WO2023059283A1 - A lateral flow test strip for detection and/or measurement of bisphenol a in breast milk - Google Patents

A lateral flow test strip for detection and/or measurement of bisphenol a in breast milk Download PDF

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WO2023059283A1
WO2023059283A1 PCT/TR2022/050577 TR2022050577W WO2023059283A1 WO 2023059283 A1 WO2023059283 A1 WO 2023059283A1 TR 2022050577 W TR2022050577 W TR 2022050577W WO 2023059283 A1 WO2023059283 A1 WO 2023059283A1
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Prior art keywords
nanofiber
bpa
lateral flow
breast milk
test strip
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PCT/TR2022/050577
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French (fr)
Inventor
Tugba AKBAY
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Marmara Universitesi Rektorlugu Ozel Kalem Birimi
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Priority claimed from TR2021/015709 external-priority patent/TR2021015709A1/en
Application filed by Marmara Universitesi Rektorlugu Ozel Kalem Birimi filed Critical Marmara Universitesi Rektorlugu Ozel Kalem Birimi
Publication of WO2023059283A1 publication Critical patent/WO2023059283A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow

Definitions

  • the present invention relates to a lateral flow test strip developed for measuring bisphenol A (BPA) in breast milk.
  • Said diagnostic test which is based on immunochromatographic methods, comprises a nanofiber, preferably a polycaprolactone/silk fibroin-based nanofiber, in the test membrane.
  • the test strip of the invention allows the detection of BPA in breast milk by virtue of color change due to the use of colloidal gold.
  • Bisphenol A 2, 2 -bis [4-hydroxyphenyl] propane
  • BPA 2, 2 -bis [4-hydroxyphenyl] propane
  • BPA is considered as an endocrine disruptor and is known to have the potential to affect infants’ health.
  • a number of studies have revealed that BPA is secreted into breast milk and colostrum at significant concentrations (1-7 ng/ml); according to these studies the mean value in colostrum is 3.41 ng/ml (Otaka H, Yasuhara A, Morita M.
  • BPA is a target for the development of diagnosis and removal methods in the related technical field. Physical absorption, membrane technology, reverse osmosis technology, etc. have been used for BPA removal. Since BPA content of wastewater cannot totally be removed with these techniques, the studies on BPA removal still continue.
  • Modi and Bellare have defined a method for removing BPA from water using hollow fiber membranes (Modi A, Bellare J. Copper sulfide nanoparticles/carboxylated graphene oxide nanosheets blended polyethersulfone hollow fiber membranes: development and characterization for efficient separation of oxybenzone and bisphenol a from water. Polymer (Guildf). 2019;163: 57- 67). Ye et al.
  • LFA Lateral Flow Test
  • the lateral flow test is a preferred method due to its high sensitivity and selectivity, low cost, long shelf life and high visual results.
  • the sensitivity of the test is important in gold nanoparticle- antibody-based lateral flow tests. Therefore, parameters such as the conjugation of gold nanoparticles and antibody, polymer structure of the membrane, capillary flow rate, and porosity of the membrane should be optimized in the design of lateral flow test strips (Paroloa C, et al. Simple paper architecture modifications lead to enhanced sensitivity in nanoparticle based lateral flow immunoassay. J Mater Chem C. 2015;3:10715-10722; Choi JR, et al. Advances and challenges of fully integrated paper-based point-of-care nucleic acid testing.
  • the pH and antibody concentration need to be optimized to ensure an effective binding of the antibody to gold nanoparticles.
  • the surfaces of gold nanoparticles have a negative charge due to the AuCh complex, and there is electrostatic repulsion between the nanoparticles.
  • the ion concentration in the solution increases, the particles get closer to each other as the ionic layer on the nanoparticle surface is compressed.
  • the repulsion is less than the attraction resulting from the London-van der Waals force, the gold nanoparticles are accumulated.
  • the color of the gold solution in turn changes from red to blue. The rate of color change may be determined as the decrease in absorbance at 520 nm and the increase in absorbance at 580 nm. If antibody molecules are physically adsorbed on the gold surface, gold nanoparticles do not aggregate.
  • US 2007/059678 Al relates to a breast milk detection device.
  • Said device includes a plurality of test strips slidably and removably attached to a test housing. Each test strip is connected to an absorbent section so that a breast milk sample deposited thereon is communicated to the test strips.
  • the device of the relevant document is directed to the detection of substances and allergens such as alcohol and peanut oil in breast milk.
  • CN 102183642 discloses a quick detection card and a detection method for bisphenol A. It is stated that said detection card belongs to the technical field of phenol content detection in polycarbonate products for food containers.
  • a test strip is arranged in a shell of the quick detection card for bisphenol A; said test strip is formed by sequentially sticking a sample pad, a colloidal gold membrane, a nitrocellulose membrane anda water absorption membrane on a support back plate.
  • the colloidal gold membrane is a glass fiber membrane containing monoclonal antibody or polyclonal antibody colloidal gold markers specific to bisphenol A.
  • the present invention provides a lateral flow test strip for the detection and/or measurement of bisphenol A in breast milk and a preparation method thereof.
  • the present invention provides a lateral flow test strip comprising a nanofiber in the test membrane for detecting and/or measuring bisphenol A (BPA) in breast milk.
  • BPA bisphenol A
  • the nanofiber comprised in the lateral flow test strip according to the present invention is polycaprolactone/silk fibroin (PCL/SF) nanofiber.
  • the core portion of the PCL/SF nanofiber may comprise polycaprolactone and the shell portion of the PCL/SF nanofiber may comprise mixture of polycaprolactone/silk fibroin.
  • the present invention provides a method for preparing the lateral flow test strip according to the present invention. Said method comprises the following steps:
  • the nanofiber prepared in the first step of the method is polycaprolactone/silk fibroin (PCL/SF) nanofiber.
  • PCL/SF polycaprolactone/silk fibroin
  • the electrospinning process performed in the lateral flow test strip preparation method of the present invention is preferably coaxial electrospinning process.
  • the process of immobilizing the BPA antibody on the nanofiber performed in the method of the present invention is preferably performed with physical adsorption.
  • the process of immobilizing the colloidal gold-BPA antibody conjugate on the glass fiber performed in the method of the present invention is preferably performed with impregnation method.
  • the present invention provides a method for the detection and/or measurement of bisphenol A in breast milk, comprising the following steps:
  • Said method preferably also comprises the step of assessing obtained test results by means of a software.
  • the present invention provides the use of the lateral flow test strip according to the present invention for the detection and/or measurement of bisphenol A in breastmilk.
  • the present invention provides a kit comprising:
  • Figure 1 shows the design of the lateral flow test strip.
