Design and Development of a Field Applicable Gold Nanosensor for

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Design and Development of a Field Applicable Gold Nanosensor for the Detection of Luteinizing Hormone Ajit Zambre,† Nripen Chanda,† Sudhirdas Prayaga,§ Rosana Almudhafar,⊥ Zahra Afrasiabi,*,⊥ Anandhi Upendran,*,¶,‡ and Raghuraman Kannan*,† Departments of †Radiology and ‡Physics, University of Missouri, Columbia, Missouri 65212, United States § Antibody Research Corporation, St. Charles, Missouri 63304, United States ⊥ Department of Life and Physical Sciences, Lincoln University, Jefferson City, Missouri 65101, United States ¶ Nanoparticle BioChem. Inc., Columbia, Missouri 65211, United States S Supporting Information *

ABSTRACT: In this paper, we describe a novel strategy for the fabrication of a nanosensor for detecting luteinizing hormone (LH) of sheep using a gold nanoparticle-peptide conjugate. A new peptide sequence “CDHPPLPDILFL” (leutinizing hormone peptide, LHP) has been identified, using BLAST and Clustal W analysis, to detect antibody of LH (sheep). LHP has been synthesized and characterized, and their affinity toward anti-LH was established using enzyme linked immunosorbant assay (ELISA) technique. The thiol group in LHP directly binds with gold nanoparticles (AuNPs) to yield AuNP-LHP construct. Detailed physicochemical analysis of AuNP-LHP construct was determined using various analytical techniques. Nanosensor using gold nanoparticle peptide conjugate was developed on the basis of competitive binding of AuNP-LHP and LH toward anti-LH. Nitrocellulose membrane, precoated with anti-LH, was soaked in the mixture of AuNPLHP and sample of analysis (LH). In the absence of LH (sheep), anti-LH coated on the membrane binds with AuNP-LHP, leading to a distinctive red color, while in the presence of LH, no color appeared in the membrane due to the interaction of antiLH with LH thereby preventing the binding of AuNP-LHP with membrane bound anti-LH. The sensor assay developed in this study can detect LH (sheep) up to a minimal concentration of ∼50 ppm with a high degree of reproducibility and selectivity. The gold-nanoparticle-peptide based nanosensor would be a simple, portable, effective, and low cost technique for infield applications.

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in the absence of a male. Such a sensor would be highly beneficial and could result in increased use of artificial insemination by small farm family operations. Since neither ewes nor goats always have overt behavioral or physically identifiable changes that signal the optimum time to breed, a physiological change must be detected. One of the most common measurable changes that occurs prior to estrus and ovulation is the presence of luteinizing hormone (LH) in the blood. A LH surge occurs prior to ovulation and sets the time for ovulation.2 It is important to recognize that the sensor to be developed for detecting the presence of LH should not utilize complicated equipment or chemistry. More importantly, it should be user-friendly for the farming community. LH concentration in sheep’s blood is one of the most common and measurable parameters prior to estrus and ovulation.3−5 At lower concentrations of LH in ewe, the chances of success for artificial insemination decreases drastically. As such, the detection and measurement of concentrations of LH in sheep

here were 7 500 000 head of sheep and goats in the United States in 2003. This number grew by 15% in 2005 and only 1% in 2006.1 The supply of goat is primary income source for many small farmers across the nation. For these reasons, it is vital to concentrate on two important aspects of sheep and goat production. First, an important determinant to the economics of production success is the genetic quality of the animals. Males represent 50% of the genetics of a flock or herd: therefore, use of high quality males is important to increase genetic quality in the herd/flock. Purchasing high quality males is often cost-prohibitive for small farm family operations, and this cost limits their ability to compete with larger farms. One alternative to owning an expensive male is to artificially inseminate females using semen from genetically superior males since semen is much less expensive than high quality rams and bucks. This approach would eliminate the need for keeping males in the herd/flock. There are, however, some problems in attempting to use artificial insemination when males are not present. The second important aspect to address, especially in sheep, is determining the appropriate time to inseminate. The farming community is interested in the development of a sensor to determine the exact time to breed © 2012 American Chemical Society

Received: August 11, 2012 Accepted: September 24, 2012 Published: September 24, 2012 9478

