Platform for direct treponemal and nontreponemal lateral flow assay for syphilis

Abstract

Embodiments relate to compositions, methods, and systems designed to screen and detect antibodies for pathogen detection. Embodiments particularly relate to systems that utilize multilayered plasmonic nanoparticles for enhanced detection sensitivity. Embodiments further relate to systems designed to simultaneously detect treponemal and non-treponemal antibodies for syphilis diagnoses.

Description

Atty. Ref. No. 0073605-001100PLATFORM FOR DIRECT TREPONEMAL AND NONTREPONEM AL LATERAL FLOW ASSAY FOR SYPHILISCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This patent application is related to and claims the benefit of priority of U.S. Provisional Application 63 / 712,599, filed on October 28, 2024. The entire contents of this application is incorporated by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH DEVELOPMENT

[0002] This invention was made with government support under Contract No. 75D30122C15492 awarded by the Center for Disease Control / DHHS. The Government has certain rights in the invention.FIELD

[0003] Embodiments relate to compositions, methods, and systems designed to screen and detect antibodies for pathogen detection. Embodiments particularly relate to systems that utilize multilayered plasmonic nanoparticles for enhanced detection sensitivity. Embodiments further relate to systems designed to simultaneously detect treponemal and non-treponemal antibodies for syphilis diagnoses.BACKGROUND

[0004] Sexually transmitted infections (STIs), including syphilis, continue to pose a significant global and national public health challenge, with recent data indicating a disturbing rise in cases. In the United States, surveillance reports from the Centers for Disease Control and Prevention (CDC) reveal that over 2.4 million cases of chlamydia, gonorrhea, and syphilis were reported inAtty. Ref. No. 0073605-0011002023, with syphilis experiencing the most notable surge, an increase of approximately 80% from 2018 to 2022. Worldwide, the World Health Organization (WHO) estimates that in 2022 alone, more than 8 million new syphilis cases were documented, including 700,000 cases of congenital syphilis resulting from vertical transmission. These increasing trends highlight the urgent need for more effective, accessible, and rapid diagnostic tools to control disease transmission and prevent severe health consequences.

[0005] Syphilis, caused by the bacterium Treponema pallidum subspecies pallidum, advances through multiple stages (e.g., primary, secondary, latent, and tertiary) each with distinct clinical features. The early stages are highly contagious, but many individuals remain asymptomatic or are unaware of their infection. If untreated, syphilis can lead to severe complications such as neurological damage, cardiovascular disease, and adverse pregnancy outcomes. The management of syphilis relies heavily on accurate and timely diagnosis, which currently depends on serological testing. Conventional laboratory methods, including non-treponemal tests like RPR and VDRL, and treponemal-specific assays such as TPHA and FTA-ABS, are effective but limited by the need for laboratory infrastructure, delayed results, and their inability to differentiate between active and past infections reliably. Direct detection techniques like darkfield microscopy and nucleic acid amplification tests are limited by their technical complexity, sample requirements, and variable sensitivity at different disease stages.

[0006] Recent advances have introduced point-of-care (POC) diagnostic platforms, notably lateral flow immunoassays (LFAs), which offer rapid, user-friendly, and inexpensive testing options. Several FDA-cleared POC tests, for example, detect treponemal antibodies and deliver results within minutes. However, these assays are generally limited to identifying antibodiesAtty. Ref. No. 0073605-001100 indicative of prior or current infection without distinguishing active disease, and they often require follow-up laboratory testing for confirmation using non-treponemal titers.

[0007] These diagnostic limitations underscore the need for innovative solutions that can simultaneously detect both non-treponemal and treponemal antibodies in a single, rapid test, providing a more accurate assessment of active infection.SUMMARY

[0008] The present disclosure relates to innovative advancements in point-of-care diagnostics through the development of a highly sensitive, multiplexed LFA platform for syphilis detection. The present disclosure has the following objectives: the use of multilayered plasmonic nanoparticles as colorimetric reporters, and the integration of simultaneous detection of both non-treponemal (NT) and treponemal (TT) antibodies within a single assay. Individually, each of these components addresses critical limitations in current syphilis diagnostics, and their combination offers a powerful solution to improve sensitivity, specificity, and clinical utility.

[0009] The first objective involves the engineering of multilayered plasmonic nanoparticles composed of a central core, an inner shell, and an outer shell. This multilayered structure may leverage strong plasmonic properties while maintaining chemical stability and biocompatibility, resulting in nanoparticles with significantly enhanced optical extinction cross-sections. These properties enable brighter, more distinct visual signals and improved detection limits in LFAs, allowing for the reliable identification of low-abundance biomarkers and early-stage infections. The customizable optical features of these multilayered nanoparticles can be tuned to optimize detection wavelengths, further increasing assay sensitivity and contrast.

[0010] The second objective addresses the critical need for rapid, accurate, and comprehensive serological testing for syphilis by enabling the simultaneous detection of NT and TT antibodiesAtty. Ref. No. 0073605-001100 on a single platform. Traditional methods often require multiple steps, separate tests, or delayed laboratory results, which hinder timely diagnosis and treatment. The invention’s integrated approach consolidates both antibody types into one assay, providing a more complete clinical picture, distinguishing active infections from past exposures, while reducing testing complexity and turnaround time. By combining these two themes, the invention offers a robust, sensitive, and user-friendly point-of-care diagnostic tool capable of delivering laboratory-grade performance in diverse clinical and resource-limited settings.

[0011] Together, the development of multilayered plasmonic nanoparticles as enhanced optical reporters and the integration of dual-marker detection within a single assay represent a significant leap forward in diagnostic platforms. Embodiments of the present disclosure therefore aim to facilitate immediate, accurate diagnosis and effective disease management, thereby addressing critical gaps in current testing methodologies and supporting public health efforts to control and reduce the spread of diseases such as syphilis.

[0012] In an exemplary embodiment, an apparatus for detecting antibodies related to one or more pathogens of interest includes a testing strip including, in sequence: a sample application region configured to receive a sample collected from a subject, wherein the sample is configured flow through the testing strip; a conjugate region having multilayered plasmonic nanoparticles functionalized with antibody capture probes configured to capture antibodies related to the one or more pathogens of interest; a first testing region configured to detect the presence of antibodies related to a first pathogen, wherein the first testing region has first immobilized capture probes bound to the testing strip, wherein the first immobilized capture probes are configured to capture antibodies related to the first pathogen; optionally, a second testing region configured to detect the presence of antibodies related to a second pathogen, wherein the secondAtty. Ref. No. 0073605-001100 testing region has second immobilized capture probes bound to the testing strip, wherein the second immobilized capture probes are configured to capture antibodies related to the second pathogen; and a control region having third immobilized capture probes bound to the testing strip, wherein the third immobilized capture probes are configured to capture the antibody capture probes.

[0013] In some embodiments, the multilayered plasmonic nanoparticles comprise a central metal core, an inner metal shell surrounding the central metal core, and an outer metal shell surrounding the inner metal shell.

[0014] In some embodiments, the central metal core is composed of a first metal, the inner metal shell is composed of a second metal, and the outer metal shell is composed of the first metal.

[0015] In some embodiments, the first metal and the second metal are different metals selected from the group consisting of gold, silver, copper, nickel, palladium, platinum, cobalt, rhodium, and iridium.

[0016] In some embodiments, the first metal and the second metal are different metals selected from gold and silver.

[0017] In some embodiments, the first metal is gold and the second metal is silver.

[0018] In some embodiments, as the sample flows through the conjugate region, the multilayered plasmonic nanoparticles functionalized with the antibody capture probes migrate with the sample as it flows through the testing strip.

