Biorecognition Elements Research Articles

Highly intensive and long-lasting chemiluminescence “Three-in-One” Hemin/Fe3O4@MoS2 hybrid nanozyme powered lab-on-fiber device for Ochratoxin A immunoassay

A novel long-lasting chemiluminescent (CL) “Three-in-One” Hemin/Fe3O4 @MoS2 hybrid nanozyme powered lab-on-fiber (CN-LOF) device was developed for Ochratoxin A (OTA) detection. The “Three-in-One” nanozymes could catalyze luminol-H2O2 system to produce robust and long-lasting CL under pH range of 6.5–9.0 and temperature range of 0–70 ℃, such excellent catalytic activity was mainly originated from the synergistic effect of MoS2, Fe3O4 and hemin and the nanozyme’s elegant 2D/3D structure. In the reaction system, the nanozymes could continuously catalyze H2O2 to generate O2•− and 1O2 species, which was the main reason for the production of persistent CL. Using OTA antibodies labeled nanozyme for CL signal amplification while a reusable functionalized optical fiber as a biorecognition and separation element and also as a CL transducer, the CN-LOF could quantify OTA with high sensitivity and stability within 20 min. The obtained linear detection range for OTA was 0.69–65.85 ng/mL and the LOD was 0.16 ng/mL under optimal conditions. The proposed CN-LOF method had been successfully applied to the detection of OTA in underground water and milk samples, and the detection results were highly consistent with those of LC-MS, demonstrated the CN-LOF method had great application potential in the analysis of environmental samples and the detection of foodborne biotoxins.

Biorecognition elements appended gold nanoparticle biosensors for the detection of food-borne pathogens - A review

Food-borne diseases emanated from bacterial contamination pose serious threats to human food safety and well-being. Microbes such as Clostridium perfringens, Escherichia coli (E. coli), Listeria monocytogenes (L. monocytogenes), Norovirus, Pseudomonas aeruginosa (P. aeruginosa), Salmonella ssp, Shigella ssp, Staphylococcus aureus (S. aureus), are key food-borne pathogens, hazardous to human health. Conventional methods undertaken for the detection of the microbes include colorimetry, electrochemical analysis, surface-enhanced Raman spectroscopy (SERS), fluorescent spectroscopy, and lateral flow assay. The pathogen discovery methods employ gold nanoparticles (Au-NPs), upheld by their specific optical properties, conductivity, biocompatibility, and high surface area-to-volume ratio. Au-NPs double up as colorimetric nanoparticles, enzyme mimics, fluorescence resonance energy transfer (FRET) quenchers and signal enhancers. In conjugation with biomolecules like nucleic acids, aptamers, antibodies, cells and phages, Au-NPs can discretely discern and bind to target pathogens. This treatise presents a comprehensive review of recent (2015–2021) developments on biorecognition elements appended to Au-NPs, and their deployment in identification of food-borne microorganisms.

Flavocytochrome b2-Mediated Electroactive Nanoparticles for Developing Amperometric L-Lactate Biosensors.

L-Lactate is an indicator of food quality, so its monitoring is essential. Enzymes of L-Lactate metabolism are promising tools for this aim. We describe here some highly sensitive biosensors for L-Lactate determination which were developed using flavocytochrome b2 (Fcb2) as a bio-recognition element, and electroactive nanoparticles (NPs) for enzyme immobilization. The enzyme was isolated from cells of the thermotolerant yeast Ogataea polymorpha. The possibility of direct electron transfer from the reduced form of Fcb2 to graphite electrodes has been confirmed, and the amplification of the electrochemical communication between the immobilized Fcb2 and the electrode surface was demonstrated to be achieved using redox nanomediators, both bound and freely diffusing. The fabricated biosensors exhibited high sensitivity (up to 1436 A·M-1·m-2), fast responses, and low limits of detection. One of the most effective biosensors, which contained co-immobilized Fcb2 and the hexacyanoferrate of gold, having a sensitivity of 253 A·M-1·m-2 without freely diffusing redox mediators, was used for L-Lactate analysis in samples of yogurts. A high correlation was observed between the values of analyte content determined using the biosensor and referenced enzymatic-chemical photometric methods. The developed biosensors based on Fcb2-mediated electroactive nanoparticles can be promising for applications in laboratories of food control.

