell such as silica (SiO2) or an organic shell such as a polymer shell. It is not intended to be theoretically constrained, but rather that such encapsulation helps stabilize the Raman reporter molecule-functionalized nanoparticles, thereby reducing the variability of the SERS signal in different assays and improving reproducibility. This encapsulation also allows for the use of more types of Raman reporter molecules, thus expanding the SERS capability of the immunoassay analyzer. Furthermore, encapsulation helps protect the nanoparticles from the surrounding environment, such as substances that can cause nanoparticle aggregation.

[0040] Encapsulation can occur on a single nanoparticle, such as a nanoparticle functionalized with a single Raman reporter molecule. In this case, a protective shell, such as an inorganic shell, can encapsulate the single nanoparticle.

[0041] Alternatively or additionally, encapsulation can occur on multiple nanoparticles and / or clusters of nanoparticles, for example, on clusters of nanoparticles functionalized with Raman reporter molecules. In this case, the protective shell, such as an inorganic shell, can encapsulate multiple nanoparticles and / or clusters of nanoparticles, such as clusters of two (dimers), three (trimers), or four (tetramers).

[0042] SERS active particles may include moiety that can bind to K18.

[0043] Typically, SERS active particles may include (recombinant) anti-K18 antibodies.

[0044] SERS active particles may include nanoparticles such as gold nanoparticles, which are functionalized with Raman reporter molecules such as DIPY, or modified with anti-K18 antibodies, such as coating.

[0045] SERS active particles may include nanoparticles such as gold nanoparticles, which are functionalized with Raman reporter molecules such as DIPY, encapsulated with a protective shell such as SiO2, and modified with anti-K18 antibodies, for example, coated.

[0046] The average size of the nanoparticles, such as the encapsulated nanoparticles, can be, for example, an average diameter of about 20-200 nm, such as about 60-70 nm, such as about 65 nm.

[0047] For a single nanoparticle, the average size of the nanoparticle, such as the average diameter of an encapsulated nanoparticle, may be about 60-70 nm.

[0048] For dimer nanoparticles (i.e., a cluster of two nanoparticles), the average size of the nanoparticles, such as the average diameter of the encapsulated nanoparticles, can be about 80-100 nm.

[0049] For trimer nanoparticles (i.e., a cluster of three nanoparticles), the average size of the nanoparticles, such as the average diameter of the encapsulated nanoparticles, can be about 120-140 nm.

[0050] For tetrameric nanoparticles (i.e., a cluster of four nanoparticles), the average size of the nanoparticles, such as the average diameter of the encapsulated nanoparticles, can be about 160-180 nm.

[0051] However, it should be understood that the size of the nanoparticles depends at least in part on the size of the selected core nanoparticles, with an average size, for example, an average diameter of about 20-50 nm. Typically, the average size of gold core nanoparticles, for example, an average diameter of about 40 nm. Typically, encapsulation with a silica shell can typically increase the average size of the nanoparticles, for example, an average diameter of about 10 nm or more. Typically, functionalization with an anti-K18 antibody can typically increase the average size of the nanoparticles, for example, an average diameter of 5-10 nm.

[0052] Nanoparticles, such as antibody-functionalized individual nanoparticles, can have a size, for example, a diameter of about 70-200 nm, such as about 75-100 nm.

[0053] LFIA may include a testing component.

[0054] The test section may include an anti-K18 antibody. Typically, the test section may include an anti-K18 antibody immobilized on a capillary element (e.g., a nitrocellulose membrane). Accordingly, as the sample flows from a first end of the immunoassay apparatus to its second end, any SERS-bound active particles present in the original sample that are bound to K18 will bind to the anti-K18 antibody and be immobilized on the test section.

[0055] Immunoassays, such as LFIA, can be configured to detect and / or measure one or more other biomarkers. In this case, the immunoassay may be referred to as a multiplex immunoassay.

[0056] The test section may include one or more antibodies, such as one or more other antibodies, configured to bind one or more other biomarkers, such as one or more biomarkers other than K18. In this case, the multiplex immunoassay may include a single test section capable of binding K18 and one or more other target biomarkers.

[0057] Alternatively or additionally, an immunoassay, such as an LFIA, may include one or more additional test sections. These additional test sections, or each additional test section, may include one or more antibodies, configured to bind one or more other biomarkers, such as one or more biomarkers other than K18. In this case, the multiplex immunoassay may include multiple test sections, including one test section capable of binding K18 and one or more other test sections capable of binding one or more other target biomarkers.

[0058] LFIA may include a control portion.

[0059] The control portion may include at least one antibody capable of binding to antibodies deposited on the binding pad and / or antibodies disposed within and / or attached to the SERS active particles. The control portion may include at least one antibody capable of binding to anti-K18. Typically, the control portion may include at least one antibody capable of binding to anti-K18 immobilized on a capillary element (e.g., a nitrocellulose membrane). Accordingly, any SERS active particles not immobilized on the test portion will bind to the antibody on the control portion and will be immobilized on the control portion. Preferably, this binding can generate a visual signal, such as a color change. This provides a convenient way to ensure that the LFIA functions as intended.

[0060] Typically, the test section can be placed between the conjugate pad and the control section.

[0061] Typically, the control section can be placed between the test section and the wicking or absorption element.

[0062] Immunoassays, such as LFIA, can be housed in a cassette, such as a plastic box. The cassette can at least partially enclose the immunoassay.

[0063] Advantageously, the box can be made of a material that reduces or avoids Raman background risk. Advantageously, the box can be made of natural and / or biodegradable polymers such as polylactic acid (PLA).

[0064] The cartridge may include a first opening configured to allow sample deposition on a sample receiving portion. The first opening may be aligned with the sample receiving portion.

[0065] The box may include a second opening configured to allow measurement of the test section, for example, using a SERS device such as a handheld Raman reader. The second opening may be aligned with the test section.

[0066] The box may include a third opening configured to allow observation of the control portion. The third opening may be aligned with the control portion.

[0067] The first opening, the second opening, and the third opening can be different or they can be combined. For example, the second opening and the third opening can form a single opening.

[0068] According to a second aspect, a system is provided configured to quantitatively measure cytokeratin-18 (K18) in a sample, wherein the system includes: An immunoassay analyzer, comprising: The sample section is configured to hold the sample; SERS active particles, configured to bind to cytokeratin-18 (K18) in the sample; and In the testing section, the configuration was used to immobilize the SERS active particles that had already been bound to K18; A surface-enhanced Raman spectroscopy (SERS) device configured to measure and / or detect K18 at or near the test section.

[0069] The immunoassay instrument may be the immunoassay instrument according to the first aspect.

[0070] Advantageously, this system allows for the measurement of K18 levels in a sample, for example, by quantitative detection.

[0071] This system allows for the measurement, for example, of K18 levels in test samples via quantitative detection.

[0072] This system can further allow, for example, the measurement of K18 levels in control samples by quantitative detection.

[0073] The SERS device can be configured to generate radiation at a predetermined wavelength. Advantageously, the SERS device can be configured to generate radiation at a wavelength of about 785 nm. This wavelength was found to avoid background interference from samples such as blood or serum, which can occur at different wavelengths, such as about 633 nm.

[0074] The system, such as a SERS device, may also include a display section arranged to display information generated by the device. The display section may show measurement results, such as, for example, the K18 level of the test section and / or the control section.

[0075] The SERS device can be a portable or handheld SERS device, or a handheld Raman reader.

[0076] A SERS device may include a radiation generator configured to generate radiation of an appropriate wavelength. For example, a SERS device may include a radiation generator configured to generate laser radiation with a wavelength of about 785 nm.

[0077] The features described in the first aspect of the invention are equally applicable to the second aspect, and for the sake of brevity, they will not be repeated here.

[0078] According to a third aspect, a method for detecting cytokeratin-18 (K18) in a sample is provided, the method comprising: Provide an immunoassay analyzer, which includes: The sample holding section is configured to hold samples. SERS active particles, configured to bind to cytokeratin-18 (K18) in the sample; and In the testing section, the configuration was used to immobilize SERS active particles that had been bound to K18; Provide the sample in the sample receiving section; and SERS analysis was performed in the testing section.

[0079] The method may include providing a liquid sample, such as a blood sample, in the sample receiving portion.

[0080] This method may include performing SERS analysis in the test section using a SERS device, such as a portable or handheld SERS device or a handheld Raman reader.

[0081] Advantageously, the method of the present invention allows for the measurement of K18 levels in a sample, for example, by quantitative detection.

[0082] The method may also include performing SERS analysis in the control section using a SERS device, such as a portable or handheld SERS device or a handheld Raman reader.

[0083] This method may include comparing signals obtained when measuring the test portion and the control portion.

[0084] This method may include using a ratio of spectral intensities, such as the intensity of a single peak, obtained from SERS analysis of the test and control portions.

[0085] This method may include using principal component analysis (PCA) to compare the full spectrum obtained from the test and control portions to derive a ratio.

