Chapter-3 Gold nanoparticle based immuno-dipstick biosensing...

Gold nanoparticle based immuno-dipstick biosensing for analysis of vitamin B12 Chapter 3

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Chapter-3

Gold nanoparticle

based

immuno-dipstick biosensing

for analysis of vitamin B12


99

3.1. INTRODUCTION

Developing field applicable methodology for detection of various analytes in

food samples at a high sensitivity is crucial for ensuring food safety and quality

control. An immunological method is one of the promising tool for the development

of easy-handling biosensors. Besides, immunoassay methods are sensitive, cost-

effective, easy to perform, and require a small sample volume (Sherry, 1997).

However, such techniques often require long reaction times and involve multiple

steps (Paek et al., 2000). The utilization of these immunoassays has been mostly

confined to laboratories equipped with tools and devices for analysis. Therefore,

there is need to develop rapid and field applicable techniques for analysis. The

convenience and speed of the test have been achieved by a novel concept of

immunodipstick that depends on the transportation of a reactant to its binding

partner immobilized on a membrane surface (Lisa et al., 2009). It combines

several benefits including a user friendly format, short assay time, long-term

stability and cost-effectiveness. These characteristics make it ideally suited for on-

site screening by people who are not skilled analysts (Cho et al., 2005). In the

present work, we report on a sensitive and rapid immunodipstick colloidal gold-

antibody probe for the detection of vitamin B12 in various food samples.

Metal nanoparticles have attracted tremendous attention due to their

unusual behaviors compared with corresponding bulk materials (Daniel, 2004).

Gold nanoparticles (GNPs) are one of the popular nanomaterials, widely used in

biosensing applications. It has unique optical and electronic properties because of

their high surface area that make them suitable in bioassays (Liu and Wong,

2009). GNPs based immunoassay has been developed and applied in various

research fields such as for the detection of hormones (pregnancy, fertility),

pesticides (Lisa et al., 2009) and bacteria (Idegami et al., 2008). Moreover, their

optical properties make them well-suited for optical detection. They are readily

bioconjugated to various ligands, such as antibodies, DNA, and aptamers. GNPs

are more stable and easy to use than the conventional systems utilizing

fluorescence or enzymatic labels. Moreover, nanoscale surfaces of GNPs are


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appropriate for accelerating the antibody–antigen recognitions, which enhances

the immunoassay signals (Richars, 1996). After the antigen–antibody reaction,

the red color caused by the accumulation of GNPs at that location, appears on

the membrane (An et al., 2001). Through these detection processes, the results

are obtained within few minutes after the sample introduction.

Vitamin B12 is one such analyte need to be detected at this hour. Vitamin

B12 belongs to the B vitamin group and prevents pernicious anemia, which is

caused by vitamin B12 deficiency. Conventional methods are time-consuming,

tedious, less safe, less sensitive, and expensive. So there is an urgent need for

the development of onsite, rapid, simple, efficient and cost effective method for the

detection of vitamin B12 in food samples. We used hens as immunization hosts to

produce vitamin B12 IgY antibody against a derived form of vitamin B12 by

immunizing a hen with vitamin B12 conjugated to a carrier protein, Keyhole Limpet

Hemocyanin as described in chapter section 1.3.1. The sensitivity and specificity

of IgY antibodies was checked using ELISA. In present work, vitamin B12 IgY

antibodies in conjugation with GNPs were utilized for the detection of the vitamin

B12 as dipstick format. GNPs of definite size were synthesized and bioconjugated

for dipstick based immunoassay.

3.2. MATERIALS AND METHODS

3.2.1. Materials.

All the reagents were analytical grade and used without further purification.

Double distilled water (DDW) was used throughout this work. Nitrocellulose

membrane (NC) (dimensions: 6 X 0.5cm) containing polymer strips of definite

thickness (0.45 μm) was procured from Advanced Micro Devices Pvt. Ltd, Ambala

Cantt, Punjab, India. Vitamin B12, Bovine serum albumin (BSA), Gold (III)

chloride,tri sodium citrate, EDC [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide]

and gelatin were procured from Sigma Aldrich chemicals, USA. Skim milk powder

procured from Lobachemie Pvt. Ltd. Bangalore, India. Vitamin B12 IgY antibody


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production and Bovine serum albumin –vitaminB12 conjugate were prepared in

Central Food Technological Research Institute, Mysore, India.