  • the breast milk sample (1), sample pad (2), conjugation pad (3), test membrane (4), absorbent pad (5), test line (6a), and control line (6b) are shown on the figure.
  • the test line comprises the BPA primary antibody
  • the control line comprises the BPA secondary antibody.
  • Figures 2-A to 2-F show SEM images of prepared coaxial nanofibers (PCL: Polycaprolactone, SF: Silk fibroin, DMF: Dimethylformamide, DCM: Dichloromethane).
  • PCL Polycaprolactone
  • SF Silk fibroin
  • DMF Dimethylformamide
  • DCM Dichloromethane
  • Figure 3 shows FTIR spectroscopy results of the hybrid nanofibers (Shell: Polycaprolactone /silk fibroin, Core: PCL).
  • the peaks of 2946 and 1720 cm 1 show the characteristic peaks of PCL; and the peaks of 3250, 1650, 1530 and 1240 cm 1 show the characteristic peaks of silk fibroin.
  • Figure 4 shows the studies carried out to determine the optimum pH and concentration for the conjugation of gold-antibody.
  • the horizontal column shows the antibody concentrations, and the vertical column shows the pH values of the gold solution.
  • Figure 5 shows the calibration curves and signal intensity ofwater BPA levels on (a) NC membrane and (b) coaxial PCL/SF nanofiber membrane (C: Color intensity of the control line, T: Color intensity of the test line, NC: Nitrocellulose, PCL: polycaprolactone, SF: silk fibroin).
  • Figure 6 shows the calibration curves and signal intensity of breast milk BPA levels on (a) NC membrane and (b) coaxial PCL/SF nanofiber membrane (C: Color intensity of the control line, T: Color intensity of the test line, NC: Nitrocellulose, PCL: polycaprolactone, SF: silk fibroin).
  • BPA is a chemical known to pass to the breast milk in significant concentrations and adversely affect the development of infants and children. Although methods have been developed for the removal and detection of BPA in the related technical field, the vast majority of these methods are carried out in vitro. Another problem with the development of a test strip for breast milk is that breast milk is a body fluid with different characteristics compared to the other body fluids. Since it is opaque and creates a background, it mostly requires pre-treatment.
  • the purpose of the present invention is to provide a highly sensitive lateral flow test strip for the detection and/or measurement of bisphenol A (BPA) in breast milk.
  • BPA bisphenol A
  • conventional nitrocellulose test membrane in the lateral flow test strip is coated with a nanofiber material, preferably with a nanofiber the inner portion (core) of which consists of polycaprolactone (PCL) and the outer portion (shell) of which consists of mixture of PCL/silk fibroin (SF).
  • the nanofiber material is coated on a support material (nitrocellulose, nonwoven, etc.).
  • Said nanofiber is preferably a polycaprolactone and silk fibroin- based nanofiber.
  • polycaprolactone/silk fibroin-based nanofiber is included as a test membrane in a test strip for the first time.
  • the novel lateral flow test strip according to the present inventionfor detecting and/or measuring bisphenol A in breast milk is advantageous as it is a product that allows in situ detection. Using said test strip, it will be possible for breastfeeding mothers to detect the BPA content in their milk in an effective, quick and reliable manner without a need for any pre-sorting process.
  • the present invention provides a method for the preparation of the lateral flow test strip of the invention, comprising the following steps:
  • the nanofiber prepared in the method of the present invention is preferably a nanofiber comprising polycaprolactone and silk fibroin (PCL/SF).
  • PCL/SF nanofiber is included in a preparation method of lateral flow test strip for the first time with the present invention and is advantageous in that it allows for adjustment of the reaction time required to detect bisphenol A in breast milk, more clear observation of the color change and much more sensitive detection of change in bisphenol A concentration compared to the conventional method.
  • nanofiber prepared in the method of the present invention may be prepared using normal or coaxial electrospinning, it is preferably prepared by coaxial electrospinning.
  • BPA antibody is immobilized on nanofiber, preferably silk fibroin and caprolactone-based nanofiber.
  • nanofiber-based immunosorbent surface is obtained.
  • the primary and secondary antibodies are successfully immobilized on the prepared nanofiber.
  • the antigen (BPA)/antibody (BPA antibody) complex is detected based on the color change principle due to colloidal gold.
  • the BPA antibody is physically immobilized on the nanofiber, preferably by physical adsorption.
  • the nanofiber is then coated on nitrocellulose and used as a test membrane in LFA. Color changes in the test membrane are assessed as the signal intensity of BPA.
  • the gold nanoparticle-antibody conjugate is prepared under conditions which were optimized with the studies carried out within the scope of the present invention. Provision of a sensitive conjugate enhances the recognition of the antibody-coated gold nanoparticles.
  • the conjugation conditions of colloidal gold and antibody were determined as described in detail in the Examples below.
  • the process of immobilizing the colloidal gold-BPA antibody conjugate on the glass fiber in the method of the present invention is preferably performed with impregnation method.
  • Said colloidal gold comprises gold nanoparticles.
  • the present invention provides a method for the detection and/or measurement of bisphenol A in breast milk. Said method comprises the following steps:
  • test results obtained from the test strip of the present invention can be interpreted by a software which is developed specifically for BPA in breast milk and is configured to determine the BPA concentration in the breast milk sample.
  • Said software is based on the principle of correlating the color change in the test membrane with the BPA concentration.
  • Assessment of the test results by a software is advantageous in that it prevents user-induced interpretation errors.
  • the present invention provides the use of the lateral flow test strip according to the present invention for the detection and/or measurement of bisphenol A in breastmilk.
  • the present invention also provides a kit comprising:
  • NC nitrocellulose
  • the core portion of the coaxial nanofiber was prepared using PCL, and the shell portion was prepared using mixture of PCL/SF.
  • PCL solution (15% weight/volume) was dissolved in dimethylformamide (DMF) by stirring for 3 hours at room temperature.
  • DMF dimethylformamide
  • PCL/SF solution (10% weight/volume); powdered SF (Hydrolysis SF Powder, 500-10.000 g/mol), PCL (60.000 g/mol), DMF and methylene chloride (DCM) were used.
  • PCL/SF solution containing 1:1 PCL:SF by weight was prepared with a 1:4 mixture of DMF:DCM by stirring atroom temperature for 24 hours.
  • Hybrid nanofiber was obtained by electrospinning using a coaxial needle for forming the outer shell (PCL/SF) and core (PCL) of the coaxial nanofiber. Electrospinning syringe was connected to a pump at a flow rate of 1 ml/hr for PCL and 2 ml/hr for PCL/SF. Drum rotation speed was setto 100 rpm. PCL (core)-PCL/SF (shell) coaxial nanofibers were obtained at 27 kV with a distance of 15 cm from the nozzle to the collector. Electrospinning was carried out in air at 30°C for 1 hour.
