DESC:FIELD OF INVENTION
[0001] The present disclosure relates generally to the field of in-vitro methodologies and systems incidental to investigative biochemistry. In particular, the present disclosure pertains to a lateral flow assay (LFA) device for detecting biomarkers for intrapartum and neonatal complications and method thereof.

BACKGROUND
[0002] Rupture of membranes (ROM) is a condition that occurs when the amniotic sac, which surrounds and protects the foetus in the uterus, ruptures (breaks) before the onset of labor. This adverse event is considered preterm premature rupture of membranes (P-PROM) if it happens before 37 weeks of gestation.
[0003] Rupturing of the amniotic sac causes leakage of amniotic fluid from the uterus and leads furthermore to various complications for both the expecting mother and the yet unborn baby. For the expecting mother, complications include an increased risk of infection (such as chorioamnionitis), placental abruption, and umbilical cord prolapse. And for the yet-unborn baby, complications include preterm birth, neonatal infection, neonatal sepsis (NeoS), respiratory distress syndrome, and fetal growth restriction.
[0004] The frequency of intra-amniotic infection in patients with pre-term premature rupture of membranes (P-PROM) is 20-40%. The risk of chorioamnionitis with PROM has been reported to be less than 10% and to increase to up to 40% for latency periods exceeding 24 hours. In contrast, when amniocentesis is performed at the time of the onset of labor, the prevalence of intra-amniotic infection as high as 75%. PROM has been recognized as a leading cause of preterm deliveries and early onset of neonatal sepsis (EONeoS) .
[0005] The global prevalence of PROM varies depending on factors such as geographical region, socioeconomic status, access to healthcare, and prevalence of risk factors such as infections and preterm birth. A background survey reveals the prevalence of PROM to be 13.7% in Ethiopia and 7.5% in Uganda. India has reported an average PROM incidence rate of 9.8%, with 20 to 30 years old expectant mothers being most susceptible, where genitourinary infections are among the most potent predisposing factors for PROM. The prevalence of PROM is more in developing countries, where access to healthcare services may be limited and socioeconomic disparities are more pronounced. At the other end, due to existence of robust healthcare systems and access to prenatal care, the prevalence of PROM is much lower in developed countries, however still remains a significant concern, particularly in populations with specific risk factors or high rates of preterm birth. Hence, there is an acute worldwide need for some effective means to diagnose, thus pre-empt and therefore mitigate the risks due to PROM and its associated complications.
[0006] Conventionally, the diagnosis of rupture of membranes is usually made based on clinical examination and the presence of characteristic signs and symptoms, such as fluid leakage from the vagina and confirmation of amniotic fluid using pH testing or microscopic examination. However, early diagnosis ofPROM is difficult due to slow fluid leakage or without the classic “gush of fluid” or any bleeding that can complicate the early preterm delivery. In cases where the diagnosis is uncertain, additional tests may be performed, such as ultrasound or Ferning test. However, these methodologies are more or less vague and depend squarely on the presence and experience level of skilled medical practitioners, hence remain subjective besides being largely out of reach of the most vulnerable populations especially in developing countries. Therefore, it would be very advantageous to have some easy-to-implement way which is accurate as well as affordable and field-deployable, for early detection of PROM.
[0007] Actim PROM, Amisure ROM test and AMINOQUICK are notable among commercially available kits for screening or monitoring of the indications subject hereof. However, these tests are intended only for the screening of rupture of membrane in pregnant women who present with signs. Moreover, the presence of blood on the vaginal swab can interfere with the accuracy of these tests, leading to false or inconclusive results. Additionally, the above diagnostic kits are very costly and are only recommended to be used upon appearance of symptoms in the patients.
[0008] Therefore, there is a need for easy-to-implement device or assay, which is accurate as well as affordable and field-deployable, for early detection of intrapartum and neonatal complications including P-PROM, chorioamnionitis or intra-amniotic inflammation, and neonatal sepsis.

