Development of a sensitive surface plasmon resonance immunosensor for detection of 2,4-dinitrotoluene with a novel oligo (ethylene glycol)-based sensor surface

A surface plasmon resonance (SPR) immunosensor for detection of 2,4-dinitrotoluene (2,4-DNT), which is a signature compound of 2,4,6-trinitrotoluene-related explosives, was developed by using a novel oligo (ethylene glycol) (OEG)-based sensor surface. A rabbit polyclonal antibody against 2,4-DNT (anti-DNPh-KLH-400 antibody) was prepared, and the avidity for 2,4-DNT and recognition capability were investigated by indirect competitive ELISA. The sensor surface was fabricated by immobilizing a 2,4-DNT analog onto an OEG-based self-assembled monolayer formed on a gold surface via an OEG linker. The fabricated surface was characterized by Fourier-transform infrared-refractive absorption spectrometry (FTIR-RAS). The immunosensing of 2,4-DNT is based on the indirect competitive principle, in which the immunoreaction between the anti-DNPh-KLH-400 antibody and 2,4-DNT on the sensor surface was inhibited in the presence of free 2,4-DNT in solution. The limit of detection for the immunosensor, calculated as three times the standard deviation of a blank value, was 20 pg mL⁻¹, and the linear dynamic range was found to be between 1 and 100 ng mL⁻¹. Additionally, the fabricated OEG-based surface effectively prevented non-specific adsorption of proteins, and the specific response to anti-DNPh-KLH-400 antibody was maintained for more than 30 measurement cycles.     

Surface plasmon resonance-based immunoassay for procalcitonin

A surface plasmon resonance (SPR) biosensor has been developed for rapid immunoassay of procalcitonin (PCT) with high detection sensitivity and reproducibility. The 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)-activated protein A (PrA), diluted in 1% (v/v) 3-aminopropyltriethoxysilane (APTES) was dispensed on a KOH-treated Au-coated SPR chip, resulting in the covalent binding of PrA in 30 min. This “single-step” PrA immobilization strategy led to the oriented binding of the anti-PCT antibody (Ab) on a PrA-functionalized gold (Au) chip. The leach-proof immobilization procedure is five-fold faster than conventional counterparts, enabling high detection specificity and reproducibility. The IA detects 4–324 ng mL⁻¹ of PCT with a limit of detection (LOD) and a limit of quantification (LOQ) of 4.2 ng mL⁻¹ and 9.2 ng mL⁻¹, respectively. It was capable of detecting PCT in real sample matrices and patient samples with high precision. The Ab-bound SPR chips were stable for more than five weeks.     

Sensitivity enhancement of SPR biosensor with silver mirror reaction on the Ag/Au film

Based on well-known silver mirror reaction the Ag film was formed on Au film modified by self-assembled monolayer (SAM) of 1,6-hexanedithiol (HDT). The sensitivity of the biosensor based on this Ag/Au film is enhanced compared to that based on Au film. When the surface plasmon resonance (SPR) biosensor based on this Ag/Au film was used to determine human IgG, the range of concentrations of human IgG that could be determined is 0.30-40.00 μg mL⁻¹. The lowest concentration (0.30 μg mL⁻¹) that could be detected was about 8 times lower than that obtained by the biosensor without modification by Ag film (2.50 μg mL⁻¹), which demonstrated that the biosensor based on Ag/Au film could make the resonant wavelength move to longer wavelength following with the sensitivity enhancement of the SPR biosensor.     

A self-assembled fusion protein-based surface plasmon resonance biosensor for rapid diagnosis of severe acute respiratory syndrome

A surface plasmon resonance (SPR)-based biosensor was developed for simple diagnosis of severe acute respiratory syndrome (SARS) using a protein created by genetically fusing gold binding polypeptides (GBPs) to a SARS coronaviral surface antigen (SCVme). The GBP domain of the fusion protein serves as an anchoring component onto the gold surface, exploiting the gold binding affinity of the domain, whereas the SCVme domain is a recognition element for anti-SCVme antibody, the target analyte in this study. SPR analysis indicated the fusion protein simply and strongly self-immobilized onto the gold surface, through GBP, without surface chemical modification, offering a stable and specific sensing platform for anti-SCVme detection. AFM and SPR imaging analyses demonstrated that anti-SCVme specifically bound to the fusion protein immobilized onto the gold-micropatterned chip, implying that appropriate orientation of bound fusion protein by GBP resulted in optimal exposure of the SCVme domain to the assay solution, resulting in efficient capture of anti-SCVme antibody. The best packing density of the fusion protein onto the SPR chip was achieved at the concentration of 10 μg mL⁻¹; this density showed the highest detection response (906 RU) for anti-SCVme. The fusion protein-coated SPR chip at the best packing density had a lower limit of detection of 200 ng mL⁻¹ anti-SCVme within 10 min and also allowed selective detection of anti-SCVme with significantly low responses for non-specific mouse IgG at all tested concentrations. The fusion protein provides a simple and effective method for construction of SPR sensing platforms permitting sensitive and selective detection of anti-SCVme antibody.     

