Label‐ and modification‐free‐based in situ selection of bovine serum albumin specific aptamer

Systematic evolution of ligands by exponential enrichment is a traditional approach to select aptamer, which has a great potential in biosensing field. However, chemical modifications of DNA library or targets before selection might block the real recognition and binding sites between aptamers and their targets. In this study, a label‐ and modification‐free‐based in situ selection strategy was developed to overcome this limitation. The strategy is an attempt to screen bovine serum albumin aptamers according to the principle of electrophoretic mobility shift assay, and allowed single‐stranded DNA sequence to be fully exposed to interact with bovine serum albumin which was mixed with the agarose gel beforehand. After eight rounds of selection, specific aptamer with low dissociation constant (K_d) value of 69.44 ± 7.60 nM was selected and used for subsequent establishment of fluorescence biosensor. After optimization, the optimal aptasensor exhibited a high sensitivity toward bovine serum albumin with a limit of detection of 0.24 ng/mL (linear range from 1 to 120 ng/mL). These results indicated that the label‐ and modification‐free‐based in situ selection strategy proposed in this work could effectively select specific aptamer to develop aptasensor for sensitive detection of bovine serum albumin or other targets in actual complicated samples.     

A comparison of four protocols for the immobilization of an aptamer on graphite composite electrodes

We have performed a comparative study on four protocols for the immobilization of the thrombin aptamer on a graphite-epoxy composite electrode with the aim to identify the most practical method for designing the corresponding impedimetric aptasensor. The protocols included (a) physical adsorption, (b) avidin-biotin affinity interaction, (c) electrochemical activation and covalent bonding via amide groups, and (d) electrochemical grafting using 4-carboxybenzenediazonium coupling. The properties of the sensing surface were probed by electrochemical impedance measurements in the presence of the (ferri/ferro)hexacyanide redox couple. An increase in the interfacial charge transfer resistance (R_ct) was noted in all cases after the aptamer-thrombin interaction had occurred. The selectivity of the aptasensor over common serum proteins was also systematically investigated. Physical adsorption resulted in the lowest detection limit of the probe (4.5 pM), while avidin-biotin interaction resulted in highest selectivity and reproducibility exhibiting a 4.9 % relative standard deviation at pM thrombin concentration levels.

Electrochemical Detection of Activated Protein C Using an Aptasensor Based on PAMAM Dendrimer Modified Pencil Graphite Electrodes

Electrochemical aptasensing of APC was carried out using PAMAM dendrimer modified pencil graphite electrodes (PGEs) for the first time herein. Poly(amidoamine) dendrimer having 16 succinamic acid surface groups (generation 2, G2‐PS) modified PGEs were developed, and then were utilized for APC monitoring using differential pulse voltammetry, electrochemical impedance spectroscopy and cyclic voltammetry. The selectivity of single‐use aptasensor was tested against to other proteins; BSA and THR as well as to the affinity of APC binding to different DNA aptamer, or oligonucleotide. Voltammetric APC detection was also explored in a diluted fetal bovine serum resulting with a detection limit DL as 1.5 µg/mL.     

Graphene Oxide Modified Chemically Activated Graphite Electrodes for Detection of microRNA

In our study, graphene oxide (GO) modified graphite electrodes were used for sensitive and selective impedimetric detection of miRNA. After chemical activation of pencil graphite electrode (PGE) surface using covalent agents (CA), GO modification was performed at the surface of chemically activated PGE. Then, CA‐GO‐PGEs were applied for impedimetric miRNA detection. The microscopic and electrochemical characterization of CA‐GO‐PGEs was performed by scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS). The optimization of experimental conditions; such as GO concentration, DNA probe concentration and miRNA target concentration was performed by using EIS technique. After the hybridization occurred between miRNA‐34a RNA target and its complementary DNA probe, the hybrid was immobilized onto the surface of CA‐GO‐PGEs. Then, the impedimetric detection of miRNA‐DNA hybridization was performed by EIS. The selectivity of our assay was also tested under the optimum experimental conditions.     