OBJECTS OF THE PRESENT DISCLOSURE
[0009] Some of the objects of the present disclosure, which at least one embodiment herein satisfies are as listed herein below.
[0010] An object of the present invention is to provide a field-deployable, and rapid in-vitro assay for the qualitative screening of biomarkers associated with intrapartum and neonatal complications, including P-PROM, chorioamnionitis or intra-amniotic inflammation, and neonatal sepsis.
[0011] Another object of the above invention is to develop a lateral flow assay (LFA)-based device for differential interpretation of individual or overlapping biomarkers corresponding to distinct clinical conditions affecting maternal and neonatal health.
[0012] Another object of the above invention is to provide a method for detecting pre-term premature rupture of membranes (P-PROM) in pregnancy, Chorioamnionitis, and neonatal sepsis (NeoS) from the biological test sample.

SUMMARY
[0013] This summary is provided to introduce a selection of concepts in a simplified form that is further described below in the detailed description section. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.
[0014] Aspects of the present disclosure relates to in-vitro methodologies and systems incidental to investigative biochemistry, specifically to in-vitro assay and a lateral flow assay (LFA)-based diagnostic device for the simultaneous and early detection of biomarkers associated with intrapartum and neonatal complications, including but not limited to pre-term premature rupture of membranes (P-PROM), neonatal sepsis (NeoS), and chorioamnionitis or intra-amniotic inflammation. The device provides qualitative and field-deployable platform for rapid diagnosis at the point of care.
[0015] Accordingly, in an aspect, the present disclosure provides a lateral flow assay (LFA) device for detecting biomarkers for intrapartum and neonatal complications from a test sample, including a lateral flow immunoassay assembly (LFIA) configured to detect the biomarkers for pre-term premature rupture of membranes (P-PROM) in pregnancy, Chorioamnionitis, and neonatal sepsis (NeoS) from the test sample. The lateral flow immunoassay assembly includes a sample pad positioned at a first end of the lateral flow immunoassay assembly configured to receive the test sample and provide controlled movement of the test sample through the lateral flow immunoassay assembly; a conjugate release matrix positioned adjacent and downstream to the sample pad, and configured to bind to the biomarkers in the test sample, wherein the conjugate release matrix is pre-impregnated with bio-functionalized gold nanoparticles (AuNPs) and latex nanoparitcles conjugated with capture antibodies specific to the biomarkers; a chromatography membrane positioned adjacent and downstream to the conjugate release matrix, and configured to facilitate capillary driven flow of the test sample, the chromatography membrane includes one or more test lines impregnated with detection antibodies specific to the biomarkers and a control line impregnated with a control antibody specific to the biomarkers; an absorbent pad positioned above the chromatography membrane and at a second end of the lateral flow immunoassay assembly, and configured to capture excess and processed test sample. The lateral flow assay (LFA) device also includes a housing unit having a top portion and a base portion, and configured to encase the lateral flow immunoassay assembly. The housing unit includes a sample reservoir embedded within the top portion of the housing unit and aligned with the sample pad, and configured to allow loading the test sample; and an observation window embedded within the top portion of the housing unit and aligned with the one or more test lines and the control line on the chromatography membrane, and configured to allow visualization of the test result.
[0016] In another aspect, the present disclosure provides a method for detecting intrapartum and neonatal complications from a test sample in an in-vitro environment, including steps of:
a) sampling 10 to 50 µL of biological fluids to serve as a test sample;
b) loading the test sample into the lateral flow assay (LFA) device (100) of claim 1 to initiate the lateral flow immunoassay;
c) allowing the test sample to flow through the LFA device (100) and visualizing the development of one or more test lines (112, 114, and 116) and a control line (118); and
d) determining the presence or absence of biomarkers related to intrapartum and neonatal complications in the test sample, to detect P-PROM, Chorioamnionitis, and NeoS by comparing the developed test lines with a reference table.
[0017] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments.