  • the present invention it was aimed to improve the surface properties of the test membrane and thus the antibody immobilization conditions, and to reduce the capillary flow rate by coating the NC with coaxial electrospun nanofiber.
  • the reason for using PCL in the test membrane is to increase the mechanical properties of PCL/SF-based shell portion of the coaxial nanofiber.
  • the mixture of PCL/SF was used for increasing hydrophobicity and thereby reducing the fluid flow. As the amount of coaxial nanofiber on the NC membrane increases, the hydrophobicity in the coated region increases.
  • Figures 2d to 2f show SEM images of the prepared coaxial nanofiber at different voltages. Homogeneous and bead- free nanofibers were obtained with a core of 15% PCL and a shell of 10% PCL-SF (1:4 DMF:DCM) at a voltage of 27 kV ( Figure 2f).
  • the breast milk samples were obtained voluntarily from healthy breastfeeding mothers.
  • the Medical Ethics Committee of Marmara University approved the breast milk collection and experimental stages of the studies of the invention. Collected breast milk samples were stored in BPA-free breast milk bags (Lansinoh-Breastmilk Storage Bags, Japan) at -20°C for a maximum of 3 months after BPA determination.
  • BPA concentrations of breast milk samples were determined using ELISA method (Ecologenia Abraxis, Japan).
  • a stock solution of 100.000 ng/ml BPA (Sigma-Aldrich, St. Louis, Missouri) was prepared with 10% methanol. BPA-free water was used in the preparation of BPA solutions. Doped water samples in the range of 0-10 ng/ml were prepared by diluting the stock solution of BPA.
  • Control samples were selected and pooled from breast milk samples containing BPA in the range of 0-0.5 ng/ml. Breast milk samples with a BPA level above 0.5 ng/ml were not used. The breast milk pool was divided and stored at -20°C in BPA-free storage bags. Doped breast milk samples in the range of 0-10 ng/ml BPA were prepared by diluting the stock solution with the control breastmilk samples.
  • Conjugation conditions of gold nanoparticles and antibody compatible with the prepared coaxial nanofiber were determined by optimizing pH and antibody concentration, as well as sucrose and albumin concentrations.
  • pH 9 was found to be suitable for the optimal conjugation of BPA antibody and gold nanoparticles.
  • Isoelectric point for the immunoglobulins of various species is in the range of pH 8 to 9.
  • pH value optimization should be tested for the conjugation.
  • Optimum antibody concentration is critical to stabilize the gold nanoparticles without aggregation.
  • optimum BPA antibody concentration was found to be 50 pg/ml. Usually, a concentration which is 50% higher than the critical concentration is used for the antibody-gold nanoparticle conjugation.
  • BSA is used as a secondary stabilizer to stabilize the surface of the gold nanoparticle and prevents flocculation. After antibody binds to the surface of the gold nanoparticles, itprevents non-specific binding of other molecules to the remaining cavities. Sucrose is used to prevent the surface of the colloidal gold from adhering to the test membrane when the membrane is dried.
  • sample pad (2) The components of the lateral flow test strip (sample pad (2), conjugation pad (3), membrane (4), and absorbent pad (5)) were combined on adhesive rear surface of the card and prepared for sample application.
  • Conjugate pad (0.3 x 0.5 cm) was prepared by immobilizing colloidal gold-BPA antibody conjugate on a glass fiber.
  • Colloidal gold-BPA antibody conjugate was diluted with 20 mM phosphate buffer solution (PBS) containing sucrose and BSA (Sigma-Aldrich) (1:1, volume/volume).
  • BPA antibody was also immobilized as a spot on hybrid nanofiber by physical adsorption method to form the test membrane (0.3 x 2.5 cm) (Zhao K, He W, Bi f et al. Development of a lateral flow immunochromatographic assay for the rapid diagnosis of Orf virus infections. J Virol Methods. 2016;236:10-17).
  • Hybrid nanofiber was treated with PBS containing BSA and dried. Cellulose fiber (Millipore) was used as absorbent pad.
  • test and control lines (6a and 6b) were prepared as shown in Figure 1.
  • test line comprises BPA primary antibody
  • control line comprises the BPA secondary antibody.
  • 50 pl of water and breast milk samples (1) were used as samples on the prepared lateral flow test strips.
  • the results of using hybrid nanofibers as the test membranes were compared with those prepared using NC membranes (Sartorius, France, pore size 0.45 pm).
  • RGB values were detected at test and control points using Image) software (Schneider C, Rasband W, Eliceiri K. NIH image to ImageJ:25 years of image analysis. Nat Methods. 2012;9:671- 675).
  • Image Image
  • all colors have a value ranging from 0 (black) to 255 (white). When the value of all three components in the RGB system is 255, it is pure white; when the RGB value is 0, it is pure black. Dark colors have a lower value than light colors in the RGB system. Color intensities in test and control lines are presented as RGB values.
  • Capillary flow rate is important as it is another optimizing parameter in LFAs.
  • Capillary flow rate is the rate at which the sample moves across the membrane. It decreases linearly as the porosity decreases. Therefore, within the scope of the present invention, the lateral flow rates of water and breast milk samples were determined as explained in detail below.
  • BPA in water and breast milk samples were characterized by the detectable concentrations in the range of 0-10 ng/ml.
  • BPA in 0-2-4-6-8-10 ng/ml water and breast milk samples was run on the lateral flow test strips in which the test membrane is (a) NC or (b) coaxial electrospun nanofiber coated NC.
  • Lateral flow rate of water samples in the test strips comprising NC membrane or coated NC membrane was 0.054 cm/s.
  • Pink (dark) color appeared at BPA concentration of 2 ng/ml in the water samples on both NC and coated NC test membrane.
  • the intensity of pink (dark) color remained unchanged at the BPA concentrations above 8 ng/ml when PCL/SF-based test membrane is used.
  • NC-based test membrane is used as the test membrane, the intensity of pink (dark) color remained unchanged at BPA concentrations above 4 ng/ml ( Figure 5).
  • the porosity of NC was increased by means of nanofiber coating as it was aimed to reduce the flow rate of breast milk on test strip. Electrospun-coated NC delayed the flow, thereby increasing the interaction ratio between BPA and the antibody thereof, and irreversibly trapping BPA antibody in the lateral flow test strip. Since the effective analyte concentration of the sample is inversely proportional to the square of the flow rate, the flow rate decreased with the coaxial electrospun coated NC membrane.
  • the polymer structure of the test membrane demonstrates the protein binding properties of the membrane.