[0086] This method may include using linear regression to generate spectral slopes obtained from the test and control portions, plotted against each other. In this case, a larger slope indicates a higher concentration. The slopes of the test / control spectra for each sample can then be used to generate a calibration curve of "slope versus K18 concentration".

[0087] This method may include the preparation of SERS-active particles.

[0088] This method may include the preparation of metal nanoparticles, such as gold nanoparticles. Nanoparticle preparation may typically involve adding trisodium citrate to an aqueous solution of sodium tetrachloroaurate(III) dihydrate.

[0089] This method may include modifying nanoparticles with Raman reporter molecules.

[0090] This method may include functionalizing nanoparticles with 4,4-bipyridine (DIPY). Functionalization with symmetric and / or multifunctional Raman reporter molecules such as DIPY can promote and / or induce aggregation between nanoparticles. This can lead to an enhancement of the electromagnetic field between NPs, which is commonly referred to as a “hot spot.” If a Raman reporter molecule is present at the hot spot, the resulting SERS signal can be enhanced. Therefore, advantageously, functionalizing nanoparticles with Raman reporter molecules can generate one or more clusters of nanoparticles, such as one or more clusters of two (dimer), three (trimer), or four (tetramer) nanoparticles.

[0091] This method may include encapsulating nanoparticles, such as nanoparticles modified with Raman reporter molecules.

[0092] This method may include encapsulating nanoparticles with a protective shell, such as SiO2. Without being bound by theory, it is believed that such encapsulation can help stabilize Raman reporter molecule-functionalized nanoparticles, thereby reducing the variability of SERS signals in different assays and improving reproducibility. This encapsulation may also allow the use of more types of Raman reporter molecules, thus expanding the SERS capability of the immunoassay analyzer.

[0093] This method may include functionalizing SERS active particles with a portion capable of binding to K18. This method may also include functionalizing SERS active particles with a (recombinant) anti-K18 antibody.

[0094] This method may include the preparation of an immunoassay instrument.

[0095] This method may include preparing a binding pad.

[0096] The method may include providing an integrative pad. The integrative pad may be made of a fibrous material, such as cellulose fiber or glass fiber, typically glass fiber.

[0097] This method may include treating the binding pad, preferably before depositing SERS active particles.

[0098] The method may include treating the conjugation pad with a sucrose solution, such as a sucrose solution with a concentration of about 0.1% to 5%, for example, about 1% (w / v). The sucrose solution may further include a surfactant, such as a nonionic surfactant, like polysorbate, for example, polysorbate 20 (Tween® 20). The concentration of the surfactant may be about 0.1% to 2%, for example, about 0.5% (w / v).

[0099] Observations have shown that once SERS active particles deposit on the conjugate pad, they aggregate, limiting their ability to flow through the immunoassay apparatus, for example, to their second end. Pre-treatment of the conjugate pad with a sucrose solution was found to resolve this issue. Without being bound by theory, it is proposed that when SERS active particles are deposited and dried after sucrose treatment, sucrose molecules form a layer around the SERS active particles, thereby stabilizing their biological structure. Furthermore, when the sample enters the conjugate pad, the sugar molecules dissolve rapidly, carrying the particles into the flow.

[0100] This method may include depositing SERS-active particles on the binding pad.

[0101] In one embodiment, the method may include treating the binding pad with a sucrose solution at a concentration of about 1% and polysorbate 20 at a concentration of about 0.5% (w / v).

[0102] The method may include preparing at least one sample accommodating element.

[0103] The method may include providing at least one sample accommodating element.

[0104] The at least one sample accommodating element may be made of fibrous materials such as cellulose fibers or glass fibers.

[0105] The method may include providing a first sample containment element, such as a first sample filter membrane. The method may include treating the first sample containment element with a first treatment agent. The first treatment agent may include sodium chloride. The method may include treating the first sample containment element with a NaCl solution, for example, a NaCl solution with a concentration of about 0.25% to 2.5%, such as about 1% (w / v). Advantageously, using a NaCl-treated membrane can cause the aggregation of cellular blood components. This can effectively convert blood into plasma in the sample containment portion. This allows whole blood to be used as a sample for the subject, thereby avoiding the need for time-consuming serum or plasma extraction. It has been found that too low a NaCl concentration results in suboptimal aggregation, while too high a concentration leads to instability of nanoparticles in the conjugate pad.

[0106] The method may include providing a second sample receiving element, such as a second sample filter membrane. The method may include treating the second sample receiving element with a second treatment agent. The second treatment agent may include a surfactant, such as a nonionic surfactant like polysorbate, for example, polysorbate 20 (Tween® 20). The method may include treating the second sample receiving element with a surfactant solution, for example, a surfactant solution with a concentration of about 1% to 10%, such as about 5% (w / v). Advantageously, it has been found that treating the second sample receiving element with a surfactant such as Tween® 20 reduces surface tension, which helps increase the diffusion of samples such as blood or plasma and the wetting of the fiber network, thereby enabling faster assays. It is not desirable to be bound by theory, but it is also thought that this reduces protein adhesion to the fibers, thereby improving the sensitivity of the instrument.

[0107] The method may include applying a first sample receiving element, for example, to a backing / the backing, preferably to a first end of the measuring instrument or its vicinity.

[0108] The method may include applying a second sample receiving element, for example, to a backing / the backing, preferably adjacent to a first sample receiving element. The first sample receiving element may at least partially overlap with the second sample receiving element.

[0109] The method may include applying a bonding pad to, for example, a backing / the backing, preferably adjacent to at least one sample receiving element, such as adjacent to a second sample receiving element. At least one sample receiving element, such as the second sample receiving element, may at least partially overlap with the bonding pad.

[0110] This method may include depositing SERS-active particles on the binding pad.

[0111] This method may include preparing a test section / the test section. The method may include depositing and / or immobilizing anti-K18 antibodies on a capillary element, such as a nitrocellulose membrane. Accordingly, as the sample flows from a first end of the immunoassay apparatus to its second end, any SERS-bound active particles present in the original sample that are bound to K18 will bind to the anti-K18 antibody and thus be immobilized on the test section.

[0112] The method may include preparing a control portion / the control portion. The method may include depositing and / or immobilizing at least one antibody capable of binding to an antibody deposited on a nitrocellulose membrane. Accordingly, any SERS-active particles not immobilized in the test portion will bind to at least one antibody on the control portion and will be immobilized on the control portion. Preferably, this binding produces a visual signal, such as a color change. This provides a convenient way to ensure that the LFIA functions as intended.

[0113] According to standard LFIA, the test section can be placed between the conjugate pad and the control section, while the control section can be placed between the test section and the wicking or absorbent element.

[0114] The method may include, for example, applying a wicking or absorption element to the backing, preferably at or near the second end of the measuring instrument.

[0115] The features described in any aspect of the invention are equally applicable to all other aspects, and for the sake of brevity, will not be repeated here. For example, the features described in relation to the composition are equally applicable to the method, and vice versa. Attached Figure Description

[0116] Embodiments of the present invention will be described with reference to the accompanying drawings, in which: Figure 1 This is a schematic diagram of an immunoassay instrument according to the first embodiment; Figure 2 An immunoassay apparatus according to a second embodiment is shown; Figures 3a to 3d illustrate implementation of the kit for use with an immunoassay analyzer; Figure 4 An embodiment of a portable SERS reader configured for use with an immunoassay analyzer is shown; Figure 5 shows the SERS spectra and measurement results obtained from the LFIA run test line of the K18-specified sample, namely (A) the SERS spectrum of the test line, and (B) the test line and control line at 1612 cm⁻¹. -1 A graph of peak intensity, (C) an example of linear regression analysis, and (D) the results of the calibration curve; Figure 6 and Figure 13 The diagrams illustrating the characterization of Au-DIPY-SiO2-Ab NP synthesis are shown. Figure 7 The 1612 cm value of DIPY obtained from SERS analysis of the test line is shown. -1 The relationship between peak intensity and K18 spiking concentration (ng / mL); Figure 8 shows graphs illustrating the K18 concentrations in patient samples with and without DILI as measured by (A) K18 ELISA and (B) the SERS-LFIA POC DILI test of this study. Figure 8(C) shows the receiver operating characteristic curve (ROC). Figure 9 The relationship between the concentration of spiked K18 and the peak height ratio is shown, along with the linear regression slope analysis; Figure 10 Visual analysis results of LFIA test strips used in clinical trials for samples without DILI (A) and with DILI (B) are shown. Figure 11 shows the visual analysis of (A) and (B) detection studies conducted in capillary blood, and (C) the SERS analysis measurement results; Figure 12 This diagram illustrates the aggregation caused by the functionalization of Raman reporter molecules. Figure 14 TEM images showing individual NPs encapsulated in silica, as well as encapsulated NP clusters (dimers, trimers, and tetramers). Detailed Implementation

[0117] Throughout this disclosure, unless the context otherwise requires, several terms will be used, the meanings of which are as follows. The specific terminology used herein to define compounds, particularly those described according to the invention, is generally based on the IUPAC rules concerning chemical compounds, specifically the IUPAC Compendium of Chemical Terminology (Gold Book). To avoid ambiguity, if any IUPAC rule conflicts with the definitions provided herein, the definitions herein shall prevail. Furthermore, if a compound structure conflicts with the name provided for that structure, the structure shall prevail.