BRIEF DESCRIPTION OF DRAWINGS
[0018] The accompanying drawings are included to provide a further understanding of the present disclosure and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure.
[0019] FIG. 1 illustrates an exemplary lateral flow assay (LFA) device, in accordance with an embodiment of the present disclosure.
[0020] FIG. 2 illustrates an exemplary lateral flow immunoassay assembly (LFIA) a) top view and b) isometric view, in accordance with an embodiment of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION
[0021] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present invention.
[0022] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
[0023] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
[0024] In some embodiments, numbers have been used for quantifying weight percentages, ratios, and so forth, to describe and claim certain embodiments of the invention and are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
[0025] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
[0026] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
[0027] Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”
[0028] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
[0029] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[0030] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified.
[0031] The description that follows, and the embodiments described therein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present invention. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.
[0032] It should also be appreciated that the present invention can be implemented in numerous ways, including as a system, a method or a device. In this specification, these implementations, or any other form that the invention may take, may be referred to as processes. In general, the order of the steps of the disclosed processes may be altered within the scope of the invention.
[0033] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
[0034] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements a, b, and c, and a second embodiment comprises elements b and d, then the inventive subject matter is also considered to include other remaining combinations of a, b, c, or d, even if not explicitly disclosed.
[0035] The terms “lateral flow assay device” or “lateral flow immunoassay device” or “LFA” or “device” are used herein interchangeably with same meaning throughout the specification.
[0036] The terms “test sample” or “biological test sample” or “biological sample” or “sample” are used herein interchangeably with same meaning throughout the specification.
[0037] The terms “biomarkers” or “analyte” or “analyte of interest” are used herein interchangeably with same meaning throughout the specification.
[0038] Aspects of the present disclosure relates to in-vitro methodologies and systems incidental to investigative biochemistry, specifically to in-vitro assay and a lateral flow assay (LFA)-based diagnostic device for the detection of biomarkers associated with intrapartum and neonatal complications. The device is visual sandwich-type lateral flow immunoassay device (VSLFID) and provides qualitative and field-deployable platform for rapid diagnosis at the point of care. The device provides simultaneous and early detection of multiple clinical conditions such as PROM, chorioamnionitis/intra-amniotic inflammation, and neonatal sepsis risks within a single test platform.
[0039] Referring to FIG. 1, the present disclosure provides a lateral flow assay (LFA) device 100 for detecting biomarkers for intrapartum and neonatal complications from a test sample. The disclosure more specifically relates to a non-invasive, qualitative assay, and a field-deployable LFA device, for in-vitro detection of biomarkers associated with pre-term premature rupture of membranes (P-PROM), chorioamnionitis or intra-amniotic inflammation, and neonatal sepsis.
[0040] Referring to FIG. 2a and 2b, the LFA device 100 includes a lateral flow immunoassay assembly (LFIA) 102 configured to detect the biomarkers for pre-term premature rupture of membranes (P-PROM) in pregnancy, Chorioamnionitis, and neonatal sepsis (NeoS) from the test sample. In one embodiment, the LFIA 102 or LFIA strip has a length ranging from 50 to 70 mm, preferably 65 mm, and more preferably 58 mm. In certain embodiments, the LFIA strip 102 has a width ranging from 2 to 6 mm, preferably 4 mm.
[0041] The lateral flow immunoassay assembly (LFIA) 102 includes a sample pad 104 positioned at a first end of the lateral flow immunoassay assembly 102 configured to receive the test sample and provide controlled movement of the test sample through the lateral flow immunoassay assembly 102. In one embodiment, the sample pad 104 is of glass fiber and includes FR-1type of blood separation membrane with a thickness of 570 µm. The FR-1 type of blood separation membrane eliminates the need for pre-processing and ensuring plasma-only flow for improved assay reliability.
[0042] The LFIA 102 also include a conjugate release matrix 106 positioned adjacent and downstream to the sample pad 104, and configured to bind to the biomarkers in the test sample. The conjugate release matrix 106 is pre-impregnated with bio-functionalized gold nanoparticles (AuNPs) and latex nanoparticles conjugated with capture antibodies specific to the biomarkers. In one embodiment, the latex is natural latex or synthetic latex.
[0043] In one embodiment, the conjugate release matrix 106 is made of polyester with high holding and absorption capacity. The conjugate release matrix 106 has a thickness ranging from 325 to 485 µm. The conjugate release matrix 106 is pre-impregnated via passive adsorption and covalent latex conjugation technique.