Enhanced surface plasmon resonance by Au nanoparticles immobilized on a dielectric SiO2 layer on a gold surface

This paper introduces strategies for enhancement of a surface plasmon resonance (SPR) signal by adopting colloidal gold nanoparticles (AuNPs) and a SiO₂ layer on a gold surface. AuNPs on SiO₂ on a gold surface were compared with an unmodified gold surface and a SiO₂ layer on a gold surface with no AuNPs attached. The modified surfaces showed significant changes in SPR signal when biomolecules were attached to the surface as compared with an unmodified gold surface. The detection limit of AuNPs immobilized on a SPR chip was 0.1 ng mL⁻¹ for the prostate-specific antigen (PSA), a cancer marker, as measured with a spectrophotometer. Considering that the conventional ELISA method can detect ∼10 ng mL⁻¹ of PSA, the strategy described here is much more sensitive (∼100 fold). The enhanced shift of the absorption curve resulted from the coupling of the surface and particle plasmons by the SiO₂ layer and the AuNPs on the gold surface.     

Preparation and application of triangular silver nanoplates/chitosan composite in surface plasmon resonance biosensing

This paper reports the utilization of triangular silver nanoplates (TSNPs) to enhance the sensitivity of surface plasmon resonance (SPR) biosensor. TSNPs modified with 3-mercaptopropinic acid (MPA) were simply mixed with chitosan and glutaraldehyde to form TSNPs/chitosan composite. The composite was deposited on Au film as immobilization substrate for SPR biosensor. The novel structures of TSNPs are preserved against etching by MPA and chitosan polymer. Moreover, chitosan cross-linked by glutaraldehyde enables antibody to be immobilized on fabricated substrate directly via Schiff alkali reaction. In the optimized conditions, the resulting biosensor based on TSNPs/chitosan composite shows a satisfactory response to bovine IgG in the concentration range of 0.075–40.00 μg mL⁻¹. While the biosensor based on chitosan without TSNPs shows a response in the concentration range of 0.6–40 μg mL⁻¹ and the biosensor based on Au film shows a response in the concentration range of 2.5–40 μg mL⁻¹. The experiment results show that the sensitivity of SPR biosensor based on TSNPs/chitosan composite was significantly enhanced and the immobilization procedure of antibody was simplified.     

Rapid detection of nivalenol and deoxynivalenol in wheat using surface plasmon resonance immunoassay

A surface plasmon resonance (SPR) immunoassay using a monoclonal antibody was developed to measure nivalenol (NIV) and deoxynivalenol (DON) contamination in wheat. A highly sensitive and stable DON-immobilized sensor chip was prepared, and an SPR detection procedure was developed. The competitive inhibition assay used a monoclonal antibody that cross-reacts with NIV and DON. The half maximal inhibitory concentration (IC₅₀) values of the SPR assay were 28.8 and 14.9 ng mL⁻¹ for NIV and DON, respectively. The combined responses of NIV and DON in wheat were obtained using a simultaneous detection assay in a one-step cleanup procedure. NIV and DON were separated using a commercial DON-specific immunoaffinity column (IAC) and their responses were obtained using an independent detection assay. Spiked tests using these toxins revealed that recoveries were in the range 91.5-107% with good relative standard deviations (RSDs) (0.40-4.1%) and that detection limits were 0.1 and 0.05 mg kg⁻¹ for NIV and DON, respectively. The independent detection using IAC showed detection limits of 0.2 and 0.1 mg kg⁻¹ for NIV and DON, respectively. SPR analysis results were correlated with those obtained using a conventional LC/MS/MS method for wheat co-contaminated with NIV and DON. These results suggested that the developed SPR assay is a practical method to rapidly screen the NIV and DON co-contamination of wheat and one of a very few immunoassays to detect NIV directly.     