[0118] The term “comprising” or variations thereof should be understood herein to imply that the element, integer or step, or group of elements, integers or steps, is included, but does not exclude any other element, integer or step, or group of elements, integers or steps.

[0119] The term “consisting of” or variations thereof shall be understood to imply the inclusion of the stated element, integer or step, or group of elements, integers or steps, and exclude any other element, integer or step, or group of elements, integers or steps.

[0120] In this document, the term "about" when modifying numbers or values ​​refers to values ​​that differ from the specified value by ±5%. For example, if the specified temperature range is about 5°C to about 13°C, then it includes temperatures from 4.75°C to 13.65°C.

[0121] Unless the context otherwise requires, references to the physical state of a substance (such as liquid or solid) refer to the state of the substance at 25°C and atmospheric pressure.

[0122] Figure 1 An immunoassay apparatus 10 according to a first embodiment is shown.

[0123] In this embodiment, the immunoassay device 10 is in the form of a lateral flow immunoassay (LFIA). This provides a convenient point-of-care (POC) device that requires minimal training or clinical expertise.

[0124] Figure 2 An immunoassay apparatus 110 according to a second embodiment is shown. Figure 2 Immunoassay 110 and Figure 1 The measuring instrument 10 is roughly similar to the one in the middle. The similar parts are represented by similar numerical symbols, but with "100" added. Figure 1 The measuring instrument 10 in the diagram is shown in the figure, while Figure 2 The measuring instrument 110 in the middle is a prototype manufactured in the laboratory.

[0125] The immunoassay instrument 10 extends between the first end 11 and the second end 12.

[0126] The LFIA 10 includes a backing (not shown) disposed on the underside of the LFIA 10. The backing can be made of any suitable material such as polyvinyl chloride.

[0127] LFIA 10 includes a capillary element 15, in the form of a capillary membrane. The capillary membrane 15 is configured to allow wicking of fluid, for example, from a first end 11 to a second end 12. The capillary element 15 is typically made of a cellulose material such as a nitrocellulose membrane. The capillary element 15 is disposed on top of a backing.

[0128] The LFIA 10 includes a wicking or absorption element 50 disposed at or near the second end 12. The wicking or absorption element 50 is configured to draw a sample through a capillary element 15 from the first end 11 to the second end 12 by capillary action.

[0129] The LFIA has a sample receiving portion 20 configured to receive a sample, preferably a liquid sample such as bodily fluids. The liquid sample may include, or may be, blood, serum, plasma, etc. Conveniently, the sample may include or may be blood, such as whole blood. This improves the convenience and efficiency of the point of care.

[0130] In this embodiment, the sample holding portion 20 includes two sample holding elements, in the form of a first sample pad 21 and a second sample pad 22, which are disposed at or near the first end 11 of the immunoassay instrument 10.

[0131] Sample pads 21 and 22 are made of fibrous material, in this embodiment, glass fiber. Advantageously, the sample pads act as filter membranes and are configured to remove and / or block one or more blood components, such as cellular material. To achieve this, sample pads 21 and 22 have been treated accordingly.

[0132] The first sample pad 21 has been treated with a first treatment agent, in this embodiment sodium chloride, specifically an aqueous solution of NaCl with a concentration of about 1% (w / v). Advantageously, it has been found that using a sample membrane treated with NaCl leads to the aggregation of cellular blood components. When a blood sample is applied to the first sample pad 21, this effectively converts the blood into plasma. This allows whole blood to be used as the subject's sample, thus avoiding the need for time-consuming serum or plasma extraction. It has been found that too low a NaCl concentration results in suboptimal aggregation, while too high a concentration leads to instability of the nanoparticles in the binding pad 30.

[0133] The second sample pad 22 has been treated with a second treatment agent, which in this embodiment is a surfactant, specifically an aqueous solution of polysorbate 20 (Tween® 20) at a concentration of about 5% (w / v). Advantageously, it has been found that treating the second sample pad with Tween® 20 reduces surface tension, thereby helping to increase the diffusion of samples such as blood or plasma, and increasing the wetting of the fiber network on the capillary membrane 15, allowing the instrument 10 to proceed more quickly. It is not desirable to be bound by theory, but it is also thought that this reduces protein adhesion to the fibers, thereby improving the sensitivity of the instrument.

[0134] like Figure 2 As shown, the first sample pad and the second sample pad 22 partially overlap.

[0135] The LFIA includes a conjugate pad 30, which is configured adjacent to the sample receiving portion 20. In this embodiment, as... Figure 2 As best shown in the diagram, the second sample pad 120 partially overlaps with the bonding pad 130.

[0136] The bonding pad 30 is made of a fibrous material, which in this embodiment is glass fiber.

[0137] The binding pad 30 is configured to contain SERS active particles configured to bind to cytokeratin-18 (K18) in the sample. The binding pad 30 is provided with SERS active particles configured to bind to K18 in the sample.

[0138] Advantageously, the conjugation pad 30 has been treated prior to the deposition of SERS active particles. In particular, the conjugation pad has been treated with an aqueous sucrose solution at a concentration of about 1% (w / v) and a surfactant, in this embodiment, the surfactant being polysorbate 20 (Tween® 20) at a concentration of about 0.5% (w / v).

[0139] Observations showed that once SERS active particles deposited on the conjugate pad 30, they aggregated, limiting their ability to flow through the immunoassay analyzer 10, for example, to its second end 12 during use. Pre-treatment of the conjugate pad 30 with a sucrose solution resolved this problem. Without being bound by theory, it is believed that when the SERS active particles are deposited and dried after sucrose treatment, sucrose molecules form a layer around the SERS active particles, thereby stabilizing their biological structure. Furthermore, when the sample enters the conjugate pad 30, the sugar molecules dissolve rapidly, carrying the particles into the flow.

[0140] like Figure 1 and Figure 2 As shown, the capillary element 15 extends between the conduit 30 and the wicking or absorbent element 50. The conduit 30 overlaps with the capillary element 15. The wicking or absorbent element 50 also overlaps with the capillary element 15.

[0141] The LFIA 10 includes a test section 40. The test section 40 has an anti-K18 antibody immobilized on a nitrocellulose membrane 15. Accordingly, when a sample flows from the first end 11 of the immunoassay apparatus 10 to its second end 12, any SERS-bound active particles present in the original sample that are bound to K18 will bind to the anti-K18 antibody and thus be immobilized on the test section 40.

[0142] LFIA 15 includes a control portion 45. The control portion 45 has at least one antibody capable of binding to antibodies deposited on the binding pad. The control portion 45 also has at least one anti-K18 antibody capable of binding to the nitrocellulose membrane 15. Accordingly, any SERS-active particles not immobilized in the test portion 40 will bind to at least one antibody on the control portion 45 and will be immobilized on the control portion 45. Preferably, this binding produces a visual signal, such as a color change. This provides a convenient way to ensure that LFIA 10 functions as intended.

[0143] According to standard LFIA, the test section 40 is positioned between the bonding pad 30 and the control section 45, while the control section 45 is positioned between the test section 40 and the wicking or absorption element 50.

[0144] Figures 3(a) to 3(d) illustrate and Figure 1 or Figure 2 The embodiment of the box 260 used together with the immunoassay instruments 10 and 110.

[0145] Conveniently, LFIA 10,110 is disposed within box 260, which in this embodiment is a plastic box. Advantageously, box 260 is made of a material that reduces or avoids the risk of Raman background. Advantageously, box 260 is made of a natural polymer, which in this embodiment is polylactic acid (PLA).

[0146] The box has a first or lower portion 261 configured to engage with a second or upper portion 262 to enclose LFIA 10, 110.

[0147] The second portion 262 of the cartridge 260 has a first opening 265 aligned with the sample receiving portion 20 of the measuring instrument 10 to allow sample deposition on the sample receiving portion 20, for example, the first sample pad 21.

[0148] The second portion 262 of the box 260 has a second opening 266 that is aligned with the test portion 40 and the control portion 45 to allow measurement of the test portion 40 and / or the control portion using a handheld Raman reader, and / or to allow observation of the control portion 45, as best shown in FIG3(d).

[0149] Figure 4 An embodiment of a portable SERS reader 370 is shown, configured to work with... Figure 1 or Figure 2 The immunoassay instruments 10 and 110 are used together.

[0150] SERS reader 370 includes an adapter portion 375 configured as a housing 260. Conveniently, the adapter is designed as a retaining housing so that the laser of reader 370 is aligned with the test portion 40 and also with the control portion 45.