3.2.2. Instruments.

Spectral analysis of GNPs was recorded in the range from 300 to 800 nm

using spectrophotometer UV-1601 (Shimadzu, Japan), and a characteristic

surface plasmon resonance (SPR) peak was noted. Particle size characterization

of synthesized GNPs was recorded using Transmission Electron Microscopy

(TEM) (Jeol 2100, USA). Chrystal characterization of synthesized GNPs powder

were recorded in the range from 30 to 80 degree (2θ) using Philips PW 1140

diffractometer (Bragg-Brantoto Geometry, Brucker, USA). The characteristic peaks

were noted and compared with standard peaks. Fourier-transform infrared (FTIR)

spectra, measured by KBr pellets containing GNPs, were obtained in the range of

4000–400 cm−1 using Avatar 360 FTIR spectrometer (Nicolet Instrument Corp.,

Madison, MI). Fluorimetric assay was done with the help of Spectrofluorometer

RF-5301 PC (Shimadzu, Japan). ELISA was performed in microtitre plate

(Tarsons, India) and ELISA-based analysis was carried out using a Versa Max

tunable micro-plate reader (Molecular Devices, USA). Agarose gel electrophoresis

was performed using Genei apparatus (Genei, Banglore, India).

3.2.3. Experimental set-up.

The principle is competitive binding between the conjugate and antibody in

which BSA-B12 conjugate was immobilized on NC membrane strips followed by

dipping in eppendorf tubes containing immunocomplex of free vitamin B12 and

GNPs-conjugated IgY antibodies, and the color development was monitored.

Concentration of vitamin B12 was indirectly proportional to color development on

the NC membrane containing strips. Schematic diagram of the GNPs-based

dipstick immunoassay is shown in (Scheme 3.1).


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Scheme 3.1. Scheme showing the color development in NC strip in the absence

and presence of vitamin B12.

3.2.4. GNPs Synthesis.

An aqueous solution of monodisperse quasi-spherical GNPs were prepared

by modified turkevitch et al., method (Turkevitch et al., 1951; Xia et al., 2010). A

total of 45 mL of Milli-Q water was taken in a reaction flask and refluxed for 10 min

with 5 mL of 0.1% Tetrachloroauric acid (HAuCl4), 2 mL of 1% trisodium citrate

and 42.5 μL of silver nitrate. Tetrachloroauric acid, trisodium citrate and silver

nitrate were mixed together in a separate beaker and incubated for 5min and

added drop by drop into the reaction flask. The reduction of gold metal ions (Au3+)

to yield GNPs (Auo) was confirmed by the appearance of dark cherry red color.

Colloidal GNPs were stored at 4°C. Colloidal gold solution was centrifuged then


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dried and subjected to characterization using various physico-chemical

techniques.

3.2.5. Bioconjugation of GNPs with proteins.

GNPs synthesized as above were conjugated with vitamin B12 antibodies

(IgY) by reacting 10 mL colloidal gold solution of pH 9.0 with 400 µg of IgY. IgY

was added drop by drop with gentle stirring. After overnight incubation at 4°C the

mixture was centrifuged at 10,000 rpm for 30 min at 4ºC. The pellet was

resuspended in minimum amount of storage buffer (1mM phosphate buffer of pH

7.4 with 0.05% Tween-20). The binding of GNPs to IgY was confirmed by the

absorption spectrum analysis of the pure GNPs and GNPs conjugated IgY in the

range of 300–700 nm.

3.2.5.1. Agarose gel electrophoresis.

To find out the bioconjugation efficiency of GNPs with proteins (BSA and

IgY) agarose gel electrophoresis was performed. A total of 50µL of concentrated

GNPs, GNP-BSA and GNP-IgY were loaded in wells of 1% agarose gel in 1X tris-

borate-EDTA buffer and ran for 2-3 h with a power supply of 80 V. Gel images

were captured on a SONY digital camera (Coolpix) equipped with a 6 mega pixel

CCD chip and transferred to computer using standard camera software.

3.2.5.2. Flocculation assay.

Flocculation assay was done to determine and optimize the minimum

amount of IgY needed to stabilize the GNPs. Varying concentrations of IgY were

prepared from the stock solution. To 1 ml of colloidal gold solution 100 µL of IgY

dilution was added and incubated for 15 min. Flocculation was induced by adding

100µL of 10% NaCl and the absorbance was scanned from 400 to 800 nm. The

amount of IgY required to prevent flocculation was found at the point where

absorbance is nearly constant with increasing concentrations of IgY. To determine


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the amount of IgY bound to GNPs different concentrations of IgY was prepared

and absorbance was measured for each dilution at 280 nm. One milliliter of

colloidal gold solution was added to each dilution and incubated with mild shaking

at RT for 2 h. After incubation the mixture was centrifuged at 12,000 rpm for 30

min and the supernatant was collected separately. Absorbance of the collected

supernatant was measured at 280 nm and subtracted from the initial absorbance

to find the amount of IgY bound to GNPs.