Enhanced sensitivity of self-assembled-monolayer-based SPR immunosensor for detection of benzaldehyde using a single-step multi-sandwich immunoassay

This paper describes the fabrication and sensing characteristics of a self-assembled monolayer (SAM)-based surface plasmon resonance (SPR) immunosensor for detection of benzaldehyde (BZ). The functional sensing surface was fabricated by the immobilization of a benzaldehyde–ovalbumin conjugate (BZ–OVA) on Au-thiolate SAMs containing carboxyl end groups. Covalent binding of BZ–OVA on SAM was found to be dependent on the composition of the base SAM, and it is improved very much with the use of a mixed monolayer strategy. Based on SPR angle measurements, the functional sensor surface is established as a compact monolayer of BZ–OVA bound on the mixed SAM. The BZ–OVA-bound sensor surface undergoes immunoaffinity binding with anti-benzaldehyde antibody (BZ-Ab) selectively. An indirect inhibition immunoassay principle has been applied, in which analyte benzaldehyde solution was incubated with an optimal concentration of BZ-Ab for 5 min and injected over the sensor chip. Analyte benzaldehyde undergoes immunoreaction with BZ-Ab and makes it inactive for binding to BZ–OVA on the sensor chip. As a result, the SPR angle response decreases with an increase in the concentration of benzaldehyde. The fabricated immunosensor demonstrates a low detection limit (LDL) of 50 ppt (pg mL⁻¹) with a response time of 5 min. Antibodies bound to the sensor chip during an immunoassay could be detached by a brief exposure to acidic pepsin. With this surface regeneration, reusability of the same sensor chip for as many as 30 determination cycles has been established. Sensitivity has been enhanced further with the application of an additional single-step multi-sandwich immunoassay step, in which the BZ-Ab bound to the sensor chip was treated with a mixture of biotin-labeled secondary antibody, streptavidin and biotin–bovine serum albumin (Bio–BSA) conjugate. With this approach, the SPR sensor signal increased by ca. 12 times and the low detection limit improved to 5 ppt with a total response time of no more than ca. 10 min. Figure A single-step multi-sandwich immunoassay step increases SPR sensor signal by ca. 12 times affording a low detection limit for benzaldehyde of 5 ppt     

Single- and multi-analyte determination of gonadotropic hormones in urine by Surface Plasmon Resonance immunoassay

Single- and multi-analyte detection of two gonadotropic hormones (follicle stimulating hormone (hFSH) and luteinizing hormone (hLH)) was achieved by a Surface Plasmon Resonance (SPR) immunoassay on untreated human urine samples. Multi-analyte detection was accomplished using two alternative formats which are based in the individual or simultaneous immobilization of the hormones on the sensor surface. The lowest detection limit for both hormones in urine was found to be 1 ng mL⁻¹, which in international units (IU) in terms of the World Health Organization (WHO) standards represents 8 mIU mL⁻¹ of hLH and 14 mIU mL⁻¹ of hFSH, respectively. The reliability of the assay was demonstrated by intra- and inter-assay variabilities <6%, chip-to-chip variabilities <5%, recoveries in the range of 80-120% and stability of the sensor response through more than 100 measurements. The sensitivity of this biosensing methodology renders it in a useful technique for the diagnosis of reproductive disorders, as well as for fertility monitoring.     

Fast and sensitive detection of ochratoxin A in red wine by nanoparticle-enhanced SPR

Herein, we present a fast and sensitive biosensor for detection of Ochratoxin A (OTA) in a red wine that utilizes gold nanoparticle-enhanced surface plasmon resonance (SPR). By combining an indirect competitive inhibition immunoassay and signal enhancement by secondary antibodies conjugated with gold nanoparticles (AuNPs), highly sensitive detection of low molecular weight compounds (such as OTA) was achieved. The reported biosensor allowed for OTA detection at concentrations as low as 0.75 ng mL⁻¹ and its limit of detection was improved by more than one order of magnitude to 0.068 ng mL⁻¹ by applying AuNPs as a signal enhancer. The study investigates the interplay of size of AuNPs and affinity of recognition elements affecting the efficiency of the signal amplification strategy based on AuNP. Furthermore, we observed that the presence of polyphenolic compounds in wine samples strongly interferes with the affinity binding on the surface. To overcome this limitation, a simple pre-treatment of the wine sample with the binding agent poly(vinylpyrrolidone) (PVP) was successfully applied.     