[0151] Advantageously, the irradiation device of the reader 370 is configured to irradiate the measuring instrument in the form of a laser bar. Accordingly, at least most of the test portion and / or most of the control portion can be irradiated by the laser, which can improve sensitivity and / or reproducibility.

[0152] The SERS device is configured to generate irradiation at a predetermined wavelength, which in this embodiment is approximately 785 nm. This wavelength has been found to avoid background interference from samples such as blood or serum, which can occur at different wavelengths, such as approximately 633 nm.

[0153] It should be understood that the embodiments of the present invention are provided as examples only, and various modifications can be made to the embodiments of the present invention without departing from the scope of the present invention.

[0154] Example experiment Material Sodium tetrachloroaurate(III) dihydrate, sodium citrate trihydrate, 4,4'-bipyridine, (3-aminopropyl)trimethoxysilane, sodium silicate solution, sodium tetraborate, boric acid, Tween® 20, sucrose, sodium chloride, and PBS tablets were all purchased from Merck (UK). Bovine serum albumin was purchased from VWR. Recombinant anti-cytokeratin 18 antibody BSA without azide (capture antibody and detection antibody) and goat anti-rabbit IgG H&L were all purchased from Abcam (UK). Nitrocellulose membrane FF170HP, Whatman® MF1 bound glass fiber filter (binding pad), Whatman® Standard 14 (sample pad), and Whatman® CF6 test strip pad and test strip (absorbent pad) were all purchased from Cytivia (UK). 3D printer filament, 2.85mm diameter, black, PLA, was purchased from Farnell (UK).

[0155] instrument All extinction measurements were performed using a Cary 300 Bio UV-Vis spectrometer. Particle size was obtained using the Malvern® Zetasizer Nano series. Solution spectra were collected using a CBex Raman spectrometer with an excitation wavelength of 785 nm and a laser power of 80 mW. SERS of the LFIA test and control lines was performed using a Wasatch® photonic spectrometer with laser excitation of 785 nm and a laser power of 3.6 mW. Test and control lines were added to the antibody using a Claremont Bio® fully automated side-flow reagent dispenser. Strips were cut to 5 mm using a CM5000 high-speed cutter. 3D printing of the kit was performed using an Ultimaker S5.

[0156] Synthesis of gold nanoparticles Gold nanoparticles (Au NPs) were synthesized in 500 mL batches. Sodium tetrachloroaurate(III) dihydrate (60.5 mg) was added to 500 mL of double-distilled deionized water in a round-bottom flask. The solution was then heated to 98 °C with continuous stirring. Subsequently, trisodium citrate (57.5 mg) was added to the solution, and the solution color changed from yellow to red, indicating the formation of Au NPs. The solution was cooled and stirred overnight, and then characterized by extinction spectroscopy and DLS.

[0157] Raman spectroscopy report on molecular functionalization and silica encapsulation Au NPs were functionalized with the Raman reporter molecule 4,4-bipyridine (DIPY) and then encapsulated in a silica (SiO2) shell. 100 mL of Au NPs were added to a 250 mL Erlenmeyer flask. DIPY (120 µL, 100 mM) was added to the Au NPs in 20 µL increments, stirring for 1 min between each addition to allow for surface functionalization. Then, 150 µL, 3 mM of 3-(aminopropyl)trimethoxysilane was added to the solution, followed immediately by 1.5 mL of sodium silicate. The solution was heated to 98 °C and stirred for 30 min, then cooled and stirred overnight. The resulting Au-SiO2 NPs were subsequently characterized using extinction spectroscopy, DLS, and SERS with a 785 nm excitation laser.

[0158] It was discovered that by synthesizing such relatively large 100 mL batches, Au NPs with larger volumes of Raman reporter molecules could be synthesized. By using Au-SiO2 NPs with the same SERS activity in 1000 LFIA test strips, the differences in SERS signals between test strips were significantly reduced, thereby improving reproducibility. Furthermore, the stability of Au-SiO2 NPs was much higher than that of bare Au NPs, and the SERS signal retention time was longer because the Raman reporter molecule was essentially fixed in place within the SiO2 shell and could not leave the surface.

[0159] Antibody functionalization To establish the K18 detection function, Au-SiO2 NPs were coated with recombinant anti-cytokeratin 18 capture antibody (AB). Borate buffer (10 mM, pH 9, 100 µL) was added to 1 mL of Au-SiO2 NPs. Recombinant anti-cytokeratin 18 antibody (4 µL, 1 mg / mL) was added to the solution, and the mixture was shaken for 2 hours. Then, bovine serum albumin (100 µL, 1% solution) was added, and the solution was shaken for another 30 minutes. Subsequently, the Au-SiO2-ABNPs were characterized using extinction spectroscopy, DLS, and SERS with a 785 nm excitation laser. The Au-SiO2-Ab NPs were then centrifuged at 4000 RPM for 20 minutes, the supernatant was removed, and the mixture was resuspended in 100 µL of double-distilled deionized water. They were stored in a refrigerator until use.

[0160] Preparation of lateral flow test strips Add line Using the Claremont® reagent dispenser, add the recombinant anti-cytokeratin detection 18 antibody (test line) and goat anti-rabbit antibody (control line) to the nitrocellulose portion of the lateral flow test strip and allow it to dry overnight.

[0161] Membrane preparation Sample pad A treated blood filtration membrane was added to the starting end of the LFIA test strip to ensure optimal separation before the serum or blood matrix came into contact with the binding pad and nitrocellulose portion of the test strip. The first membrane (1 cm wide) was treated with NaCl (1% w / v solution), and the second membrane (1.5 cm wide) was treated with Tween 20 (5% w / v solution). After treatment, both membranes were dried in an oven (37°C, 1 hour).

[0162] Advantageously, the chemical functionalization of the sample pad membrane was found to improve blood separation. High salt concentrations cause changes in cell shape and tight packing, leading to aggregation. This effectively converts blood into plasma, which can then flow onto the nitrocellulose test strip. It was found that higher NaCl concentrations (5% w / v) resulted in NP instability and affected assay performance; therefore, a concentration of 1% was used. The surfactant Tween® 20 was used in the second membrane. It reduces surface tension, thereby enhancing plasma diffusion and wetting of the fiber network, allowing for timely assays. It also reduces protein adhesion to the fibers, which will improve assay sensitivity.

[0163] Combination pad The conjugate pads (1 cm wide) were treated with a solution of sucrose (1% w / v) and Tween 20 (0.5% w / v) prepared in water and dried in an oven (37°C, 1 hour). The conjugate pads were then cut into 5 mm strips, and Au-SiO2-ABNP (8 µL) was added to each strip. The pads were then dried in an oven at 37°C for 1 hour.

[0164] It was found that, without treatment of the conjugate pad, dried NPs aggregated on the conjugate pad and did not flow onto nitrocellulose for assay. Through studies of various treatments, it was found that treatment with a sucrose solution helped maintain the protein structure. Furthermore, when Au-SiO2-Ab NPs were dried in the presence of sucrose, it was believed that sucrose molecules formed a layer around them, thereby stabilizing their biological structure. Additionally, when the sample entered the conjugate pad, the sugar molecules dissolved rapidly, carrying the particles into the flow.

[0165] Limit of detection in spiked serum samples Detection limits were studied on healthy human serum spiked with clinically relevant concentrations of K18. Samples containing 25 µL of healthy human serum and 75 µL of buffer (5% Tween 20 in 10 mM PBS) were mixed and spiked with K18 at concentrations of 0, 5, 10, 25, 50, 100, 200, 350, 500, and 750 ng / mL, respectively. The samples were then added to the sample port of the cartridge and run for 20 minutes prior to analysis. Calibration was performed on human serum from three healthy donors to construct calibration curves including the mean and standard deviation.

[0166] SERS analysis Analyze the test and control lines in the LFIA using a handheld Raman reader. Slide the box into the adapter and align it with the laser lines, as shown. Figure 4 As shown. Analysis was performed using 785 nm laser excitation, 3.5 mW laser power, 1 second integration, and 5 average alignments. Linear regression analysis was used to derive the slope of the test relative to the control for each sample. For each concentration, a graph of the average slope versus concentration was plotted to obtain a calibration curve. The error for each concentration is the standard deviation among the three donors. Using these results, a calibration model was created using R software to determine the concentration of K18 in samples from unknown patients.

[0167] Early experiments were conducted using a handheld Raman reader with 633 nm laser excitation. However, a large background was observed in the resulting SERS spectra due to fluorescence from the serum. This obscured the peaks from the Raman reporter molecule and reduced the quantification results. To reduce the background, a handheld Raman reader with 785 nm laser excitation was used. Advantageously, this eliminated the observed background, making the SERS spectra of the Raman reporter molecule clearly visible.