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is important from an economic (and farming community) point of view.5 Current method of analysis of LH involves immunoassay using antibodies specific toward LH.6 Much of the earlier research work was focused on development of a solid-phase radioimmunoassay for LH. These methods utilized antibody-coated polystyrene tubes for capturing LH, followed by counting the radioactive 125I attached with the tube.7 Even though this method is highly accurate and efficient, the analysis requires skilled labor and a sophisticated laboratory setting. It is worthy to note that LH production in sheep follows a different mechanism compared to that of humans, and because of this difference, LH production in sheep rises very sharply, within a span of 12 h. Given this scenario, it is not presently feasible for a farmer to utilize LH as a factor for initiating the artificial insemination at the appropriate time. Thus, there is a strong rationale to develop a portable, colorimetric sensor for detecting LH under field conditions. Gold nanoparticles (AuNPs) have been used extensively in the design of biosensors.8−15 AuNPs possess excellent userfriendly characteristics that include ease of surface functionalization, high extinction coefficients, and unique distancedependent plasmonic absorption.16−23 There are several literature reports on using antibody conjugated AuNPs as sensors to detect the antigen of interest with high level of specificity and selectivity.24−28 However, utilization of peptide conjugated AuNPs for the design and development of sensors is very rare. Of relevance to the current study, Rusling and coworkers have reported AuNP-peptide conjugates to detect IgE antibodies that are specific for major peanut allergen, Arachis hypogaea 2 (Ara h2).29 In this study, a Ara h2-avid peptide sequence, chosen from the major IgE-binding eptiope from Ara h2, is attached on the surface of AuNPs. There are several advantages on utilizing peptides that include the following: (i) synthesizable under laboratory conditions; (ii) ease of surface conjugation to gold via thiol molecules; (iii) high specificity toward biomolecule of interest; and most importantly, (iv) highly stable and easily portable. These advantages are very well recognized by several research groups.18,30−32 Therefore, in the present study, we have chosen peptide conjugated gold nanoparticles to identify LH. Additionally, peptides are robust and can be stored in the farm for an extended period of time. The overall goal of the sensor developed in this study is to utilize it in a farm setting, where a freezer for storing antibody or scientific instruments are likely to be unavailable. Even though the study utilizes advanced nanotechnology for developing the sensor, the final “strip-assay” based sensor requires only simple “mixing and shaking” to identify LH. Herein, we report the identification of a new peptide sequence, luteinizing hormone peptide (LHP), with high specificity toward anti-LH by standard enzyme linked immunosorbant assay (ELISA), its conjugation to AuNPs, detailed characterization of the nanoconjugate (AuNP-LHP), utility of AuNP-LHP to detect LH, and as a “proof-of-concept” of the detection of LH (spiked) in sheep’s blood (Scheme 1). The results reported in this paper include: (i) identification, synthesis, and characterization of LHP (LH-peptide; “CDHPPLPDILFL”) sequence; (ii) raising polyclonal antibody against LHP (anti-LHP) in rabbits for confirming the biospecificity and quality control; (iii) confirmation of the LHP affinity toward anti-LH and anti-LHP by conventional ELISA techniques; (iv) conjugation of LHP to the surface of AuNPs and standard physicochemical characterization of the resulting conjugate AuNP-LHP; (v) detailed ELISA studies to

Scheme 1. Schematic Showing the Process of Nanosensor for the Detection of LH Using AuNP-LHP

confirm the binding affinities of the conjugate, AuNP-LHP, toward native polyclonal antibody of sheep lutenizing hormone (anti-LH) and polyclonal antibody raised against native peptide (anti-LHP); (vi) competitive ELISA and immunostrip assay studies using the AuNP-LHP to explore its potential for use as a nanosensor; and (vii) identification of LH in sheep blood by spiking experiments.

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EXPERIMENTAL SECTION Synthesis and Characterization of Gold Nanoparticle Conjugated LH-Peptide (AuNP-LHP). Citrate stabilized gold nanoparticles (CAuNP) were synthesized and concentrated by diafiltration.33 One equivalent (5.0 mg) of PEG-750 (thiolated methoxy polyethylene glycol MW = 750 Da, Rapp Polymer, Germany) was added to concentrated CAuNP solution (1 equivalent of Au concentration (1.32 mg) as determined by UV−visible absorbance measurements, OD525 = 3.95 units). The reaction mixture was stirred for 2 h at room temperature followed by the addition of 0.5 equivalents of luteinizing hormone peptide (LHP, 4.5 mg in 1 mL of water). The mixture was stirred overnight (∼8 h) at room temperature to obtain the LHP conjugated gold nanoparticles (AuNP-LHP). After successful conjugation, the unbound peptide was separated by centrifugation at 15 000 rpm for 20 min at 7 °C. The pellet obtained was further washed two times with HPLC grade water and finally dried under vacuum. ELISA Binding Studies. A stock solution of LHP (10 μg/ mL) or LH (sheep) (10 μg/mL) and AuNP-LHP (10 μg/mL) in 0.1 M NaHCO3, pH 9.6, was prepared to facilitate maximum binding to polystyrene enzyme immune-assay (EIA) plate. LHP (400 ng) and AuNP-LHP were immobilized in two sets of wells and were coated. Ten μL of anti-LHP in a serial 10-fold dilution was incubated for 20 min at 37 °C. The binding of native luteinizing hormone protein from sheep (LH) and a rabbit polyclonal antibody to LH sheep protein (anti-LH) were used as controls. After washing, 50 μL of goat anti-Rabbit IgG-HRP (Antibody Research Corporation) was added to all the wells, incubated for 15 minutes at 37 °C, and washed 3 times with PBS containing 0.05% Tween-20. To all the wells was added 50 μL of TMB (tetra methyl benzidine) and 1 component of substrate followed by the addition of 50 μL of 1 M HCL, and they were monitored at 450 nm using a microplate reader. The 9479