A highly selective and sensitive cocaine aptasensor based on covalent attachment of the aptamer-functionalized AuNPs onto nanocomposite as the support platform

Based on the conformational changes of the aptamer-functionalized gold nanoparticles (AuNPs) onto MWCNTs/IL/Chit nanocomposite as the support platform, we have developed a sensitive and selective electrochemical aptasensor for the detection of cocaine. The 5′-amine-3′-AuNP terminated aptamer is covalently attached to a MWCNTs/IL/Chit nanocomposite. The interaction of cocaine with the aptamer functionalized AuNP caused the aptamer to be folded and the AuNPs with negative charge at the end of the aptamer came to the near of electrode surface therefore, the electron transfer between ferricyanide (K₃Fe(CN)₆) as redox probe and electrode surface was inhibited. A decreased current of (K₃Fe(CN)₆) was monitored by differential pulse voltammetry technique. In an optimized condition the calibration curve for cocaine concentration was linear up to 11 μM with detection limit (signal-to-noise ratio of 3) of 100 pM. To test the selectivity of the prepared aptasensor sensing platform applicability, some analgesic drugs as the interferes were examined. The potential of the aptasensor was successfully applied for measuring cocaine concentration in human blood serum. Based on our experiments it can be said that the present method is absolutely beneficial in developing other electrochemical aptasensor.     

Electrochemiluminescence Aptasensor for the MUC1 Protein Based on Multi‐functionalized Graphene Oxide Nanocomposite

The electrochemiluminescence (ECL) aptasensor was prepared for the detection of Mucin 1 based on its specific recognition by aptamer immobilized on multi‐functionalized graphene oxide nanocomposite, which was prepared with N‐(4‐aminobutyl)‐N‐ethylisoluminol (ABEI) and aptamer chemically bound to the surface of magnetic GO (nanoFe₃O₄@GO). ABEI and aptamer acted as the electrochemiluminophore and the capture device for Mucin 1 respectively. NanoFe₃O₄@GO brought multi‐functionalized graphene oxide nanocomposite attracted on the surface of magnetic glass carbon electrode through magnetism, enabled all the ABEI immobilized electrochemically active due to its good conductivity and thus then facilitated the sensitive detection of Mucin 1. In addition, the ECL aptasensor can be prepared through a one‐step process. Under optimal conditions, the ECL intensity of the aptasensor decreased proportionally to the logarithmic concentrations of Mucin 1 in the range of 0.005–1000 ng mL^?1. This aptasensor displays good specificity, stability, reproducibility and application. This method has a large potential because such a multi‐functionalized graphene oxide nanocomposite also may be applied to other ECL‐based aptasensors.     

Development of Electrochemical Impedance Spectroscopy Based Malaria Aptasensor Using HRP‐II as Target Biomarker

Current demand for a stable, low cost and sensitive malaria sensor has prompted to explore novel recognition systems that can substitute widely used protein based labile biorecognition elements to be used in point of care diagnostic devices. Here, we report a novel ssDNA aptamer of 90 mer sequence developed by SELEX process against HRP‐II, a specific biomarker for Plasmodium falciparum strains. High stability of the secondary structure of the isolated aptamer was discerned from its free energy of folding of −20.40 kcal mole⁻¹. The binding constant (K_d) of the aptamer with HRP‐II analysed by isothermal titration calorimetry was ∼1.32 μM. Circular dichroism studies indicated B form of the aptamer DNA. The aptamer was chemically immobilized on a gold electrode surface through a self‐assembled monolayer of dithio‐bis(succinimidyl) propionate to produce the aptasensor. The step wise modification of the layers over the gold electrode during fabrication of the aptasensor was confirmed by cyclic voltammetry. The aptasensor was then challenged with different concentration of HRP‐II and analysed the interaction signals through electrochemical impedance spectroscopy. The impedance signal behaved reciprocally with the increasing concentrations of the target in the sample from which a dynamic range of 1 pM–500 pM (R²=0.99) and LOD of ∼3.15 pM were discerned. The applicability of the developed aptasensor to detect HRP‐II in mimicked real sample was also validated.     