[0168] Conventionally, the SERS intensity of a single peak from the test line is plotted against the concentration of the target protein to construct a calibration curve. However, this method was found to yield reproducible results, and individual SERS values ​​differed between different serum samples. By utilizing both the test and control lines for SERS analysis and applying test / control analysis, much more reproducible signals were obtained between serum samples. The differences in SERS signals on the test line are believed to be caused by several different factors, such as the formation of the protein crown and the flow of different serum samples on the device. However, since both the test and control lines are affected by these factors, test / control line analysis effectively cancels out signal differences.

[0169] Different methods were used to compare the test / control lines. The intensity of individual peaks on both the test and control lines was used to calculate the ratio, while principal component analysis (PCA) was employed to compare the entire spectra obtained from the test and control lines to derive another ratio. However, the best results were obtained using a linear regression method, which yielded the slope of the test / control spectra plotted relative to each other. A steeper slope indicated a higher concentration. Therefore, a calibration curve of "slope versus K18 concentration" was constructed using the test / control slopes for each sample.

[0170] Clinical research A blinded, preclinical study was conducted on 100 patient samples to evaluate the sensitivity and specificity of the SERS-LFIA POC DILI test. The samples consisted of 50 samples with DILI and 50 samples without DILI, with the presence of DILI determined by measuring K18 concentration using a K18 ELISA method. Patient serum samples were thawed, and 25 µL was added to 75 µL of buffer. This sample was then added to the sample port of the cartridge containing the LFIA test strip. The test was run for 20 minutes, and the test and control lines were analyzed using a handheld Raman reader. Each sample was operated on and analyzed three times by three separate users. To determine the K18 concentration in the patient samples, the SERS of the test and control lines was analyzed using linear regression to obtain the slope, which was then plotted on a calibration curve previously constructed using an R script to derive the K18 concentration.

[0171] Advantageously, using an R script to perform linear regression on the original patient sample SERS data and subsequently using that value to determine the K18 concentration from a calibration curve allows for the automated determination of K18 levels, thus eliminating human error when working with complex data. It is also designed to provide K18 concentrations immediately after analysis without complex data processing. Therefore, this approach is beneficial in point-of-care applications.

[0172] Statistical analysis Preclinical data were analyzed to determine sensitivity, specificity, and ROC-AUC.

[0173] Detection limit in spiked capillary blood samples 20 µL of capillary blood was collected from the donor's finger using a lancet and added to 80 µL of K18-spiked buffer (samples of 0, 25, 50, 100, 200, 350, and 750 ng / mL were prepared). This buffer contained 5% Tween 20 and 0.38% sodium citrate in 10 mM PBS. The sample was added to the sample port and run for 20 minutes, after which the test and control lines were analyzed using a handheld Raman reader. Linear regression analysis was used to derive the slope of the test spectrum relative to the control spectrum for each sample. For each concentration, the average slope versus concentration was plotted to obtain a calibration curve. The error for each concentration is the standard deviation among the three donors.

[0174] result Au-DIPY-SiO2-Ab NP Synthesis To develop a SERS-LFIA POC DILI assay that will provide consistent and reproducible results, SERS-active Au NPs were first considered. A major problem in quantitative SERS analysis is the variation in SERS signals between different batches of Au NPs, as even small differences in concentration, size, and shape can lead to significant variations in SERS intensity. To eliminate this problem, a large quantity of Au NPs (20 L) was synthesized to provide core Au NPs for multiple tests. In fact, this would be sufficient for 200,000 tests if used for large-scale production. A large glass container was filled with 20 L of distilled water and sodium tetrachloroaurate. The container was covered with an oil jacket providing controlled heating up to 98 °C and a large paddle for continuous stirring of the solution. Sodium citrate was then added, and the solution changed from yellow to red, indicating that the Au NPs had been successfully reduced, capped, and formed. Next, the Au NPs were SERS-functionalized by adding the Raman reporter molecule 4,4-bipyridine (DIPY). This was carried out in a small volume (100 mL in an Erlenmeyer flask with continuous stirring), but it can be scaled up to 4 L to prepare large quantities of Au NPs with the same SERS signal. A predetermined SERS intensity was set for each batch of synthesized Au-DIPY NPs: DIPY at 1612 cm⁻¹ when analyzed using 785 nm laser excitation. -1 The peak value is 25,000 au. This is another method to maintain the repeatability of the SERS signal in multiple tests.

[0175] When functionalized via symmetrical and / or multifunctional Raman reporter molecules such as DIPY, functionalization can promote and / or induce aggregation between nanoparticles. This can lead to an enhancement of the electromagnetic field between nanoparticles (NPs), commonly referred to as a "hot spot." If a Raman reporter molecule is present at the hot spot, the resulting SERS signal can be enhanced. In this embodiment, DIPY is a symmetrical molecule with two pyridine groups, such as... Figure 12 As shown. Figure 12 The diagram shows the initial gold NP 310, which is functionalized with dipyles to generate functionalized NP 311. At low concentrations, dipylene molecules typically lie flat on the NP surface. However, at higher concentrations, more dipylene molecules bind to the NP surface, causing them to adopt an "upright" configuration, usually perpendicular to the surface. In this orientation, dipylene has "free" nitrogen facing away from the NP orientation, which can bind to another NP. This can induce the formation of dimers 312 where NPs are close to each other. The same principle can lead to the formation of larger clusters, such as... Figure 12 Examples such as trimer 313 or tetramer 314 are schematically shown. Figure 14TEM images show a single NP321 encapsulated in silica, as well as encapsulated NP clusters (dimer 322, trimer 323, and tetramer 324).

[0176] To improve the lifetime of Au-DIPY NPs, they were encapsulated in a thin silica layer. This prevented the desorption of DIPY and the aggregation of Au NPs over time, both of which would hinder quantification. The resulting gold-DIPY-silica shell nanoparticles (Au-DIPY-SiO2NPs) were highly stable in solution, and the SERS signal was maintained for several months. To enhance K18 detection capabilities, Au-DIPY-SiO2NPs were encapsulated in K18-capturing antibodies using an electrostatic method to prepare Au-DIPY-SiO2-AbNPs. This method was prepared on a 1 mL scale, with the pH of the Au NPs adjusted to 9 before the antibody was added. pH 9 protonated the lysine groups on the antibody, allowing them to be electrostatically attracted to the negatively charged surface of silica. Figure 12 A schematic diagram showing the encapsulated single NP (321) and the encapsulated dimer (322), trimer (323) and tetramer (324).

[0177] A schematic diagram of the final Au-DIPY-SiO2-Ab NP used in the above test is shown below. Figure 1 As shown.

[0178] The synthesis of Au-DIPY-SiO2-Ab NP was characterized at each step using UV / Vis extinction spectroscopy, dynamic light scattering, and Raman spectroscopy excited by a 785 nm laser. The results are shown in Figure 6 below. Figure 13 And in Table 1.

[0179] Table 1: Characterization data of Au-DIPY-SiO2-Ab NP synthesis

[0180] As expected, characteristic changes in localized surface plasmon resonance (LSPR) were observed after the addition of SiO2 and Ab, indicating a change in the refractive index around the Au NPs due to the addition of SiO2 and Ab. In this case, based on the size measurements of individual nanoparticles, the size of the Au NPs increased from 55 nm to 65 nm for the Au-DIPY-SiO2 NPs and 86 nm for the Au-DIPY-SiO2-Ab NPs. This again demonstrates the successful addition of SiO2 and Ab. Finally, the SERS signal of DIPY remained detectable after the addition of Ab, thus providing a strong SERS signal on both the test and control lines of the test strip. To maintain consistency and reproducibility among multiple SERS-LFIAs, extinction rate, size, and SERS criteria were set for each batch of synthesized Au-DIPY-SiO2-Ab NPs. This is important because although each batch has the same Au NP core, only 1 mL of Au-DIPY-SiO2-Ab NP can be synthesized each time, so there may be differences between different batches. The characterization and standards of the 6 batches of Au-DIPY-SiO2-Ab NP are shown in Table 2 below, indicating that very small differences were observed between the batches, and that the method for preparing stable Au-DIPY-SiO2-Ab NP is reproducible.

[0181] Table 2: Characterization of multiple batches of Au-SiO2-Ab NP

[0182] Figure 13 The diagram shows a comparison of the extinction spectra of NPs before and after encapsulation. The black spectrum, representing the NP before encapsulation, has a narrow peak at 530 nm, representing a single, unfunctionalized NP. When dipy is added and the NPs are encapsulated, the resulting spectrum (shown in red) has two peaks. The main peak at 535 nm represents a single encapsulated NP. The peak position shifts from 530 nm to 535 nm, indicating a change in refractive index due to the addition of the encapsulation shell. The second peak appears at 750 nm and is most clearly seen in the magnified image. This peak only appears after encapsulation, indicating cluster formation. Although it doesn't appear to be a large peak, it indicates the presence of trimers and dimers, and that a mixture of clusters has been formed.

[0183] SERS-LFIA Test As described above, the embodiments of the test strips and related boxes are as follows: Figure 2 As shown in Figure 3. Box 260 was printed using PLA, a plastic chosen because of its low Raman scattering properties.