[0019] In some embodiments, as the sample flows through the testing strip, if antibodies related to the one or more pathogens of interest are present in the sample, the multilayered plasmonic nanoparticles functionalized with the antibody capture probes capture the antibodies as the sample flows through the conjugate region; if antibodies related to the first pathogen are presentAtty. Ref. No. 0073605-001100 in the sample, the first immobilized capture probes capture the antibodies related to the first pathogen as the sample flows through the first testing region; if antibodies related to the second pathogen are present in the sample, the second immobilized capture probes capture the antibodies related to the second pathogen as the sample flows through the second testing region; and the third immobilized capture probes capture the antibody capture probes functionalized to the plasmonic nanoparticles.

[0020] In some embodiments, the multilayered plasmonic nanoparticles effectuate a color change when immobilized on the testing strip.

[0021] In an exemplary embodiment, an apparatus for detecting non-treponemal (NT) antibodies and treponemal (TT) antibodies, the apparatus includes a testing strip including, in sequence: a sample application region configured to receive a sample collected from a subject, wherein the sample is configured flow through the testing strip; a conjugate region having multilayered plasmonic nanoparticles functionalized with antibody capture probes configured to capture the NT antibodies and the treponemal TT antibodies; a first testing region configured to detect the presence of NT antibodies, wherein the first testing region has NT antigens bound to the testing strip and configured to capture NT antibodies; a second testing region configured to detect the presence of TT antibodies, wherein the second testing region has TT antigens bound to the testing strip and configured to capture TT antibodies; and a control region having immobilized capture probes bound to the testing strip, wherein the immobilized capture probes are configured to capture the antibody capture probes.

[0022] In some embodiments, the multilayered plasmonic nanoparticles comprise a central metal core, an inner metal shell surrounding the central metal core, and an outer metal shell surrounding the inner metal shell.Atty. Ref. No. 0073605-001100

[0023] In some embodiments, the central metal core is composed of a first metal, the inner metal shell is composed of a second metal, and the outer metal shell is composed of the first metal.

[0024] In some embodiments, the first metal and the second metal are different metals selected from the group consisting of gold, silver, copper, nickel, palladium, platinum, cobalt, rhodium, and iridium.

[0025] In some embodiments, the first metal and the second metal are different metals selected from gold and silver.

[0026] In some embodiments, the first metal is gold and the second metal is silver.

[0027] In some embodiments, as the sample flows through the conjugate region, the multilayered plasmonic nanoparticles functionalized with the antibody capture probes migrate with the sample as it flows through the testing strip.

[0028] In some embodiments, as the sample flows through the testing strip, if NT antibodies and / or TT antibodies are present in the sample, the multilayered plasmonic nanoparticles functionalized with the antibody capture probes capture the NT antibodies and / or TT antibodies as the sample flows through the conjugate region; if NT antibodies are present in the sample, the NT antigens capture the NT antibodies as the sample flows through the first testing region; if TT antibodies are present in the sample, the TT antigens capture the TT antibodies as the sample flows through the second testing region; and the immobilized capture probes capture the antibody capture probes functionalized to the plasmonic nanoparticles.

[0029] In some embodiments, the multilayered plasmonic nanoparticles effectuate a color change when immobilized on the testing strip.Atty. Ref. No. 0073605-001100

[0030] Other details, objects, and advantages of our compositions, methods, and systems will become apparent as the following description of certain exemplary embodiments thereof proceeds.BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The above and other objects, aspects, features, advantages, and possible applications of embodiments of the present innovation will be more apparent from the following more particular description thereof, presented in conjunction with the following drawings. Like reference numbers used in the drawings may identify like components.

[0032] FIG. l is a schematic illustration of an exemplary lateral flow assay system designed to simultaneously detect non-treponemal antibodies (NT) and treponemal antibodies (TT) for syphilis diagnoses. The presence of NT antibodies may be indicated by a test (Tl) line and the presence of TT antibodies may be indicated by a test (T2) line, along with a control (C) line.

[0033] FIG. 2 is a schematic representation of an exemplary test strip based on the multilayered plasmonic nanoparticles.

[0034] FIG. 3 is a schematic representation of an exemplary synthesis process for 15 nm and 40 nm gold nanoparticles.

[0035] FIG. 4 is a schematic representation of an exemplary synthesis process for gold-silver nanoparticles and gold-silver-gold nanoparticles.

[0036] FIG. 5 is a graph showing normalized UV-vis spectra of the three kinds of colloidal solutions: gold nanoparticles, gold-silver nanoparticles, and gold-silver-gold nanoparticles.

[0037] FIG. 6 is a graph showing DLS measurement of synthesized nanoparticles.

[0038] FIG. 7 shows TEM images of as-synthesized gold-silver-gold nanoparticles.

[0039] FIG. 8 shows HAADF micrographs of the gold-silver-gold nanoparticles.Atty. Ref. No. 0073605-001100

[0040] FIG. 9 shows an HR-TEM image of the gold-silver-gold nanoparticles with lattice spacing calculations.

[0041] FIG. 10 is an optical image of the synthesized 1. gold nanoparticles, 2. gold-silver nanoparticles, and 3. gold-silver-gold nanoparticles.

[0042] FIG. 11 is a graph showing extinction spectra of the synthesized nanoparticles.

[0043] FIG. 12 shows hyperspectral dark-field image of gold nanoparticles.

[0044] FIG. 13 shows hyperspectral dark-field image of gold-silver-gold nanoparticles.

[0045] FIG. 14 shows a scattering spectrum of gold nanoparticles and gold-silver-gold nanoparticles.

[0046] FIG. 15 demonstrates the number of gold particles needed to generate a visible signal.

[0047] FIG. 16 demonstrates the number of gold-silver-gold particles needed to generate a visible signal.

[0048] FIG. 17 shows biotin BSA-coated NC membrane with Streptavidin as a capture ligand (different concentrations). Biotin tagged gold nanoparticles and gold-silver-gold nanoparticles.

[0049] FIG. 18 shows biotin BSA-coated NC membrane with Streptavidin as a capture ligand (different concentrations). Biotin tagged gold nanoparticles and gold-silver-gold nanoparticles.

[0050] FIG. 19 shows an exemplary combination assay with sensitivity up to 1: 1 titer as opposed to conventional gold nanoparticles (1 :4 titer) based on the gold-silver-gold nanoparticles.

[0051] FIG. 20 shows preliminary results obtained from 40 serum samples using an exemplary assay. Sample numbers are indicated in Table 1. The figure shows results from 12 samples, in which the assay successfully detected low RPR titers (1 : 1 and 1 :2).

[0052] FIG. 21 is a graph showing test line (Tl) intensity comparison between gold-silver-gold nanoparticles and standard 40 nm gold nanoparticles.Atty. Ref. No. 0073605-001100

[0053] FIG. 22 is a graph showing test line (T2) intensity comparison between gold-silver-gold nanoparticles and standard 40 nm gold nanoparticles.

[0054] FIG. 23 shows results from 6 additional samples were reactive only toward the Abbott treponemal test and non-reactive in the non-treponemal test.

[0055] FIG. 24 shows a specificity study of the proposed LFA using the two most common blood-borne pathogens, HIV and HCV. The LFA showed a signal only for syphilis-positive samples, with no signal detected for the other cases. T1 : treponema, T2: non-treponema and C: control.DETAILED DESCRIPTION

[0056] The following description is of exemplary embodiments and methods of use that are presently contemplated for carrying out the present invention. This description is not to be taken in a limiting sense, but is made merely for the purpose of describing the general principles and features of various aspects of the present invention. The scope of the present invention is not limited by this description.