Biosensors with Metal Ion-Phosphate Chelation Interaction for Molecular Recognition.

Biosensors show promising prospects in the assays of various targets due to their advantages of high sensitivity, good selectivity and rapid response. Molecular recognition is a key event of biosensors, which usually involves the interaction of antigen-antibody, aptamer-target, lectin-sugar, boronic acid-diol, metal chelation and DNA hybridization. Metal ions or complexes can specifically recognize phosphate groups in peptides or proteins, obviating the use of biorecognition elements. In this review, we summarized the design and applications of biosensors with metal ion-phosphate chelation interaction for molecular recognition. The sensing techniques include electrochemistry, fluorescence, colorimetry and so on.

A label-free electrochemical biosensor for highly sensitive detection of GM2A based on gold nanoparticles/conducting amino-functionalized thiophene polymer layer

In this study, a new immunosensor based on an indium tin oxide (ITO) substrate functionalized with gold nanoparticles (GNPs)/amino-functionalized thiophene polymer P(ThiAmn) multilayer was fabricated for impedimetric determination of GM2 activator protein (GM2A). To engineer the biosensor, a relatively simple approach composed of GNPs electrodeposition and ThiAmn electropolymerization was utilized. The use of GNPs and P(ThiAmn) increased the substrate surface area, which was beneficial to immobilize a large amount of anti-GM2A biorecognition elements. The designed bifunctional layer served as a promising matrix material and provided an innovative sensor fabrication. Electrochemical techniques were employed to investigate the specific immuno-interaction process between biorecognition anti-GM2A antibodies and GM2A antigens. Apart from these techniques, spectral techniques were utilized for the characterization of different modified electrode surfaces. Under optimum conditions, GM2A was determined in a linear concentration range from 0.0185 to 111 pg/mL with a detection limit (LOD) of 5.8 fg/mL by electrochemical impedance spectroscopy (EIS) technique. This biosensor exhibited good reproducibility, long storage-stability, and excellent specificity for GM2A antigens. Additionally, this immunosensor was applied to quantify GM2A in commercial serum samples, and satisfactory results were obtained.

Allosteric probe-triggered isothermal amplification to activate CRISPR/Cas12a for sensitive electrochemiluminescence detection of Salmonella

We report an electrochemiluminescence (ECL) sensor for Salmonella detection based on allosteric probe as a bio-recognition element and CRISPR/Cas12a as a signal amplification strategy. In the presence of Salmonella, the structure switching occurs on allosteric probes, resulting in their hybridization with primers to trigger isothermal amplification. Salmonella is then released to initiate the next reaction cycle accompanying by generating a large amount of dsDNA, which are subsequently recognized by CRISPR-gRNA for activating the trans-cleavage activity of Cas12a. Furthermore, the activated Cas12a can indiscriminately cut the ssDNA which is bound to the electrode, enabling the release of the ECL emitter porphyrinic Zr metal − organic framework (MOF, PCN-224) and exhibiting a decreased ECL signal accordingly. The linear range is 50 CFU·mL−1–5 × 106 CFU·mL−1 and the detection limit is calculated to be 37 CFU·mL−1. This method sensitively detects Salmonella in different types of real samples, indicating it is a promising strategy for Salmonella detection

Nanomaterial interfaces designed with different biorecognition elements for biosensing of key foodborne pathogens.

Foodborne diseases caused by pathogen bacteria are a serious problem toward the safety of human life in a worldwide. Conventional methods for pathogen bacteria detection have several handicaps, including trained personnel requirement, low sensitivity, laborious enrichment steps, low selectivity, and long-term experiments. There is a need for precise and rapid identification and detection of foodborne pathogens. Biosensors are a remarkable alternative for the detection of foodborne bacteria compared to conventional methods. In recent years, there are different strategies for the designing of specific and sensitive biosensors. Researchers activated to develop enhanced biosensors with different transducer and recognition elements. Thus, the aim of this study was to provide a topical and detailed review on aptamer, nanofiber, and metal organic framework-based biosensors for the detection of food pathogens. First, the conventional methods, type of biosensors, common transducer, and recognition element were systematically explained. Then, novel signal amplification materials and nanomaterials were introduced. Last, current shortcomings were emphasized, and future alternatives were discussed.