  • the background formed by the breast milk on the surfaces due to the structural properties thereof was determined by comparing the signal intensity thereof with the signal intensity of water. Water samples were found to have higher signal intensity as compared to the breast milk samples. Although the detection limit was 2 ng/ml in both coaxial PCL/SF nanofiber and nitrocellulose (NC) test membranes, the color intensity was observed to increase with increasing BPA concentration in coaxial PCL/SF nanofiber. In other words, coaxial PCL/SF nanofiber provided a higher color intensity and more visible results than NC membrane.
  • a novel and sensitive lateral flow test strip and a sensitive in situ method for the detection and/or measurement of BPA in breast milk have been developed using a nanofiber material, preferably coaxial PCL/SF nanofiber coating, in the test membrane of the lateral flow test strip.

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Abstract

The present invention relates to a lateral flow test strip developed for detection and/or measurement of bisphenol A in breast milk. Said test, which is based on immunochromatographic methods, comprises a nanofiber, preferably a polycaprolactone/silk fibroin-based nanofiber, in the test membrane. The test strip of the invention allows the detection and/or measurement of bisphenol A in breast milk by virtue of color change due to the use of colloidal gold. The present invention further relates to a method for preparing said test strip.

Description

A LATERAL FLOW TEST STRIP FOR DETECTION AND/OR MEASUREMENT OF BISPHENOL A IN BREAST MILK
Technical Field
The present invention relates to a lateral flow test strip developed for measuring bisphenol A (BPA) in breast milk. Said diagnostic test, which is based on immunochromatographic methods, comprises a nanofiber, preferably a polycaprolactone/silk fibroin-based nanofiber, in the test membrane. The test strip of the invention allows the detection of BPA in breast milk by virtue of color change due to the use of colloidal gold.
Background of the Invention
Bisphenol A (BPA, 2, 2 -bis [4-hydroxyphenyl] propane) is a chemical which has been used in several applications such as safety equipments, toys, feeding bottles, food boxes and dental filling pastes since the 1950s. It is known to adversely affect preimplantation embryos or fetuses and alter postnatal development. BPA is considered as an endocrine disruptor and is known to have the potential to affect infants’ health. A number of studies have revealed that BPA is secreted into breast milk and colostrum at significant concentrations (1-7 ng/ml); according to these studies the mean value in colostrum is 3.41 ng/ml (Otaka H, Yasuhara A, Morita M. Determination of Bisphenol a and 4-nonylphenol in human milk using alkaline digestion and cleanup by solid-phase extraction. Anal Sci. 2003;19:1663-1666; Kuruto-Niwa R, Tateoka Y, Usuki Y, Nozawa R. Measurement of bisphenol Aconcentrations in human colostrum. Chemosphere. 2007; 66:1160- 1164).
For above-mentioned reasons, BPA is a target for the development of diagnosis and removal methods in the related technical field. Physical absorption, membrane technology, reverse osmosis technology, etc. have been used for BPA removal. Since BPA content of wastewater cannot totally be removed with these techniques, the studies on BPA removal still continue. For example, Modi and Bellare have defined a method for removing BPA from water using hollow fiber membranes (Modi A, Bellare J. Copper sulfide nanoparticles/carboxylated graphene oxide nanosheets blended polyethersulfone hollow fiber membranes: development and characterization for efficient separation of oxybenzone and bisphenol a from water. Polymer (Guildf). 2019;163: 57- 67). Ye et al. have revealed a photocatalytic technique using rhombohedral a-Fe20s for the degradation of BPA (Ye C, et al. Controllable synthesis of rhombohedral a -Fe203 efficient for photocatalytic degradation of bisphenol a. J Water Process Eng. 2019;27:205-210). In another study, a powdered activated carbon/chitosan/polyvinyl alcohol hydrogel was produced for the removal of BPA from water (Zhou A, et al. Fabrication ofhydrophobic/hydrophilic bifunctional adsorbent for the removal of sulfamethoxazole and bisphenol A in water. J Environ Chem Eng. 2020;8: 104161).
In addition to the removal of BPA, the development of tests allowing precise BPA measurement is also an important area of research. The methods for the detection of BPA in water and breast milk are based on analytic techniques, including liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography (HPCL) and enzyme-linked immunosorbent assay (ELISA). These methods are ideal for measuring BPA in vitro, however they are insufficient for in situ BPA measurement. Therefore, a number of sensor techniques are continued to be developed in the related technical field (Ragavan KV, et al. Sensors and biosensors for analysis of bisphenol-a. TrAC-Trends Anal Chem. 2013;52:248-260). While in all the other methods listed above, milk samples have to be subjected to pre-treatment by skilled persons, the milk can be analyzed without any pre-treatment when using the sensor technique.
Polymers have an important role in the development of numerous molecular diagnostic and biosensor devices for sensing a medical or environmental analyte. Lateral Flow Test (LFA), which is one of these sensor techniques, is generally used to detect and measure analytes in complex mixtures such as blood and urine. The lateral flow test is also used in other areas such as food safety, drug screening tests and microbiological diagnostic tests, as well as for the detection of pregnancy. These tests are generally used as a qualitative method.
The lateral flow test is a preferred method due to its high sensitivity and selectivity, low cost, long shelf life and high visual results. The sensitivity of the test is important in gold nanoparticle- antibody-based lateral flow tests. Therefore, parameters such as the conjugation of gold nanoparticles and antibody, polymer structure of the membrane, capillary flow rate, and porosity of the membrane should be optimized in the design of lateral flow test strips (Paroloa C, et al. Simple paper architecture modifications lead to enhanced sensitivity in nanoparticle based lateral flow immunoassay. J Mater Chem C. 2015;3:10715-10722; Choi JR, et al. Advances and challenges of fully integrated paper-based point-of-care nucleic acid testing. TrAC-Trends Anal Chem. 2017;93:37- 50; Millipore EMD. Rapid Lateral Flow Test Strips: Considerations for Product Development. Billerica, MA, USA: EMD Millipore Corporation; 2013). In lateral flow tests, color test lines/dots, which are visible depending on the presence of the material to be identified, are generated in the observation window. Before starting the test, there is no visible line/dot in this area, visible color test lines/dots appear due to the formation of antigen-antibody complex. Therefore, studies on lateral flow tests mostly focused on antibody purification, synthesis of different gold nanoparticles, and binding of antibody to the surface of gold nanoparticle (Farrell BO. Immunoassays C. Lateral flow immunoassay. Japanese J Med Mycol. 2016;57(3):J 125).
For the lateral flow test, the pH and antibody concentration need to be optimized to ensure an effective binding of the antibody to gold nanoparticles. The surfaces of gold nanoparticles have a negative charge due to the AuCh complex, and there is electrostatic repulsion between the nanoparticles. When the ion concentration in the solution increases, the particles get closer to each other as the ionic layer on the nanoparticle surface is compressed. When the repulsion is less than the attraction resulting from the London-van der Waals force, the gold nanoparticles are accumulated. The color of the gold solution in turn changes from red to blue. The rate of color change may be determined as the decrease in absorbance at 520 nm and the increase in absorbance at 580 nm. If antibody molecules are physically adsorbed on the gold surface, gold nanoparticles do not aggregate.
