Biorecognition Events Research Articles

Immuno-Rolling Circle Amplification (Immuno-RCA): Biosensing Strategies, Practical Applications, and Future Perspectives.

In the rapidly evolving field of life sciences and biomedicine, detecting low-abundance biomolecules, and ultraweak biosignals presents significant challenges. This has spurred a rapid development of analytical techniques aiming for increased sensitivity and specificity. These advancements, including signal amplification strategies and the integration of biorecognition events, mark a transformative era in bioanalytical precision and accuracy. A prominent method among these innovations is immuno-rolling circle amplification (immuno-RCA) technology, which effectively combines immunoassays with signal amplification via RCA. This process starts when a targeted biomolecule, such as a protein or cell, binds to an immobilized antibody or probe on a substrate. The introduction of a circular DNA template triggers RCA, leading to exponential amplification and significantly enhanced signal intensity, thus the target molecule is detectable and quantifiable even at the single-molecule level. This review provides an overview of the biosensing strategy and extensive practical applications of immuno-RCA in detecting biomarkers. Furthermore, it scrutinizes the limitations inherent to these sensors and sets forth expectations for their future trajectory. This review serves as a valuable reference for advancing immuno-RCA in various domains, such as diagnostics, biomarker discovery, and molecular imaging.

Distinguishable Magnetic Reporter Coordination with Buoyancy-Magnetism Separation for Immobilization-Free Dual-Target Electrochemical Immunosensing.

Simultaneous sensitive and precise determination of multibiomarkers is of great significance for improving detection efficiency, reducing diagnosis and treatment expenses, and elevating survival rates. However, the development of simple and portable biosensors for simultaneous determination of multiplexed targets in biological fluids still faces challenges. Herein, a unique and versatile immobilization-free dual-target electrochemical biosensing platform, which combines distinguishable magnetic signal reporters with buoyancy-magnetism separation, was designed and constructed for simultaneous detection of carcinoembryonic (CEA) and α-fetoprotein (AFP) in intricate biological fluids. To construct such distinguishable magnetic signal reporters with signal transduction, amplification, and output, secondary antibodies of CEA and AFP were respectively functionalized on methylene blue (MB) and 6-(ferrocenyl)hexanethiol (FeC) modified Fe3O4@Au magnetic nanocomposites. Meanwhile, a multifunctional flotation probe with dual target recognition, capture, and isolation capability was prepared by conjugating primary antibodies (Ab1-CEA, Ab1-AFP) to hollow buoyant microspheres. The target antigens of CEA and AFP can trigger a flotation-mediated sandwich-type immunoreaction and capture a certain amount of the distinguishable magnetic signal reporter, which enables the conversion of the target CEA and AFP quantities to the signal of the potential-resolved MB and FeC. Thus, the MB and FeC currents of magnetically adsorbed distinguishable magnetic reporters can be used to determine the CEA and AFP targets simultaneously and precisely. Accordingly, the proposed strategy exhibited a delightful linear response for CEA and AFP in the range of 100 fg·mL-1-100 ng·mL-1 with detection limits of 33.34 and 17.02 fg·mL-1 (S/N = 3), respectively. Meanwhile, no significant nonspecific adsorption and cross-talk were observed. The biosensing platform has shown satisfactory performance in the determination of real clinical samples. More importantly, the proposed approach can be conveniently extended to universal detection just by simply substituting biorecognition events. Thus, this work opens up a new promising perspective for dual and even multiple targets and offers promising potential applications in clinical diagnosis.

How Biorecognition Affects the Electronic Properties of Reduced Graphene Oxide in Electrolyte‐Gated Transistor Immunosensors

AbstractAmbipolar electrolyte‐gated transistors (EGTs) based on reduced graphene oxide (rGO) have been demonstrated as ultra‐sensitive and highly specific immunosensors. However, the physics and chemistry ruling the device operation are still not fully unraveled. In this work, the aim is to elucidate the nature of the observed sensitivity of the device. Toward this aim, a physical–chemical model that, coupled with the experimental characterization of the rGO‐EGT, allows one to quantitatively correlate the biorecognition events at the gate electrode and the electronic properties of rGO‐EGT is proposed. The equilibrium of biorecognition occurring at the gate electrode is shown to determine the apparent charge neutrality point (CNP) of the rGO channel. The multiparametric analysis of the experimental transfer characteristics of rGO‐EGT reveals that the recognition events modulate the CNP voltage, the excess carrier density Δn, and the quantum capacitance of rGO. This analysis also explains why hole and electron carrier mobilities, interfacial capacitance, the curvature of the transfer curve, and the transconductances are insensitive to the target concentration. The understanding of the mechanisms underlying the transistor transduction of the biorecognition events is key for the interpretation of the response of the rGO‐EGT immunosensors and to guide the design of novel and more sensitive devices.