3.2.5.3. Critical flocculation concentration (CFC).

CFC of GNP-IgY was found by adding 100 µl of increasing concentrations

of NaCl (0.01-1 M) to 1 ml of bioconjugates (GNP-IgY). The mixture was incubated

at RT for 1 h with mild shaking. Aggregation was assessed by monitoring changes

in the characteristic GNPs plasmon frequency. The threshold NaCl concentration

that caused the aggregation of particles in colloidal gold solution was determined

as CFC.

3.2.5.4. Fluorimetric assay.

Spectrofluorimetric assay was done to determine the binding constant of

IgY to GNPs. 100 µg/ml of IgY solution was mixed with different concentrations of

GNPs and the mixture was incubated at RT for 2 hrs. Fluorescence of

bioconjugate (GNP-IgY) solution was measured between 300 and 500 nm with an

excitation at 290 nm. Binding constant was calculated as reported previously

(Tedesco et al., 2004).

3.2.5.5. Fourier-transform infrared spectroscopy (FTIR).

FTIR spectra of IgY as well as GNPS-IgY were measured using Avatar 360

FTIR spectrometer in KBr. The IgY and GNPs-IgY were dried in lyophilizer prior to

their use for FTIR analysis. Spectra was obtained at RT.


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3.2.6. Enzyme-linked Immunosorbent Assay (ELISA) for vitamin B12

determination.

ELISA was done as reported in our previous publication and absorbance

was measured at 405 nm in an ELISA microplate reader as described in chapter 1

Section 1.2.6.

3.2.7. GNP-based immunodipstick assay

3.2.7.1. Immobilization of BSA-vitamin B12 on dipstick.

Three different concentrations of BSA-vitamin B12 were immobilized from

0.5, 1.0, and 2.0 µg/µl by direct spotting on to the surface of NC membrane and

dried at room temperature (RT) for 30 min. The immobilized strips were blocked by

5% skim milk powder solution at 37°C for 1 h followed by washing with 1 mM

phosphate buffer (pH 7.4). Strips were dipped in optimized concentration of GNPs-

IgY. The intensity of color development was observed.

3.2.7.2. Optimization of GNP-IgY concentration.

An optimized concentration of BSA-vitamin B12 was immobilized by direct

spotting on to the surface of NC membrane and dried at RT for 30 min. The

immobilized strips were blocked by 5% skim milk powder solution at 37°C for 1 h

followed by washing with 1 mM phosphate buffer (pH 7.4). The strips were dipped

in different dilutions of GNPs-IgY 1:5, 1:10, 1:20 from the stock of 0.4 mg/ml to a

final volume of 400 μl. The intensity of color development was observed.

3.2.7.3. Detection of vitamin B12 using immunodipstick.

An optimized concentration of BSA-vitamin B12 was immobilized by direct

spotting on to the surface of NC membrane and dried at RT for 30 min. The

immobilized strips were blocked by 5% skim milk powder solution at 37°C for 1 h

followed by washing with 1 mM phosphate buffer (pH 7.4). The strips were dipped

in optimized concentration of GNPs-IgY containing 100 μl of different


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concentration of vitamin B12 from 1 to 1000 ng/ml to a final volume of 400 μl. The

intensity of color development was observed.

3.2.8. Spiked real sample analysis.

Three different fruit drinks and three different labeled energy drinks were

bought from local market. Fruit drinks were fortified with three different

concentrations of vitamin B12 from 1, 10 and 100 ng/ml for its accuracy before and

after addition of vitamin B12. Reproducibility of developed GNPs-based

immunodipstick was checked and well compared with conventional ELISA method.

3.3. RESULTS AND DISCUSSION

3.3.1. Preparation and characterization of GNPs

During the preparation of colloidal gold, tetrachloroauric acid was reduced

due to the transfer of electrons from the carboxyl group of tri-sodium citrate and

Au3+ ion leads to the formation of Au0. This metallic gold then nucleates and grows

to form GNPs, and is subsequently capped and stabilized by the citrate ions. The

concentration of tri-sodium citrate used in its synthesis dictates the size of GNPs

and reduces gold chloride to GNPs (Wang et al.,2005). The change in color of the

gold colloid is shown in (Figure 3.1). The color of the solution changes from pale

yellow to metallic cherry red color. Minute concentration of silver nitrate was

added in order to get uniform nucleation during the preparation of GNPs.