US 2007/059678 Al relates to a breast milk detection device. Said device includes a plurality of test strips slidably and removably attached to a test housing. Each test strip is connected to an absorbent section so that a breast milk sample deposited thereon is communicated to the test strips. The device of the relevant document is directed to the detection of substances and allergens such as alcohol and peanut oil in breast milk.
CN 102183642 discloses a quick detection card and a detection method for bisphenol A. It is stated that said detection card belongs to the technical field of phenol content detection in polycarbonate products for food containers. A test strip is arranged in a shell of the quick detection card for bisphenol A; said test strip is formed by sequentially sticking a sample pad, a colloidal gold membrane, a nitrocellulose membrane anda water absorption membrane on a support back plate. Herein, the colloidal gold membrane is a glass fiber membrane containing monoclonal antibody or polyclonal antibody colloidal gold markers specific to bisphenol A.
In the light of the information presented above, it can be seen that there is a need for an in situ, but not in vitro, measurement and detection of BPA in the related technical field. In particular, easy testing of the breast milk for its BPA content, which is known to be harmful to infants, will provide a great development and improvement in the related field. For this purpose, the present invention provides a lateral flow test strip for the detection and/or measurement of bisphenol A in breast milk and a preparation method thereof.
Brief Description of the Invention
In an aspect, the present invention provides a lateral flow test strip comprising a nanofiber in the test membrane for detecting and/or measuring bisphenol A (BPA) in breast milk.
Preferably, the nanofiber comprised in the lateral flow test strip according to the present invention is polycaprolactone/silk fibroin (PCL/SF) nanofiber. The core portion of the PCL/SF nanofiber may comprise polycaprolactone and the shell portion of the PCL/SF nanofiber may comprise mixture of polycaprolactone/silk fibroin.
In another aspect, the present invention provides a method for preparing the lateral flow test strip according to the present invention. Said method comprises the following steps:
- preparing a nanofiber by electrospinning,
- immobilizing the BPA antibody on the nanofiber,
- obtaining a test membrane (4) by coating prepared nanofiber on nitrocellulose,
- preparing a colloidal gold-BPA antibody conjugate,
- preparing a conjugation pad (3) by immobilizing obtained colloidal gold-BPA antibody conjugate on glass fiber,
- combining the sample pad (2), the conjugation pad (3), the test membrane (4) and the absorbent pad (5), and
- obtaining the lateral flow test strip.
Preferably, the nanofiber prepared in the first step of the method is polycaprolactone/silk fibroin (PCL/SF) nanofiber.
The electrospinning process performed in the lateral flow test strip preparation method of the present invention is preferably coaxial electrospinning process.
The process of immobilizing the BPA antibody on the nanofiber performed in the method of the present invention is preferably performed with physical adsorption.
The process of immobilizing the colloidal gold-BPA antibody conjugate on the glass fiber performed in the method of the present invention is preferably performed with impregnation method. In another aspect, the present invention provides a method for the detection and/or measurement of bisphenol A in breast milk, comprising the following steps:
- providing a breast milk sample, and
- determining the amount of BPA in the breast milk sample with the lateral flow test strip according to any one of claims 1 to 3.
Said method preferably also comprises the step of assessing obtained test results by means of a software.
In a further aspect, the present invention provides the use of the lateral flow test strip according to the present invention for the detection and/or measurement of bisphenol A in breastmilk.
In a further aspect, the present invention provides a kit comprising:
- the lateral flow test strip according to any one of claims 1 to 3, and
- a software configured to determine the BPA concentration in a breast milk sample.
Brief Description of the Figures
Figure 1 shows the design of the lateral flow test strip. The breast milk sample (1), sample pad (2), conjugation pad (3), test membrane (4), absorbent pad (5), test line (6a), and control line (6b) are shown on the figure. Herein, the test line comprises the BPA primary antibody, and the control line comprises the BPA secondary antibody.
Figures 2-A to 2-F show SEM images of prepared coaxial nanofibers (PCL: Polycaprolactone, SF: Silk fibroin, DMF: Dimethylformamide, DCM: Dichloromethane).
Figure 3 shows FTIR spectroscopy results of the hybrid nanofibers (Shell: Polycaprolactone /silk fibroin, Core: PCL). The peaks of 2946 and 1720 cm 1 show the characteristic peaks of PCL; and the peaks of 3250, 1650, 1530 and 1240 cm 1 show the characteristic peaks of silk fibroin.
Figure 4 shows the studies carried out to determine the optimum pH and concentration for the conjugation of gold-antibody. The horizontal column shows the antibody concentrations, and the vertical column shows the pH values of the gold solution.
Figure 5 shows the calibration curves and signal intensity ofwater BPA levels on (a) NC membrane and (b) coaxial PCL/SF nanofiber membrane (C: Color intensity of the control line, T: Color intensity of the test line, NC: Nitrocellulose, PCL: polycaprolactone, SF: silk fibroin). Figure 6 shows the calibration curves and signal intensity of breast milk BPA levels on (a) NC membrane and (b) coaxial PCL/SF nanofiber membrane (C: Color intensity of the control line, T: Color intensity of the test line, NC: Nitrocellulose, PCL: polycaprolactone, SF: silk fibroin).
Detailed Description of the Invention
BPA is a chemical known to pass to the breast milk in significant concentrations and adversely affect the development of infants and children. Although methods have been developed for the removal and detection of BPA in the related technical field, the vast majority of these methods are carried out in vitro. Another problem with the development of a test strip for breast milk is that breast milk is a body fluid with different characteristics compared to the other body fluids. Since it is opaque and creates a background, it mostly requires pre-treatment.
For these reasons, the purpose of the present invention is to provide a highly sensitive lateral flow test strip for the detection and/or measurement of bisphenol A (BPA) in breast milk. For this purpose, conventional nitrocellulose test membrane in the lateral flow test strip is coated with a nanofiber material, preferably with a nanofiber the inner portion (core) of which consists of polycaprolactone (PCL) and the outer portion (shell) of which consists of mixture of PCL/silk fibroin (SF).
The use of nitrocellulose in test membranes is common in the related technical field. Within the scope of the present invention however, the nanofiber material is coated on a support material (nitrocellulose, nonwoven, etc.). Said nanofiber is preferably a polycaprolactone and silk fibroin- based nanofiber. With the present invention, polycaprolactone/silk fibroin-based nanofiber is included as a test membrane in a test strip for the first time.