Nanomaterials and Their Recent Applications in Impedimetric Biosensing.

Impedimetric biosensors measure changes in the electrical impedance due to a biochemical process, typically the binding of a biomolecule to a bioreceptor on the sensor surface. Nanomaterials can be employed to modify the biosensor's surface to increase the surface area available for biorecognition events, thereby improving the sensitivity and detection limits of the biosensor. Various nanomaterials, such as carbon nanotubes, carbon nanofibers, quantum dots, metal nanoparticles, and graphene oxide nanoparticles, have been investigated for impedimetric biosensors. These nanomaterials have yielded promising results in improving sensitivity, selectivity, and overall biosensor performance. Hence, they offer a wide range of possibilities for developing advanced biosensing platforms that can be employed in various fields, including healthcare, environmental monitoring, and food safety. This review focuses on the recent developments in nanoparticle-functionalized electrochemical-impedimetric biosensors.

Architectural Effect on Self-Assembly and Biorecognition of Randomly Grafted Linear and Branched Polymers at Liquid Crystal-Water Interfaces.

Because of simple synthetic strategies, randomly functionalized amphiphilic polymers have gained much attention. Recent studies have demonstrated that such polymers can be reorganized into different nanostructures, such as spheres, cylinders, vesicles, etc., similar to amphiphilic block copolymers. Our study investigated the self-assembly of randomly functionalized hyperbranched polymers (HBP) and their linear analogues (LP) in solution and at the liquid crystal-water (LC-water) interfaces. Regardless of their architecture, the designed amphiphiles self-assembled into spherical nanoaggregates in solution and mediated the ordering transitions of LC molecules at the LC-water interface. However, the amount of amphiphiles required for LP was 10 times lower than that required for HBP amphiphiles to mediate the same ordering transition of LC molecules. Further, of the two compositionally similar amphiphiles (linear and branched), only the linear architecture responds to biorecognition events. The architectural effect can be attributed to both of these differences mentioned above.

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.

Tetrahedral DNA Nanostructure-Engineered Paper-Based Sensor with an Enhanced Antifouling Ability for Photoelectrochemical Sensing.

Herein, a newly designed two-in-one tetrahedral DNA (TDN) nanostructure with an antifouling surface and backbone-rigidified interfacial tracks was developed for highly sensitive and selective detection of miRNA-182-5p. The well-regulated TDN tracks were assembled onto the surface of the TiO2/MIL-125-NH2-functionalized paper electrode, which efficiently avoided the obstacle of DNA strand tangling and decreased the probability of suspension during the walking process, thus greatly promoting the moving efficiency of DNA walkers. More interestingly, the TDN-modified sensing interfaces demonstrated incomparable antifouling ability against protein samples and interfering miRNAs due to the strong hydrophilic capacity and special molecular conformations, which addressed the dilemma of low sensitivity from traditional antifouling coating strategies. As a proof of concept, the designed bifunctional tetrahedron-modified paper-based photoelectrochemical sensor was successfully used to quantify miRNA-182-5p with a low detection limit of 0.09 fM and high specificity and was validated for monitoring of miRNA-182-5p in real samples. This TDN-engineered biointerface could be used as a universal platform for tracking various targets by substituting the biorecognition events, providing great promise for bioanalysis and clinical diagnosis.

Cutting-Edge Advancements in EIS Technologies for Rapid Detection of Pathogenic Bacteria in Water

The emerging research surrounding biosensors has seen an unprecedented rise in recent years: from innovative methods of exploiting the bio-recognition event to groundbreaking first-generation designs, work around biosensors is playing a more substantial role in all facets of modern biotechnology. This mini-review explores a type of biosensor that analyzes bio-recognition events through a technique known as electrochemical impedance spectroscopy (EIS). The EIS technique belongs to the electrochemical class of biosensors and is used to examine analyte-electrode interactions through the transfer of electrons. While the technique has proved effective in detecting several virulent bacteria, this review will primarily focus on E. coli O157:H7, L.monocytogenes, and S. Typhimurium. These three pathogens are all highly contagious and capable of causing severe infections and thus must be carefully managed in essential resources such as water. Though existing methods are effective, there are ways EIS biosensing can be further enhanced in terms of accuracy and precision. At the end of the manuscript, we further overview the state-of-the-art challenges and opportunities in EIS.