Figure 3.1. Change in color of the gold sol during preparation.


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3.3.2. Spectrophotometry.

The size and concentration of GNPs were calculated using the UV-vis

spectra of GNPs as shown below (Haiss et al., 2007).

From the spectrophotometric data the following values were obtained,

λSPR = 504nm

A450=0.914

ASPR=1.495

Diameter of the GNPs = (ASPR/ A450)

The ratio of absorbance of GNPs at the SPR peak (ASPR) to the absorbance

at 450 nm (A450) was calculated and found to be 1.6356 which corresponds to a

GNPs size (diameter) of 14-16 nm, so average size of 15 nm is taken for further

studies. The concentration of the GNPs was calculated using the formula

C = (A450/ε450)

where, ε450 is the molar decadic extinction coefficient at 450 nm. And the

average value of ε450 was found to be 2.203 x 108 M-1 cm-1. By substituting the

value in the above equation we get the concentration of the gold sol as 4.19 x 10-9

M. Average size and concentration of the gold sol were found to be 15 nm and

4.19 x 10-9 M. Size and shape of the resulted GNPs were further confirmed using

TEM micrograph (Figure 3.2) with diameter of about < 20 nm and provided strong

evidence of formation of uniform quasi-spherical GNPs during synthesis. The

surface plasmon resonance (SPR) peak of freshly synthesized GNPs was

observed at 504 nm as shown in (Figure 3.2).


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Figure 3.2. Graph showing SPR peak of GNPs and shift in SPR peak after GNP

conjugation with IgY. The relative SPR was measured spectrophotometrically

between 300 and 700 nm. Inset shows TEM micrographs of citrate capped GNPs.

Particles are approx.< 20 nm in diameter, and are fairly quasi spherical. The scale

bar in the inset corresponds to 20nm.

3.3.3. X-ray diffraction (XRD).

The precipitate obtained after centrifugation of colloidal gold was confirmed

for its crystalline nature using XRD (Figure 3.3). Several peaks are observed,

these being at 38.2°, 44.5°, 64.7° and 77.6°, which correspond to the {111}, {200},

{220} and {311} facets of the face-centred cubic (FCC) crystal structure of gold,

respectively. The observation of diffraction peaks for the GNPs indicates that

these are crystalline in this size range, while its broadening is related to the


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particles in the nanometer size regime. Similar observation was made by few

groups (Sun et al., 2005, Luo, 2006).

Figure 3.3. XRD pattern of precipitate obtained from centrifuging colloidal gold

thus formed.

3.3.4. Bioconjugation of GNPs with proteins

3.3.4.1. Spectral analysis.

The synthesized GNPs were capped with citrate layer, which imparts them

a net negative charge that can be targeted for bioconjugation based on charge-

coupled interactions (Scheme 3.2). Electrostatic interactions of GNPs are possible

with antibodies (Frens, 1973). As the isoelectric point of antibodies is around pH

9.0 where they exist in zwitter-ion conditions, the positive amino terminals of IgY

were targeted for conjugation with GNPs by adjusting the pH of GNPs to pH 9.0

with 0.1 M K2CO3. Further, the conjugation of GNPs to IgY was confirmed by the

spectrum analysis of the pure and conjugated GNPs in the range of 300–700 nm

and observed a red shift in the peak of about 6 nm from 504 to 510 nm (Figure


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3.2). This shift is due to the conjugation of double bonds lowering the energy

required for the electron transitions and hence causing an increase in the

wavelength. The shift in absorption peak is an evidence for the confirmation of

GNPs bioconjugation with IgY. It was reported that nanoparticles can show shift in

their absorption as a result of bioconjugation due to the change in their overall

structure and spatial closeness of biomolecules and nanoparticles (Vinayaka et al.,

2009).

Scheme 3.2. Scheme showing the nucleation of GNPs and its electrostatic

conjugation with IgY.

3.3.4.2. Agarose gel electrophoresis.

Agarose gel electrophoresis was done to confirm the bioconjugation of

GNPs with IgY and BSA. The bands in the 1% agarose gel showed a marked

difference in the movement of bioconjugates. GNPs are poorly negatively charged

molecules so remain static in the well-1 and well-4 against the electrophoretic

separation. Whereas the bioconjugates were strongly charged due to conjugation

with proteins and showed marked difference in the movement in the gel according

to the molecular weight of the protein. GNP-IgY (well 2 and well 5) moved half the


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way through the gel in comparison with GNP-BSA (Well 3 and well 6) (Figure 3.4).

