Description:FIELD OF THE INVENTION
The present invention broadly relates to veterinary diagnostic device. Particularly, the present invention relates to a point-of-care multiplexed lateral-flow immunochromatographic device for early detection of pregnancy in livestock species using urine samples (collected from 21st day post-insemination). A method of its manufacturing and use is disclosed. The present invention offers on-strip simultaneous determination of PdG/E1S/pH level in colourimetric visualization format within 3-5 minutes in non-invasive instrument-free manner. This multiplexed lateral-flow testing format enhances sensitivity and specificity beyond conventional single-analyte pregnancy tests and is tailored for on-farm use without laboratory infrastructure.

BACKGROUND OF THE INVENTION
Livestock reproductive management critically depends on timely, accurate pregnancy diagnosis to optimize herd productivity, reduce economic losses associated with late or missed diagnoses, and guide fiscal and feeding strategies. Conventional bovine assays fall broadly into two categories: laboratory‐based hormone immunoassays (e.g., estrone or progesterone ELISAs) requiring centralized facilities, trained technicians, and cold‐chain logistics; and field tests that often rely on single‐parameter colorimetric reactions or agglutination methods with limited sensitivity, specificity, and user‐interpretation reliability. Neither approach simultaneously addresses the need for multiplexed biomarker detection, rapid results, and instrument‐free operation under diverse on‐farm conditions. Conventional methods, including progesterone and PdG assays in blood, milk, or feces, require laboratory settings or specialized training and equipment.

A reference may be made to US20120225438A1 that discloses a test kit for detecting animal pregnancy from blood/plasma/serum samples, which involves invasive and complex procedure.

Estrone, a principal estrogen metabolite excreted in bovine urine, provides a reliable pregnancy indicator from approximately Day 28 post–artificial insemination. However, immunoassay‐only lateral‐flow devices historically suffer from occasional false positives due to cross‐reactive steroids and variable urinary matrix effects. Single-marker field tests often face sensitivity and specificity limitations due to hormonal fluctuations and cross-reactivity. Complementary chemical detection of estrone sulphate via barium chloride precipitation adds orthogonal confirmation but has been restricted to solution‐phase assays, precluding integration into conventional strip formats. Furthermore, most commercial lateral flow assays include only a single test line and a control line, lacking the capacity for simultaneous multi‐analyte readout necessary to improve diagnostic confidence in decentralized settings. Therefore, there is a need of exploring alternative approaches to develop a cost-effective easy-to-use lateral flow test strip format that can bridge current diagnostic gaps, enabling rapid, reliable, and instrument-free pregnancy detection on livestock farms.

A lateral flow device (LFD) is a simple, rapid, and portable diagnostic tool that uses capillary action to detect the presence of a specific substance (analyte) in a liquid sample. The device consists of a strip with several zones, including a sample pad, a reagent pad, a detection zone with immobilized antibodies, and an absorbent pad, all made from porous nitrocellulose membranes. When a sample is applied, it moves across the strip, and if the target analyte is present, a visible signal, often a coloured line, develops in the detection zone, indicating a positive result within minutes.

The conventional or commercially available lateral flow devices (test strips) have several limitations towards material (antibody, reagent, chemical) selection, biomarker design, application/use, diagnosis results, and manufacturing/fabrication complexity, it is required to devise an improved approach, especially integrating immunoassay and chemical precipitation detection techniques, and incorporating multiple biomarkers in a single strip with enhanced sensitivity and specificity. Moreover, it is desired to develop a point-of-care multiplexed lateral-flow immunochromatographic device for early detection of pregnancy in livestock species using urine samples, and method of its manufacturing and use; which includes all the advantages of the conventional/existing techniques/methodologies and overcomes the deficiencies of such techniques/methodologies.

OBJECT OF THE INVENTION
It is an object of the present invention to detect livestock pregnancy instantly using their urine samples collected just from Day 21 post insemination without any costly and large laboratory set-up.

It is another object of the present invention to offer on-strip simultaneous determination of PdG/E1S/pH level in colourimetric visualization format within 3-5 minutes in non-invasive manner.

It is one more object of the present invention to design a stable, field-robust, and cost-effectively manufacturable PdG/E1S/pH biomarker based lateral flow testing strip that can significantly lower false positives and negatives compared to single-marker strip, enhancing confidence in on-farm decision-making.

It is a further object of the present invention is to devise an easy-to-use point-of-care multiplexed lateral-flow immunochromatographic device for early detection of pregnancy in livestock species using urine samples, and method of its manufacturing and use.