[0057] Embodiments relate to compositions, methods, and systems that utilize multilayered plasmonic nanoparticles for screening and detecting antibodies for pathogen detection. Embodiments may particularly relate to systems, such as lateral flow assays, designed to simultaneously detect one or more of non-treponemal (NT) antibodies and treponemal (TT) for syphilis diagnoses.Plasmonic Nanoparticles

[0058] In the context of lateral flow assays, plasmonic nanoparticles may be designed to act as reporter probes to provide a visible signal that indicates the presence and / or concentration of a target analyte. The color and brightness of spots on a testing strip are primarily determined byAtty. Ref. No. 0073605-001100 the extinction cross-section of the nanoparticles, which is the sum of their absorption and scattering properties.

[0059] In exemplary embodiments, plasmonic nanoparticles are multilayered plasmonic nanoparticles including a core composed of a first metal and a shell composed of a second metal. The shell may surround or encase the core, thus creating a core-shell layered architecture.

[0060] In exemplary embodiments, plasmonic nanoparticles are multilayered plasmonic nanoparticles including a central core composed of a first metal, an inner shell composed of a second metal, and an outer shell composed of a third metal. The inner shell may surround or encase the central core, and the outer shell may surround or encase the inner shell, thus creating a core-shell-shell layered architecture.

[0061] The multilayered plasmonic nanoparticles can be used to improve the sensitivity of lateral flow assays. For example, the multilayered structure can leverage strong plasmonic properties while maintaining chemical stability and biocompatibility, resulting in a broader, color (e.g., red)-shifted scattering profile that enhances signal contrast on white or light colored testing strips.

[0062] The first metal, second metal, and / or third metal may each be a transition metal. In particular, the first metal, second metal, and / or third metal may each be selected from the group consisting of gold, silver, copper, nickel, palladium, platinum, cobalt, rhodium, iridium, and / or the like.

[0063] In some embodiments, the first metal and the third metal may be the same. For example, the core and the outer shell may be composed of one metal, and the inner shell may be composed of a second metal.Atty. Ref. No. 0073605-001100

[0064] In alternative embodiments, the first metal and the second metal may be the same. For example, the core and the inner shell may be composed of one metal, and the outer shell may be composed of a second metal.

[0065] In alternative embodiments, the second metal and the third metal may be the same. For example, the inner shell and the outer shell may be composed of one metal, and the core may be composed of a second metal.

[0066] In one embodiment, The first metal, second metal, and third metal may each be gold or silver. For example, the core may be composed of gold, the inner shell may be composed of silver, and the outer shell may be composed of gold. Alternatively, the core may be composed of silver, the inner shell may be composed of gold, and the outer shell may be composed of silver.Lateral Flow Assay

[0067] Embodiments further relate to a lateral flow assay system and method configured to receive and analyze a sample to determine if the sample includes antibodies related to pathogen(s) of interest. For example, the system may be configured to determine if a sample includes NT and / or TT antibodies. The lateral flow assay system and method can be used as a point-of-care (POC) test, for example as a rapid lab test.

[0068] As seen in FIG. 1 and FIG. 2, a lateral flow system 100 may include a testing strip 102. The testing strip 102 may include a sample application region 104, a conjugate region 106, at least one testing region (e g., a first testing region 108a and a second testing region 108b), and a control region 110. A sample collected from a subject, or a solution including a sample collected from a subject, may be placed on or at the sample application region 106 and flow through the testing strip 102 (e.g., across the length of the testing strip 102) thereafter. The sample application region 104 may therefore be designated as the beginning of the test strip 102. TheAtty. Ref. No. 0073605-001100 conjugate region 106 may be positioned between the sample application region 104 and the at least one testing region. Similarly, the at least one testing region may be positioned in between the conjugate region 106 and the control region 110. Accordingly, the sample may flow from the sample application region 104, then to the conjugate region 106, then to the at least one testing region, and then to the control region 110.

[0069] Exemplary methods and systems for screening and detecting pathogen(s) of interest may include collecting a sample from a subject. The sample may be collected using any suitable means, including but not limited to, an oral swab, a nasal swab, a cervical swab, a blood collecting swab, urine collection, or any other suitable means for collecting nucleic acid from the subject. The sample may be collected using any suitable instrument, including but not limited to, a cotton swab or any other suitable instrument for collecting nucleic acid from the subject.

[0070] The collected sample may optionally be introduced to a sensing solution to form an aqueous mixture. In some embodiments, the sensing solution may include a nucleic acid extraction buffer configured to extract nucleic acids from the collected sample. In alternative embodiments, nucleic acids may not be extracted from the collected sample. Extraction of nucleic acid and amplification of nucleic acid may be performed but are not requirements for using the lateral flow system.

[0071] In some embodiments, the aqueous mixture may be incubated prior to application to the testing strip 102. Incubation may include maintaining the aqueous mixture for a period of time under predetermined conditions. For example, the aqueous mixture may be incubated for at least 5 minutes at or near a predetermined temperature, such as room temperature, 37°C, etc.

[0072] The sample or aqueous mixture may be placed at or near the sample application region104. The sample or aqueous mixture may then flow via capillary action through the test strip 102Atty. Ref. No. 0073605-001100 in a flow direction and towards the conjugate region 106, the testing regions 108a and 108b, and the control region 110. The speed of the flow may be affected by a number of factors, such as the types, quality and size of the flow strip used.

[0073] Plasmonic nanoparticles, such as the multilayered plasmonic nanoparticles described above, may be positioned at the conjugate region 106 of the testing strip 102. The plasmonic nanoparticles may be functionalized with an antibody capture probe configured to bind to antibodies related to pathogen(s) of interest. For example, if antibodies related to a first pathogen of interest are present in the sample applied to the testing strip 102, as the sample flows through the conjugate region 106, the plasmonic nanoparticles functionalized with the antibody capture probes will bind to the antibodies and continue flowing through the testing strip. Similarly, if antibodies related to a second pathogen of interest are present in the sample applied to the testing strip 102, as the sample flows through the conjugate region 106, the plasmonic nanoparticles functionalized with the antibody capture probes will bind to the antibodies and continue flowing through the testing strip. Alternatively, if no antibodies related to any pathogens of interest are present in the sample applied to the testing strip 102, the plasmonic nanoparticles will nevertheless flow through the testing strip with the antibody capture probes attached thereto.

[0074] In specific embodiments, the plasmonic nanoparticles may be functionalized with an antibody capture probe configured to bind to both NT and TT antibodies. For example, if NT antibodies are present in the sample applied to the testing strip 102, as the sample flows through the conjugate region 106, the plasmonic nanoparticles functionalized with the antibody capture probes will bind to the NT antibodies and continue flowing through the testing strip. Similarly, if TT antibodies are present in the sample applied to the testing strip 102, as the sample flows through the conjugate region 106, the plasmonic nanoparticles functionalized with the antibodyAtty. Ref. No. 0073605-001100 capture probes will bind to the TT antibodies and continue flowing through the testing strip. Alternatively, if neither NT nor TT antibodies are present in the sample applied to the testing strip 102, the plasm onic nanoparticles will flow through the testing strip with the antibody capture probes attached thereto but unbound to NT or TT antibodies.

[0075] The antibody capture probe can be any molecule, compound, antibody, etc. capable of attaching to the nanoparticle and capturing both NT and TT antibodies. In some embodiments, the antibody capture probe is anti-IgG antibodies, anti-IgM antibodies, or biotin.