Since breast milk passes rapidly through the conventional test membrane, it cannot react, making it difficult to obtain a clear result. When the passage of the sample through the test membrane is reduced by using a nanofiber, preferably polycaprolactone/silk fibroin-based nanofiber, time for the reaction (BPA-BPA antibody interaction) to occur is prolonged. In other words, with the nanofiber coating used in the present invention, it is aimed to reduce the flow rate of breast milk through the test membrane, to prolong the reaction time, and thereby to detect the presence and/or amount of BPA with high accuracy.
The novel lateral flow test strip according to the present inventionfor detecting and/or measuring bisphenol A in breast milk is advantageous as it is a product that allows in situ detection. Using said test strip, it will be possible for breastfeeding mothers to detect the BPA content in their milk in an effective, quick and reliable manner without a need for any pre-sorting process. In another aspect, the present invention provides a method for the preparation of the lateral flow test strip of the invention, comprising the following steps:
- preparing a nanofiber by electrospinning,
- immobilizing the BPA antibody on the nanofiber,
- obtaining a test membrane (4) by coating prepared nanofiber on nitrocellulose,
- preparing a colloidal gold-BPA antibody conjugate,
- preparing a conjugation pad (3) by immobilizing obtained colloidal gold-BPA antibody conjugate on glass fiber,
- combining the sample pad (2), the conjugation pad (3), the test membrane (4) and the absorbent pad (5), and
- obtaining the lateral flow test strip.
The nanofiber prepared in the method of the present invention is preferably a nanofiber comprising polycaprolactone and silk fibroin (PCL/SF). PCL/SF nanofiber is included in a preparation method of lateral flow test strip for the first time with the present invention and is advantageous in that it allows for adjustment of the reaction time required to detect bisphenol A in breast milk, more clear observation of the color change and much more sensitive detection of change in bisphenol A concentration compared to the conventional method.
While the nanofiber prepared in the method of the present invention may be prepared using normal or coaxial electrospinning, it is preferably prepared by coaxial electrospinning.
Also within the scope of the invention, BPA antibody is immobilized on nanofiber, preferably silk fibroin and caprolactone-based nanofiber. In other words, a nanofiber-based immunosorbent surface is obtained. The primary and secondary antibodies are successfully immobilized on the prepared nanofiber. The antigen (BPA)/antibody (BPA antibody) complex is detected based on the color change principle due to colloidal gold.
The BPA antibody is physically immobilized on the nanofiber, preferably by physical adsorption. The nanofiber is then coated on nitrocellulose and used as a test membrane in LFA. Color changes in the test membrane are assessed as the signal intensity of BPA.
The gold nanoparticle-antibody conjugate is prepared under conditions which were optimized with the studies carried out within the scope of the present invention. Provision of a sensitive conjugate enhances the recognition of the antibody-coated gold nanoparticles. The conjugation conditions of colloidal gold and antibody were determined as described in detail in the Examples below. The process of immobilizing the colloidal gold-BPA antibody conjugate on the glass fiber in the method of the present invention is preferably performed with impregnation method. Said colloidal gold comprises gold nanoparticles.
In another aspect, the present invention provides a method for the detection and/or measurement of bisphenol A in breast milk. Said method comprises the following steps:
- providing a breast milk sample, and
- determining the amount of BPA in the breast milk sample with the lateral flow test strip according to any one of claims 1 to 3.
The test results obtained from the test strip of the present invention can be interpreted by a software which is developed specifically for BPA in breast milk and is configured to determine the BPA concentration in the breast milk sample. Said software is based on the principle of correlating the color change in the test membrane with the BPA concentration. Assessment of the test results by a software is advantageous in that it prevents user-induced interpretation errors.
In another aspect, the present invention provides the use of the lateral flow test strip according to the present invention for the detection and/or measurement of bisphenol A in breastmilk.
The present invention also provides a kit comprising:
- the lateral flow test strip according to any one of claims 1 to 3, and
- a software configured to determine the BPA concentration in a breast milk sample.
Studies for the preparation of the lateral flow test strip of the present invention and the tests performed are described in detail in the Examples below.
EXAMPLES
1. Coating of nitrocellulose (NC) membrane by coaxial electrospinning
The core portion of the coaxial nanofiber was prepared using PCL, and the shell portion was prepared using mixture of PCL/SF. PCL solution (15% weight/volume) was dissolved in dimethylformamide (DMF) by stirring for 3 hours at room temperature. In order to prepare PCL/SF solution (10% weight/volume); powdered SF (Hydrolysis SF Powder, 500-10.000 g/mol), PCL (60.000 g/mol), DMF and methylene chloride (DCM) were used. PCL/SF solution containing 1:1 PCL:SF by weight was prepared with a 1:4 mixture of DMF:DCM by stirring atroom temperature for 24 hours. Hybrid nanofiber was obtained by electrospinning using a coaxial needle for forming the outer shell (PCL/SF) and core (PCL) of the coaxial nanofiber. Electrospinning syringe was connected to a pump at a flow rate of 1 ml/hr for PCL and 2 ml/hr for PCL/SF. Drum rotation speed was setto 100 rpm. PCL (core)-PCL/SF (shell) coaxial nanofibers were obtained at 27 kV with a distance of 15 cm from the nozzle to the collector. Electrospinning was carried out in air at 30°C for 1 hour.
With the present invention, it was aimed to improve the surface properties of the test membrane and thus the antibody immobilization conditions, and to reduce the capillary flow rate by coating the NC with coaxial electrospun nanofiber. The reason for using PCL in the test membrane is to increase the mechanical properties of PCL/SF-based shell portion of the coaxial nanofiber. The mixture of PCL/SF was used for increasing hydrophobicity and thereby reducing the fluid flow. As the amount of coaxial nanofiber on the NC membrane increases, the hydrophobicity in the coated region increases.
2. Scanning Electron Microscopy (SEM)
The morphology of the coaxial nanofiber was studied using SEMoscope Tabletop Compact SEM model (INOVENSO, Turkey). Nanofiber images obtained by applying different conditions (polymer preparation and voltage) are presented in Figure 2. A beaded structure was observed when formic acid was used in the preparation of the shell portion of the coaxial nanofiber (Figure 2a). DCM and DMF solutions were mixed in different ratios to prepare the shell portion of the coaxial nanofiber (Figure 2b and 2c). The beads were observed again on the nanofibers obtained by mixing DMF and DCM at ratio of 1:2 and 1:3. No beads were formed when a 1:4 mixture of DMF:DCM was used. After the solvent mixing conditions were determined, the effect of different electric field strengths was tested to obtain the best electrospinning conditions. Figures 2d to 2f show SEM images of the prepared coaxial nanofiber at different voltages. Homogeneous and bead- free nanofibers were obtained with a core of 15% PCL and a shell of 10% PCL-SF (1:4 DMF:DCM) at a voltage of 27 kV (Figure 2f).