SUMMARY OF THE INVENTION
In one aspect, the present invention provides a device for determining livestock pregnancy in urine samples. The device comprises three isolated detection flow channels formed on nitrocellulose membranes affixed on a plastic support base in form of a lateral flow test strip packed in a cassette having at least one urine sample receiving region and at least one result visualization window. The first detection channel is designed to detect pregnanediol-3-glucuronide (PdG) level in the urine sample, and comprises: a scavenger band loaded with 0.75 mg/mL anti-pregnanediol-3-glucuronide antibody conjugated bovine serum albumin solution (anti-PdG-BSA conjugate) at an application rate of 0.9 μL/cm; a test line loaded with 0.5 nanomole anti pregnanediol-3-glucuronide antibody conjugated gold nanoparticle solution (anti PdG-AuNP conjugate) at an application rate of 10–15 μL per cm, and 1 mg/mL anti mouse IgG solution at an application rate of 0.8 μL/cm; and a control line loaded with 1 mg/mL anti chicken IgY solution at an application rate of 0.8 μL/cm. The second detection channel is configured to detect estrone sulfate (E1S) level in the urine sample, and comprises: a scavenger band loaded with 0.6 mg/mL anti-estrone sulfate antibody conjugated bovine serum albumin solution (anti-E1S-BSA conjugate) at an application rate of 0.8–1 μL/cm; a test line loaded with 0.2-4.6 nanomole anti-estrone sulfate antibody conjugated gold nanoparticle solution (anti-E1S-AuNP conjugate) at an application rate of 10-15 μL/cm; and a control line loaded with 1 mg/mL goat anti‑mouse IgG solution at an application rate of 0.8 μL/cm. The third detection channel is configured to detect pH level in the urine sample, and comprises: a test line loaded with 0.04% w/v bromophenol blue solution at an application rate of 3 μL/cm; and a control line loaded with 0.05 M citrate buffer with 3-5 pH at an application rate of 1 μL/cm.

Other aspects, advantages, and salient features of the present invention will become apparent to those skilled in the art from the following detailed description, which delineate the present invention in different embodiments.

BRIEF DESCRIPTION OF DRAWINGS
These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying figures.

Fig. 1 illustrates structural arrangement of various components/parts of the device for determining livestock pregnancy in urine samples, in accordance with an embodiment of the present invention.

Fig. 2 illustrates alternative structural arrangement of various components/parts of the device for determining livestock pregnancy in urine samples, in accordance with an embodiment of the present invention.

Fig. 3 illustrates various method steps/parameters as employed in manufacturing/fabrication of the device, in accordance with an embodiment of the present invention.

List of reference numerals
100 pregnanediol-3-glucuronide (PdG) detection channel (first flow channel)
200 estrone sulfate (E1S) detection zone (second flow channel)
300 pH detection zone (third flow channel)
400 plastic support base/card
500 cassette
502 urine sample receiving region/opening
504 result visualization window/opening
SP sample pad
CP conjugate pad
AP absorbent pad
S scavenger band
T test line
C control line
Z1 first detection zone
Z2 second detection zone
Z3 third detection zone

DETAILED DESCRIPTION OF THE INVENTION
Various embodiments described herein are intended only for illustrative purposes and subject to many variations. It is understood that various omissions and substitutions of equivalents are contemplated as circumstances may suggest or render expedient, but are intended to cover the application or implementation without departing from the scope of the present invention. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

The use of terms “including,” “comprising,” or “having” and variations thereof herein are meant to encompass the items listed thereafter and equivalents thereof as well as additional items. Further, the terms, “an” and “a” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced items. The term ‘application rate’ used herein refers to quantity of materials (in fluid, semi-fluid, or powder/particle format) applied/loaded/deposited per unit of area of a porous substrate/layer/membrane. The term ‘livestock’ used herein refers to animals like cows, buffalos, bovines, goats, and sheep, which are reared for diary and meat. The term ‘urine samples’ used herein refers to raw urine of livestock or mixed with some testing chemical such as BaCl2.

Hormones like pregnanediol-3-glucuronide (PdG) level and estrone sulfate (E1S) concentration, and associated pH value in urine of non-pregnant livestock usually remain in range of 2–5 ng/ml, 5–20 ng/ml, and 6.0–7.0, respectively. During pregnancy the values of these three parameters are significantly changed. For example, the PdG level increases to three to four times the concentration of non-pregnant livestock within 21 days after conception, and this level continue to increase (i.e., 10–15 ng/ml), potentially reaching a peak around 30 ng/ml about 30 days before calving, then declining in the weeks leading up to birth. Further, the E1S concentration may increase to range of 30–80 ng/ml in pregnant livestock urine. Similarly, the pH value may increase upto 8.0–8.5 in pregnant livestock urine, which is often associated with a reduction in total urinary protein concentration (<100 mg/dL), reflecting physiological changes in renal handling of proteins during pregnancy. These elevated and predictable patterns of PdG, E1S, and pH values make them reliable markers for early pregnancy detection in the livestock. The present invention aims at designing a cost-effective and easily manufacturable lateral flow test strip that can reliably detect PdG/E1S/pH level, and display indication (in form of specific colour lines/bands) in real-time for confirming whether the livestock is pregnant or non-pregnant. The urine sample is to be taken 21st day post-insemination onwards for performing pregnancy test using the proposed device. Additionally, for pH testing, the urine samples undergo pretreatment with pinch (0.5-1 mg/ml) of BaCl₂.