[0076] The first testing region 108a may correspond to a first pathogen of interest and may include first immobilized capture probes, such as immobilized antibodies or antigens. The first immobilized capture probes may be immobilized at the first testing region 108a such that the capture probes may not flow with the aqueous mixture as it flows through the testing strip 102. The first immobilized capture probes are configured to capture the antibodies related to the first pathogen of interest. Accordingly, in embodiments wherein the mixture has antibodies related to the first pathogen of interest and plasmonic nanoparticles bound to said antibodies (e.g., after passing through the conjugate region 106), the first immobilized capture probes may bind to the antibodies, thereby immobilizing the antibodies and plasmonic nanoparticles at the first testing region 108a.

[0077] In specific embodiments, the first testing region 108a may correspond to NT antibodies and may include first immobilized capture probes, such as NT antigens. The NT antigens are configured to capture the NT antibodies. Accordingly, in embodiments wherein the mixture has NT antibodies and plasmonic nanoparticles bound to said antibodies (e.g., after passing through the conjugate region 106), the NT antigens may bind to the NT antibodies, thereby immobilizing the NT antibodies and plasmonic nanoparticles at the first testing region 108a.Atty. Ref. No. 0073605-001100

[0078] The second testing region 108b may correspond to a second pathogen of interest and may include second immobilized capture probes, such as immobilized antibodies or antigens. The second immobilized capture probes may be immobilized at the second testing region 108b such that the capture probes may not flow with the aqueous mixture as it flows through the testing strip 102. The second immobilized capture probes are configured to capture the antibodies related to the second pathogen of interest. Accordingly, in embodiments wherein the mixture has antibodies related to the second pathogen of interest and plasmonic nanoparticles bound to said antibodies (e.g., after passing through the conjugate region 106), the second capture probes may bind to the antibodies, thereby immobilizing the antibodies and plasmonic nanoparticles at the second testing region 108b.

[0079] In specific embodiments, the second testing region 108b may correspond to TT antibodies and may include second immobilized capture probes, such as TT antigens. The TT antigens are configured to capture the TT antibodies. Accordingly, in embodiments wherein the mixture has TT antibodies and plasmonic nanoparticles bound to said antibodies (e.g., after passing through the conjugate region 106), the TT antigens may bind to the TT antibodies, thereby immobilizing the TT antibodies and plasmonic nanoparticles at the second testing region 108b.

[0080] As described above, the plasmonic nanoparticles, and particularly the multilayered plasmonic nanoparticles, are configured to effectuate a color change when immobilized at a region. In some embodiments, when the plasmonic nanoparticles are immobilized at the first testing region 108a, the plasmonic nanoparticles may effectuate a color change at the first testing region 108a. In some embodiments, the color change may appear as a visible line or mark at the first testing region 108a. Therefore, the presence of a line or mark at the first testing region 108aAtty. Ref. No. 0073605-001100 signals the presence of a first pathogen of interest (or in some embodiments, the presence of NT antibodies). However, no line or mark at the first testing region 108a signals the absence of the first pathogen of interest (or NT antibodies) in the sample.

[0081] Similarly, when the plasmonic nanoparticles are immobilized at the second testing region 108b, the plasmonic nanoparticles may effectuate a color change at the second testing region 108b. In some embodiments, the color change may appear as a visible line or mark at the second testing region 108b. Therefore, the presence of a line or mark at the second testing region 108b signals the presence of a second pathogen of interest (or in some embodiments, the presence of TT antibodies). However, no line or mark at the first testing region 108b signals the absence of the first pathogen of interest (or TT antibodies) in the sample.

[0082] The control region 110 may include third immobilized capture probes, such as immobilized antibodies. The third immobilized capture probes may be immobilized at the control region 110 such that the capture probes may not flow with the aqueous mixture as it flows through the testing strip 102. The third immobilized capture probes are configured to capture the nanoparticles flowing through the testing strip 102 by binding to the antibody capture probes functionalized to the plasmonic nanoparticles. It is understood that, even if antibodies of interest are present in the sample and bind to plasmonic nanoparticles, at least some of the plasmonic nanoparticles may nevertheless not bind to the antibodies of interest and may flow with the aqueous mixture as it flows through the testing strip 102 past the testing regions 108a and / or 108b. These remaining plasmonic nanoparticles may be captured by the third immobilized capture probes via the antibody probes and immobilized at the control region 110.

[0083] The third immobilized capture probes can be any molecule, compound, antibody, etc. capable of capturing the antibody capture probe. In some embodiments, the third immobilizedAtty. Ref. No. 0073605-001100 capture probes are IgG antibodies, IgM antibodies, or streptavidin. For example, the antibody capture probe may be anti-IgG antibodies and the third immobilized capture probes may be IgG antibodies. As another example, the antibody capture probe may be anti-IgM antibodies and the third immobilized capture probes may be IGM antibodies. As another example, the antibody capture probe may be biotin and the third immobilized capture probes may be streptavidin.

[0084] As the plasmonic nanoparticles may be configured to effectuate a color change when reacted and immobilized at a region, when the plasmonic nanoparticles are immobilized at the control region 110, the nanoparticles may effectuate a color change at the control region 110. In some embodiments, the color change may appear as a visible line or mark at the control region 110. All proper tests and samples should result in a line or mark at the control region 110, such that the control region 110 ensures the system 100 is working properly.

[0085] A negative test (e.g., a sample without NT and TT antibodies) may result in a line or mark only at the control region. The control region 110 may be designated by a “C.” A test positive for NT antibodies but negative for TT antibodies may result in a line or mark at the first testing region and a line of mark at the control region 110. The first testing region 108a may be designated by a “T1 ” A test positive for TT antibodies but negative for NT antibodies may result in a line or mark at the second testing region and a line of mark at the control region 110. The second testing region 108b may be designated by a “T2 ” A test positive for both of NT and TT antibodies may result in a line or mark at the first testing region, a line or mark at the second testing region, and a line of mark at the control region.

[0086] As can be appreciated by the above, the lateral flow system 100 may be configured to detect the presence of one or both of NT and TT antibodies The lateral flow system 100 may therefore serve as a one step, simultaneous detection method for syphilis in a POC setting.Atty. Ref. No. 0073605-001100

[0087] As there is an ongoing and immediate need to develop approaches that are low-cost, rapid, do not require the use of advanced equipment, and can be used as a screening tool for the diagnosis of syphilis at POC, it is contemplated that embodiments described herein may provide one or more advantages over currently available screening and detecting techniques. For example, embodiments described herein: (i) do not need prior nucleic acid extraction; (ii) do not demand the use of advanced equipment (e g., centrifuge, thermocycler, etc.); (iii) do not use conventional pH sensitive dyes; and / or (iv) has a short turnaround time. In some embodiments, the presently described system provides for rapid turnaround time for detection of syphilis.

[0088] The detection of syphilis may be performed within about 5, 10, 15, 20, 25, or 30 minutes. In one embodiment, detection may be completed within about 10 minutes of applying a sample to a testing strip.