The molecular weight of IgY is 190 KDa and BSA is 66 KDa.

Figure 3.4. Agarose gel electrophoresis band of GNPs and bioconjugates.

3.3.4.3. Flocculation assay.

Concentration, pH and appropriate functional group are very critical

parameters for bioconjugation of any biomolecules to nanoparticles (Vinayaka et

al., 2009). To determine the minimum amount of IgY required for nanoparticles

stabilization, flocculation assay was done for varying IgY concentration for citric

acid-reduced GNPs. Inset of Figure 3.5 shows the absorption spectrum of different

concentrations of IgY bioconjugated with GNP. The stability of GNPs can be

maintained by the adsorption of IgY on it preventing the salt-induced aggregation.

Most of the GNPs get aggregated at IgY concentration less than 30 µg/ml

indicating that the lower concentration of IgY are not enough to shield the GNPs.

Whereas, IgY concentration more than 30 µg/ml GNPs are not getting aggregated

indicating the complete shielding of GNPs. Thus, among all the concentration of

IgY 30 µg/ml was selected as optimum because visually and graphically distinct


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color change was observed. The color change from red to blue of the GNPs is

mainly due to varying interparticle plasmon coupling during aggregation and

resulting in plasmon band shift. The full saturation of the colloidal gold surface

increases the chance of an antibody–antigen interaction after collision with the

protein and improves the stability of colloidal particles by shielding its surface

against coagulation (Chandler et al., 2001). Figure 3.5 shows the absorption

spectra for IgY containing solutions before and after reaction with the GNPs. At

low IgY concentration, the absorbance of the supernatant of the centrifuged IgY

solution after treating with colloidal GNPs essentially decreases to zero. Only after

saturation of the GNPs surfaces with the IgY molecules, the noticeable

absorbance of supernatant is observed. For a fixed IgY concentration (1 mg/ml),

the amount of IgY adsorbed on GNPs was found to be 30 μg/ml. The amount of

IgY molecules per mL of GNPs (15 nm) were calculated by measuring the

difference of absorbance of the IgY solution before and after incubation with

GNPs.


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Figure 3.5. The graph shows the amount of IgY molecules adsorb on per ml of

gold particle (~15 nm) was calculated by measuring the difference of absorbance

of the IgY solution before and after incubation with GNPs.Inset shows the

flocculation assay determining the amount of IgY (µg/ml) required to stabilize the

GNPs.

3.3.4.4. Critical flocculation concentration.

GNPs are highly polarizable materials with a large Hamaker constant (Liu et

al., 2007). They are prone to aggregation in high ionic strength solutions in which

van der waals attraction is stronger than the electrostatic and steric repulsion

provided by surface-bound ligand. For confirmation of stability of GNP-IgY

complex, CFC was determined, as the threshold concentration of the electrolyte

(NaCl) in the colloidal gold that caused rapid aggregation of particles. The CFC is

indicated by a large decrease and/or red shift of the maxima as a result of

aggregation of GNPs. A CFC of 1M of NaCl was determined for the citrate-capped

GNPs (Figure 3.6). This confirmed the good stability of citrate-capped GNPs

toward electrolyte (NaCl)-induced aggregation. The CFC value provides a critical

parameter for the development and functioning of dipstick while analyzing vitamin

B12 in food samples.


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Figure 3.6. UV–vis spectra of GNPs (∼15 nm) incubated with varying

concentrations of NaCl. The CFC is indicated by a large decrease and red shift of

the plasmon absorption band.

3.3.4.5. Determination of Binding constant (Kb) of GNPs-IgY by Fluorimetric

assay.

Specific non-covalent binding of nanoparticles to biological

macromolecules, such as proteins and other molecules due to quenching of

fluorescence intensity of tryptophan residues of protein molecules enabled the

determination of binding constant (Kb) of proteins (IgY) to GNPs. Figure 3.7 shows

the relative fluorescence intensity of tryptophan residues of IgY with different

concentrations of GNPs. The fluorimetric assay has been chosen due to the high

intensity of the technique in probing the intrinsic fluorescence of the tryptophan

residue of IgY molecules, which is quenched by the binding of GNPs to the

specific sites (Tedesco et al., 2004). This provides a strategy to investigate the

interaction between GNPs and IgY through the evaluation of specific parameters

that clearly describe the binding process as the binding constant (Kb). Increased


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concentration of GNPs gradually reduces the emission intensity of IgY. Binding

constant (Kb) was determined using the fluorimetric assay.

Binding constant is useful in determination of stability of the bioconjugate.