According to an embodiment of the present invention, as shown in Fig. 1-2, the device for determining livestock pregnancy in urine samples is depicted. The device is formed of nitrocellulose membranes in form of a lateral flow test strip. The device comprises at least one sample pad (SP) at upstream end, at least one absorbent pad (AP) at downstream end, and at least one conjugate pad (CP) positioned therebetween. The urine samples (US) flow in a specific direction i.e., from the sample pad (SP) towards the absorbent pad (AP) passing through the conjugate pad (CP). The sample pad (SP) soaks the urine samples to be tested. The conjugate pad (CP) detects/binds target molecules present in the urine as flown from the sample pad (SP). The absorbent pad (AP) soaks the residual urine as discarded from the conjugate pad (CP). The device especially comprises three dedicated and isolated detection channels such as a first flow channel (100) designed for detecting the PdG level in the urine sample (US), a second flow channel (200) designed for detecting the E1S level in the urine sample (US), and a third flow channel (300) designed for detecting the pH level in the urine sample (US). All the pads are formed of nitrocellulose membranes arranged in partly overlapping fashion and affixed on a plastic support base (400) to give a shape of the lateral flow test strip packed in a cassette (500). The cassette (500) has at least one urine sample receiving region (502) at the sample pad side and at least one result visualization window (504) at the conjugate pad side. Alternatively, the pads with the base are transparently laminated except the urine sample receiving region.

According to an embodiment of the present invention, as shown in Fig. 1, the three detection channels (100, 200, 300) are sequentially formed/patterned in three nitrocellulose membrane-based zones (Z1, Z2, Z3). These three zones (Z1, Z2, Z3) are isolated from one another using wax based hydrophobic barriers (HB), adhered on a single plastic base/card (400) in a series, and packed in a single cassette (500). Each zone has one sample pad (SP) at the upstream end, one absorbent pad (AP) at the downstream end, and one conjugate pad (CP) positioned therebetween (with opposite end overlapping). Each conjugate pad contains one detection channel. The cassette (500) has a sample receiving opening (502) at each sample pad (SP) region, and a result visualization window/opening (504) at each conjugate pad (CP) region. The urine samples are simultaneously dropped in three sample pads through three sample receiving openings (502) of three zones to see the test results at three conjugate pads through three visualization windows of three zones. Three absorbent pads absorb/soak the residual urine discarded from corresponding three conjugate pads of corresponding detection zones.

According to an embodiment of the present invention, as shown in Fig. 2, the three detection channels (100, 200, 300) are formed/patterned in parallel fashion on a single nitrocellulose conjugate pad (CP) that is positioned (with opposite end overlapping) between a nitrocellulose sample pad (SP) at the upstream end and a nitrocellulose absorbent pad (AP) at the downstream end. The detection channels on the conjugate pad are isolated from one another using wax based hydrophobic barriers (HB). Here the three pads are affixed on a single plastic strip (400), and packed in a single cassette (500). The cassette (500) has a common sample receiving opening (502) at the sample pad region, and a common result visualization window (504) at the conjugate pad region. The urine sample is dropped in the common sample receiving opening (502) at the sample pad side to see the test results in the common result visualization window (504) at the conjugate pad side. The absorbent pad absorb/soak the residual urine discarded from the conjugate pad.

According to an embodiment of the present invention, the PdG detection (first flow) channel (100) comprises a scavenger band (S), a test line (T), and a control line (C). The scavenger band (S) is loaded/impregnated with 0.75 mg/mL anti-pregnanediol-3-glucuronide antibody conjugated bovine serum albumin solution (anti-PdG-BSA conjugate) at an application rate of 0.9 μL/cm. The test line (T) is loaded with 0.5 nanomole anti pregnanediol-3-glucuronide monoclonal antibody conjugated gold nanoparticle solution (anti PdG-AuNP conjugate) at an application rate of 10–15 μL per cm, and 1 mg/mL anti mouse IgG solution at an application rate of 0.8 μL/cm. The control line (C) is loaded/impregnated with 1 mg/mL anti chicken IgY solution at an application rate of 0.8 μL/cm.