Fabrication of highly catalytic silver nanoclusters/graphene oxide nanocomposite as nanotag for sensitive electrochemical immunoassay

Silver nanoclusters and graphene oxide nanocomposite (AgNCs/GRO) is synthesized and functionalized with detection antibody for highly sensitive electrochemical sensing of carcinoembryonic antigen (CEA), a model tumor marker involved in many cancers. AgNCs with large surface area and abundant amount of low-coordinated sites are synthesized with DNA as template and exhibit high catalytic activity towards the electrochemical reduction of H₂O₂. GRO is employed to assemble with AgNCs because it has large specific surface area, super electronic conductivity and strong π-π stacking interaction with the hydrophobic bases of DNA, which can further improve the catalytic ability of the AgNCs. Using AgNCs/GRO as signal amplification tag, an enzyme-free electrochemical immunosensing protocol is designed for the highly sensitive detection of CEA on the capture antibody functionalized immunosensing interface. Under optimal conditions, the designed immunosensor exhibits a wide linear range from 0.1 pg mL⁻¹ to 100 ng mL⁻¹ and a low limit of detection of 0.037 pg mL⁻¹. Practical sample analysis reveals the sensor has good accuracy and reproducibility, indicating the great application prospective of the AgNCs/GRO in fabricating highly sensitive immunosensors, which can be extended to the detection of various kinds of low abundance disease related proteins.     

Sensitive and real-time fiber-optic-based surface plasmon resonance sensors for myoglobin and cardiac troponin I

A sensor to detect markers of cardiac muscle cell death at less than 3 ng ml⁻¹ and in less than 10 min has been achieved. This fiber-optic-based surface plasmon resonance (SPR) sensor is being applied to detect myoglobin (MG) and cardiac troponin I (cTnI) in HEPES buffered saline solution. An in vivo sensor for the early detection of the onset of myocardial infarction (MI) will greatly enhance the patient care. MG and cTnI are two biological markers released from dying cardiac muscle cells during an MI, and their detection at biologically-relevant levels can be diagnostic of MI. Antibodies specific to an antigen of interest are attached to a carboxymethylated dextran layer on a gold SPR surface. With the method developed, the lower limit of detection (LOD) for MG is 2.9 ng ml⁻¹ at 25 °C. The biological level for MG reaches 15-30 ng ml⁻¹ in patient blood after myocardial damage. A Langmuir adsorption isotherm describes the binding well. For cTnI, a lower detection limit of 1.4 ng ml⁻¹ was achieved in preliminary tests. cTnI levels are in the range of 1-3 ng ml⁻¹ in patient blood after myocardial damage. The antibody reaction with the carboxymethylated dextran surface was optimized by modifying the reaction pH, the temperature, and the dextran chain length.     

Improved porous silicon microarray based prostate specific antigen immunoassay by optimized surface density of the capture antibody

Enriching the surface density of immobilized capture antibodies enhances the detection signal of antibody sandwich microarrays. In this study, we improved the detection sensitivity of our previously developed P-Si (porous silicon) antibody microarray by optimizing concentrations of the capturing antibody. We investigated immunoassays using a P-Si microarray at three different capture antibody (PSA – prostate specific antigen) concentrations, analyzing the influence of the antibody density on the assay detection sensitivity. The LOD (limit of detection) for PSA was 2.5 ng mL⁻¹, 80 pg mL⁻¹, and 800 fg mL⁻¹ when arraying the PSA antibody, H117 at the concentration 15 μg mL⁻¹, 35 μg mL⁻¹, and 154 μg mL⁻¹, respectively. We further investigated PSA spiked into human female serum in the range of 800 fg mL⁻¹ to 500 ng mL⁻¹. The microarray showed a LOD of 800 fg mL⁻¹ and a dynamic range of 800 fg mL⁻¹ to 80 ng mL⁻¹ in serum spiked samples.     