Aptamer-Based Point-of-Care Devices: Emerging Technologies and Integration of Computational Methods.

Recent innovations in point-of-care (POC) diagnostic technologies have paved a critical road for the improved application of biomedicine through the deployment of accurate and affordable programs into resource-scarce settings. The utilization of antibodies as a bio-recognition element in POC devices is currently limited due to obstacles associated with cost and production, impeding its widespread adoption. One promising alternative, on the other hand, is aptamer integration, i.e., short sequences of single-stranded DNA and RNA structures. The advantageous properties of these molecules are as follows: small molecular size, amenability to chemical modification, low- or nonimmunogenic characteristics, and their reproducibility within a short generation time. The utilization of these aforementioned features is critical in developing sensitive and portable POC systems. Furthermore, the deficiencies related to past experimental efforts to improve biosensor schematics, including the design of biorecognition elements, can be tackled with the integration of computational tools. These complementary tools enable the prediction of the reliability and functionality of the molecular structure of aptamers. In this review, we have overviewed the usage of aptamers in the development of novel and portable POC devices, in addition to highlighting the insights that simulations and other computational methods can provide into the use of aptamer modeling for POC integration.

Aptamer-Based Electrochemical Microfluidic Biosensor for the Detection of Cryptosporidium parvum.

Cryptosporidium parvum is a high-risk and opportunistic waterborne parasitic pathogen with highly infectious oocysts that can survive harsh environmental conditions for long periods. Current state-of-the-art methods are limited to lengthy imaging and antibody-based detection techniques that are slow, labor-intensive, and demand trained personnel. Therefore, the development of new sensing platforms for rapid and accurate identification at the point-of-care (POC) is essential to improve public health. Herein, we propose a novel electrochemical microfluidic aptasensor based on hierarchical 3D gold nano-/microislands (NMIs), functionalized with aptamers specific to C. parvum. We used aptamers as robust synthetic biorecognition elements with a remarkable ability to bind and discriminate among molecules to develop a highly selective biosensor. Also, the 3D gold NMIs feature a large active surface area that provides high sensitivity and a low limit of detection (LOD), especially when they are combined with aptamers,. The performance of the NMI aptasensor was assessed by testing the biosensor's ability to detect different concentrations of C. parvum oocysts spiked in different sample matrices, i.e., buffer, tap water, and stool, within 40 min detection time. The electrochemical measurements showed an acceptable LOD of 5 oocysts mL-1 in buffer medium, as well as 10 oocysts mL-1 in stool and tap water media, over a wide linear range of 10-100,000 oocysts mL-1. Moreover, the NMI aptasensor recognized C. parvum oocysts with high selectivity while exhibiting no significant cross-reactivity to other related coccidian parasites. The specific feasibility of the aptasensor was further demonstrated by the detection of the target C. parvum in patient stool samples. Our assay showed coherent results with microscopy and real-time quantitative polymerase chain reaction, achieving high sensitivity and specificity with a significant signal difference (p < 0.001). Therefore, the proposed microfluidic electrochemical biosensor platform could be a stepping stone for the development of rapid and accurate detection of parasites at the POC.

Recycled additive manufacturing feedstocks with carboxylated multi-walled carbon nanotubes toward the detection of yellow fever virus cDNA

Recycled additive manufacturing sensing platforms are fabricated with carboxylated multi-walled carbon nanotubes (COOH-MWCNT) with exhibit enhanced electrochemical biosensor performance allowing for the enhanced direct coupling of the biorecognition element to the COOH-MWCNT for the preparation of an electrochemical genosensor for the detection of yellow fever virus cDNA. Bespoke additive manufacturing filaments was produced using recycled poly(lactic acid) (rPLA, 65 wt%), polyethylene succinate (PES, 10 wt%), carbon black (CB, 15 wt%), and COOH-MWCNT (10 wt%) which exhibits enhanced electrochemical performance over that of commercial filament. A bespoke all-in-one additive manufactured electroanalytical cell is proposed, with the working, reference and counter electrodes in addition to a modification rim that allows for the facile production of biosensors through the application of droplets. The genosensor was applied to the detection of yellow fever Virus cDNA using anodic square wave voltammetry; a linear dynamic range (LDR) of 0.5–15 µM with an R2 of 0.9995, sensitivity of 177 ± 2 µA µM−1, limit of detection (LOD) of 0.138 µM, and limit of quantification (LOQ) of 0.859 µM were obtained. This work highlights how bespoke additive manufacturing filament production can enhance biosensing platforms, whilst using recycled feedstock to improve end-product sustainability.