3. Fourier Transform Infrared Spectroscopy (FT-IR)
The chemical structure of the nanofiber was determined by FTIR-IR Tracer (SHIMADZU, Japan). The spectra were recorded in a wavelength range of 600 to 4.000 cm 1. C-H planar vibration band at 2946 cm 1 and C=O tension at 1720 cm 1 were observed in the FT-IR spectrum of the nanofiber. These peaks prove the presence of PCL in the structure of the coaxial nanofiber. Also in the FT-IR spectrum, there was a NH stretch at 3250 cm 1, and the absorption bands of amide I (C=O stretch) at 1650 cm 1, amide II (secondary NH bending, p-sheet structure) at 1530 cm 1 and amide III (CN and NH functional groups) at 1240 cm 1. These peaks indicate the presence of SF in the coaxial nanofiber (Figure 3). 4. Collection and storage of breast milk samples
The breast milk samples were obtained voluntarily from healthy breastfeeding mothers. The Medical Ethics Committee of Marmara University approved the breast milk collection and experimental stages of the studies of the invention. Collected breast milk samples were stored in BPA-free breast milk bags (Lansinoh-Breastmilk Storage Bags, Japan) at -20°C for a maximum of 3 months after BPA determination.
5. BPA determination in water and breast milk samples
BPA concentrations of breast milk samples were determined using ELISA method (Ecologenia Abraxis, Japan).
6. Preparation of BPA solution and water samples
A stock solution of 100.000 ng/ml BPA (Sigma-Aldrich, St. Louis, Missouri) was prepared with 10% methanol. BPA-free water was used in the preparation of BPA solutions. Doped water samples in the range of 0-10 ng/ml were prepared by diluting the stock solution of BPA.
7. Preparation of breast milk samples
Control samples (blank samples) were selected and pooled from breast milk samples containing BPA in the range of 0-0.5 ng/ml. Breast milk samples with a BPA level above 0.5 ng/ml were not used. The breast milk pool was divided and stored at -20°C in BPA-free storage bags. Doped breast milk samples in the range of 0-10 ng/ml BPA were prepared by diluting the stock solution with the control breastmilk samples.
8. Preparation of a colloidal gold-antibody conjugate
The optimum conditions (pH, antibody concentration) for the conjugation of gold nanoparticles (30 nm, Sigma-Aldrich, St. Louis, MO) and antibody (BPA rabbit polyclonal antibody, Origene, MD) were determined by the method of Zhou et al. (Zhou C, et al. Rapid detection of chloramphenicol residues in aquatic products using colloidal gold immunochromatographic assay. Sensors. 2014;14:21872-21888). Conjugation conditions of the gold nanoparticles and BPA antibody were determined with a slight modification to the method described by Zhao et al. (Zhao K, He W, Bi f et al. Development of a lateral flow immunochromatographic assay for the rapid diagnosis of Orf virus infections. J Virol Methods. 2016;236:10-17). Colloidal gold nanoparticles (30 nm) and BPA antibody (50 pg/ml) were incubated for 2 hours at room temperature. Bovine serum albumin (BSA) (10%) was then added to this mixture and incubated at room temperature for 10 minutes. The mixture was centrifuged (l.OOOxg) for 10 minutes at +4°C, and the supernatant was centrifuged (3.000xg) for 10 minutes at +4°C. The pellet was suspended in 0.01 M Borate buffer (pH 8) containing 1% BSA and 5% sucrose. Conjugate Control and Go kit (Expedon, UK) was used to check whether the antibody binds to the surface of the gold nanoparticles. Obtained gold nanoparticle-antibody conjugates were stored at +4°C for use in the lateral flow test
As shown in Figure 4, the pink (dark) color remained unchanged in the solutions at pH 5 and 6 and in the BPA antibody concentrations of 15.6 pg/ml and higher. AtpH 7, 8 and 9, 31.5 pg/ml of antibody was found to be sufficient for the conjugation of BPA antibody and gold nanoparticles. In this study, gold nanoparticles (30 nm) and 50 pg/ml BPA antibody were conjugated at pH 9.
Conjugation conditions of gold nanoparticles and antibody compatible with the prepared coaxial nanofiber were determined by optimizing pH and antibody concentration, as well as sucrose and albumin concentrations. In studies within the scope of the present invention, pH 9 was found to be suitable for the optimal conjugation of BPA antibody and gold nanoparticles. Isoelectric point for the immunoglobulins of various species is in the range of pH 8 to 9. However, pH value optimization should be tested for the conjugation. Optimum antibody concentration is critical to stabilize the gold nanoparticles without aggregation. Within the scope of the present invention, optimum BPA antibody concentration was found to be 50 pg/ml. Usually, a concentration which is 50% higher than the critical concentration is used for the antibody-gold nanoparticle conjugation. BSA is used as a secondary stabilizer to stabilize the surface of the gold nanoparticle and prevents flocculation. After antibody binds to the surface of the gold nanoparticles, itprevents non-specific binding of other molecules to the remaining cavities. Sucrose is used to prevent the surface of the colloidal gold from adhering to the test membrane when the membrane is dried.
9. Preparation of the lateral flow test strip
The components of the lateral flow test strip (sample pad (2), conjugation pad (3), membrane (4), and absorbent pad (5)) were combined on adhesive rear surface of the card and prepared for sample application. Glass fiber (Millipore, MA) based sample pad (0.3 x 2 cm) was incubated with tris (hydroxymethyl) aminomethane (TRIS) (Sigma-Aldrich, St Louis, MO) buffer (pH 9) containing Tween-20 at room temperature. Conjugate pad (0.3 x 0.5 cm) was prepared by immobilizing colloidal gold-BPA antibody conjugate on a glass fiber. Colloidal gold-BPA antibody conjugate was diluted with 20 mM phosphate buffer solution (PBS) containing sucrose and BSA (Sigma-Aldrich) (1:1, volume/volume). BPA antibody was also immobilized as a spot on hybrid nanofiber by physical adsorption method to form the test membrane (0.3 x 2.5 cm) (Zhao K, He W, Bi f et al. Development of a lateral flow immunochromatographic assay for the rapid diagnosis of Orf virus infections. J Virol Methods. 2016;236:10-17). Hybrid nanofiber was treated with PBS containing BSA and dried. Cellulose fiber (Millipore) was used as absorbent pad. This part functions as a waste collection part The size of the paper-based absorbent pads was 0.3 x 1.5 cm. Test and control lines (6a and 6b) were prepared as shown in Figure 1. Herein, test line comprises BPA primary antibody, and control line comprises the BPA secondary antibody. 50 pl of water and breast milk samples (1) were used as samples on the prepared lateral flow test strips. The results of using hybrid nanofibers as the test membranes were compared with those prepared using NC membranes (Sartorius, France, pore size 0.45 pm).