[0184] To begin the assay, the serum sample is diluted in running buffer and added to the sample port located directly above the blood filtration membrane. The first membrane is pretreated with 1% NaCl to remove red blood cells from the hemolyzed serum sample, which could interfere with LFIA performance. The sample is then allowed to flow onto a second filtration membrane pretreated with 5% Tween® 20. Tween® 20 reduces the surface tension of the serum, thereby increasing its diffusion and wettability to the fiber network, allowing the assay to proceed promptly. It also reduces protein adhesion to the fibers, thus improving the sensitivity of the assay. The “filtered” sample is then allowed to flow onto the conjugation pad, where Au-DIPY-SiO2-Ab NP is stored. To ensure effective rehydration of the conjugate by the sample, it must be dried uniformly and stably, which is achieved by treating the pad with 1% sucrose and 0.5% Tween® 20. Sucrose is beneficial because when drying Au-DIPY-SiO2-Ab NP, sucrose molecules form a layer around it, thereby stabilizing its biological structure. Furthermore, when the sample flows onto the binding pad, sucrose molecules dissolve rapidly, carrying Au-DIPY-SiO2-Ab NPs into the flow. Once rehydrated, Au-DIPY-SiO2-Ab NPs bind to K18 in the sample and flow onto the nitrocellulose membrane to interact with the test and control lines. If K18 is present, the sandwich immunoassay form and NPs on the test line are immobilized, producing the characteristic red / purple lines. Au-DIPY-SiO2-Ab NPs that do not participate in the immunoassay flow towards the control line and are immobilized, or continue to flow and are collected by the absorbent pad.

[0185] To evaluate the sensitivity and range of SERS-LFIA, a limit of detection (LOD) study was conducted. Clinically relevant concentrations of K18 (0–750 ng / mL) were spiked into serum samples (25 μL) from healthy donors, diluted in running buffer (75 μL), and used for the sample port of the kit. LOD studies were performed using serum from three healthy donors, in triplicate. Serum is primarily composed of diglycerides, triglycerides, phospholipids, fatty acids, steroids, and steroid derivatives, which can form protein crowns on the surface of Au-DIPY-SiO2-Ab NPs, thus affecting the binding properties of the assay. The concentration of each component also varies from donor to donor; therefore, we assessed and considered any possible variations by using different donors. Visual results from the triplicate LOD studies concluded that all three donors exhibited the same visual trend. As pre-selected, only a control line (or a very weak test line) appeared when K18 was not spiked in the serum sample. The appearance of the test line increased with increasing K18 concentration. At concentrations above 200 ng / mL of K18, the test lines were very strong, making it difficult to determine which test showed the highest amount of K18 added. Therefore, to perform quantification and construct calibration curves, the tests were analyzed using a handheld Raman reader with 785 nm laser excitation.

[0186] SERS analysis To obtain quantitative information from the test, it was analyzed using a handheld Raman reader, such as... Figure 4 As shown and discussed above, the test or control line is aligned with the 785 nm laser excitation, and three SERS measurements are performed on each line. Conventionally, when a handheld Raman reader is used in conjunction with an LFIA, point illumination and a fast raster scan line are employed. However, this design uses line illumination and allows for detection of the entire line in a single 5-second acquisition. It should be understood that when using a laser in A&E, it must be operated at 3R level (below 4 mW). Therefore, a laser power of 3.6 mW was used to analyze the test strip to meet this requirement. Due to the strong SERS signal obtained from the DIPY, no loss of sensitivity was observed. Overall, this device allows the end user to safely detect SERS signals from an LFIA from the cartridge using a handheld Raman reader equipped with a laser leakage-free adapter without removing it from the cartridge.

[0187] Detection limit study in serum To construct a platform capable of predicting K18 concentration in patient samples, a robust calibration curve of K18 concentration relative to SERS readings is required. Triple LOD studies were analyzed using a handheld Raman reader, yielding the average SERS signal from the test line as shown below. Figure 5AAs shown. Generally, the SERS signal increases with increasing K18 concentration; however, there are significant differences in SERS signals between samples with the same K18 concentration but run using different donors. When plotting the spiked K18 concentration against the average SERS intensity and adding error bars to show the standard deviation between three replicates, the results are not ideal, as shown... Figure 7 As shown. Larger error bars indicate variations in SERS, which may be due to the different components of donor serum affecting the binding of K18 to Ab. Clearly, the concentration of K18 in unknown patient samples cannot be determined by using only the SERS intensity of the test line.

[0188] However, LFIA has a built-in internal standard in the form of a control line, and it has also been SERS-tested. Figure 5B In the diagram, we show the SERS spectra recorded at 1612 cm⁻¹ from the control and test lines at each K18 concentration. -1 The peak height at the test line. We have already shown that the SERS signal is generally enhanced; however, we now demonstrate that the SERS signal of the control line weakens with increasing K18 concentration. This is not unexpected, as a larger proportion of Abs on the NPs participate in the sandwich immunoassay at higher K18 concentrations, leading to reduced Ab binding and NP fixation on the control line, resulting in a lower SERS signal. Using this, a calibration curve is constructed by comparing the test and control lines. This also takes into account any differences in protein crown formation. For example, if a serum sample with a high K18 concentration forms a larger protein crown around the NPs, this will adversely affect the binding of ABs to K18. Despite the high concentration of K18, this will result in a lower SERS signal on the test line. However, the binding of Abs to the Abs on the control line will also be weakened, and the SERS signal will also decrease. Therefore, using a comparative method will effectively eliminate the binding problem. This method also normalizes any differences in SERS signal between different batches of Au-DIPY-SiO2-Ab NPs. If the SERS signal of one batch is slightly higher, the signals of both the test and control lines will increase, again eliminating the difference.

[0189] There are many methods for comparing test and control lines. We evaluated and compared two different methods to determine whether they reduced donor-to-donor error and to evaluate which method produced a calibration curve with r² closest to 1. The ratio obtained by the first method was derived by obtaining 1612 cm⁻¹ of DIPY from the test line. -1 The peak height is calculated by dividing it by the height of the same peak on the control line. This method can be applied to multiple peaks in a Dipy spectrum, but only utilizes a portion of the SERS spectrum. The second method uses linear regression, which does not calculate the ratio, but instead calculates the slope of the test spectrum compared to the control spectrum. It compares the entire spectrum of the test and control lines. Figure 8C An example of linear regression analysis is shown for test strips run with 0 and 750 ng / mL K18. SERS spectra of the test and control strips were plotted, showing a slope of 0.37 at 0 ng / mL and 3.93 at 750 ng / mL. Calibration curves of K18 concentration versus ratio or slope were plotted, as shown below. Figure 9 As shown. Both methods yielded good results and exhibited linear fit within the range of 0-750 ng / mL. The r² value of the linear regression method was more favorable, with an average error of 15% between serum samples, which was 3% lower than the error calculated using the peak ratio method. Therefore, the linear regression method was chosen to construct the calibration curve of spiked K18 concentration versus slope (test / control), as shown. Figure 5D As shown.

[0190] Figure 5A The SERS spectrum is shown from an LFIA test line run using samples spiked with K18. The spectrum is the average of three LFIAs performed using serum from three different donors. Figure 5B The bar chart shows the LFIA performed using samples spiked with K18, with the dipy at 1612 cm⁻¹ obtained from the test (green) and control (orange) samples. -1 Peak height. The height represents the mean, and the error bars represent the standard deviation, which was obtained by using three test lines and control lines of LFIA performed using serum from three different donors. Figure 5C Linear regression analysis was performed on an LFIA run using a sample spiked with 0 ng / mL K18 and another run using a sample spiked with 750 ng / mL K18. The plot compares the intensity of the control SERS spectrum with the intensity of the test SERS spectrum. A straight line was fitted, and the gradient rate (slope) was derived. Figure 5D Calibration curves of K18 spiked concentration versus slope (test / control). Mean and error bars obtained from slopes obtained from three LFIAs using three different serum donors. SERS spectra obtained using 785 nm laser excitation, 3.6 mW, 1 second integration, and 5 averaged acquisitions.

[0191] Overall, SERS-LFIA produced linear calibration curves between 0–750 ng / mL with small errors between different serum donors. This is primarily attributed to linear regression analysis, which reduced saturation at higher concentrations and normalized the signals from the test and control lines. The wide range of LFIA allows for direct detection of patient samples with high K18 concentrations without dilution, a significant advantage in POC applications. However, the wide range reduces the instrument's sensitivity, with a detection limit of 35 ng / mL for the calibration curve. Narrowing the range to 0–200 ng / mL yields a calculated LOD of 25 ng / mL. In preclinical trials, we opted to sacrifice sensitivity for a wide range, enabling the assessment of a broader range of K18 concentrations without sample dilution.