Amplified impedimetric aptasensor based on gold nanoparticles covalently bound graphene sheet for the picomolar detection of ochratoxin A

An amplified electrochemical impedimetric aptasensor for ochratoxin A (OTA) was developed with picomolar sensitivity. A facile route to fabricate gold nanoparticles covalently bound reduced graphene oxide (AuNPs–rGO) resulted in a large number of well-dispersed AuNPs on graphene sheets with tremendous binding sites for DNA, since the single rGO sheet and each AuNP can be loaded with hundreds of DNA strands. An aptasensor with sandwich model was fabricated which involved thiolated capture DNA immobilized on a gold electrode to capture the aptamer, then the sensing interface was incubated with OTA at a desired concentration, followed by AuNPs–rGO functionalized reporter DNA hybridized with the residual aptamers. By exploiting the AuNPs–rGO as an excellent signal amplified platform, a single hybridization event between aptamer and reporter DNA was translated into more than 10⁷ redox events, leading to a substantial increase in charge-transfer resistance (R_ct) by 7∼ orders of magnitude compared with that of the free aptamer modified electrode. Such designed aptasensor showed a decreased response of R_ct to the increase of OTA concentrations over a wide range of 1 pg mL⁻¹–50 ng mL⁻¹ and could detect extremely low OTA concentration, namely, 0.3 pg mL⁻¹ or 0.74 pM, which was much lower than that of most other existed impedimetric aptasensors. The signal amplification platform presented here would provide a promising model for the aptamer-based detection with a direct impedimetric method.     

DNA Electrochemical Aptasensor for Detecting Fumonisins B1 Based on Graphene and Thionine Nanocomposite

In this paper, a novel aptasensor was designed by with the dual amplification of Au nanoparticles (AuNPs) and graphene/thionine nanocomposites (GS‐TH) for sensitive determination of fumonisins B₁ (FB₁). AuNPs is modified at the electrode surface to increase the electrical conductivity and fabricate specific recognition interface for FB₁ through the hybridization of capture DNA and its aptamer. Large number of TH molecules were loaded at the surface of graphene sheet to served as electrochemical probe and increase its electrochemical signal due to the excellent conductivity and large surface area of graphene sheet. This type of nanocomposites is then assembled to the single strand section of FB₁ aptamer at electrode surface by π–π stacking interactions between them, leading to an enhanced electrochemical signal. After the specific combination between FB₁ aptamer and its target (FB₁) in solution, GS–TH was released from electrode surface, resulting in a decreased electrochemical signal. The result demonstrated that the decreased currents were proportional to the FB₁ concentration in the range of 1–10⁶ pg/mL with a detection limit of 1 pg/mL. Besides, the developed aptasensor was also applied successfully for the determination of FB₁ in feed samples. The result shows this aptasensor has a higher sensitivity and selectivity.     

Chitosan/Nitrogen Doped Reduced Graphene Oxide Modified Biosensor for Impedimetric Detection of microRNA

The development of a low‐cost and disposable biosensor platform for the sensitive and rapid detection of microRNAs (miRNAs) is of great interest for healthcare, pharmaceuticals, and medical science. We designed an impedimetric biosensing platform using Chitosan (CHIT)/nitrogen doped reduced graphene oxide (NRGO) conductive composite to modify the surface of pencil graphite electrodes (PGE) for the sensitive detection of miRNAs. An initial optimisation protocol involved investigation of the effect of NRGO concentration and miR 660 DNA probe concentration on the response of the modified electrode. After the optimization protocol, the sequence‐selective hybridization between miR 660 DNA probe and its RNA target was evaluated by measuring changes on charge transfer resistance, R_ct values. Moreover, the selectivity of impedimetric biosensor was tested in the presence of non‐complementary miRNA (NC) sequences, such as miR 34a and miR 16. The hybridization process was examined both in phosphate buffer (PBS) and in PBS diluted fetal bovine serum (FBS:PBS) solutions. The biosensor demonstrated a detection limit of 1.72 μg/mL in PBS and 1.65 μg/mL in FBS:PBS diluted solution. Given the easy, quick and disposable attributes, the proposed conductive nanocomposite biosensor platform shows great promise as a low‐cost sensor kit for healthcare monitoring, clinical diagnostics, and biomedical devices.     