[0044] As used herein the term “passive adsorption technique” refers to non-covalent immobilization of biomolecules onto a surface through physical interactions such as hydrophobic interactions, electrostatic (ionic) interactions, Van der Waals forces, and hydrogen bonding. As used herein the term “covalent latex conjugation technique” refers to a technique used to attach biomolecules, such as proteins, antibodies, or nucleic acids, to nanoparticles through stable covalent bonds.
[0045] In one embodiment, the AuNPs include a size ranging from 22 to 34 nm. In one embodiment, the latex nanoparticles include a size ranging from 300 to 400 nm.
[0046] In one embodiment, the bio-functionalized gold nanoparticles (AuNPs) and the latex nanoparticles are present in a ratio of 1:5.
[0047] The LFIA 102 includes a chromatography membrane 108 positioned adjacent and downstream to the conjugate release matrix 106, and configured to facilitate capillary driven flow of the test sample. In one embodiment, the chromatography membrane 108 is made of transparent polyester of 100µm thickness backed by nitrocellulose membrane of 110 µm thickness with 5-8 µm pore size. The chromatography membrane 108 includes one or more test lines 112, 114, and 116 impregnated with detection antibodies specific to the biomarkers and a control line 118 impregnated with a control antibody specific to the biomarkers. In an embodiment, the chromatographic membrane 108 has a length ranging from 20 to 30 mm, preferably 25 mm.
[0048] The LFIA 102 includes an absorbent pad 110 positioned above the chromatography membrane 108 and at a second end of the lateral flow immunoassay assembly 102, and configured to capture excess and processed test sample and running buffer. In an embodiment, the absorbent pad 110 is made of cellulose fiber with thickness ranging from 1100 to 1200 µm. In one embodiment, the absorbent pad 110 has a thickness of 1150 µm.
[0049] The LFA device 100 further includes a housing unit 120 configured to encase the lateral flow immunoassay assembly 102. In an embodiment, the housing unit 120 is a compact and portable cuboid hollow housing made of plastic or polyvinyl chloride.The housing unit 120 includes a sample reservoir 122 embedded within a top surface 124 housing unit 120 and aligned with the sample pad 104, and configured to allow loading the test sample. The sample reservoir 122 has a diameter ranging from 2 to 6 mm. In one embodiment, the sample reservoir 122 has a diameter of 4 mm.
[0050] The housing unit 120 includes an observation window or viewing port 126 embedded within the top surface 124 of the housing unit 120 and aligned with the one or more test lines 112, 114, and 116 and the control line 118 on the chromatography membrane 108. The observation window 126 configured to allow visualization of the test result. For example, the observation window 126 may be disposed over a portion of a LFIA 102 within the housing 120 that gives a visible “positive” or “negative” indication (e.g., by color change, line formation, graphics, and so forth). In one embodiment, the observation window 126 has a length ranging from 15 to 20 mm, preferably 16.8 mm.
[0051] In certain embodiments, size and shape of the LFA device 100 may generally vary. For instant, the LFA device 100 may have a length ranging from 60 to 80 mm, preferably 70 mm. In one embodiment, the LFA device 100 may have a width ranging from 10 to 30 mm, preferably 20 mm.
[0052] In various embodiments, the biomarkers includes but not limited to insulin-like growth factor-binding protein-1, placental protein-14, placental alpha microglobulin-1, matrix metalloproteinase-8, serum amyloid A, proadrenomedullin, visfatin, resistin, presepsin, and procalcitonin, hepcidin, progranulin, pentraxin-3, Interleukin-6, and Interleukin-8. In one embodiment,
[0053] In certain embodiments, the test sample is a biological fluid selected from a group consisting of urine, blood, cervicovaginal secretions, vaginal secretions, or vaginal washing fluids. In one embodiment, LFA device requires the test sample in a volume ranging from 10 to 50 µL for the detection of intrapartum infections. In one embodiment, LFA device requires 50 µL volume of the test sample, preferably 35 µL volume of the test sample, more preferably, 20 µL volume of the test sample, or most preferably 10 µL volume of the test sample.
[0054] In certain embodiments, the one or more test lines 112, 114, and 116 impregnated with detection antibodies specific to biomarkers includes a first test line 112 located adjacent to the conjugate release matrix (106), and configured to detect biomarkers of P-PROM. The one or more test lines 112, 114, and 116 also includes a second test line 114 located downstream from the first test line 112, and configured to detect biomarkers of Chorioamnionitis. The one or more test lines 112, 114, and 116 also includes a third test line 116 located downstream from the second test line 114, and configured to detect biomarkers of NeoS.
[0055] In various embodiments, the first test line 112 is impregnated with the detection antibody including anti-insulin like growth factor-binding protein-1 (IGFB-1), anti-placental protein-14, and anti-placental alpha macroglobulin-1 in a concentration of 0.5 to1 mg/mL.
[0056] In certain embodiments, the second test line 114 is impregnated with the detection antibody including anti-interleukin-6, anti-interleukin-8, and anti-matrix metalloproteinase-8 in a concentration of 0.2 to 0.8 mg/mL.
[0057] In certain embodiments, the third test line 116 is impregnated with the detection antibody including anti-procalcitonin, anti-serum amyloid A, and anti-interleukin-6 in a concentration of 0.3to 0.5 mg/mL.