According to an embodiment of the present invention, the E1S detection (second flow) channel (200) comprises a scavenger band (S), a test line (T), and a control line (C). The scavenger band (S) is loaded/impregnated with 0.6 mg/mL anti-estrone sulfate antibody conjugated bovine serum albumin solution (anti-E1S-BSA conjugate) at an application rate of 0.8–1 μL/cm. The test line (T) is loaded/impregnated with 0.2-4.6 nanomole anti-estrone sulfate antibody conjugated gold nanoparticle solution (anti-E1S-AuNP conjugate) at an application rate of 10-15 μL/cm. The control line (C) is loaded/impregnated with 1 mg/mL goat anti‑mouse IgG solution at an application rate of 0.8 μL/cm.

According to an embodiment of the present invention, the pH detection (third flow) channel (300) comprises a test line (T) and a control line (C). The test line (T) is loaded/impregnated with 0.04% w/v bromophenol blue solution at an application rate of 3 μL/cm. The control line (C) is loaded/impregnated with 0.05 M citrate buffer with 3-5 pH at an application rate of 1 μL/cm.

According to an embodiment of the present invention, the scavenger band (S), the test line (T), and the control line (C) in each of the detection channels (100, 200, 300) are patterned in parallel in a direction transverse to a sample (US) flow direction.

According to an embodiment of the present invention, as shown in Fig. 3, the method of manufacturing a device for determining livestock pregnancy in urine samples is depicted. The method comprises steps of: defining (S1) three (first, second, third) isolated detection channels (100, 200, 300) on at least one nitrocellulose membrane through hydrophobic barriers (HB) (e.g. using paraffin wax printing technique); applying (S2) PdG level detection materials (conjugates) on the first detection channel (100) through a BioDot dispenser; loading (S3) E1S level detection materials (conjugates) on the second detection channel (200) through the BioDot dispenser; depositing (S4) pH level indicator on the third detection channel (300) through the BioDot dispenser; affixing (S5) the membrane on a plastic strip (400) to form a lateral flow test strip followed by drying in a hot air dryer at 37 °C with <30% relative humidity for 2 hours; and encasing (S6) the strip (400) in a cassette (500) having at least one urine sample receiving region (502) and at least one result visualization window (504).

According to an embodiment of the present invention, in the channel defining step (S1), the three detection channels (100, 200, 300) are patterned in three nitrocellulose membrane-based zones (Z1, Z2, Z3) which are isolated from one another through the hydrophobic barriers (HB). These three detection zones are serially connected and affixed onto a single plastic base/card (400) to form a single strip. Each zone has a sample pad (SP) at upstream end, an absorbent pad (AP) at downstream end, and a conjugate pad (CP) with one detection channel placed therebetween. The conjugate pad of the first detection zone (Z1) is designed to detect PdG level in the urine samples, and has a scavenger band (S), a test line (T), and a control line (C). The conjugate pad of the second detection zone (Z2) is designed to detect E1S level in the urine samples, and has a scavenger band (S), a test line (T), and a control line (C). The conjugate pad of the third detection zone (Z3) is designed to detect pH level in the urine samples, and has a test line (T) and a control line (C).

According to an embodiment of the present invention, in the channel defining step (S1), the three detection channels (100, 200, 300) are patterned in parallel, and isolated through the hydrophobic barriers (HB) in a single nitrocellulose conjugate pad (CP) that is positioned between a nitrocellulose sample pad (SP) at the upstream end and a nitrocellulose absorbent pad (AP) at the downstream end. The first detection channel (100) has a scavenger band (S), a test line (T), and a control line (C). The second detection channel (200) has a scavenger band (S), a test line (T), and a control line (C). The third detection channel (300) has a test line (T) and a control line (C).

According to an embodiment of the present invention, in the PdG level detection material applying step (S2), anti-PdG antibody and BSA are mixed at a weight ratio of 1:1 in sterile water or alkaline water having 7-10 pH to obtain the anti-PdG-BSA conjugate that is used to form the scavenger band (S). Anti PdG antibody and AuNP taken at a weight ratio of 1:1, 5% w/v of trehalose or sucrose, 0.5–1% v/v of bovine serum albumin (BSA), and 0.05–0.5% v/v of Tween 20 are mixed in phosphate buffered saline (PBS) or borate solvent to obtain the anti PdG-AuNP conjugate that is used to create the test line (T). Then, anti chicken IgY is mixed in sterile water or alkaline water having 7-10 pH to obtain the anti chicken IgY solution that is used to create the control line (C).