[0089] The system is further capable of achieving high specificity for NT and / or TT antibodies with minimal or no cross-interference from other bacterial species.EXAMPLE

[0090] Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0091] In the quest for more effective colorimetric reporters compared to traditional gold nanoparticles, the below example developed and analyzed a multilayered plasmonic nanoparticle, consisting of a gold core, silver inner shell, and gold outer shell. As a practical example, the multilayered plasmonic nanoparticles were used to improve the sensitivity of lateralAtty. Ref. No. 0073605-001100 flow immunoassays for detecting syphilis. The Au-Ag-Au structure took advantage of silver’s strong plasmonic properties while maintaining gold’s chemical stability and bioconjugation compatibility, resulting in a broader, red-shifted scattering profile that enhances signal contrast on nitrocellulose membranes. The multilayered plasmonic nanoparticles enabled visual detection of streptavidin at 25 pg / mL, representing a threefold improvement over conventional gold nanoparticles, and showed superior analytical sensitivity in detecting both treponemal (TT) and non-treponemal (NT) antibodies. In clinical validation with 40 patient samples, the ML-PNP- based LFA exhibited high agreement with reference laboratory methods (RPR and TP-PA), including reliable detection of low-titer samples (1 : 1 RPR), and showed no cross-reactivity with HIV-1 or HCV

[0092] Principle of Detection: In the context of lateral flow assays, plasmonic nanoparticles are designed to serve as optical reporter probes, producing a visible signal when interacting with incident light that indicates the presence or concentration of the target analyte. The strength of this signal, shown by the brightness and color of the test and control lines, is directly related to the nanoparticle’s optical extinction cross-section (cre%t), which is the sum of absorption and scattering properties of the NPs. Conventional CLFIAs employing gold nanoparticles smaller than 60 nm display characteristic red hues because absorption dominates for these small metal nanoparticles due to their localized surface plasmon resonance (LSPR) near 520 nm. However, the optical extinction coefficient of sub-60 nm gold nanoparticles is relatively low (108- 109M'1cm'1), which limits the maximum attainable signal strength and makes it harder to distinguish low-concentration analytes from background noise. Additionally, the narrow LSPR band of these particles offers limited color contrast against the nitrocellulose membrane, further decreasing their visual detectability.Atty. Ref. No. 0073605-001100

[0093] Efforts to overcome these limitations have focused on increasing particle size up to 100 nm to boost the extinction coefficient (up to 1011M-1cm-1). As particle size increases, light scattering becomes more significant, resulting in brighter signals. However, when the nanoparticle diameter exceeds about 100 nm, gravitational sedimentation and particle aggregation become more prominent, causing reduced colloidal stability, uneven migration through the membrane, and lower assay reproducibility. Collectively, these issues limit the sensitivity and reliability of traditional lateral flow assays, especially in applications that require detecting low analyte levels or high-contrast visual results. Therefore, recent research has focused on designing plasmonic nanoparticles with higher optical extinction coefficients.

[0094] According to Mie theory, the optical extinction coefficient (crezt) depends on the nanoparticle radius R, the excitation wavelength A, and the real (£r) and imaginary (g£) parts of the metal’s dielectric function, along with the dielectric constant of the surrounding medium £m. The extinction cross-section (cre%t) for spherical particles can be calculated using the Equation (1):

[0095] Equation (1) on 1 highlights that the extinction behavior strongly depends on the electronic structure and dielectric response of the plasmonic material. Metals with a low imaginary part of the dielectric constant (f£) exhibit reduced optical losses and more pronounced plasmonic oscillations, leading to stronger scattering and absorption bands. Among noble metals, silver (Ag) intrinsically exhibits sharper and stronger LSPR features than gold (Au) because of its lower st value and a higher plasma frequency. Consequently, silver nanoparticles demonstrateAtty. Ref. No. 0073605-001100 molar extinction coefficients up to an order of magnitude higher (=4 O10to 1011M-1cm-1) compared with gold nanoparticles of equivalent diameter («108to 109M-1cm-1). Moreover, silver nanoparticles possess an LSPR band that can be tuned across a broader range of the visible spectrum, offering enhanced color contrast and brightness on nitrocellulose membranes. Despite these optical advantages, gold nanoparticles face challenges in aqueous and biological environments due to susceptibility to oxidation and less inert surfaces, which complicates bioconjugation and promotes aggregation or dissolution. In contrast, gold nanoparticles offer superior chemical and colloidal stability, supported by strong gold-thiol chemistry, enabling reliable biomolecule functionalization. Nonetheless, their comparatively lower molar extinction coefficient and brightness necessitate size increases that may impair colloidal behavior and assay performance.

[0096] A gold-silver-gold (Au-Ag-Au) multilayer nanoparticle provides a rationally engineered plasmonic architecture that combines the advantages of both metals to overcome inherent trade-offs. The Au core serves as a stable, chemically inert, and well-characterized substrate for shell deposition, offering a robust plasmonic foundation. Surrounding this core, the intermediate Ag shell markedly enhances plasmonic intensity by promoting electron oscillations at the metal-dielectric interface and shifting the LSPR toward longer wavelengths, thereby increasing optical brightness and color contrast. Finally, the outer Au shell functions as a protective barrier that prevents silver oxidation while ensuring compatibility with biomolecular conjugation through established Au-thiol chemistry. Beyond protection, this outer Au layer maintains biocompatibility, stabilizes the colloid under physiological conditions, and facilitates functionalization with capture antibodies without diminishing the plasmonic enhancementAtty. Ref. No. 0073605-001100 contributed by the silver shell. By exploiting the interplay between material composition and electromagnetic field localization, the Au-Ag-Au multilayer nanoparticles should achieve superior extinction coefficients without exceeding the particle diameter stability limit (-100 nm). This multilayer design enhances light-matter interactions via dielectric coupling between gold and silver layers, resulting in a broadened and intensified LSPR band in the visible spectrum.

[0097] Nanoparticle Synthesis: Multilayered plasmonic nanoparticles, comprising a gold (Au) core, an inner silver (Ag) shell, and an outer gold (Au) shell, were synthesized using a seed- mediated method and galvanic replacement reaction (FIG. 3 and FIG. 4). In the standard synthesis of the multilayered plasmonic nanoparticles, a four-step bottom-up approach was employed. First, approximately 15 nm citrate-stabilized gold nanoparticles were synthesized following our previously reported method. 4.2 mb of these 15 nm gold nanoparticles were used as seeds for the next step of synthesizing 40 nm gold nanoparticles. Next, 1 mb of 25 mM hydroquinone was added to a solution containing gold (III) chloride and 1% sodium citrate, while stirring vigorously to promote the seed-mediated growth of approximately 40 nm gold nanoparticles. In this step, hydroquinone functioned as a mild reducing agent, converting Au (III) ions to Au (0) atoms and supporting controlled particle growth, while citrate acted as a stabilizing ligand to prevent nanoparticle aggregation. Next, a silver shell was deposited onto the gold nanoparticle surface through the reduction of silver nitrate. Briefly, 50 mb of the 40 nm gold nanoparticle suspension was mixed with 0.25 mb of 0.1 M L-ascorbic acid, which served as a reducing agent to convert Ag(I) ions into metallic silver. Then, 1.25 mb of 10 mM silver nitrate was added dropwise via a syringe pump at a controlled rate, ensuring uniform nucleation and shell growth of silver. The ascorbic acid also aided in maintaining colloidal stability during shell formation. The solution was stirred at room temperature (RT) for 1 hour to obtain gold-silverAtty. Ref. No. 0073605-001100 nanoparticles. Finally, the outermost gold layer was deposited by galvanic replacement. Briefly, 50 mL of the gold-silver nanoparticles solution was heated to boiling, and 8 mb of 1 mM aqueous gold (III) chloride was added slowly, dropwise, via a syringe pump at a controlled rate. During this process, the less noble silver in the shell was partially oxidized and replaced by gold atoms deposited on the surface, resulting in gold-silver gold plasmonic nanoparticles.