High sensitivity detection of 16s rRNA using peptide nucleic acid probes and a surface plasmon resonance biosensor

A signal enhancing method allowing highly sensitive detection of E. coli 16s rRNA was developed using peptide nucleic acid (PNA) as a capture probe and a surface plasmon resonance (SPR) sensor as a detector. 16s rRNA has been used as a genetic marker for identification of organisms, and can be analyzed directly without PCR amplification due to the relatively high number of copies. PNA has a neutral backbone structure, therefore hybridization with 16s rRNA results in the ionic condition being changed from neutral to negative. A cationic Au nanoparticle was synthesized and used for signal amplification by ionic interaction with 16s rRNA hybridized on the PNA probe-immobilized SPR sensor chip. This method resulted in a detection limit of E. coli rRNA of 58.2 ± 1.37 pg mL⁻¹. Using this analytical method, Staphylococcus aureus was detected without purification of rRNA.     

A surface plasmon resonance probe without optical fibers as a portable sensing device

A surface plasmon resonance (SPR) sensor integrating a small sensor probe, a laser emission diode, a photo detector, and a polarizer was developed as a portable sensing device. The sensor probe was made with a glass cylinder, 50 mm long and 1.5 mm in diameter, that was connected directly to a beam splitter without optical fibers. The SPR spectrum obtained with this probe system showed a 10% reflectivity minimum at 690 nm. Shifts of the SPR spectrum induced by refractive index (RI) changes in the sample were measured by detecting the reflection light intensity at 670 nm. When the sensitivity was compared using a BIAcore™ SPR instrument, the lowest sensor response of 1 mV observed with the SPR probe system coincided with 1.4 × 10⁻⁶ of the RI changes. The RI resolution of the SPR probe was estimated with experimentally evaluated noise on the signal, and, consequently, it was concluded that the RI resolution was 1.2 × 10⁻⁵. Moreover, immunoreaction was demonstrated with adsorbed bovine serum albumin (BSA) and anti-BSA antibody as an analyte. As a result, 50 ng mL⁻¹ of the lower detection limit was estimated.     

Multichannel SPR biosensor for detection of endocrine-disrupting compounds

A surface plasmon resonance (SPR) biosensor for simultaneous detection of multiple organic pollutants exhibiting endocrine-disrupting activity, namely atrazine, benzo[a]pyrene, 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-nonylphenol, is reported. The biosensor utilizes a multichannel SPR sensor based on wavelength modulation of SPR and wavelength division multiplexing (WDM) of sensing channels, antibodies as biorecognition element and a competitive immunoassay detection format. An analysis time of 45 min (including 30-min incubation of the sample with antibodies) and limits of detection as low as 0.05, 0.07, 0.16 and 0.26 ng mL⁻¹ are demonstrated for benzo[a]pyrene, atrazine, 2,4-D and 4-nonylphenol, respectively. The biosensor is also shown to be regenerable and suitable for repeated use.     

Ultrasensitive detection of endotoxins using computationally designed nanoMIPs

Novel molecularly imprinted polymer nanoparticles (nanoMIPs) were designed for endotoxin from Escherichia coli 0111:B4, using computational modeling. The screening process based on binding energy between endotoxin and each monomer was performed with 21 commonly used monomers, resulting in the selection of itaconic acid, methacrylic acid and acrylamide as functional monomers due to their strong binding interaction with the endotoxin template. The nanoMIPs were successfully synthesized with functional groups on the outer surface to aid in the immobilization onto sensor surface. The solid phase photopolymerization approach used for the synthesis of nanoMIPs ranging from 200 to 235 nm in diameter. The limit of detection and K_D were significantly improved when endotoxin samples were prepared using a novel triethylamine method. This improved the efficiency of gold nanoparticle functionalization by targeting the subunits of the endotoxin. Compared to the vancomycin MIP control, the endotoxin MIPs displayed outstanding affinity and selectivity towards the endotoxin with K_D values in the range of 4.4–5.3 × 10⁻¹⁰ M, with limits of detection of 0.44 ± 0.02 ng mL⁻¹ as determined by surface plasmon resonance (SPR) sensor when itaconic acid was used as the functional monomer. The MIP surface can be regenerated >30 times without significant loss of binding activity making this approach highly cost effective for expensive analyte templates. The combination of molecular modeling and solid phase synthesis enabled the successful synthesis of nanoMIPs capable of recognition and ultrasensitive detection of endotoxins using the highly sensitive SPR biosensor with triethylamine method.     