According to an embodiment of the present invention, in the E1S level detection material loading step (S3), anti-E1S antibody and BSA are mixed at a weight ratio of 1:1 in sterile water or alkaline water having 7-10 pH to obtain the anti-E1S-BSA conjugate that is used to create the scavenger band (S). Anti-E1S antibody and AuNP taken a weight ratio of 1:1 is mixed in sterile water or alkaline water having 7-10 pH to obtain the anti-E1S-AuNP conjugate that is used to create the test line (T). Then, goat anti‑mouse IgG is mixed in sterile water or alkaline water having 7-10 pH to obtain the goat anti‑mouse IgG solution that is used to create the control line (C).

According to an embodiment of the present invention, in the pH indicator depositing step (S4), bromophenol blue is mixed in 0.05 M citrate buffer with pH 3-5 the bromophenol blue solution that is used to create the test line (T). The 0.05 M citrate buffer with 3-5 pH is used to create the control line (C).

In the PdG detection channel (100), the anti-PdG-BSA conjugate of the scavenger band (S) selectively captures the conjugated PdG-BSA while the PdG concentration is ≤ 5 ng/mL (non-pregnancy stage), thereby preventing downstream binding and eliminating false positives. While the PdG concentration is above 5 ng/mL (pregnancy stage), the scavenger band becomes saturated allowing the excess conjugated PdG-BSA to migrate to the test line (T), where the immobilized anti-PdG antibodies capture the PdG complexes and generate a visible pink-red signal proportional to PdG concentration. The control line (C), containing immobilized anti-chicken IgY, binds the chicken IgY–AuNP tracer irrespective of PdG levels, thereby confirming reagent integrity, capillary flow, and assay validity. The pink-red colouration of both test and control lines results from localized aggregation of gold nanoparticles (through localized surface plasmon resonance effects), wherein presence of both lines indicates pregnancy, presence of only the control line indicates non-pregnancy, and absence of the control line renders the assay invalid.

In the E1S detection channel (200), the anti-E1S–BSA conjugate of the scavenger band (S) captures the conjugated E1S–BSA while the E1S concentrations is ≤ 5 ng/mL, thereby retaining the majority of conjugates in non-pregnant or very early-stage animals and preventing unspecific binding at the test line. When the E1S concentration exceeds 5 ng/mL (typically 5–80 ng/mL in pregnant urine), the scavenger band becomes saturated, permitting the excess conjugated E1S–BSA to migrate to the test line (T), where the immobilized E1S–BSA conjugates competitively bind the antibody–AuNPs, generating an inversely proportional pink-red signal whose intensity decreases as E1S concentration increases. The control line (C), coated with goat anti-mouse IgG, binds the mouse IgG–AuNP conjugates independent of analyte presence, thereby validating sample migration, reagent functionality, and assay integrity. The pink-red coloration of both test and control lines results from the localized aggregation of gold nanoparticles (through localized surface plasmon resonance effects), with diagnostic interpretation being: a weak or absent test line with a visible control line indicates high E1S (pregnancy positive), a strong test line with a visible control line indicates low E1S (non-pregnant), and absence of the control line renders the test invalid.

In the pH detection channel (300), urine samples undergo pinch (0.5-1 mg/ml) of BaCl₂ pretreatment, which precipitates sulfates and clarifies the matrix, thereby allowing reliable colorimetric protein evaluation. The test line (T), impregnated with bromophenol blue, functions as a pH- and protein-sensitive indicator: at pH >5.0 with protein concentration <100 mg/dL, the test line develops a blue-green colouration, confirming a physiologically valid sample consistent with pregnancy status. Conversely, when urine exhibits pH <5.0 with protein concentration >100 mg/dL, the test line turns yellow, reflecting acidic or protein-rich conditions that correlate with non-pregnancy. The control line (C) ensures reagent stability, membrane integrity, and complete fluid migration, with absence of this line rendering the assay invalid. The observed colour shift arises from pH-dependent ionization of bromophenol blue, which transitions from yellow (acidic/protein >100 mg/dL) to blue-green (neutral to alkaline/protein <100 mg/dL). Thus, diagnostic interpretation is as follows: blue-green test line with control line = pregnancy positive, yellow test line with control line = pregnancy negative, and absence of control line = invalid assay. The colour visualization conditionings are shown in Table 1 to indicate diagnostic interpretations.