[0098] Nanoparticle Characterization: The structural and optical properties of the synthesized nanoparticles were analyzed using a combination of transmission electron microscopy (TEM), UV-Vis spectroscopy, energy dispersive X-ray spectroscopy (EDS), dynamic light scattering (DLS), and hyperspectral imaging. Initially, approximately 15 nm gold nanoparticles were synthesized to be used as seeds in the synthesis of multilayered nanoparticles. The UV-Vis spectrum, particularly the absorption at 520 nm, confirmed the formation of roughly 15 nm gold nanoparticles (FIG. 5). Particle size distribution was confirmed using DLS (FIG. 6). For optical signal readout in conventional CLFIAs, the optimal gold nanoparticle size ranges from 33 to 52 nm. Therefore, in this study, the ~15 nm gold nanoparticles were used as seeds to synthesize 40 nm gold nanoparticles, which were selected as cores for constructing the multilayered plasmonic nanoparticles. Particle size distribution for 40nm gold nanoparticles were confirmed using DLS (FIG. 6). These 40 nm particles were then coated with a silver shell.

[0099] FIGS. 7-9, respectively, shows representative low-magnification and high-magnification TEM images of the multilayered plasmonic nanoparticles that were obtained from a standard synthesis.

[0100] TEM images showed that the multilayered nanoparticles had a polyhedral shape. The distinctive polyhedral or octahedral shape observed in the synthesized gold-silver-gold multilayered plasmonic nanoparticles is directly attributed to the interplay of crystallographicAtty. Ref. No. 0073605-001100 facet selectivity, controlled shell deposition, and galvanic replacement dynamics. During the seed-mediated growth process, citrate-stabilized gold cores provide a smooth template, but as silver is deposited, nucleation preferentially occurs at high-energy facets and edges, imparting initial anisotropy to the shell. Subsequently, when the outer gold layer is introduced via galvanic replacement, gold atoms selectively replace silver at specific crystallographic facets, particularly the higher-energy surfaces leading to further evolution toward well-defined polyhedral geometries. This process is reinforced by thermal diffusion and ligand effects, which encourage atomic reorganization along facet directions, resulting in a stable multilayered structure with pronounced edges and corners. The combined influence of these mechanistic factors yields nanoparticles with the observed polyhedral or frame-like morphology, which are highly desirable for their enhanced surface area and plasmonic properties.

[0101] Optical Images: The as-synthesized solution of multilayered plasmonic nanoparticles is shown in FIG. 10, where the scattered light appears primarily as dark red.

[0102] UV Vis and Extinction Spectra: Moreover, the extinction spectra revealed that the plasmon peak of gold nanoparticles is blue-shifted upon the growth of the silver layer and then red-shifted upon the growth of the outer gold shell in forming the final structure, as shown in FIG. 11. The plasmon peaks of the gold nanoparticles (red line) and the gold-silver nanoparticles (yellow line) are centered at 524 and 420. However, in the case of the gold-silver-gold multilayered nanoparticles (red line), the plasmon beak was broader, which is due to the larger extinction cross-section as hypothesized.

[0103] PLS Measurement: Furthermore, the size of these multilayered plasmonic nanoparticles was less than 60 nm, making them ideal for the later flow assays (FIG. 6).Atty. Ref. No. 0073605-001100

[0104] Zeta Potential Measurements: Under neutral pH, the zeta potential of citrate- stabilized AuNPs is -39.4 ± 5.1 mV, while that of multilayered plasmonic nanoparticles is -28.4 ± 6.5 mV. AA is less ionized than the strongly ionized Cit ligand (AA versus Cit3), which might be a reason for weakly ionized multilayered plasmonic nanoparticles when compared to gold nanoparticles.

[0105] NT A Measurements: The number of particles measured by performing an NTA analysis were approximately 1.2 E+l 1 particles / mL.

[0106] Hyperspectral Imaging: Hyperspectral imaging was employed to study the scattering properties. Hyperspectral darkfield imaging showed that the multilayered plasmonic nanoparticles exhibited a broad and distinct color profile compared to the conventional gold nanoparticles (FIGS. 12 and 13). FIG. 14 demonstrates that the multilayered nanoparticles exhibit not only a red-shifted scattering peak (658 nm vs. 555 nm for gold nanoparticles), but also a noticeably broader scattering band. This combination of peak shift and spectral broadening results from the plasmonic coupling within the multilayered structure, yielding higher and more tunable optical signal intensity suitable for sensitive lateral flow assay detection.

[0107] XPS: The surface properties of multilayered plasmonic nanoparticles were characterized by X-ray photoelectron spectroscopy (XPS). The XPS survey spectrum taken for the multilayered plasmonic nanoparticles was very similar to the spectrum of initial gold nanoparticles, except for the emergence of Ag characteristic peaks. XPS spectra for gold nanoparticles at a higher resolution of the Au 4f peaks showed that the Au shell was primarily composed of Au (0). Moreover, the XPS spectra for a gold-silver core-shell nanoparticles showed that they contained Ag, Au, and that the Au signal was much lower than the Ag signal,Atty. Ref. No. 0073605-001100 as well as lower than what was observed on the gold nanoparticles sample. Both observations are consistent with a very thin (2-4 nm) layer of Ag on top of an Au core.

[0108] Multilayered Plasmonic Nanoparticles as Colorimetric Reporters in Lateral Flow Assays: Conventional CLFIAs using 30 - 40 nm gold nanoparticles as reporter probes have fundamental limitations, such as low capture rates (<5%) and weak signal -to-background ratios, resulting in relatively low sensitivity. To determine whether multilayered plasmonic nanoparticles could be used as reporter probes in lateral flow assays and overcome these gold nanoparticle limitations, we compared their performance on a nitrocellulose membrane. We first determined the minimum number of particles needed to generate a visibly detectable signal by drop-casting serial dilutions of gold nanoparticles (FIG. 15) and multilayered plasmonic nanoparticles (FIG. 16) at specific concentrations. Nominal calculations indicated that approximately 1.25 x 106particles were enough to produce a visible signal with multilayered plasmonic nanoparticles, compared to 7.25 x 106particles for standard gold nanoparticles, demonstrating a lower detection threshold for multilayered plasmonic nanoparticles. Next, to directly compare the performance of multilayered plasmonic nanoparticles and gold nanoparticles in the lateral flow assay format, we used the well-characterized biotin-streptavidin conjugate pairing system, which is known to exhibit extremely high binding affinity. Both reporters (gold nanoparticles and multilayered plasmonic nanoparticles) were functionalized with biotin; streptavidin served as the target analyte, and biotinylated bovine serum albumin (biotin- BSA) was immobilized as the capture ligand (FIGS. 17 and 18). Test spots on the nitrocellulose membrane were prepared by applying 0.5 pL of 5 mg / mL biotinylated BSA, and lateral flow assay strips were assembled as described above. Strips were then challenged with streptavidin standards ranging from 0.1 pg / mL to 100 pg / mL, while a fixed input of each reporter (1 OD,Atty. Ref. No. 0073605-001100 biotin-conjugated gold nanoparticles or multilayered plasmonic nanoparticles) was applied. Colorimetric reporters flow along the nitrocellulose membrane after binding to the target analyte (streptavidin) via capillary force. They are then captured by the capture ligand (biotin-BSA), leading to the accumulation of nanoparticles at the test spot. Accumulation of a sufficient number of reporters converts the color at the test spot to red in the case of gold nanoparticles and dark red / brownish in the case of multilayered plasmonic nanoparticles, indicating a positive result and the presence of the target analyte. In both cases, 1 OD generated a readily discernible test spot, and signal intensity decreased with decreasing streptavidin concentration. Notably, multilayered plasmonic nanoparticles enabled visual detection down to 25 pg / mL streptavidin, whereas gold nanoparticles were limited to 75 pg / mL, representing a threefold improvement. These results establish the fundamental basis that multilayered plasmonic nanoparticles can serve as robust colorimetric reporters for ultrasensitive detection of target analytes in a lateral flow assay.