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RESULTS AND DISCUSSION As a first step toward developing LH sensor, we focused our research on identifying a peptide sequence that has high affinity toward anti-LH (antibody). A short 12 amino acid peptide sequence (LHP) (Figure S1, Supporting Information) of the native sheep hormone LH has been identified, synthesized, purified, and conjugated with AuNPs. The synthesized LHP has been characterized by HPLC (Figure S2, Supporting Information) and ES-MS analysis (Figure S3, Supporting Information). The rabbit polyclonal antibody to the synthesized peptide (anti-LHP) was raised and used as controls for the binding studies. The synthetic details of LHP conjugation to AuNPs, binding affinity of the nanoconjugate, and the initial studies to explore the use of gold nanoconjugate as nanosensor to detect LH are described in detail below. Synthesis and Characterization of Gold Nanoparticle Conjugated Peptide (AuNP-LHP). AuNP-LHP was synthesized by conjugating AuNPs with LHP as shown in Scheme 2.

ELISA studies were representative measurements from three independent experiments, and the readings were plotted as a graph of fold dilution versus absorbance. The plate details are presented in the Supporting Information (Table S2). Competitive Inhibition ELISA. In a 96 well plate (Corning Costar), LHP (2 μg/mL, 100 μL) was coated in wells. 50 μL of AuNP-LHP was added, starting at 1 mg/mL in a serial 10-fold dilution fashion along with 50 μL of 1 μg/mL of anti-LHP to monitor competitive inhibition. After washing, 50 μL of goat anti-rabbit-IgG-HRP was added to all the wells and incubated for 15 min at 37 °C. To all the wells was added 50 μL of TMB and 1 component of substrate followed by the addition of 50 μL of 1 M HCL, and they were monitored at 450 nm using a microplate reader. The readings were plotted as a graph of fold dilution versus absorbance. The binding of LHP against antiLHP was used as a control for this experiment. The competitive inhibition ELISA plate details are presented in the Supporting Information (Table S3). Immunostrip Assay. One μL of different concentrations of anti-LH (0.6, 1.2, and 1.8 μg/μL) or anti-LHP (1.6, 3.2, 4.8 μg/ μL) was spotted on two, 10 mm × 100 mm nitrocellulose membrane (NC) strips. The spots were air-dried at room temperature for 20 min, and nonspecific sites on membrane strips were blocked by incubating the membrane with 2.5% bovine serum albumin (BSA) (w/v) in 1× PBS and 0.05% Tween-20 (v/v) for 2 h at 25 °C. After blocking, the strips were washed with 1× PBS and incubated with 1.5 mL of 0.5 mg/mL AuNP-LHP solution. Ruby red color developed within 5 min in all anti-LH/LHP spots on the NC membrane. Competitive Inhibition Immunostrip Assay. The antiLH (0.6 μg/μL) was spotted on each of the five nitrocellulose membrane strips (10 mm × 100 mm). The spots were air-dried at room temperature for 20 min, and nonspecific sites on membrane strips were blocked by incubating the membrane with 2.5% BSA (w/v) in 1× PBS and 0.05% Tween-20 (v/v) for 2 h at 25 °C. After blocking, the strips were washed twice with 1× PBS and incubated with AuNP-LHP (1.5 mL of 0.50 mg/mL) mixed with varying concentrations of LH (0, 20, 50, 70, 100, 200, 300 μg in 1.5 mL). Intense ruby red color formation was observed in strips incubated with AuNP-LHP solution in which LH was not present. LH Identification in Sheep Blood. One mL of blood was withdrawn from a sheep’s leg. Blood was immediately mixed with EDTA to prevent clumping. Each assay was performed in triplicate. Typically, 1 μL of different concentrations of anti-LH (0.6, 1.2, and 1.8 μg/μL) or anti-LHP (1.6, 3.2, 4.8 μg/μL) was spotted on two, 10 mm × 100 mm nitrocellulose membrane (NC) strips. The spots were air-dried at room temperature for 20 min, and nonspecific sites on membrane strips were blocked by incubating the membrane with 2.5% BSA (w/v) in 1× PBS and 0.05% Tween-20 (v/v) for 2 h at 25 °C. For each run, 0.2 mL of sheep’s blood was mixed with 1.3 mL of AuNP-LHP (0.5 mg/mL). To these vials, increased concentrations of LH (20 μg (13.33 ppm), 50 μg (33.33 ppm), and 70 μg (46.66 ppm)) were added. The strips were immersed in the vials for 15 min. As expected, within 15 min, a ruby red color appeared in the strips immersed in vials containing 20 μg and 50 μg LH; whereas, a solution containing 70 μg of LH did not show any color. Strips incubated with vials that did not contain sheep’s blood were used as controls. These strips showed intense red color. The field trials were conducted during a nonbreeding season.