10. Interpretation of the lateral flow test results and comparison of the test strips
For quantitative measurements, high resolution test photos were generated (using a Canon EOS 700D camera with 18-55 lens) to read the BPA levels in the LFA, and the images were exported as jpeg files. RGB values were detected at test and control points using Image) software (Schneider C, Rasband W, Eliceiri K. NIH image to ImageJ:25 years of image analysis. Nat Methods. 2012;9:671- 675). In the RGB system, all colors have a value ranging from 0 (black) to 255 (white). When the value of all three components in the RGB system is 255, it is pure white; when the RGB value is 0, it is pure black. Dark colors have a lower value than light colors in the RGB system. Color intensities in test and control lines are presented as RGB values.
Capillary flow rate is important as it is another optimizing parameter in LFAs. Capillary flow rate is the rate at which the sample moves across the membrane. It decreases linearly as the porosity decreases. Therefore, within the scope of the present invention, the lateral flow rates of water and breast milk samples were determined as explained in detail below.
Lateral flow test for BPA in water and breast milk samples were characterized by the detectable concentrations in the range of 0-10 ng/ml. BPA in 0-2-4-6-8-10 ng/ml water and breast milk samples was run on the lateral flow test strips in which the test membrane is (a) NC or (b) coaxial electrospun nanofiber coated NC. Lateral flow rate of water samples in the test strips comprising NC membrane or coated NC membrane was 0.054 cm/s. Pink (dark) color appeared at BPA concentration of 2 ng/ml in the water samples on both NC and coated NC test membrane. The intensity of pink (dark) color remained unchanged at the BPA concentrations above 8 ng/ml when PCL/SF-based test membrane is used. When NC-based test membrane is used as the test membrane, the intensity of pink (dark) color remained unchanged at BPA concentrations above 4 ng/ml (Figure 5).
Breast milk samples were run on (a) NC-based test strip in 0.036 cm/s and (b) coated NC-based test strip in 0.022 cm/s. The reason for the increased flow rate is the reduced pore size of the test membrane, which is composed of hybrid nanofibers. The intensity of pink (dark) color did not change as the breast milk BPA concentration increased in both test strips, and the intensity of pink (dark) colors was greater in the test strip made of PCL/SF nanofiber (Figure 6). PCL/SF coating caused a decrease of 0.054 cm/s in breast milk flow rate as compared to uncoated NC membrane. This decrease increases the biomolecular interaction between BPA in breast milk and BPA antibody in the test membrane. Therefore signal intensity of the test line increased. The porosity of NC was increased by means of nanofiber coating as it was aimed to reduce the flow rate of breast milk on test strip. Electrospun-coated NC delayed the flow, thereby increasing the interaction ratio between BPA and the antibody thereof, and irreversibly trapping BPA antibody in the lateral flow test strip. Since the effective analyte concentration of the sample is inversely proportional to the square of the flow rate, the flow rate decreased with the coaxial electrospun coated NC membrane. The polymer structure of the test membrane demonstrates the protein binding properties of the membrane.
The background formed by the breast milk on the surfaces due to the structural properties thereof was determined by comparing the signal intensity thereof with the signal intensity of water. Water samples were found to have higher signal intensity as compared to the breast milk samples. Although the detection limit was 2 ng/ml in both coaxial PCL/SF nanofiber and nitrocellulose (NC) test membranes, the color intensity was observed to increase with increasing BPA concentration in coaxial PCL/SF nanofiber. In other words, coaxial PCL/SF nanofiber provided a higher color intensity and more visible results than NC membrane. Consequently, with the present invention, a novel and sensitive lateral flow test strip and a sensitive in situ method for the detection and/or measurement of BPA in breast milk have been developed using a nanofiber material, preferably coaxial PCL/SF nanofiber coating, in the test membrane of the lateral flow test strip.

Claims

1. A lateral flow test strip for detecting and/or measuring bisphenol A (BPA) in breast milk, wherein the test membrane comprises a nanofiber.
2. The lateral flow test strip according to claim 1, wherein said nanofiber is polycaprolactone/silk fibroin (PCL/SF) nanofiber.
3. The lateral flow test strip according to claim 2, wherein the core portion of said nanofiber comprises polycaprolactone and the shell portion of said nanofiber comprises a mixture of polycaprolactone/silk fibroin.
4. A method for preparing the lateral flow test strip according to any one of the preceding claims, wherein the method comprises the steps of:
- preparing a nanofiber by electrospinning,
- immobilizing the BPA antibody on the nanofiber,
- obtaining a test membrane (4) by coating prepared nanofiber on nitrocellulose,
- preparing a colloidal gold-BPA antibody conjugate,
- preparing a conjugation pad (3) by immobilizing obtained colloidal gold-BPA antibody conjugate on glass fiber,
- combining the sample pad (2), the conjugation pad (3), the test membrane (4) and the absorbent pad (5), and
- obtaining the lateral flow test strip.
5. The method according to claim 4, wherein the nanofiber prepared in the first step of the method is polycaprolactone/silk fibroin (PCL/SF) nanofiber.
6. The method according to claim 4, wherein electrospinning is axial electrospinning.
7. The method according to claim 4, wherein the step of immobilizing the BPA antibody on the nanofiber is performed by physical adsorption.
8. The method according to claim 4, wherein the step of immobilizing the colloidal gold-BPA antibody conjugate on the glass fiber is performed by impregnation method.
9. A method for detecting and/or measuring bisphenol A in breast milk, comprising the steps of: providing a breast milk sample, and determining the amount of BPA in the breast milk sample with the lateral flow test strip according to any one of claims 1 to 3.
10. The method according to claim 9, wherein the method further comprises the step of assessing obtained test results by means of a software.
11. Use of the lateral flow test strip according to any one of claims 1 to 3 in the detection and/or measurement of bisphenol A in breast milk.
12. A kit comprising:
- the lateral flow test strip according to any one of claims 1 to 3, and
- a software configured to determine the BPA concentration in a breast milk sample.
PCT/TR2022/050577 2021-10-08 2022-06-13 A lateral flow test strip for detection and/or measurement of bisphenol a in breast milk WO2023059283A1 (en)

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GÜREL‐GÖKMEN BEGÜM, TASLAK HAVA DUDU, ÖZCAN OZAN, İPAR NECLA, TUNALI‐AKBAY TUĞBA: "lateral flow test strip for quick and facile determination of bisphenol A in breast milk", JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART B: APPLIED BIOMATERIALS, vol. 109, no. 10, 1 October 2021 (2021-10-01), US , pages 1455 - 1464, XP093061092, ISSN: 1552-4973, DOI: 10.1002/jbm.b.34805 *

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