Detection of thrombin using electrogenerated chemiluminescence based on Ru(bpy)3(2+)-doped silica nanoparticle aptasensor via target protein-induced strand displacement

A sensitive and selective aptasensor using tri(2,2′-bipyridyl)ruthenium(II)-doped silica nanoparticles (Ru(bpy)₃²⁺-doped SNPs) as DNA tags for detection of thrombin is developed based on the target protein-induced strand displacement of the DNA probe. For the proposed aptasensor, the aptamer was assembled on the surface of the Au electrode through Au-S binding. The hybridization event between the DNA probe labeled by the Ru(bpy)₃²⁺-doped SNPs and the aptamer was evaluated by electrogenerated chemiluminescence (ECL) measurements. Then, the DNA probe was displaced by thrombin and the binding event between the thrombin and the aptamer was monitored by ECL measurements again. The difference of ECL intensity (ΔI_ECL) of the two events could be used to quantify the thrombin. Other proteins, such as bovine serum albumin and bovine hemoglobin, had almost negligible ΔI_ECL. Under the optimal conditions, the ΔI_ECL was linearly related to the concentration of the thrombin in the range of 10 fM to 10 pM and the detection limit was down to 1.0 fM since SNPs containing a large number of Ru(bpy)₃²⁺ molecules were labeled on the DNA probe.     

Preparation of Epirubicin Aptasensor Using Curcumin as Hybridization Indicator: Competitive Binding Assay between Complementary Strand of Aptamer and Epirubicin

The present work explains the fabrication of a novel electrochemical aptasensor for identifying and measuring the epirubicin (Epi) by using curcumin (Cur) as an anticancer electrochemical indicator. The aptasensor prepared by immobilizing the thiolated aptamer on the surface of graphite screen‐printed electrode modified with gold nanoparticles/functionalized multiwall carbon nanotubes, ionic liquid and chitosan nanocomposite (AuNPs/FMWCNTs‐IL‐Chit/SPE). To evaluate the willingness of aptamer to interaction with Epi in the presence of complementary strand DNA, competitive binding assay between the complementary strand of aptamer and Epi were used. Cur tends to bound to the grooves of two strands DNA. With increasing the concentration of Epi in the range of 0.007–7.0 μM, the peak current of Cur decreased, due to the formation of aptamer‐Epi complex and decreasing the amount of complementary strand DNA. Through the control experiments, we examined the response of fabricated aptasensor for some anticancer drugs, which have a structure similar to the Epi. The results showed that using the thiol‐terminated aptamer as a recognition layer led to a sensor with a high tendency for Epi compared to other anticancer drugs.     

基于电化学双共价键法构建可再生高灵敏的电化学发光适体传感器检测可卡因

基于点击化学和重氮盐法的双共价键固定化方法,制备了一种高灵敏、可重复使用的电化学发光(ECL)适体传感器. 该方法以可卡因为分析物,以可卡因适体为分子识别物质,以钌联吡啶衍生物为ECL信号物质. 采用电化学方法在玻碳电极表面重氮化叠氮苯胺,通过点击反应连接炔基功能化的钌联吡啶衍生物标记可卡因适体,获得适体传感器. 该传感器在共反应剂存在下,产生弱的电化学发光信号,可卡因存在下,电化学发光信号增加. 基于此,建立了“信号增强”型检测可卡因的电化学发光分析新方法. 电化学发光信号与可卡因浓度在0.1 nmol·L^-1 ~ 100 nmol·L^-1范围内呈良好的线性关系,检出限为60 pmol·L^-1. 该传感器具有良好的稳定性,可重复多次使用. 该双共价键法在构建ECL传感器方面具有很好的应用前景.     

A label free aptasensor for Ochratoxin A detection in cocoa beans: An application to chocolate industries

Contamination of food by mycotoxin occurs in minute/trace quantities. Nearly 92.5% of the cocoa samples present Ochratoxin A (OTA) levels at trace quantity. Hence, there is a necessity for a highly sensitive and selective device that can detect and quantify these organic toxins in various matrices such as cocoa beans. This work reports for the first time, a facile and label-free electrochemical impedimetric aptasensor for rapid detection and quantitation of OTA in cocoa beans. The developed aptasensor was constructed based on the diazonium-coupling reaction mechanism for the immobilization of anti-OTA-aptamer on screen printed carbon electrodes (SPCEs). The aptasensor exhibited a very good limit of detection (LOD) as low as 0.15 ng/mL, with added advantages of good selectivity and reproducibility. The increase in electron transfer resistance was linearly proportional to the OTA concentration in the range 0.15–2.5 ng/mL, with an acceptable recovery percentage (91–95%, RSD = 4.8%) obtained in cocoa samples. This work can facilitate a general model for the detection of OTA in cocoa beans based on the impedimetric aptasensor. The analysis can be performed onsite with pre-constructed and aptamer modified electrodes employing a portable EIS set up.     