Scheme 2. Synthesis of AuNP-LHP Conjugate Showing Covalent Conjugation of LHP over the Surface of AuNPs and Formation of Second Layer via Intermolecular Hydrogen Bonding

In the first step, citrate stabilized gold nanoparticles (CAuNP) of 10 nm size were bound with an ancillary ligand, using thiol containing polyethylene glycol (MW = 750 Da), to yield AuNP-PEG conjugate. Subsequently, the thiol-PEG was exchanged with LHP. The thiol group present in LHP binds directly to the surface of AuNP (characterized by X-ray photoelectron spectroscopy (XPS) and details provided below). The non-PEG route that is a direct conjugation of CAuNP with LHP resulted in aggregation of AuNPs. The surface coating of the CAuNP by pegylation improved the charge distribution and stability to facilitate the conjugation with LHP. The conjugation of LHP to AuNPs was achieved through the covalent binding of sulfur atom to the AuNPs from the cysteine residue present in the isolated peptide. AuNP-LHP was characterized in detail through their physicochemical properties (Figure 1). UV−visible absorption spectrum of AuNP-LHP showed a characteristic surface plasmon resonance peak at 525 nm (Figure 1A). Transmission electron microscope (TEM) analysis showed a uniform size distribution of the nanoconjugate, with an average size of 15 ± 2 nm (Figure 1B,C). The size of the AuNP-LHP was further confirmed by disk centrifuge sedimentation analysis (16 ± 1 nm) (Figure 1D). The physicochemical properties of AuNP-LHP are summarized in Table 1. The hydrodynamic diameter for AuNP-LHP is 67 ± 5 nm. The high zeta potential value of AuNP-LHP (+ 60 9480

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in literature for cysteine-coated gold nanoparticles.34 The presence of two layers in the conjugate is correlated by XPS analysis. The inner layer consists of cysteine (from LHP) bound to gold atoms on the surface of nanoparticles. The leucine groups present in the inner layer of LHP are oriented away from the gold surface and electrostatically bound to another layer of LHP. The bonding between the layers is primarily mediated by hydrogen bonding of leucine carboxyl group and the protonated free amine from the secondary layer of the peptide (Scheme 2). The positive zeta potential for AuNP-LHP due to protonation of free amine present in the second layer of the peptide provides proof for the presence of H-bonding network between the layers. In XPS study, no peaks were observed in the higher energy sulfur region corresponding to oxidized sulfur. This study indirectly indicates the formation of AuNP-LHP as the exclusive product.35 (Detailed XPS interpretation is provided in Supporting Information, Figure S5 and Table S1.) In Vitro Stability Studies. In order to utilize AuNP-LHP for sensing applications, it is important that the construct is stable under various biological conditions for several hours. In vitro stability of AuNP-LHP was monitored in biologically relevant solutions such as 3% NaCl, 0.5% cysteine, 0.2 M histidine, 0.5% human serum albumin (HSA), and BSA, respectively. The stability and the identity of the nanoparticles were measured by monitoring the UV−vis absorbance at 0.5 h as well as after 24 h. The surface plasmon resonance peak of the conjugate shows minimal shift, indicating high in vitro stability and retention of the nanoparticulate size in the above mixtures (Figure 2). AuNP-LHP conjugates demonstrated excellent in

Figure 1. Physico-chemical characteristics of AuNP-LHP. (A) UV− visible absorption spectrum, (B) TEM image, (C) size distribution histogram, and (D) particle size analysis by disk centrifuge sedimentation system.