Magnetic Fe3O4@Au composite-enhanced surface plasmon resonance for ultrasensitive detection of magnetic nanoparticle-enriched α-fetoprotein

Small molecules or analytes present at low concentrations are difficult to detect directly using conventional surface plasmon resonance (SPR) techniques because only small changes in the refractive index of the medium are typically induced by the binding of these analytes. Here, we present an amplification technique using core–shell Fe₃O₄@Au magnetic nanoparticles (MNPs) for an SPR bioassay. To evaluate this amplification effect, a novel SPR sensor based on a sandwich immunoassay was developed to detect α-fetoprotein (AFP) by immobilizing a primary AFP antibody (Ab₁) on the surface of a 3-mercapto-1-propanesulfonate/chitosan-ferrocene/Au NP (MPS/CS-Fc/Au NP) film employing Fe₃O₄@Au–AFP secondary antibody conjugates (Fe₃O₄@Au–Ab₂) as the amplification reagent. The stepwise fabrication of the biosensor was characterized using UV-vis spectroscopy, electrochemical impedance spectroscopy, and cyclic voltammetry. A calibration curve of Fe₃O₄@Au–Ab₂ conjugates amplification for AFP detection was obtained to yield a correlation in the range of 1.0–200.0 ng mL⁻¹ with a detection limit of 0.65 ng mL⁻¹, and a significant increase in sensitivity was therefore afforded through the use of Fe₃O₄@Au–Ab₂ conjugates as an amplifier. This magnetic separation and amplification strategy has great potential for the detection of other biomolecules of interest with low interference and high sensitivity by changing the antibody label used in the Fe₃O₄@Au–antibody conjugates.     

An ultrasensitive streptavidin-functionalized carbon nanotubes platform for chemiluminescent immunoassay

An ultrasensitive chemiluminescent (CL) immunoassay system was developed for the detection of tumor marker. This sandwich CL assay method was for the first time designed based on a highly efficient streptavidin-functionalized multi-walled carbon nanotubes (MWCNTs) platform. The glass slide was firstly silylanized with 3-gycidoxypropyltrimethoxysilane (GPTMS) to generate surface epoxy group functionality. Subsequently, the MWCNTs/chitosan solution was mixed with streptavidin solution, and a certain amount of the resulting suspension was dropped on the surface of the epoxy-activated glass substrate to form a firm streptavidin-functionalized MWCNTs platform. The biofunctionalized-MWCNTs platform shows large reactive surface area and excellent biocompatibility. The capture antibody can be efficiently immobilized on the biosensing platform surface based on the highly selective recognition of streptavidin to biotinylated antibody. Using α-fetoprotein (AFP) as model analyte, the proposed method exhibits wide linear range of 0.001–0.1 ng mL⁻¹ with a low detection limit down to 0.52 pg mL⁻¹. The CL immunoassay system displays 7.9-fold increase in the detection sensitivity compared to the immunosensor without using MWCNTs. Moreover, the resulting immunosensor demonstrates excellent specificity, good reproducibility, and acceptable stability. This streptavidin-functionalized MWCNTs platform opens a novel and promising avenue for fabricating ultrasensitive CL immunoassay system.     

Surface plasmon resonance-based immunosensor with oriented immobilized antibody fragments on a mixed self-assembled monolayer for the determination of staphylococcal enterotoxin B

An immunosensor based on surface plasmon resonance (SPR) with a mixed self-assembled monolayer (SAM) was developed to determine staphylococcal enterotoxin B (SEB). The SAM on a gold surface was fabricated by adsorbing a mixture of 16-mercapto-1-hexadecanoic acid (16-MHA) and hexanethiol at various molar ratios. Initially, full-length anti-SEB was randomly immobilized onto the SAM to form the immunosensing surface. Through optimization of surface functionalization and anti-SEB immobilization, the SPR sensors can be applied to the determination of SEB in a linear range of 0.01?~?1.0 μg.mL^?1. Furthermore, a smaller antibody fragment (F(ab)’) was generated and immobilized randomly (via amino groups) or in an oriented manner (via ?SH groups) to form the immunosensing surface. The oriented immobilization of F(ab)’ led to a 50% increase in the antigen binding efficiency compared to randomly immobilized covalent F(ab’) fragments. The resulting calibration curve showed higher sensitivity. In addition, the specificity and applicability of the proposed immunosensor to milk samples were also demonstrated. Furthermore, the sensor can be regenerated using 0.1 M HCl, and 70% of the initial response was maintained over 3 cycles.