[0192] Clinical research To determine the sensitivity and selectivity of the SERS-LFIA POC DILI test, a blinded clinical study was conducted. One hundred serum samples from patients who had overdosed on paracetamol were analyzed, with 50 samples showing DILI and the other 50 not. Each patient sample was independently operated and analyzed by three different users. This was to assess whether the device's performance depended on whether the user was a trained spectrophotographer, or whether scientists from different disciplines (or medical specialties) could obtain the same results. The three replicate operations and analyses also evaluated the performance of the SERS-LFIA test itself and the variability in the use of the same sample on different test strips.

[0193] First, an initial visual assessment of LFIA was performed by asking each analyst whether they believed the appearance test based on the test line indicated a non-DILI patient sample or a DILI patient sample. Results are as follows... Figure 10 As shown in the figure, this diagram represents a visual analysis of the LFIA test strips used in clinical trials. Figure 10 A shows the results obtained from three analysts using samples without DILI. Figure 10 B shows the results from samples with DILI. If the box is red, the analyst considers the patient sample to indeed have DILI; if the box is green, the analyst considers the patient not to have DILI. Analyst 1 has a sensitivity of 90% and a specificity of 80%; Analyst 2 has a sensitivity of 88% and a specificity of 72%; Analyst 3 has a sensitivity of 94% and a specificity of 80%.

[0194] Overall, the instrument achieved visual sensitivity and specificity of 90% and 77%, respectively, calculated as an average of the sensitivity and specificity achieved by each user. This result indicates good visual potential but also suggests a false positive problem, as approximately one-fifth of non-DILI samples showed test lines that could indicate a positive result. This could be due to several factors. First, if we observe the calibration curve, lines above 10 ng / mL are visually apparent. Based on our experience with SARS-CoV-2 testing, these test lines indicate a positive result; therefore, even if we have a ULN below 30 ng / mL (as with ELISA), we would still consider DILI to have occurred. Another important factor to note is that these samples are real clinical samples collected from clinical trials, and their performance may differ from that of the healthy serum donors used to construct the calibration curve. Preclinical trial samples can exhibit a range of ages, sexes, ethnicities, and general health conditions, which can also affect assay performance. The appearance of serum samples also varies; some samples contain particulate matter, while others show hemolysis. This can lead to NP aggregation on the binding pad, resulting in nonspecific binding and false positive results.

[0195] Next, the LFIA was analyzed using a handheld Raman reader. The slope (test / control) was calculated, and the K18 concentration was determined using the calibration curve constructed in the previous section. To reduce human error in data storage, slope calculation, and K18 determination, a patient information plugin was added to the Wasatch® software, and an R script was built to automate the K18 assay. This workflow was designed to more accurately reflect how the SERS-LFIA POC DILI test is performed in the A&E department to provide results immediately after analysis. The K18 concentrations determined using the SERS-LFIA POC DILI test were then analyzed to determine their sensitivity and specificity. The results are shown in Figure 8, which illustrates the K18 concentrations in patient samples with and without DILI, as measured by 8A – using the K18 ELISA and 8B – the SERS-LFIA POC DILI test. Figure 8C The receiver operating characteristic (ROC) curve is shown, with an area under the curve of 0.947 within the 95% confidence interval.

[0196] Figure 8A The table shows the K18 concentrations in 100 patient samples with and without disseminated intravascular coagulation (DILI), as measured using a K18 ELISA. The K18 concentrations in non-DILI samples ranged from 4 to 28 ng / mL, while those in DILI samples ranged from 73 to 1903 ng / mL, a significant difference. Similarly... Figure 8BThe K18 concentration was constructed using a geometric mean K18 concentration derived from three analyst measurements obtained using the SERS-LFIA assay. Again, we observed differences between the two groups, with some samples without DILI showing higher readings and some with DILI showing lower readings. To calculate sensitivity and selectivity values ​​using the SERS-LFIA-POC DILI assay, a new ULN was calculated to determine which K18 concentration cutoff provides the highest combination of sensitivity and selectivity.

[0197] When the ULN was set at 91 ng / mL, the specificity was 94% and the sensitivity was 82%. A ULN of 59 ng / mL produced 94% sensitivity and 82% specificity. These figures demonstrate the great potential of SERS LFIA for detecting DILI and improve upon results obtained from visual analysis.

[0198] Receiver operating characteristic curve (ROC) as follows Figure 8C As shown, the area under the curve (AUC) was 0.946 within the 95% confidence interval. Using this ROC-AUC analysis, the SERS-LFIA POC DILI test performed comparably to the reference standard ELISA in identifying patients with DILI. Table 3 summarizes the clinical performance of K18 as measured by ELISA and our test. The ELISA performance level cited herein has been approved by the US FDA for its formal biomarker certification process. The SERS-LFIAPOC DILI test's performance is comparable to the reference standard assay, indicating its potential use in this application.

[0199] Table 3: ROC-AUC obtained from the K18 clinical study

[0200] Using blood for testing and research As shown above, this test exhibits excellent sensitivity and specificity when used to determine DILI in patient serum samples. However, when used in A&E, it is desirable to avoid sample pretreatment, which means that serum is not preferred and needs to be centrifuged to remove red blood cells before use in the test.

[0201] The assay was studied using untreated spiked capillary blood to confirm its applicability for detecting K18 in this matrix. 20 µL of capillary blood was collected from healthy donors and added to 80 µL of buffer solution spiked with K18 (0, 25, 50, 100, 200, 350, and 750 ng / mL). Sodium citrate was added to the buffer solution to prevent blood clotting, which could affect assay performance. Sodium citrate acts as an in vitro anticoagulant by binding to free calcium in the blood, a cofactor essential for several steps in the coagulation cascade. Triple LOD studies were performed using capillary blood from three donors. Visual analysis of one LOD is shown below. Figure 11A middle.

[0202] Figure 11A Visual analysis of a study on the detection limits in capillary blood. Figure 11B Visual analysis in QC. Figure 11C Calibration curves of spiked K18 concentration (ng / mL) versus slope (test / control). Mean and error bars are derived from slopes obtained from three LFIAs using three different capillary blood donors (black dots – “calibration points”). Red dots (“spiked points”) represent QC spiked K18 values ​​versus slope (test / control) results. SERS spectra were collected using 785 nm laser excitation, 3.6 mW, 1 second integration, and 5 averages.

[0203] As expected, the intensity of the test line increased with increasing K18 concentration in the spiked capillary blood. The nitrocellulose section of the analyzer did indeed turn red / pink for the donor due to the removal of some red blood cells by the filter membrane. Some red blood cells did transfer onto the test strip, likely due to sample leakage or viscosity. However, the red color did not interfere with the appearance of the test or control lines. LOD analysis was performed using a handheld Raman reader. Figure 11C The calibration curve showing K18 spike concentration versus slope (test / control) is presented. The slope increases with increasing concentration, but unlike serum calibration, this curve is not linear and saturates at higher concentrations. This may be due to the complex matrix degrading the instrument's performance, as components in plasma bind to the NP surface. Nevertheless, the instrument performs well, detecting spiked K18 in capillary blood within 20 minutes without sample preparation.

[0204] To test the effectiveness of the calibration curve in detecting K18 in unknown samples, quality control (QC) samples were performed. Capillary blood (three samples from different donors) was spiked with 30 ng / mL (representing non-DILI samples) and 75, 150, and 400 ng / mL K18 (representing DILI samples), diluted in buffer, and tested. Photographs were taken after 20 minutes. Figure 11B The samples were analyzed using a handheld Raman reader to obtain the slope value for each sample. The K18 spike concentration and slope value for each sample have been plotted on [the graph / database]. Figure 11C The calibration curve shown (red dots) is used to derive the K18 concentration based on the trend line equation, and the results are shown in Table 4.

[0205] Table 4: Determination of QC K18 concentration using POC-DILI test

[0206] Generally, the K18 concentration determined using the SERS-LFIA POC-DILI test increases with increasing K18 concentration, but there is some difference from the initially spiked concentration. This could be due to several factors. For example, this method represents a real procedure occurring in A&E, so the amount of blood collected by the lancet may not be exactly 20 µL, or not all blood / buffer sample may be pipetted onto the test plate, which could lead to slight deviations in the results. Nevertheless, the test can still accurately detect DILI at higher concentrations (because they produce higher slope values), while DILI was not detected in samples of 30 ng / mL. There is also a trend in QC's, with samples with the highest K18 values ​​corresponding to the largest slopes and the largest determined K18 values. This is a first step towards introducing this platform into A&E to determine DILI in patients who have overdosed on paracetamol within a shorter timeframe than the current gold standard.

[0207] in conclusion We have successfully combined LFIA with a handheld Raman reader to develop a SERS-LFIA POCDILI assay capable of detecting clinically relevant concentrations of K18 in spiked serum. This assay was subsequently used in clinical trials to evaluate its application in determining DILI in stock serum samples collected from patients who had taken an overdose of paracetamol. DILI status was determined for each patient sample by setting a K18 concentration cutoff. When the K18 cutoff was set at 59 ng / mL, the assay exhibited a sensitivity of 94% and a specificity of 82%. The ROC-AUC was 0.95, comparable to values ​​reported in clinical trials using a K18 ELISA.