Table 1. Physicochemical Properties of AuNP-LHP size analysis [nm]

a

costruct

λmax [nm]

AuNP-LHP

525

TEMa

DCSb

DLSc

charged [mV]

15 ± 2

16 ± 1

67 ± 5

59.8 ± 3

b

Core Size by TEM. Size analysis by disc centrifugal sedimentation (CPS). cHydrodynamic size by dynamic light scattering. dZeta potential.

mV) indicates that the conjugate is highly stable in aqueous medium. The positive zeta potential value may be attributed to the protonation of the amines present in cysteine groups. The effective conjugation of the peptide to AuNPs is also reflected from the zeta potential value, as the charge of the pegylated AuNPs (−32.6 mV) is negative and changes to a highly positive value upon conjugation. The total amount of LHP conjugated to the AuNPs has been quantified by detailed HPLC analysis. HPLC of different concentrations of pure LHP was recorded, and a standard calibration curve was constructed. HPLC of the supernatant solutions obtained after conjugation of LHP to AuNPs were also recorded (Figure S4, Supporting Information). The HPLC of the supernatant solutions were compared with standard calibration curves to correlate the amount of LHP conjugated to AuNPs. From these calculations, it is estimated that ∼96% of LHP is coated on the surface of the AuNPs. Neutron activation analysis (NAA) of AuNP-LHP indicated that gold content in the peptide conjugate is ∼70% of total weight. This value is the optimum concentration of gold that is necessary in a nanoconjugate for performing ELISA and immunostrip assays. Upon increasing the concentration of gold in the construct, either aggregation or loss of sensitivity in the assays has been observed. Nature of LHP Interaction over AuNP Surface. To understand the nature of the bonding and chemical interaction between AuNPs and LHP conjugated on the surface, detailed XPS analysis has been performed. A layered structure, in which LHP is bound to the surface of AuNPs through cysteine present in the peptide, was predicted for the AuNP-LHP nanoconstruct. A similar structural possibility has been reported

Figure 2. In vitro stability studies of AuNP-LHP in various biological media of 0.5 mL of 3% NaCl, 0.5% cysteine, 0.2 M histidine, 0.5% HSA, and 0.5% BSA solutions. UV−visible absorption spectrum of these solutions after a 24 h treatment was recorded.

vitro stability under the pH 7 and 9 range, implying that these nanoparticles can be used in a wide pH range. The presence of biological media did not cause any aggregation or decomposition of nanoparticles, indicating that the stability is provided through covalent binding of the peptide to AuNPs. The stability of AuNP-LHP was also monitored in ELISA conditions to explore any dissociation of LHP from the AuNPs 9481

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residue remains unaltered and can be used as a mimic for LH toward biosensor applications. Immunostrip Assay Experiments. Three different concentrations of anti-LH (0.6, 1.2, and 1.8 μg/μL) were spotted on the nitrocellulose (NC) membrane followed by washing with PBS and incubation with AuNP-LHP. Upon incubation with AuNP-LHP, ruby red color spots developed within minutes as shown in Figure 4. Upon incubation with

in ELISA medium. The UV−visible absorption peaks and fwhm values (Δλ ∼ 65 nm) remain unaltered and clearly prove that LHP does not dissociate from the AuNPs in the ELISA medium. ELISA Binding Studies. We focused our studies on understanding the biospecificity of “LHP” toward antibody of LH through ELISA binding studies. ELISA studies were performed in detail to demonstrate the following three binding affinities: (i) binding of rabbit polyclonal native sheep hormone (anti-LH) against synthesized peptide (LHP); (ii) binding of rabbit polyclonal antibody of peptide (anti-LHP) against the native sheep hormone (LH); and (iii) binding of AuNP-LHP against anti-LH and anti-LHP. ELISA binding was monitored using a secondary antibody, anti-rabbit-IgG-HRP. The serial decrease in absorption of the secondary antibody anti-rabbitIgG-HRP was monitored and correlated to antigen−antibody binding. The plot of absorbance versus concentration of antigen for the binding study is shown in Figure 3, and the ELISA plate map is shown in Table S2 (Supporting Information).

Figure 4. Immunostrip assay of AuNP-LHP vs anti-LH. Anti-LH was spotted on a nitrocellulose membrane at three different concentrations 0.6, 1.2, and 1.8 μg/μL and was detected using AuNP-LHP. The color was developed in