[0058] In various embodiments, the control line 118 is impregnated with control antibody including anti-mouse IgG in a concentration of 0.5 to 1.5 mg/mL. In one embodiment, the control antibody is impregnated in a concentration of 1 mg/mL.
[0059] In various embodiments, the capture antibodies comprises a combination of _monoclonal antibodies against interleukin-6 and interleukin-8.
[0060] In an exemplary embodiment, the one or more test lines 112, 114, and 116 and the control line 118 are otherwise invisible to the naked eye, and can be seen only when the LFA device 100 is used successfully, with a positive determination of biomarkers being present in the test sample. However, the corresponding markings on the observation window 126 are always visible, and serve to indicate the positions on the nitrocellulose membrane, where the lateral flow immunoassay outcome, i.e. lines, should develop / appear when the LFA device 100 is used successfully, with a positive determination of biomarkers being present in the test sample.
[0061] In one embodiment, the LFA device 100 incorporates a reference table (table 1) integrated into the device’s design to facilitate qualitative analysis of biomarkers directly at the point of care. The table 1, either embedded on the device’s 100 surface or provided as a detachable card, contains a series of color or intensity reference standards corresponding to different conditions such as P-PROM, Chorioamnionitis, and NeoS. After loading the test sample, the user observes the development of test lines 112, 114, and 116 and compares their intensities with the calibrated standards on the reference table 1. This comparison enables the user to determine the presence of biomarkers in the sample without relying on external laboratory equipment or sophisticated interpretation methods.
[0062] In another aspect, the present disclosure provides a method for detecting intrapartum and neonatal complications from a test sample in an in-vitro environment, including steps of:
a) sampling 10 to 50 µL of biological fluids to serve as a test sample;
b) loading the test sample into the lateral flow assay (LFA) device (100) of claim 1 to initiate the lateral flow immunoassay;
c) allowing the test sample to flow through the LFA device (100) and visualizing the development of one or more test lines (112, 114, and 116) and a control line (118); and
d) determining the presence or absence of biomarkers related to intrapartum and neonatal complications in the test sample, to detect P-PROM, Chorioamnionitis, and NeoS by comparing the developed test lines with a reference table.
Table 1 is the reference table for interpreting presence or absence of biomarkers related to intrapartum and neonatal complications.
Test line 1 Test line 2 Test line 3 Control line Interpretation
N N N N Inconclusive, discard VSLFID
Y N N Y PROM
Y Y N Y PROM and Chorioamnionitis or intra-amniotic inflammation
Y Y Y Y PROM, inflammation and NeoS
Where, “Y” = Line visualized; “N” = No line visualized.
[0063] In an exemplary embodiment, implementation of the LFA device provided by the present disclosure can be effectively carried out at ambient temperatures ranging from 8°C to 37°C under normal atmospheric pressure within 15 minutes, thereby eliminating the need for specialized laboratory or clinical settings.
[0064] An able qualitative, point-of-care solution for in-vitro detection of biomarkers associated with P-PROM, chorioamnionitis or intra- amniotic inflammation and NeoS, is thus provided for effective and accurate differential diagnosis of membrane rupture, including risk of developing neonatal sepsis, and thus allow planning of gestational age-specific interventions to optimize intrapartum and neonatal complications outcomes and minimize serious complications for the to-be-born baby and expecting mother.
[0065] While the foregoing description discloses various embodiments of the invention, other and further embodiments of the invention may be devised without departing from the basic scope of the disclosure. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.
EXAMPLES
[0066] The present invention is further explained in the form of the following examples. However, it is to be understood that the foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention.
Example 1: Synthesis and functionalization of gold and latex nanoparticles
[0067] Gold nanoparticles and colored latex beads are commonly used as visible signal labels. For the synthesis of GNPs, 50 mL of milli-Q water was heated to a boiling point at 200°C, followed by the addition of 600 µL of a 1% auric chloride solution. The solution was heated for a duration of three minutes, following which 1 mL of a 1.2% tri-sodium citrate solution was introduced into the flask under constant stirring. The solution was subjected to continue heating until its appearance transformed from transparent to red color. After reaching the reaction’s end, the solution was left to cool to room temperature, and the flask was covered with aluminium foil to shield it from light. The GNPs were subjected to an aging process for a minimum duration of 24 hours prior to subsequent utilization. The synthesized GNPs have a size range of 20-35 nm, while the latex particles measure 200-300 nm, displaying a tunable SPR (Surface Plasmon Resonance) peak between 520 and 600 nm. The synthesized nanoparticles are characterized for various parameters listed in table 2.
Table 2: characterization parameters of gold and latex nanoparticles.
Parameters GNPs Latex
Colour Wine red Blue, green
pH 5.0 to 6.0 6.5 to 7
Peak wavelength 516 to 522 nm 580-600
Size of nanoparticles 22 to 34 nm 300-400 nm
Zeta potential -30 mV to -45 mV -26 mV to -41mV