Antibody-Conjugated Electrospun Nanofibers for Electrochemical Detection of Methamphetamine.

Multifunctional electrospun nanofibers (ENs) with improved properties have increased attention nowadays. Their insoluble forms in water with decreased hydrophobicity are desired for the immobilization of biological molecules. Also, the addition of functional groups on the backbone provides the conjugation of biomolecules onto the surface of ENs via covalent bonds to increase their stability. Here, poly(vinylidene fluoride) (PVDF) was chosen to prepare a platform, which is insoluble in water, and polyethylenimine (PEI) was used to add amine groups on the surface of ENs to bind biological molecules via covalent conjugation. So, PVDF-PEI nanofibers were prepared on a glassy carbon electrode to immobilize an antimethamphetamine antibody (Anti-METH) as a model biomolecule. The obtained PVDF-PEI/Anti-METH was used for the bioelectrochemical detection of methamphetamine (METH), a common illicit drug. Bioelectrochemical detection of METH on PVDF-PEI/Anti-METH-coated electrodes was carried out by voltammetry in the range of 2.0-50 ng/mL METH. Moreover, the effect of dansyl chloride (DNC) derivatization of METH on the sensitivity of PVDF-PEI/Anti-METH was tested. Finally, METH analysis was carried out in synthetic body fluids. The obtained results showed that PVDF-PEI ENs can be adopted as an immobilization matrix for the biorecognition elements of biobased detection systems, and the derivative of METH (METH-DNC) increased the sensitivity of PVDF-PEI/Anti-METH.

Effect of symmetry and substituents of cobalt based phthalocyanines in aptasensor design for the electrochemical impedimetric detection of the human epidermal growth factor receptor 2

Surface functionalization in biosensor design remains the major contributor to biosensor efficiency and selectivity. In electrochemical biosensor design, the surface is usually functionalized to achieve immobilization of the biorecognition element as well as electron transfer mediation for signal amplification. An asymmetric cobalt tris-(acetylphenoxy)-mono-phenoxyacrylic acid phthalocyanine (CoMPhAaPc) and a symmetric cobalt tetra-acetylphenoxy phthalocyanine were used for immobilization of HB5 aptamer and signal amplification towards the electrochemical detection of the human epidermal growth factor 2 (HER2). The efficiency of the two phthalocyanines is compared in relation to the substituents and symmetry. The two phthalocyanines were further coupled with sulphur nitrogen graphene quantum dots (SNGQDs) via π-π interactions, for enhanced activity and performance towards HER2 detection. The efficiency of the designed sensors was tested using the detection of HER2 in human serum. The best performance was observed for the SNGQDs(π)CoMPhAaPc/HB5 (amide) with limit of detection value of 0.0027 ng/mL and sensitivity of 72,253 Ωng−1mL. Considering these results, the aptasensors demonstrate great potential for improved monitoring of human epidermal growth factor receptor 2 levels for the management of breast cancer.

Generation of a high-affinity DNA aptamer for on-site screening of toxic aristolochic acid I in herbal medicines and botanical products

Aristolochic Acid I (AAI) is an environmental and foodborne toxin found in the Aristolochia and Asarum species of plants that are widespread all over the world. Therefore, there is an urgent need to develop a sensitive and specific biosensor for identifying AAI. Aptamers as a powerful biorecognition element provide the most viable options for solving this problem. In this study, we used library-immobilized SELEX to isolate an AAI-specific aptamer with a KD value of 86 ± 13 nM. To verify the practicability of the selected aptamer, a label-free colorimetric aptasensor was designed. This aptasensor exhibited a low detection limit of 225 nM. Besides, it had been further applied for the determination of AAI in real samples and the recoveries ranged from 97.9% to 102.4%. In the future, AAI aptamer will provide a promising tool for safety evaluation in various fields of agriculture, food, and medication.

Detection of Salmonella Enteritidis in Milk Using Conductometric Immunosensor Coated on Polyaniline/Zinc Oxide Nanocomposite.