[0208] This test is also used to detect K18 in capillary blood immediately after blood is drawn from a donor's finger. Clinically relevant concentrations of K18 are spiked into the blood, diluted in a buffer containing an anticoagulant, and then tested. Using a handheld Raman reader, the slope (test / control) is obtained, and a successful calibration curve is generated. The slope value of the QC is then used to determine the concentration of K18 in QC samples spiked with a known concentration. Although there are some differences between the spiked value and the measured K18 value, this test has demonstrated its ability to detect K18 and can therefore be used to determine DILI in capillary blood samples using an instrument that may be used at the point of origin (POC).

[0209] References 1.Caparrotta, TM, DJ Antoine, and JW Dear, Are some people at increased risk of paracetamol-induced liver injury? A critical review of the literature. Eur J Clin Pharmacol, 2018.74(2): p. 147-160. 2.Ferner, RE, JW Dear, and DN Bateman, Management of paracetamol poisoning. Bmj, 2011.342: p. d2218. 3. Thanacoody, HKR Large paracetamol overdose - Higher dose NAC is NOT required. British Journal of Clinical Pharmacology, 2023.89(1): p. 39-42. 4. Smilkstein, MJ, et al. Efficacy of oral N-acetylcysteine ​​in the treatment of acetaminophen overdose. Analysis of the national multicenter Study (1976 to 1985). N Engl J Med, 1988.319(24): p. 1557-62. 5. Saccomano, SJ, Acute acetaminophen toxicity in adults. NursePract, 2019.44(11): p. 42-47. 6. Dawson, AH and IM Whyte, Therapeutic drug monitoring in drugs overdose. Br J Clin Pharmacol, 1999.48(3): p. 278-83. 7. Dear, JW, et al., Risk stratification after paracetamol overdose using mechanistic biomarkers: results from two prospective cohort studies.The Lancet Gastroenterology&Hepatology, 2018.3(2): p. 104-113. 8.Dear, J.W., et al., Early detection of paracetamol toxicity using circulating liver microRNA and markers of cell necrosis. British journal ofclinical pharmacology, 2014.77(5): p. 904-905. 9.Church, R.J., et al., Candidate biomarkers for the diagnosis and prognosis of drug-induced liver injury: An international collaborative effort. Hepatology, 2019.69(2): p. 760-773. 10.Antoine, D.J., et al., Mechanistic biomarkers provide early and sensitive detection of acetaminophen-induced acute liver injury at first presentation to hospital. Hepatology, 2013.58(2): p. 777-87. 11.Clarke, J.I., N. Brillanf, and D.J. Antoine, Novel circulating- and imaging-based biomarkers to enhance the mechanistic understanding of human drug-induced liver injury. J Clin Transl Res, 2017.3(1): p. 199-211. 12.Llewellyn, H.P., et al., Evaluating the Sensitivity and Specificity of Promising Circulating Biomarkers to Diagnose Liver Injury in Humans. Toxicol Sci, 2021.181(1): p. 23-34. 13.Rupprechter, S.A.E., et al., MicroRNA-122 and cytokeratin-18 have potential as a biomarkers of drug-induced liver injury in European and African patients on treatment for mycobacterial infection. Br J ClinPharmacol, 2021.87(8): p. 3206-3217. 14.Hosseini, S., et al., Advantages, Disadvantages and Modifications of Conventional ELISA , in Enzyme-linked Immunosorbent Assay (ELISA): From A to Z , S. Hosseini, et al., Editors. 2018, Springer Singapore: Singapore. p. 67-115。

Claims

1. A lateral flow immunoassay (LFIA) device, comprising: The sample holding section is configured to hold samples. SERS active particles, configured to bind to cytokeratin-18 (K18) in the sample; and In the testing section, the configuration was set to immobilize the SERS active particles that had been combined with K18.

2. The immunoassay instrument according to claim 1, wherein, The immunoassay instrument extends between the first end and the second end.

3. The immunoassay apparatus according to any one of the preceding claims, wherein, The immunoassay apparatus includes a capillary element configured to allow wicking of fluid from a first end to a second end.

4. The immunoassay apparatus according to any one of the preceding claims, wherein, The immunoassay instrument includes a wicking or absorption element disposed at or near its second end / second end.

5. The immunoassay apparatus according to any one of the preceding claims, wherein, The sample container is configured to hold the subject's blood.

6. The immunoassay apparatus according to any of the preceding claims, wherein, The sample container is disposed and / or located at or near the first end of the immunoassay instrument.

7. The immunoassay apparatus according to any one of the preceding claims, wherein, The sample containing portion includes a first sample containing element, wherein the first sample containing element includes a first sample filter membrane treated with sodium chloride.

8. The immunoassay apparatus according to any one of the preceding claims, wherein, The sample containing portion includes a second sample containing element, wherein the second sample containing element includes a second sample filter membrane treated with a nonionic surfactant.

9. The immunoassay apparatus according to any one of the preceding claims, wherein, The immunoassay instrument includes a binding pad positioned adjacent to the sample containment portion and configured to contain the SERS active particles, the SERS active particles being configured to bind to cytokeratin-18 (K18) in the sample.

10. The immunoassay instrument according to claim 9, wherein, The binding pad has been treated with a sucrose solution, for example, a sucrose solution with a concentration of about 0.1% to 5%, or for example, about 1% (w / v), prior to the deposition of the SERS active particles.

11. The immunoassay apparatus according to any of the preceding claims, wherein, The SERS active particles include Raman reporter molecules.

12. The immunoassay apparatus according to any of the preceding claims, wherein, The SERS active particles comprise one or more nanoparticle clusters, optionally clusters of two (dimers), three (trimers), and / or four (tetramers).

13. The immunoassay apparatus according to any of the preceding claims, wherein, The SERS active particles are encapsulated in a protective shell.

14. The immunoassay instrument according to claim 13, wherein, The protective shell is an inorganic shell consisting of or composed of silicon dioxide.

15. The immunoassay apparatus according to any of the preceding claims, wherein, The SERS active particles include an anti-K18 antibody.

16. The immunoassay apparatus according to any of the preceding claims, wherein, The test includes an anti-K18 antibody.

17. The immunoassay apparatus according to any of the preceding claims, wherein, The immunoassay instrument includes a control portion, wherein the control portion includes at least one antibody capable of binding antibodies deposited on the binding pad and / or antibodies disposed within the SERS active particles and / or antibodies attached to the SERS active particles. Optionally, the control portion includes at least one antibody capable of binding anti-K18 antibodies.

18. The immunoassay apparatus according to any of the preceding claims, wherein, The immunoassay instrument is housed within a box configured to at least partially surround the immunoassay instrument.

19. A system configured to provide a quantitative measurement of cytokeratin-18 (K18) in a sample, wherein, The system includes: Lateral flow immunoassay analyzer, including: The sample section is configured to hold the sample; SERS active particles, configured to bind to cytokeratin-18 (K18) in the sample; and In the testing section, the configuration is used to immobilize the SERS active particles that have been bound to K18; A surface-enhanced Raman spectroscopy (SERS) device is configured to measure and / or detect K18 at or near the test section.

20. The system according to claim 19, wherein, The SERS device is configured to generate radiation with a wavelength of approximately 785 nm.

21. The system according to claim 19 or claim 20, wherein, The SERS device is a portable or handheld SERS device or a handheld Raman reader.

22. A method for detecting cytokeratin-18 (K18) in a sample, the method comprising: A lateral flow immunoassay analyzer is provided, the lateral flow immunoassay analyzer comprising: The sample holding section is configured to hold samples. SERS active particles, configured to bind to cytokeratin-18 (K18) in the sample; and In the testing section, the configuration was used to immobilize SERS active particles that had been bound to K18; The sample is provided in the sample receiving portion; and SERS analysis was performed in the test section.

23. The method of claim 22, further comprising performing SERS analysis at a control portion, wherein the control portion comprises at least one antibody capable of binding antibodies deposited on the binding pad and / or antibodies disposed within the SERS active particles and / or antibodies attached to the SERS active particles; and comparing and measuring the signals obtained from the test portion and the control portion.

24. The method of claim 23, further comprising using the spectral intensity ratio obtained from SERS analysis of the test portion and the control portion.

25. The method according to any one of claims 22 to 24, the method comprising preparing the SERS active particles, wherein the method comprises: Preparation of metal nanoparticles; The nanoparticles were modified with Raman reporter molecules; Nanoparticles modified with Raman reporter molecules; as well as The SERS active particles are functionalized using the portion that can bind to K18.

26. The method according to any one of claims 22 to 25, comprising preparing the test portion, the method comprising depositing and / or immobilizing anti-K18 antibody on a capillary element of the immunoassay instrument / the capillary element.

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