Example 2: Gold latex Conjugate preparation
[0068] Briefly, MAbs (monoclonal antibodies- Interleukein-6, IGFBP-1) were covalently linked to Au-latex nanoparticles. Blue and green latex beads (300 nm) were activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), then coupled to each MAb at a surface concentration of 0.8 mg/m². Antibodies stocks were prepared at 10-30 µg/mL in phosphate buffer saline, are then conjugated to the activated nanoparticles for 2 hours at 4°C. After conjugation, unreacted sites are blocked with ethanolamine, and the conjugates are purified through centrifugation. GNPs were conjugated with the (Interleukein-6 and 8 ) following the same protocol only difference in antibody (conc. 250 µg/ml) 32 µL were added to 500 µL of GNPs (1 O. D.). After 20 mins, 1% BSA is added to the solution, and thorough mixing. The tube is subsequently incubated upright at room temperature. The GNP- antibody solution is centrifuged at 13000 rpm for 40 mins, at 4oC (in a precooled centrifuge) and the supernatant is discarded. The pellet O. D. 520nm is adjusted to 1.8-1.9 by stepwise addition of conjugate storage buffer (BSA- 1%, sucrose and trehalose- 5%, tween 20- 0.025%). Equal volume of colored latex and GNPs conjugates (1:2, and 1:5) used for conjugate release matrix coating. The coated conjugate release matrix are then dried at 37oC for 1 hour and overnight in the Biosafety Cabinet.
[0069] Lateral flow assay (LFA), intended for implementation using the LFA device 100 described in the present invention, is arranged for detection of intrapartum and neonatal complications, if present, of the AoI (s) in test samples by involving the following selection of commercially-available antibodies-
i) Capture antibody – combination of monoclonal antibodies against IGFBP-1, interleukin-6 and 8
ii) Detection antibody – anti-IGFB-1, anti-placental protein-14, and anti-placental alpha macroglobulin-1 (the first test line 112); anti-interleukin-6, anti-interleukin-8, and anti-metalloproteinase-8 (the second test line 114); and anti-procalcitonin, anti-serum amyloid A, and anti-interleukin-6 (the third test line 116)
iii) Control antibody - Anti-Mouse IgG.
Example 3: Implementation of the LFA device (lateral flow assay device)
[0070] The sample is applied to the sample pad 104. After application, the sample moves through the LFA device 100 via controlling capillary flow rate. The sample first passes through the conjugate pad 106, where the sample encounters labeled antibodies or other reagents that are specific to the target biomarker (the substance being tested for). These reagents are usually attached to colored particles, nanoparticles or enzymes. If the target biomarker is present in the sample, it binds to the labeled antibodies in the conjugate pad 106. This complex (analyte-antibody complex) then moves further along with the sample as it continues to migrate. As the sample moves through the nitrocellulose membrane, the analyte-antibody complex binds to specific capture antibodies that are immobilized at the test line region (112, 114 and 116). This binding results in the accumulation of the colored particles, nanoparticles or enzymes at the test line, making a visible colored line if the target biomarker is present. Simultaneously, the sample continues to migrate to the control line 118 region. Here, another set of antibodies or reagents captures any excess labeled particles or enzymes, forming a second visible line. This control line 118 confirms that the test has been performed correctly and that the reagents are functioning properly. After a set amount of time, the presence or absence of lines at the test and control regions is assessed-using the reference table (Table 1).
Table 3 enlists the working parameters of the lateral flow assay device 100 discloses in the present disclosure.
Sample type: Biological sample / Biological specimen
Nanoparticles used: Gold nanoparticles and latex particles
Sample volume: 10-50µL
Time for visualization of results: 15 minutes
Sensitivity: 94.12%
Specificity: 92.45%