The tryptophan residue fluorescence intensity (F) scales with the GNPs

concentration [GNPs] through (F0-F)/(F-Fsat) = ([GNPs]/Kdiss)n . The binding

constant Kb was obtained by plotting log (F0-F)/(F-Fsat) versus log[GNPs], where

F0 and Fsat are the relative fluorescence intensities of the IgY alone and the IgY

saturated with GNPs, respectively. The value of log [GNPs] at log(F0-F)/(F-Fsat) = 0

equals to the logarithm of the dissociation constant (Kdiss). The reciprocal of (Kdiss)

is the binding constant Kb. Fluorescence intensity data corresponding to

nanoparticles with a core size of roughly 15 nm is shown in inset of Fig.3.7,

represent the best fit in the data using (F0-F)/(F-Fsat) = ([GNPs]/Kdiss)n. The value of

Kb obtained from the fitting of GNP-IgY was equal to 2.535X108. This showed

good binding of IgY with GNPs and provided an idea about the bioconjugation

stability.


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Figure 3.7. Graph showing the quenching of fluorescence of IgY by GNPs. Inset

shows the double logarithmic plot of the quenching of the fluorescence by GNPs

for the calculation of binding constant (Kb).

3.3.4.6. Fourier-transform infrared spectroscopy.

Figure 3.8 shows the IR spectroscopic measurements of IgY and the

bioconjugated GNPs, respectively. GNPs are good adsorbents for biomolecules

such as proteins and antibodies. The citrate-capped GNPs were used to bind IgY

to make stable GNPs-IgY complexes. The adsorption is established through

electrostatic interaction between the surface-terminated anionic groups (-COO-) on

the nanoparticles and the positively charged amino groups (-NH3+) of the lysine

residue of the protein. Apart from the electrostatic interaction, ionic/hydrogen

bonding between the –NH3+ and –COO- functionalized surface is also possible.

The typical binding energies for such bonds are in the range of 1-10 kcal mole-1. It


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is therefore obvious that lysine rich proteins of immunoglobulin (IgY antibodies)

are highly attractive toward the citrate-capped GNPs. Figure 3.8 shows the bands

at 3700-3500 cm-1 from the amide N-H stretch, 3500-3300 cm-1 from the amine

stretch, 3550-3200 broad peak from the O-H stretch, 3100-3010 cm-1 from the

alkenyl C-H stretch, 3000-2500 cm-1 from the carboxylic acid O-H stretch, 2260-

2100 cm-1 from the alkynyl CΞC stretch, 1700-1500 cm-1 from the aromatic C=C

bending, 1250-925 cm-1 from the C-O-C asymmetric and symmetric vibrations of

phospholipids and 860-680 cm-1 from the aromatic C-H bending (Sankari et al.,

2010; Infrared spectroscopy theory, 2002). However, significant difference was

observed between the unconjugated and conjugated IgY due to electrostatic

interactions between the various functional groups of IgY and GNPs. From FTIR

analysis the newly formed bonds between the IgY and GNPs-IgY was observed.


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Figure 3.8. FTIR absorbance spectra for IgY and bioconjugate (IgY-GNPs). FTIR

spectra were acquired at room temperature.


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3.3.5. Enzyme-linked immunosorbent assay.

The vitamin B12-binding capacity of the antibody was estimated by direct

ELISA in chapter 1 section 1.3.3 with limit of detection (LOD) 10 ng/ml.

3.3.6. GNPs-based immunodipstick assay

3.3.6.1. Optimization.

Nitrocellulose membranes are commonly used matrix in protein blotting.

Most proteins can be successfully blotted using a 0.45-μm pore size membrane for

proteins or vitamins of low molecular weight. When used for immunodipsticks,

nitrocellulose membranes have no problems of background and flow obstruction.

The developed assay could be used for the semi-qualitative detection of vitamin

B12 by visual observation. The concentration of BSA-B12 and GNPs-IgY conjugate

was determined to be the key factor for competitive immunoassay for visual

differentiation between the control and test samples and therefore optimized. We

investigated the effect of BSA-B12 on the intensity of the immunodipstick. Usually,

the intensity of the immunodipstick could be increased via increasing the

immobilized amount of BSA-B12 on test strip. As so, it is not possible to evaluate

vitamin B12 with low concentration. So, an optimal amount of BSA-B12 applied to

NC membrane was 1.0 μg/µl at a concentration of 1.0 mg/ml. However,

immunodipstick tests for small molecules are based on a competitive format, the

less the antibody used, the higher the sensitivity will be. So, in the second step,