Label‐free Electrochemical Aptasensor for Carcino‐embryonic Antigen Based on Ternary Nanocomposite of Gold Nanoparticles,Hemin and Graphene

In this report, a label‐free electrochemical aptasensor for carcino‐embryonic antigen (CEA) was successfully developed based on a ternary nanocomposite of gold nanoparticles, hemin and graphene nanosheets (AuNPs‐HGNs). This nanocomposite was prepared by decorating gold nanoparticles on the surface of hemin functionalized graphene nanosheets via a simple wet‐chemical strategy. The aptamer can be assembled on the surface of AuNPs‐HGNs/GCE (glassy carbon electrode) through Au‐S covalent bond to form the sensing interface. Hemin absorbed on the graphene nanosheets not only acts as a protective agent of graphene sheets, but also as an in situ probe base on its excellent redox properties. Gold nanoparticles provide with both numerous binding sites for loading CEA binding aptamer (CBA) and good conductivity to promote the electron transfer. The current changes, which are caused by CEA specifically binding on the modified electrode, are exploited for the label‐free detection of CEA in a very rapid and convenient protocol. Therefore, the method has advantages of high sensitivity, wide linear range (0.0001–10 ng mL^?1), low detection limit (40 fg mL^?1) and attractive specificity. The results illustrate that the proposed label‐free electrochemical aptasensor has a potential application in the biological or clinical target analysis for its simple operation and low cost.     

Ultrasensitive and Signal‐on Electrochemiluminescence Aptasensor Using the Multi‐tris(bipyridine)ruthenium(II)‐β‐cyclodextrin Complexes

An ultrasensitive and signal‐on electrochemiluminescence (ECL) aptasensor to detect target protein (thrombin or lysozyme) was developed using the host‐guest recognition between a metallocyclodextrin complex and single‐stranded DNA (ss‐DNA). The aptasensor uses both the photoactive properties of the metallocyclodextrins named multi‐tris(bipyridine)ruthenium(II)‐β‐cyclodextrin complexes and their specific recognition with ss‐DNA, which amplified the ECL signal without luminophore labeling. After investigating the ECL performance of different multi‐tris(bipyridine)ruthenium(II)‐β‐cyclodextrin (multi‐Ru‐β‐CD) complexes, tris‐tris(bipyridine)‐ruthenium(II)‐β‐cyclodextrin (tris(bpyRu)‐β‐CD) was selected as a suitable host molecule to construct an atasensor. First, double‐stranded DNA (ds‐DNA) formed by hybridization of the aptamer and its target DNA was attached to a glassy carbon electrode via coupling interaction, which showed low ECL intensity with 2‐(dibutylamino) ethanol (DBAE) as coreactant, because of the weak recognition between ds‐DNA and tris(bpyRu)‐β‐CD. Upon addition of the corresponding protein, the ECL intensity increased when target ss‐DNA was released because of the higher stability of the aptamer‐protein complex than the aptamer‐DNA one. A linear relationship was observed in the range of 0.01 pmol/L to 100 pmol/L between ECL intensity and the logarithm of thrombin concentrations with a limited detection of 8.5 fmol/L (S/N=3). Meanwhile, the measured concentration of lysozyme was from 0.05 pmol/L to 500 pmol/L and the detection limit was 33 fmol/L (S/N=3). The investigations of proteins in human serum samples were also performed to demonstrate the validity of detection in real clinical samples. The simplicity, high sensitivity and specificity of this aptasensor show great promise for practical applications in protein monitoring and disease diagnosis.     

Label-free electrochemical aptasensor for the detection of lysozyme

This work reports the advantages of a label free electrochemical aptasensor for the detection of lysozyme. The biorecognition platform was obtained by the adsorption of the aptamer on the surface of a carbon paste electrode (CPE) previously blocked with mouse immunoglobulin under controlled-potential conditions. The recognition event was detected from the decrease in the guanine and adenine electro-oxidation signals produced as a consequence of the molecular interaction between the aptamer and lysozyme. The biosensing platform demonstrated to be highly selective even in the presence of large excess (9-fold) of bovine serum albumin, cytochrome C and myoglobin. The reproducibility for 10 repetitive determinations of 10.0 mg L⁻¹ lysozyme solution was 5.1% and 6.8% for guanine and adenine electro-oxidation signals, respectively. The detection limits of the aptasensor were 36.0 nmol L⁻¹ (if considering guanine signal) and 18.0 nmol L⁻¹ (if taking adenine oxidation current). This new sensing approach represents an interesting and promising alternative for the electrochemical quantification of lysozyme.     