Example 4: Experimental validation of the LFA device 100 (lateral flow assay device)
[0071] The LFA device 100 has been reduced to practice and experimentally validated. Table 3 shows detection and interpretation of intrapartum and neonatal complications including P-PROM, Chorioamnionitis, and NeoS.
Table 3: Screening and detection of intrapartum and neonatal complications.
Sample type ELISA validation (ng/mL) Device validation Interpretation
PROM Chorioamnionitis NeoS Test lines detection
Standard proteins 500 200 300 T1,T2, faint T3 Analytical performance
Positive P-PROM pregnancy
Urine 100-140 30-41 10-20 No test lines
P-PROM, infection/ inflammation and mild risk of NeoS
Vaginal swab >100 130-180 23 T1, faint T2 & T3
Amniotic fluid >11000 >300 59 Dark T1,T2, faint T3
Serum >80 >250 >100 T1,T2, T3
P-PROM negative control pregnancy
Urine 10 30 10 No test lines

Mild inflammation
Vaginal swab 150±20 43 16 Very Faint T2
Amniotic fluid < 5000 < 200 40 faint T2
Serum 15±10 50-70 7 No test lines
Non- pregnant control
Urine <5 12±4 3

No test lines P-PROM, infection and sepsis are not detected
Vaginal swab 50± 18±2 16
Serum 5±2 10±4 2-5

[0072] In conclusion, the lateral flow assay device (LFD) 100 and associated method for detecting intrapartum and neonatal complications from complex biological samples is designed to operate effectively in point-of-care settings. The device 100 allows for rapid, accurate assessments within approximately 15 minutes, helping to preserve biomarker or analyte concentrations and enabling timely decision-making. The device 100 is crafted for non-dependency on costly analytical instruments or specialized laboratory setups.
[0073] The LFA device 100 requires only a low volume of the test sample between 10 to 50 µL, making it highly suitable for testing limited sample amounts. The LFA device 100 operates efficiently at ambient room temperature, ranging from 8°C to 37°C, under standard atmospheric pressure, ensuring stable performance without the need for specialized environmental controls. The device 100 utilizes visual test lines 112, 114, and 116 that can be readily interpreted by comparing with a reference table 1 embedded on the device 100, eliminating the necessity for complex ancillary equipment or electronic readers.

ADVANTAGES OF THE PRESENT DISCLOSURE
[0074] The LFA device enables simultaneous and early detection of multiple clinical conditions such as P-PROM, chorioamnionitis/intra-amniotic inflammation, and neonatal sepsis within a single test platform, enhancing diagnostic efficiency and reducing diagnostic delays.
[0075] The LFA device allows for non-invasive sampling and can be deployed at point-of-care or resource-limited settings, without the need for complex laboratory infrastructure, refrigeration, or specialized training.
[0076] The LFA device provides quick assessments (within 15 minutes), reducing the risk of biomarker degradation and enabling faster decision-making in clinical and research applications.
[0077] The LFA device can function effectively at ambient temperatures (8°C to 37°C) and standard atmospheric pressure, eliminating the need for controlled environmental conditions.
,CLAIMS:1. A lateral flow assay (LFA) device (100) for detecting biomarkers for intrapartum and neonatal complications from a test sample, comprising:
a lateral flow immunoassay assembly (LFIA) (102) configured to detect the biomarkers for pre-term premature rupture of membranes (P-PROM) in pregnancy, Chorioamnionitis, and neonatal sepsis (NeoS) from the test sample, the lateral flow immunoassay assembly (102) includes:
a sample pad (104) positioned at a first end of the lateral flow immunoassay assembly (102) configured to receive the test sample and provide controlled movement of the test sample through the lateral flow immunoassay assembly (102),
a conjugate release matrix (106) positioned adjacent and downstream to the sample pad (104), and configured to bind to the biomarkers in the test sample,
wherein the conjugate release matrix (106) is pre-impregnated with bio-functionalized gold nanoparticles (AuNPs) and latex nanoparticles conjugated with capture antibodies specific to the biomarkers,
a chromatography membrane (108) positioned adjacent and downstream to the conjugate release matrix (106), and configured to facilitate capillary driven flow of the test sample, the chromatography membrane (108) includes one or more test lines (112, 114, and 116) impregnated with detection antibodies specific to the biomarkers and a control line (118) impregnated with a control antibody specific to the biomarkers,
an absorbent pad (110) positioned above the chromatography membrane (108) and at a second end of the lateral flow immunoassay assembly(102), and configured to capture excess and processed test sample; and
a housing unit (120) configured to encase the lateral flow immunoassay assembly (102), the housing unit (120) includes:
a sample reservoir (122) embedded within a top surface (124) housing unit (120) and aligned with the sample pad (104), and configured to allow loading the test sample,
an observation window (126) embedded within the top surface (124) of the housing unit (120) and aligned with the one or more test lines (112, 114, and 116) and the control line (118) on the chromatography membrane (108), and configured to allow visualization of the test result.