IgY-GNPs conjugates containing 1:10 (12 µg in 300µl) dilution at a concentration

of 0.4 mg/ml was used for clear red visible spot. It is considered by previous

reports that the selected antibody concentration must be greater than the minimum

amount antibody concentration required for stabilization of colloidal gold (Shi et al.,

2008). Therefore, 0.4 mg/ml was used for further studies. The intensity of the color

was found increasing with time; therefore, an optimum incubation time was found

to be 20–30 min. With all the above optimal parameters, it was observed that there

was a detectable difference in the intensity of color development on NC

membrane-containing strips. A maximum intensity of color was observed in the

case where there was no vitamin B12 present in the sample and maximum GNPs-


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IgY conjugate bound to the immobilized BSA-vitamin B12. The intensity of the color

was less in the vitamin B12-containing samples due to binding of GNPs-IgY

conjugate to free vitamin B12 and was not available for binding with the

immobilized BSA-vitamin B12 on NC membrane strip as shown in Fig. 3.9. As a

result when the vitamin B12 concentration in the sample was high, less intense red

color was developed and when the concentration of vitamin B12 in the sample was

less, more intense color was developed. There was no significant difference in the

color intensity at lower concentration (1ng/ml) as compared with control. The color

intensity for vitamin B12 was found distinguishable from that of control strip on

visual observation for higher concentration than 1ng/ml. Therefore, the visual

detection limit of the assay was found to be 1 ng/ml.

Figure 3.9. Visual intensity of the GNPs based immuno-dipstick for vitamin B12

standards with various concentrations. Dipsticks showing difference in the color

intensity.

3.3.7. Cutoff value of the GNPs-based immunodipstick.

Under the optimal conditions, vitamin B12 standards were detected by using

GNPs-based immune-dipstick. The yes/no response could be provided on test and

control dipstick, which indicated that the content of vitamin B12 was below or above

the visible detection limit. In this study, the visible detection limit is defined as the

lowest vitamin B12 concentration level, which inhibited the color development on

test strip. Thus, vitamin B12 concentrations lower than 1 ng/ml were scored as

negative. These tests could be traditionally characterized with the visible detection


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limit or cutoff value set at 1 ng/ml for the detection of vitamin B12. This immuno

strip test is purely semi-qualitative method of detection in which the results were

interpreted by visual observation.

More significantly, the sensitivity rates of the developed dipstick

immunoassay were evaluated by using these data (Eqs. 1). The sensitivity rate of

a qualitative method is defined as the ability to detect truly positive (tp) samples as

positive and truly negative (tn) samples as negative, respectively (Trullols et al.,

2004). The parameters can be calculated as:

Test positives tp

Sensitivity rate = ------------------------------------------- = -----------

(1)

Total number of known positives tp+ fn

where fn are false negative for samples, respectively. The immunological

dipstick had sensitivity rate of 92 % (n = 25 for 1 ng/ml) (Table 3.1). Therefore,

limit of detection was confirmed as 1 ng/ml. Below 10 ng/ml of concentration

ELISA could not detect vitamin B12 therefore it is scored as “not detected”. The

apparent cutoff value of the immunodipstick translates into 1 ng/ml when used for

the detection of real food samples. Although this value is relatively less, it can be

tuned further by more professionally fabricated process, e.g., the use of a spotting

machine, alternative membrane materials and optimized nano gold loading of the

antibody, especially by controlling the immobilized amount of antibody on each

gold surface.


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Table 3.1. The reproducibility of GNPs based immunodipstick. (+): Positive, (-)

Negative.

Assay C [vit B12] (ng/ml) Assay Time; Experiment result (+, -)

1 2 3 4 5

Intra 0 - - - - -

1 (+-+++) (+++++) (+++++) (+++-+) (+++++)

10 + + + + +

100 + + + + +

Inter 0 - - - - -

1 (- -+++) (+++-+) (+++++) (+++++) (+++++)

10 + - + + +

100 + + + + +

3.3.8. Reproducibility and stability of the GNPs-based immuno-dipstick.

The reproducibility of the GNPs-based immuno-dipstick was monitored by

intra- and interassay precision (Table 3.1). During the measurement process, four

levels with 0, 1, 10, and 100 ng/ml vitamin B12 were used for the evaluation of

reproducibility of the GNPs-based immuno-dipstick. Each sample was assayed 5

times. The intra-assay precision was obtained by using the GNPs-based immune-

dipstick of the same batch, while that of the interassay precision was achieved of

various batches. Seen from (Table 3.1), the reproducibility of the GNPs-based

immuno-dipstick was acceptable. Moreover, consistent results were obtained with

five replicates, showing good reproducibility and strip-to-strip performance. The

stability of the GNPs-based immuno-dipstick was investigated on a 20-day period

using the same batch immuno-dipstick. The GNPs-based immuno-dipstick were

dried and stored at 4°C, and measured intermittently toward the same

concentration of vitamin B12 on every two to three days. The experimental results

could remain the 90% of the original cutoff value on the test and control strips at

18th day. Thus, the stability of GNPs-based immuno-dipstick was satisfactory.