Carbon Nanotubes Modified Graphite Electrodes for Monitoring of Biointeraction Between 6‐Thioguanine and DNA

In this present study, single‐walled carbon nanotubes (SWCNT) modified disposable pencil graphite electrodes (SWCNT‐PGEs) were developed for the electrochemical monitoring of anticancer drug, and its interaction with double stranded DNA (dsDNA). Under this aim, SWCNT‐PGEs were applied for the first time in the literature to analyse of 6‐Thioguanine (6‐TG), and also to investigate its interaction with DNA by voltammetric and impedimetric methods. The surface morphologies of PGE and SWCNT‐PGE were explored using scanning electron microscopy (SEM) and electrochemical characterization of unmodified/modified electrodes was performed by cyclic voltammetry (CV). Experimental parameters; such as, the concentration of 6‐TG and its interaction time with dsDNA were optimized by using differential pulse voltammetry (DPV). Additionally, the interaction of 6‐TG with dsDNA was studied in case of different interaction times by electrochemical impedance spectroscopy (EIS) in contrast to voltammetric results. The detection limit of 6‐TG was found to be 0.25 μM by SWCNT‐PGE.     

A Sandwich‐type Electrochemiluminescence Aptasensor for Thrombin Based on Functional Co‐polymer Electrode Using Ru(bpy)32+ Doped Nanocomposites as Signal‐amplifying Tags

Herein, a signal‐on sandwich‐type electrochemiluminescence (ECL) aptasensor for the detection of thrombin (TB) was proposed. The graphene (GR) doped thionine (TH) was electropolymerized synchronously on the bare glassy carbon electrode (GCE) to form co‐polymer (PTG) electrode. The gold nanoparticles (AuNPs) were decorated on the surface of the PTG by in‐situ electrodeposition, and the functional co‐polymer (PTG‐AuNPs) electrode was utilized as sensing interface. Then, TB binding aptamer I (TBA I) as capture probes were modified on the PTG‐AuNPs electrode to capture TB, and Ru(bpy)₃²⁺/silver nanoparticles doped silica core‐shell nanocomposites‐labeled TB binding aptamer II (RuAg/SiO₂NPs@TBA II) were used as signal probes to further bind TB, resulting in a sandwich structure. With the assistant of silica shell and AgNPs, the enrichment and luminous efficiency of Ru(bpy)₃²⁺ were significantly improved. Under the synergy of PTG‐AuNPs and RuAg/SiO₂NPs, the ECL signal was dramatically increased. The proposed ECL aptasensor displayed a wide linear range from 2 fM to 2 pM with the detection limit of 1 fM, which is comparable or better than that in reported ECL aptasensors for TB using Ru(bpy)₃²⁺ and its derivatives as the luminescent substance. The excellent sensitivity makes the proposed aptasensor a promising potential in pharmaceutical and clinical analysis.     

Beta‐lactoglobulin Electrochemical Detection Based with an Innovative Platform Based on Composite Polymer

In this work, we present a new electrochemical disposable platform based on poly(aniline‐co‐anthranilic acid) (PANI/PAA) composite polymer coupled with an aptamer for sensitive detection of β‐lactoglobulin. Firstly, PANI/PAA film was electrodeposited on the graphite screen‐printed electrode surface by cyclic voltammetry. The co‐polymer modified electrode was then employed as platform for the covalent immobilization of an amino‐modified aptamer. Various β‐lactoglobulin solutions, with a fixed amount of biotinylated oligonucleotide complementary sequence, were dropped onto the aptasensor surface. A streptavidin‐alkaline phosphatase conjugate was then employed to trace the affinity reaction. After the addition of 1‐naphthyl‐phosphate enzymatic substrate, 1‐naphthol electroactive product was detected by differential pulse voltammetry. A decrease in the signal was obtained when the target concentration was increased, in according to a signal‐off approach. After optimization of key experimental parameters, a dose‐response curve was obtained between 0.01–1.0 μg mL^?1 β‐lactoglobulin concentration range. The limit of detection of 0.053 μg L^?1 was obtained. Milk samples spiked with β‐lactoglobulin were analyzed.