2. The LFA device (100) as claimed in claim 1, wherein the biomarkers are selected from a group consisting of insulin-like growth factor-binding protein-1, placental protein-14, placental alpha microglobulin-1, matrix metalloproteinase-8, serum amyloid A, proadrenomedullin, visfatin, resistin, presepsin, and procalcitonin, hepcidin, progranulin, pentraxin-3, Interleukin-6, and Interleukin-8.

3. The LFA device (100) as claimed in claim 1, wherein the test sample is a biological fluid selected from a group consisting of urine, blood, cervicovaginal secretions, vaginal secretions, or vaginal washing fluids.

4. The LFA device (100) as claimed in claim 1, wherein the one or more test lines (112, 114, and 116) impregnated with detection antibodies specific to biomarkers comprises:
a first test line (112) located adjacent to the conjugate release matrix (106), and configured to detect biomarkers of P-PROM;
a second test line (114) located downstream from the first test line (112), and configured to detect biomarkers of Chorioamnionitis; and
a third test line (116) located downstream from the second test line (114), and configured to detect biomarkers of NeoS.
5. The LFA device (100) as claimed in claim 4, wherein the first test line (112) is impregnated with the detection antibody including anti-insulin like growth factor-binding protein-1 (IGFB-1), anti-placental protein-14, and anti-placental alpha macroglobulin-1 in a concentration of 0.5 to 1 mg/mL.

6. The LFA device (100) as claimed in claim 4, wherein the second test line (114) is impregnated with the detection antibody including anti-interleukin-6, anti-interleukin-8, and anti-metalloproteinase-8 in a concentration of 0.2 to 0.8 mg/mL.

7. The LFA device (100) as claimed in claim 4, wherein the third test line (116) is impregnated with the detection antibody including anti-procalcitonin, anti-serum amyloid A, and anti-interleukin-6 in a concentration of 0.3 to 0.5 mg/mL.

8. The LFA device (100) as claimed in claim 1, wherein the control line (118) is impregnated with control antibody including anti-mouse IgG in a concentration of 0.5 to 1.5 mg/mL.

9. The LFA device (100) as claimed in claim 1, wherein the capture antibodies comprises a combination of monoclonal antibodies against interleukin-6 and interleukin-8 , wherein the bio-functionalized gold nanoparticles (AuNPs) and the latex nanoparticles are present in a ratio of 1:5.

10. A method for detecting intrapartum and neonatal complications from a test sample in an in-vitro environment, comprising steps of:
a) sampling 10 to 50 µL of biological fluids to serve as a test sample;
b) loading the test sample into the lateral flow assay (LFA) device (100) of claim 1 to initiate the lateral flow immunoassay;
c) allowing the test sample to flow through the LFA device (100) and visualizing the development of one or more test lines (112, 114, and 116) and a control line (118); and
d) determining the presence or absence of biomarkers related to intrapartum and neonatal complications in the test sample, to detect P-PROM, Chorioamnionitis, and NeoS by comparing the developed test lines with a reference table.