123

3.3.9. Detection of vitamin B12 in real food samples.

To evaluate the possible application of the GNPs-based immuno-dipstick

for the detection of vitamin B12 in fruit and energy drinks, three spiked fruit drinks

samples and three commercially labelled vitamin B12 energy drinks were assayed

using the GNPs-based immuno-dipstick strip. Lists of experimental results

obtained from the GNPs-based immuno-dipstick and corresponding enzyme-linked

immunosorbent assay (ELISA) for spiked fruit drinks and commercially labelled

vitamin B12 energy drinks samples were shown in (Table 3.2 and 3.3),

respectively.The visual evaluation results of the immuno-dipstick were in good

agreement with those of ELISA. The concentrations of the spiked vitamin B12 were

1, 10, 100 ng/ml, respectively in fruit drinks and labelled vitamin B12 in commercial

energy drinks. Seen from Table. 3.2 and 3.3, most food samples with low

concentrations of vitamin B12 (i.e. C[vitB12] <1 ng/ml) obtained from the ELISA

were not detected and scored as negative (-) using GNPs-based immuno-dipstick.

When the concentrations of vitamin B12 were above the cutoff value (i.e. C[vitB12]

>1 ng/ml) ELISA could detect and scored as positive (+) using GNPs-based

immuno-dipstick. So, the GNPs-based immuno-dipstick could be preliminarily

applied for the determination of vitamin B12 in fruit and energy drinks.

Table 3.2. Comparison of GNPs based immuno-dipstick and ELISA for

determination of vitamin B12 in different Fruit drinks spiking at 1, 10, 100 ng/ml of

derivatized vitamin B12 (n=5). (+): Positive (C [vit B12] ≥1 ng/ml), (-): Negative (C

[vit B12] < 1 ng/ml).

Fruit drinks Spiking level (ng/ml)

GNPs based immunodipstick (+,-

) (n=5)

ELISA (n=5) (mean ± SD, ng/ml)

Mango drinks 1 - - + + - Not Detected

10 + - - + + 8.80 (± 2.30)

100 + + + + + 98.08 (± 3.60)

Apple drinks 1 + + + + - Not Detected

10 + + - + + 10.16(± 0.98)

100 + + + + + 101.54 (± 6.65)

Orange drinks 1 - - + + + Not Detected

10 + - + + + 8.6 (± 1.57)

100 + + + + + 99.9 (± 11.4)


124

Table 3.3. Comparison of GNPs based immuno-dipstick and ELISA for

determination of vitamin B12 in different energy drinks labeled at 2µg/100ml vitamin

B12 (n=5). (+): Positive (C [vit B12] ≥1 ng/ml), (-): Negative (C [vit B12] < 1 ng/ml).

Energy

drinks

Amount labeled

(µg/100 ml)

GNPs based

immunodipstick

(+,-) (n=5)

ELISA (n=5)

(mean ± SD,

µg/100 ml)

Sample 1 2 + + + + + 1.72 (± 0.42)

Sample 2 2 + + + + + 1.96 (± 0.28)

Sample 3 2 + + + + + 1.96 (± 0.10)

3.4. CONCLUSIONS

A sensitive immuno-dipstick assay for vitamin B12 was developed. The

visible detection limit of vitamin B12 is 1 ng/ml. Using quasi-spherical monodisperse

GNPs, dipstick-based immunosensor was successfully developed for the detection

of vitamin B12 in fruit and energy drinks. Characterization was done for the

bioconjugates and GNPs to study about the properties that are essential for the

dipstick assay. For validation, both the strip test and a standard ELISA method

were used to detect vitamin B12 in real samples and good agreement of the results

between them were shown. It also has other advantages like simple, effective and

doesn’t require skilled persons to operate. Because of their numerous advantages

they can be used to detect any type of analyte like microbial toxins, chemical

toxins, adulterants, nutrients, vitamins, heavy metals and microbes. Significantly,

the GNPs-based immuno-dipsticks can emerge as potential biosensor tool for

monitoring purpose.


125

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