Table 1
Channel S (Scavenger band) T (Test line) C (Control line) Interpretation
PdG detection channel (100) Captures anti-PdG–AuNP at PdG <5 ng/mL (no visible colour) Pink-red line visible at PdG >5 ng/mL Pink-red line always visible Test & Control lines visible = Pregnant;
Only Control line visible = Nonpregnant;
Control line invisible = Invalid
E1S detection channel (200) Not applicable (competitive assay) Strong pink-red line visible at E1S <5 ng/mL;
weak visible /absent line at E1S 5–80 ng/mL Pink-red line always visible Test line weakly visible/ invisible & Control line visible = Pregnant;
Test & Control lines strongly visible = Nonpregnant;
Control line invisible = Invalid
pH detection channel (300) Not applicable Blue-green at pH >5.0 with protein <100 mg/dL; Yellow at pH <5.0 with protein >100 mg/dL Reference colour band always visible Blue-green Test & Control lines visible = Valid/Pregnant;
Yellow Test & Control lines visible = Nonpregnant;
Control line invisible = Invalid

The diagnostic interpretation of the three-lane assay is based on colour development at the test (T) and control (C) lines, which arises from localized aggregation of gold nanoparticles or pH indicator ionization. In the PdG channel, a visible pink-red test line along with a control line indicates high PdG concentration (>5 ng/mL) consistent with pregnancy, whereas the presence of only the control line indicates non-pregnancy. In the E1S channel, the test line signal is inversely proportional to E1S concentration, with a weak or absent test line (alongside a control line) confirming pregnancy, and a strong test line confirming non-pregnancy. In the pH channel, the bromophenol blue indicator provides a built-in sample integrity check, where a blue-green test line with control line indicates a physiologically valid sample with protein <100 mg/dL (pregnancy positive), while a yellow test line indicates acidic or protein-rich urine (>100 mg/dL, non-pregnant). Absence of the control line in any channel invalidates the test.

Experimental analysis

For testing purpose, all the materials/parts are procured from Anand district in Gujarat. A prototype device is fabricated using three detection zones arranged in series (each zone having a sample pad, a conjugate pad, and an absorbent pad) as shown in Fig. 1 with following dimensions as shown in Table 2.
Table 2

Parts Length Width Loading Materials Application Rate
Total active length of nitrocellulose membrane/plastic base 25 mm ~4 mm (per channel) - -
sample pad 12 mm 4 mm - -
Absorbent pad 10 mm 4 mm - -
Conjugate pad 20 mm 4 mm - -
1st detection channel Scavenger band - 1.8 mm 0.75 mg/mL anti-PdG-BSA 0.9 μL/cm
Test Line - 0.5 mm 0.5 nanomole anti PdG-AuNP 0.8 μL/cm
Control line - 0.5 mm 1 mg/mL anti chicken IgY 0.8 μL/cm
2nd detection channel Scavenger band - 0.5 mm 0.6 mg/mL anti-E1S-BSA 0.9 μL/cm
Test Line - 0.5 mm 4 nanomole anti-E1S-AuNP 12 μL/cm
Control line - 0.5 mm 1 mg/mL goat anti‑mouse IgG 0.8 μL/cm
3rd detection channel Test Line - 2.0 mm 0.04% w/v bromophenol blue 3 μL/cm
Control line - 0.5 mm 0.05 M citrate buffer with 3-5 pH 0.05 M citrate buffer with 3 pH

Multiplex lateral flow strips with three independent channels (PdG, E1S, pH) are fabricated using wax-patterned nitrocellulose. Different pad overlaps and line spacings were tested (±1 mm variation). In each detection zone, the sample pad overlaps with the conjugate pad at 2 mm length, and the conjugate pad overlaps the absorbent pad at 1 mm length. The gap between scavenger band and the test line is 3 mm, and the gap between the test line and the control line is 5 mm, which was optimized through experimental trials to achieve consistent signal intensity, minimal cross-diffusion, and reproducible flow dynamics.

Around 200 urine samples collected from livestock (21–35 days post-artificial insemination), confirmed by ultrasonography, are used to check operational efficiency of the prototype device, where the device results (whether pregnant or non-pregnant) accurately match with the ultrasonography report. Further, the prototype device is compared with the conventional livestock pregnancy diagnosis approaches such as (a) single-hormone PdG LFAs, (b) E1S-only kits, and (c) commercial urine dipsticks without sample authentication. The key evaluation metrics such as sensitivity, specificity, invalid result rate, and operator error rate are recorded as shown in Table 3.