[0109] Detection of TT Antibodies: Having established that multilayered plasmonic nanoparticles function as highly sensitive colorimetric reporters, we then evaluated their ability to detect treponemal (TT) antibodies in a lateral flow assay format. TT antibodies include IgM and IgG produced against T. pallidum specific membrane lipoproteins, primarily TpN15, TpN17, and TpN47. During natural infection, seroconversion to these antigens generally occurs within 2-3 weeks and, in most cases, persists for life, even after antimicrobial therapy, unless treatment is administered very early. These serological dynamics and their persistence make TT antibodies important clinical targets at all stages of syphilis and provide a useful benchmark for evaluating the effectiveness of multilayered plasmonic nanoparticle-enabled CLFIAs. Accordingly, we designed the lateral flow assay where the test spot (Tl) was prepared with 0.3 pL of 0.5 mg / mlAtty. Ref. No. 0073605-001100 of Tpl5-Tpl7-Tp47 recombinant antigen. This multi epitope fusion protein was selected because it integrates three major immunodominant T. pallidum antigens into a single construct, offering broad reactivity with antibodies generated during both primary and latent infection. The combined antigenic presentation enhances the likelihood of detecting diverse antibody subsets, thereby improving the assay’s diagnostic sensitivity and reliability compared with single-antigen formats. Furthermore, the control spot (C) on the nitrocellulose membrane was prepared with 0.3 pL of 1 mg / mL rabbit IgG antibody, while a fixed amount of both gold nanoparticles and multilayered plasmonic nanoparticles, conjugated with 0.35 pg / mL human IgG and IgM at 1 OD, were applied at the conjugate pad. LFA strips were then challenged with 20 pL syphilis antibody-positive reference sera obtained from the CDC repository. Across the panel, multilayered plasmonic nanoparticle reporters consistently produced higher T1 intensities than gold nanoparticles (FIG. 19), with clear visual differentiation at both low and high clinical titers. Notably, gold nanoparti cl es-based strips did not show a visible T1 spot for two samples, while multilayered plasmonic nanoparticles strips maintained reliable reactivity for all the samples, indicating superior analytical sensitivity for TT antibody detection. When detectable, gold nanoparticles signals are often faint or borderline, which aligns with reduced sensitivity at lower antibody concentrations. Overall, these findings show that combining a multiepitope TT capture antigen with multilayered plasmonic nanoparticles reporters improves low-titer performance in syphilis LFA, allowing for strong, sensitive treponemal serology throughout the disease spectrum.

[0110] Detection of NT Antibodies: Once it was confirmed that the multilayered plasmonic nanoparticle-based LFA for TT antibody detection outperformed the conventional gold nanoparticle-based LFA in analytical performance, we expanded the assessment to NTAtty. Ref. No. 0073605-001100 antibodies to determine whether the platform reliably detects both serological markers that indicate the presence of syphilis, its disease stage, and treatment response. Unlike TT antibodies, which remain detectable for life following exposure, NT antibodies are directed against cardiolipin-lecithin-cholesterol complexes released during T. pallidum infection and tissue damage. Their titers correlate with disease activity and typically decline following successful antimicrobial therapy, thereby serving as quantitative markers for treatment monitoring and reinfection detection. Evaluating NT antibodies alongside TT antibodies, therefore, enables comprehensive disease characterization, differentiating active from past infection and facilitating clinical staging. Accordingly, we designed the lateral flow assay where the test spot (Tl) on the nitrocellulose membrane was prepared with 0.3 pL of 0.5 mg / ml of Venereal Disease Research Laboratory (VDRL) antigen obtained from Arlington Scientific. The VDRL antigen is a diagnostic reagent used in the VDRL test, a nontreponemal serological assay for syphilis screening. The VDRL antigen is a suspension containing a mixture of cardiolipin, cholesterol, and lecithin, which serves to detect reagin antibodies produced in response to cellular damage caused by T. pallidum infection. The test functions as both a qualitative assay, indicating a reactive (positive) or nonreactive (negative) result, and a semiquantitative assay based on antibody titration levels. When the patient’s serum or cerebrospinal fluid containing reagin antibodies is mixed with the VDRL antigen, a visible reaction known as flocculation occurs if antibodies are present. This flocculation indicates an antigen-antibody complex formation, signifying active or recent syphilis infection. Furthermore, the control spot (C) on the nitrocellulose membrane was prepared with 0.3 pL of 1 mg / mL rabbit IgG antibody, while a fixed amount of both gold nanoparticles and multilayered plasmonic nanoparticles, conjugated with 0.35 pg / mL human IgG and IgM at 1 OD, were applied at the conjugate pad. LFA stripsAtty. Ref. No. 0073605-001100 were then challenged with 20 pL syphilis reference sera obtained from the CDC repository. Across the panel, multilayered plasmonic nanoparticle reporters consistently produced higher T1 intensities than AuNPs (FIG. 19), with clear visual differentiation at both low and high clinical titers. Notably, gold nanoparticle-based strips did not show a visible T1 spot at 1 : 1 and 1 :2 titers, while multilayered plasmonic nanoparticle strips maintained reliable reactivity at these low titers, indicating superior analytical sensitivity for TT antibody detection. When detectable, gold nanoparticle signals are often faint or borderline, which aligns with reduced sensitivity at lower antibody concentrations. Thus, integrating NT antibody detection into the multilayered plasmonic nanoparticle-based LFA framework also allowed the assessment of whether the platform maintains high sensitivity and signal clarity in detecting low-titer sera, which is crucial for monitoring treatment response in late latent or previously treated cases.

[0111] Demonstration of Clinical Use: Finally, to evaluate the clinical applicability of the multilayered plasmonic nanoparticle-LFA, the assay was employed to detect both treponemal (TT) and non-treponemal (NTT) antibodies in de-identified patient serum samples sourced from the CDC, University of Washington, Seattle, and Boca Biolistics. A total of forty (N=40) serum samples, including both syphilis-positive cases and healthy controls, were analyzed to validate the assay’s performance. Sample positivity was confirmed by standard treponemal (Abbott TP- PA) and non-treponemal (RPR) serological tests (Table 1). The multilayered plasmonic nanoparticle-LFA effectively detected TT antibodies targeting the Tpl5-Tpl7-Tp47 recombinant antigen mixture, as shown by the development of representative test lines (FIG. 20). The assay’s response for syphilis-reactive samples was clearly distinguishable from that of non-reactive samples (FIG. 20). Importantly, the results from the multiplexed CLFIA were consistent with clinical non-treponemal RPR titer values, enabling detection across a broad dynamic range,Atty. Ref. No. 0073605-001100 including samples with high (1 : 128) and low (1 : 1) RPR titers (FIG. 20). The ability of the assay to identify samples with low antibody titers is especially significant, as it may facilitate early- stage syphilis diagnosis. Additionally, we quantified the signal enhancement in test line intensities using an ESEQuant Lateral Flow Reader. Quantitative comparison showed a 19-46% increase in test line intensities (across different concentrations) with the use of multilayered plasmonic nanoparticles compared to conventional gold nanoparticles (FIGS. 21 and 22). To further confirm reliability, the assay was tested with a pool of six (N=6) clinical specimens that were exclusively reactive for treponemal antigens, resulting in accurate identification of these cases (Table 1, FIG. 23). The specificity of the assay was evaluated against two common bloodborne pathogens, HIV-1 and HCV. Testing of HIV-1 positive serum (N=2) and HCV-positive sera (N=2) showed no cross-reactivity (FIG. 24). Overall, the multiplexed assay reliably distinguished syphilis-reactive from non-reactive clinical specimens (N=40) and demonstrated concordance with reference RPR and Abbott TP -PA methods (Table 1). The ability to detect low RPR titers addresses a known limitation of current serodiagnostics, indicating that multilayered plasmonic nanoparticle-LFA can deliver rapid, POC testing with sensitivity and specificity comparable to laboratory gold standards, especially for low-titer reactive samples.Atty. Ref. No. 0073605-001100Table 1 : Comparison of a standard reverse algorithm-based syphilis assay with our comboplatform (preliminary data obtained with serum samples from Boca, CDC & UW).