The demand for rapid and accurate detection methods for Salmonella Enteritidis necessitates the development of highly sensitive and specific biosensors to ensure proper monitoring of food safety and quality requirements in the food sector and to secure human health. This study focused on development of a polyaniline/zinc oxide (PANI/ZnO) nanocomposite film on a gold electrode conductometric immunosensor for detection of Salmonella Enteritidis. The sensor was modified with monoclonal anti-Salmonella Enteritidis antibodies as biorecognition elements. The fabricated sensor was able to detect and quantify the target pathogen within 30 min and showed a good detection range from 101 to 105 colony-forming units (CFU)/mL for Salmonella Enteritidis and a minimum detection limit of 6.44 CFU/mL in 0.1% peptone water. Additionally, the fabricated sensor showed good selectivity and detection limit toward the target bacterium and successfully determined Salmonella Enteritidis content in ultrahigh heat-treated skim milk samples without pretreatment of the food sample.

MicroRNA Detection via Nanostructured Biochips for Early Cancer Diagnostics.

MicroRNA (miRNA) are constituted of approximately 22 nucleotides and play an important role in the regulation of many physiological functions and diseases. In the last 10 years, an increasing interest has been recorded in studying the expression profile of miRNAs in cancer. Real time-quantitative polymerase chain reaction (RT-qPCR), microarrays, and small RNA sequencing represent the gold standard techniques used in the last 30 years as detection methods. The advent of nanotechnology has allowed the fabrication of nanostructured biosensors which are widely exploited in the diagnostic field. Nanostructured biosensors offer many advantages: (i) their small size allows the construction of portable, wearable, and low-cost products; (ii) the large surface-volume ratio enables the loading of a great number of biorecognition elements (e.g., probes, receptors); and (iii) direct contact of the recognition element with the analyte increases the sensitivity and specificity inducing low limits of detection (LOD). In this review, the role of nanostructured biosensors in miRNA detection is explored, focusing on electrochemical and optical sensing. In particular, four types of nanomaterials (metallic nanoparticles, graphene oxide, quantum dots, and nanostructured polymers) are reported for both detection strategies with the aim to show their distinct properties and applications.

Selective Aptamer Modification of Au Surfaces in a Microelectrode Sensor Array for Simultaneous Detection of Multiple Analytes

Aptamers have been employed as the biorecognition element in electrochemical aptamer-based (E-AB) biosensors, for the detection of a diverse range of analyte molecules, on electrodes with sizescales ranging from a few microns to several millimeters. Simultaneous detection of multiple different analytes requires the selective modification of multiple electrode surfaces with different aptamers. This process is typically achieved by incubating separate macroscale electrodes in a solution with the desired aptamer, which is unsuitable for microelectrode arrays in which the electrodes are closely spaced. In this work, we selectively modified electrode surfaces with thiolated aptamers of different single-stranded DNA sequences, by successive removal and addition of thiol monolayers. This was achieved by electrodesorption of thiol monolayers using controlled potential, to expose unmodified gold electrodes to be modified with a different thiolated aptamer, thus enabling multiple different aptamers to be used on the surfaces of closely spaced microelectrodes. All aptamers were methylene blue terminated, allowing redox currents to be measured and used to monitor aptamer probe packing density on the electrode surface and the selectivity of the sensors. Here, we demonstrate the microscale E-AB sensor multianalyte detection method using aptamers for target analytes, adenosine triphosphate, dopamine, and serotonin, which can ultimately be applied to perform localized simultaneous detection using electrode arrays.

RBD-Modified Polyaniline-Based Label-Free Immunosensor for Sensitive Impedimetric Detection of Anti-SARS-CoV-2 Antibodies

The emergence of the SARS-CoV-2 virus and the associated pandemic has affected the entire human population. Human susceptibility to the virus has highlighted a tremendous need for affordable diagnostic systems to manage the pandemic and monitor the effectiveness of vaccination. We have developed a simple and label-free electrochemical immunosensor for the detection of human anti-SARS-CoV-2 IgG antibodies, which consists of a supporting screen-printed carbon electrode (SPCE) modified with an electrodeposited polyaniline film and glutaraldehyde, allowing effective immobilization of the SARS-CoV-2 spike glycoprotein receptor-binding domain (RBD) as a biorecognition element. The impedimetric immunosensor showed a linear response over a wide concentration range of 0.01–10 μg mL−1, that is, 67 pM–6.7 nM, with a low detection limit of 25.9 pM. A dual working electrode configuration with a built-in negative control unit was demonstrated for practical field applications. The immunosensor was successfully used in a real serum sample from an infected patient and showed good reproducibility and fair agreement with ELISA. An optional amplification step with secondary goat anti-human IgG antibodies was demonstrated, resulting in an extended linear range and a detection limit as low as 0.93 pM.