Table 3
Parameter PdG-only LFA E1S-only LFA Urine Dipsticks (pH/protein only) Present Invention (Multiplex PdG + E1S + pH)
Analytes detected PdG only E1S only Protein/pH only PdG + E1S + pH (simultaneous)
Sample authentication None None pH only, no hormone link Integrated (BaCl₂ pretreatment + pH/protein cutoff)
Detection logic Competitive, counter-intuitive (weak test = positive) Competitive (weak test = positive) Qualitative only Inverted PdG logic (intuitive) + competitive E1S + pH integrity
Invalid result rate 12–15% 10–12% 20%+ <3%
Sensitivity (25–35 days post-AI) 80–85% 78–82% Not applicable 96%
Specificity 85–88% 82–85% Not applicable 93%
Operator interpretation errors High (due to counter-intuitive logic) Moderate Moderate Negligible (intuitive visual outputs)
Field usability Moderate Moderate High but inaccurate High, accurate, integrated

The present invention exhibits following key technical advantages:
• Multiplexed Detection: Simultaneous quantification of PdG and E1S along with pH/protein-based sample authentication from a single urine sample, unlike prior art devices that detect only one parameter.
• Inverted PdG Logic: Scavenger band design provides visible test line at high PdG levels, eliminating operator confusion common to competitive LFAs.
• Integrated Sample Integrity Check: The pH/protein lane coupled with BaCl₂ pretreatment ensures sample authenticity, preventing false results from adulterated or degraded urine.
• Improved Accuracy: Clinical validation demonstrated 94.5% overall accuracy at 25–35 days post-artificial insemination, surpassing conventional PdG/E1S LFAs.
• Reduced Invalid Results: <3% invalid rate due to control line and pH authentication, compared to >10% in prior arts.
• User-Friendly: Single dip-operation with intuitive colour interpretation (pink-red for hormone lines, blue-green/yellow for pH) allows non-technical farm workers to use it reliably.
• Manufacturing Scalability: Wax-patterned hydrophobic barriers enable roll-to-roll production at low cost, making the device commercially viable for large-scale veterinary use.
• Long shelf-life: The loading materials shows ≥18‑month ambient stability, thus making the device one-time usable within 18 months from the date of its manufacturing.

The foregoing descriptions of exemplary embodiments of the present invention have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiment was chosen and described in order to best explain the principles of the invention and its practical application, to thereby enable the persons skilled in the art to best utilize the invention and various embodiments with various modifications as are suited to the particular use contemplated. It is understood that various omissions, substitutions of equivalents are contemplated as circumstance may suggest or render expedient, but is intended to cover the application or implementation without departing from the scope of the claims of the present invention. , Claims:We claim:

1. A device for determining livestock pregnancy in urine samples, the device comprises:
a pregnanediol-3-glucuronide (PdG) detection channel (100) comprising:
a scavenger band (S) loaded with 0.75 mg/mL anti-pregnanediol-3-glucuronide antibody conjugated bovine serum albumin solution (anti-PdG-BSA conjugate) at an application rate of 0.9 μL/cm;
a test line (T) loaded with 0.5 nanomole anti pregnanediol-3-glucuronide monoclonal antibody conjugated gold solution nanoparticle solution (anti PdG-AuNP conjugate) at an application rate of 10–15 μL per cm, and 1 mg/mL anti mouse IgG solution at an application rate of 0.8 μL/cm; and
a control line (C) loaded with 1 mg/mL anti chicken IgY solution at an application rate of 0.8 μL/cm;
an estrone sulfate (E1S) detection channel (200) comprising:
a scavenger band (S) loaded with 0.6 mg/mL anti-estrone sulfate antibody conjugated bovine serum albumin solution (anti-E1S-BSA conjugate) at an application rate of 0.8–1 μL/cm;
a test line (T) loaded with 0.2-4.6 nanomole anti-estrone sulfate monoclonal antibody conjugated gold nanoparticle solution (anti-E1S-AuNP conjugate) at an application rate of 10-15 μL/cm; and
a control line (C) loaded with 1 mg/mL goat anti‑mouse IgG solution at an application rate of 0.8 μL/cm; and
a pH detection channel (300) comprising:
a test line (T) loaded with 0.04% w/v bromophenol blue solution at an application rate of 3 μL/cm; and
a control line (C) loaded with 0.05 M citrate buffer with 3-5 pH at an application rate of 1 μL/cm;
wherein all the three detection channels (100, 200, 300) are formed on nitrocellulose membranes affixed on a plastic support base (400) in form of a lateral flow test strip packed in a cassette (500) having at least one urine sample receiving region (502) and at least one result visualization window (504).
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2. The device as claimed in claim 1, wherein the three detection channels (100, 200, 300) are sequentially formed in three nitrocellulose membrane-based zones (Z1, Z2, Z3) isolated through hydrophobic barriers (HB), each zone having a sample pad (SP) at upstream end, an absorbent pad (AP) at downstream end, and a conjugate pad (CP) placed therebetween with one detection channel.

3. The device as claimed in claim 1, wherein the three detection channels (100, 200, 300) are formed as three parallel channels isolated through hydrophobic barriers (HB) in a nitrocellulose conjugate pad (CP) placed between a nitrocellulose sample pad (SP) at upstream end and a nitrocellulose absorbent pad (AP) at downstream end.