[0112] It should be understood that modifications to the embodiments disclosed herein can be made to meet a particular set of design criteria. For instance, the number of or configuration of components or parameters may be used to meet a particular objective.

[0113] It will be apparent to those skilled in the art that numerous modifications and variations of the described examples and embodiments are possible in light of the above teachings of the disclosure. The disclosed examples and embodiments are presented for purposesAtty. Ref. No. 0073605-001100 of illustration only. Other alternative embodiments may include some or all of the features of the various embodiments disclosed herein. For instance, it is contemplated that a particular feature described, either individually or as part of an embodiment, can be combined with other individually described features, or parts of other embodiments. The elements and acts of the various embodiments described herein can therefore be combined to provide further embodiments.

[0114] It is the intent to cover all such modifications and alternative embodiments as may come within the true scope of this invention, which is to be given the full breadth thereof. Additionally, the disclosure of a range of values is a disclosure of every numerical value within that range, including the end points. Thus, while certain exemplary embodiments of the apparatus and process and / or utilization and methods of making and using the same have been discussed and illustrated herein, it is to be distinctly understood that the invention is not limited thereto but may be otherwise variously embodied and practiced within the scope of the following claims.

Claims

Atty. Ref. No. 0073605-001100What is claimed is:

1. An apparatus for detecting antibodies related to one or more pathogens of interest, the apparatus comprising a testing strip including, in sequence: a sample application region configured to receive a sample collected from a subject, wherein the sample is configured flow through the testing strip; a conjugate region having multilayered plasmonic nanoparticles functionalized with antibody capture probes configured to capture antibodies related to the one or more pathogens of interest; a first testing region configured to detect the presence of antibodies related to a first pathogen, wherein the first testing region has first immobilized capture probes bound to the testing strip, wherein the first immobilized capture probes are configured to capture antibodies related to the first pathogen; optionally, a second testing region configured to detect the presence of antibodies related to a second pathogen, wherein the second testing region has second immobilized capture probes bound to the testing strip, wherein the second immobilized capture probes are configured to capture antibodies related to the second pathogen; and a control region having third immobilized capture probes bound to the testing strip, wherein the third immobilized capture probes are configured to capture the antibody capture probes.Atty. Ref. No. 0073605-0011002. The apparatus of claim 1, wherein the multilayered plasmonic nanoparticles comprise a central metal core, an inner metal shell surrounding the central metal core, and an outer metal shell surrounding the inner metal shell.

3. The apparatus of claim 2, wherein the central metal core is composed of a first metal, the inner metal shell is composed of a second metal, and the outer metal shell is composed of the first metal.

4. The apparatus of claim 3, wherein the first metal and the second metal are different metals selected from the group consisting of gold, silver, copper, nickel, palladium, platinum, cobalt, rhodium, and iridium.

5. The apparatus of claim 3, wherein the first metal and the second metal are different metals selected from gold and silver.

6. The apparatus of claim 3, wherein the first metal is gold and the second metal is silver.

7. The apparatus of claim 1, wherein as the sample flows through the conjugate region, the multilayered plasmonic nanoparticles functionalized with the antibody capture probes migrate with the sample as it flows through the testing strip.

8. The apparatus of claim 7, wherein as the sample flows through the testing strip,Atty. Ref. No. 0073605-001100 if antibodies related to the one or more pathogens of interest are present in the sample, the multilayered plasmonic nanoparticles functionalized with the antibody capture probes capture the antibodies as the sample flows through the conjugate region; if antibodies related to the first pathogen are present in the sample, the first immobilized capture probes capture the antibodies related to the first pathogen as the sample flows through the first testing region; if antibodies related to the second pathogen are present in the sample, the second immobilized capture probes capture the antibodies related to the second pathogen as the sample flows through the second testing region; and the third immobilized capture probes capture the antibody capture probes functionalized to the plasmonic nanoparticles.

9. The apparatus of claim 8, wherein the multilayered plasmonic nanoparticles effectuate a color change when immobilized on the testing strip.

10. An apparatus for detecting non-treponemal (NT) antibodies and treponemal (TT) antibodies, the apparatus comprising a testing strip including, in sequence: a sample application region configured to receive a sample collected from a subject, wherein the sample is configured flow through the testing strip; a conjugate region having multilayered plasmonic nanoparticles functionalized with antibody capture probes configured to capture the NT antibodies and the treponemal TT antibodies;Atty. Ref. No. 0073605-001100 a first testing region configured to detect the presence of NT antibodies, wherein the first testing region has NT antigens bound to the testing strip and configured to capture NT antibodies; a second testing region configured to detect the presence of TT antibodies, wherein the second testing region has TT antigens bound to the testing strip and configured to capture TT antibodies; and a control region having immobilized capture probes bound to the testing strip, wherein the immobilized capture probes are configured to capture the antibody capture probes.

11. The apparatus of claim 10, wherein the multilayered plasmonic nanoparticles comprise a central metal core, an inner metal shell surrounding the central metal core, and an outer metal shell surrounding the inner metal shell.

12. The apparatus of claim 11, wherein the central metal core is composed of a first metal, the inner metal shell is composed of a second metal, and the outer metal shell is composed of the first metal.

13. The apparatus of claim 12, wherein the first metal and the second metal are different metals selected from the group consisting of gold, silver, copper, nickel, palladium, platinum, cobalt, rhodium, and iridium.

14. The apparatus of claim 12, wherein the first metal and the second metal are different metals selected from gold and silver.Atty. Ref. No. 0073605-00110015. The apparatus of claim 12, wherein the first metal is gold and the second metal is silver.

16. The apparatus of claim 10, wherein as the sample flows through the conjugate region, the multilayered plasmonic nanoparticles functionalized with the antibody capture probes migrate with the sample as it flows through the testing strip.

17. The apparatus of claim 16, wherein as the sample flows through the testing strip, if NT antibodies and / or TT antibodies are present in the sample, the multilayered plasmonic nanoparticles functionalized with the antibody capture probes capture the NT antibodies and / or TT antibodies as the sample flows through the conjugate region; if NT antibodies are present in the sample, the NT antigens capture the NT antibodies as the sample flows through the first testing region; if TT antibodies are present in the sample, the TT antigens capture the TT antibodies as the sample flows through the second testing region; and the immobilized capture probes capture the antibody capture probes functionalized to the plasmonic nanoparticles.

18. The apparatus of claim 17, wherein the multilayered plasmonic nanoparticles effectuate a color change when immobilized on the testing strip.

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