Plasmonic genosensor for detecting hazelnut Cor a 14-encoding gene for food allergen monitoring

A plasmonic nanostructure was constructed as a biorecognition element coupled to an optical sensing platform in sandwich format, targeting the hazelnut Cor a 14 allergen-encoding gene. The analytical performance of the genosensor presented a linear dynamic range between 100 amol L−1 and 1 nmol L−1, a limit of detection (LOD) < 19.9 amol L−1, and a sensitivity of 13.4 ± 0.6 m°. The genosensor was successfully hybridized with hazelnut PCR products, tested with model foods, and further validated by real-time PCR. It reached a LOD <0.001% (10 mg kg−1) of hazelnut in wheat material (corresponding to 1.6 mg kg−1 of protein) and a sensitivity of −17.2 ± 0.5 m° for a linear range of 0.001%–1%. Herein, a new genosensing approach is proposed as a highly sensitive and specific alternative tool with potential application in monitoring hazelnut as an allergenic food, protecting the health of sensitized/allergic individuals.

Optical bi-enzyme-titania biosensor system: A new way to detect lactose

Lactose concentration is a key parameter for assessing the quality of raw and processed milk and identifying abnormal milk. Current methods for lactose determination are laborious, time-consuming and oftentimes require skilled lab workers, expensive equipment, and special reagents. In this study, an optical biosensor system based on nanosized titanium dioxide (TiO2) is presented for routine measurement of lactose in milk without sample preparation. TiO2 was obtained via sol-gel synthesis and characterized using dynamic light scattering, microelectrophoresis, X-ray diffraction, transmission electron microscopy, and nitrogen adsorption-desorption analysis. The system, whose colorimetric response was based on the chromogen-free TiO2-based detection of hydrogen peroxide (H2O2), used β-galactosidase and/or glucose oxidase as the biorecognition elements. The selectivity and sensitivity of the biosensors for lactose and glucose was investigated by diffuse reflectance spectroscopy using standard aqueous lactose and glucose solutions and whole milk samples. The results showed that the proposed biosensors had high optical signal reproducibility (RSD = 5–6%), high sensitivity (LOD = 0.005 wt%), and high selectivity. The linear dynamic ranges of lactose and glucose detection were from 0.005 wt% to 0.100 wt%, and the optimal response time of the biosensor was 25 min. The listed features and advantages of label-free detection make the biosensor attractive for simple, inexpensive, sensitive, and selective determination of lactose in milk.

Controlling the Binding Efficiency of Surface Confined Antibodies through the Design of Mixed Self‐Assembled Monolayers

AbstractA plethora of different electronic and optoelectronic devices have been developed lately, for biosensing applications (e.g., label‐free, fast, and easier to operate) based on a detecting interface accommodating the biorecognition elements, anchored by thiolate self‐assembled monolayers (SAMs) on a gold surface. Here, a surface plasmon resonance (SPR) characterization of anti‐p24 anchored on different SAMs is performed to investigate the effect of the SAM structure on the antibodies’ packing efficiency and the sensors’ analytical figures of merit. Notably, the mixed SAM deposited from a solution 10:1 of 3‐mercaptopropionic acid and 11‐mercaptoundecanoic acid (11MUA) is compared to that resulting from a solution 10:1 of ad hoc synthesized N‐(2‐hydroxyethyl)‐3‐mercaptopropanamide (NMPA)/11MUA. Despite the improvement in the anti‐p24 surface coverage registered using the 11MUA/NMPA SAM, the latter produces a significant decrease in the antibodies’ binding efficiency against human immunodeficiency virus p24 protein. To provide a molecular rationale behind the SPR data, density functional theory calculations are also undertaken. A comprehensive physical view of the main competing phenomena affecting the biorecognition events at a biofunctionalized gold detecting interface is represented here.