4. The device as claimed in claim 1, wherein the scavenger band (S), the test line (T), and the control line (C) in each of the detection channels (100, 200, 300) are patterned in parallel in a direction transverse to a sample flow direction.

5. A method of manufacturing a device for determining livestock pregnancy in urine samples, the method comprises steps of:
defining (S1) three (first, second, third) isolated detection channels (100, 200, 300) on at least one nitrocellulose membrane through hydrophobic barriers (HB);
applying (S2) on the first detection channel (100) through a BioDot dispenser:
a scavenger band (S) loaded with 0.75 mg/mL anti-pregnanediol-3-glucuronide antibody conjugated bovine serum albumin solution (anti-PdG-BSA conjugate) at an application rate of 0.9 μL/cm;
a test line (T) loaded with 0.5 nanomole anti pregnanediol-3-glucuronide monoclonal antibody conjugated gold solution nanoparticle solution (anti PdG-AuNP conjugate) at an application rate of 10–15 μL per cm, and 1 mg/mL anti mouse IgG solution at an application rate of 0.8 μL/cm; and
a control line (C) loaded with 1 mg/mL anti chicken IgY solution at an application rate of 0.8 μL/cm;
loading (S3) on the second detection channel (200) through the BioDot dispenser:
a scavenger band (S) loaded with 0.6 mg/mL anti-estrone sulfate antibody conjugated bovine serum albumin solution (anti-E1S-BSA conjugate) at an application rate of 0.8–1 μL/cm;
a test line (T) loaded with 0.2-4.6 nanomole anti-estrone sulfate monoclonal antibody conjugated gold nanoparticle solution (anti-E1S-AuNP conjugate) at an application rate of 10-15 μL/cm; and
a control line (C) loaded with 1 mg/mL goat anti‑mouse IgG solution at an application rate of 0.8 μL/cm;
depositing (S4) on the third detection channel (300) through the BioDot dispenser:
a test line (T) loaded with 0.04% w/v bromophenol blue solution at an application rate of 3 μL/cm; and
a control line (C) loaded with 0.05 M citrate buffer with 3-5 pH at an application rate of 1 μL/cm;
affixing (S5) the membrane on a plastic strip (400) to form a lateral flow test strip followed by drying in a hot air dryer at 37 °C with <30% relative humidity for 2 hours; and
encasing (S6) the strip (400) in a cassette (500) having at least one urine sample receiving region (502) and at least one result visualization window (504).

6. The method as claimed in claim 5, wherein the channel defining step (S1) comprises sequentially forming the three detection channels (100, 200, 300) in three nitrocellulose membrane-based zones (Z1, Z2, Z3) isolated through the hydrophobic barriers (HB), each zone having a sample pad (SP) at upstream end, an absorbent pad (AP) at downstream end, and a conjugate pad (CP) placed therebetween with one detection channel.

7. The method as claimed in claim 5, wherein the channel defining step (S1) comprises forming the three detection channels (100, 200, 300) as three parallel channels isolated through the hydrophobic barriers (HB) in a nitrocellulose conjugate pad (CP) placed between a nitrocellulose sample pad (SP) at upstream end and a nitrocellulose absorbent pad (AP) at downstream end.

8. The method as claimed in claim 5, wherein the applying step (S2) comprises:
mixing anti-PdG antibody and BSA at a weight ratio of 1:1 in sterile water or alkaline water having 7-10 pH to obtain the anti-PdG-BSA conjugate;
blending anti PdG antibody and AuNP at a weight ratio of 1:1, 5% w/v of trehalose or sucrose, 0.5–1% v/v of bovine serum albumin (BSA), and 0.05–0.5% v/v of Tween 20 in phosphate buffered saline (PBS) or borate solvent to obtain the anti PdG-AuNP conjugate; and
adding anti chicken IgY in sterile water or alkaline water having 7-10 pH to obtain the anti chicken IgY solution.

9. The method as claimed in claim 5, wherein the loading step (S3) comprises:
mixing anti-E1S antibody and BSA at a weight ratio of 1:1 in sterile water or alkaline water having 7-10 pH to obtain the anti-E1S-BSA conjugate;
blending anti-E1S antibody and AuNP a weight ratio of 1:1 in sterile water or alkaline water having 7-10 pH to obtain the anti-E1S-AuNP solution; and
adding goat anti‑mouse IgG mixed in sterile water or alkaline water having 7-10 pH to obtain the goat anti‑mouse IgG solution.

10. The method as claimed in claim 5, wherein the depositing step (S4) comprises mixing bromophenol blue mixed in 0.05 M citrate buffer with pH 3-5 the bromophenol blue solution.