CRISPR/Cas‐Powered Biosensing

AbstractCRISPR/Cas system is to be a powerful tool to construct the nucleic acid diagnostic platform. Specific recognition and cleavage of CRISPR/Cas systems enable selective recognition of analytes and physicochemical signal translation of biorecognition events. Given the diversity of Cas effectors, we outline various types of Cas effectors and their associated single‐guide RNA (sgRNA), protospacer adjacent motif (PAM) sequence, or protospacer flanking sequence (PFS), as well as the characteristics of the CRISPR/Cas complex in nucleic acid detection. In light of this, we discuss the coupling of CRISPR/Cas systems to various biosensing strategies (e.g., amplification strategy, electronic methods, or optical methods) and point‐of‐care testing (POCT) (e.g., paper‐based lateral flow assays, field‐effect transistor, microfluidics devices, and solid‐state nanopore sensors) that could enable the specificity and sensitivity of the diagnostic platform for biosensing applications. Additionally, we also discuss the new frontiers and the challenges to be addressed in this field.

Cobalt dual-atom clusters with strong chemiluminescent response for analyzing pathogenic bacteria by using cell wall bind domain as recognizer

As new type of catalysts, dual or multiple atomic clusters have displayed extremely high atom utilization rate and superior catalytic activity. In the present work, a metal binuclear atom pair with unique cobalt coordination environment of Co2-O8 were prepared on the supports of MOFs MIL-53(Al), and named as Co dual-atom clusters (CoDACs). Fascinatingly, CoDACs with desirable water dispersibility display very high catalytic efficiency for redox-based chemiluminescent (CL) reaction by generating abundant reactive oxygen species. The catalysts boosted the CL signal of luminol-H2O2 system for 1192 times as their concentration were 1.0 μg mL-1, thus can be applied as superior CL probes for tracing biorecognition events. To verify their application potential, CoDACs were used to establish a CL method for analyzing methicillin-resistant Staphylococcus aureus (MRSA) strains by adopting cell wall bind domain of bacteriophage P108 as a recognizer. The method displays a dynamic range of 6.1 × 102 - 6.1 × 107 CFU mL-1 and a detection limit of 220 CFU mL-1. The results for analysis of MRSA in biological, drug and food samples manifest their reliability for real application. To our best knowledge, it is the first study focusing on DACs as efficient catalysts for CL reaction.

Unraveling the binding mode of a methamphetamine aptamer: A spectroscopic and calorimetric study

Nucleic-acid aptamers are bio-molecular recognition agents that bind to their targets with high specificity and affinity and hold promise in a range of biosensor and therapeutic applications. In the case of small-molecule targets, their small size and limited number of functional groups constitute challenges for their detection by aptamer-based biosensors because bio-recognition events may both be weak and produce poorly transduced signals. The binding affinity is principally used to characterize aptamer-ligand interactions; however, a structural understanding of bio-recognition is arguably more valuable in order to design a strong response in biosensor applications. Using a combination of nuclear magnetic resonance, circular dichroism, and isothermal titration calorimetry, we propose a binding model for a new methamphetamine aptamer and determine the main interactions driving complex formation. These measurements reveal only modest structural changes to the aptamer upon binding and are consistent with a conformational-selection binding model. The aptamer-methamphetamine complex formation was observed to be entropically driven, apparently involving hydrophobic and electrostatic interactions. Taken together, our results exemplify a means of elucidating small molecule-aptamer binding interactions, which may be decisive in the development of aptasensors and therapeutics and may contribute to a deeper understanding of interactions driving aptamer selection.

A rationally designed triple-qualitative and double-quantitative high precision multi-signal readout sensing platform

Single mode sensing is more likely suffers from false positive or negative results than multi-signal readout assay. By using a layer-by-layer modified ITO/Au/BiOI electrode as the matrix and core/shell upconversion nanoparticles (UCNPs) as the signal labels, a triple-qualitative (smartphone based naked-eye readout, photoelectrochemical (PEC) and fluorescence (FL)) and double-quantitative (PEC and FL) multi-signal readout sensing platform was designed for high-precision assay of prostate-specific antigen (PSA), a biomarker for prostate cancer diagnosis. Based on the PSA bio-recognition events, the PEC mode sensing was achieved, while the specific enzymatically active of PSA for 5-Carboxyfluorescein (FAM) labeled helix peptide (CGHSSKLQK-FAM) enabled the FL sensing. Besides, under the excitation of 980 nm near-infrared (NIR) laser, the signal antibody@UCNPs bioconjugates can emit visible light for smartphone based naked-eye readout. Compared with the single-signal method, the accuracy of PSA serodiagnosis was obviously improved. Moreover, the above procedure could be extended to the single-molecule fluorescence counting method, the limit of detection for PSA can reach 0.005 ag/mL (S/N = 3). This work combined the advantages of different techniques, minimized the misdiagnose detection, and provides a rational design idea for multi-signal readout sensing platform.

Plasmonic nanocrystals on polycarbonate substrates for direct and label-free biodetection of Interleukin-6 in bioengineered 3D skeletal muscles

Abstract The development of nanostructured plasmonic biosensors has been widely widespread in the last years, motivated by the potential benefits they can offer in integration, miniaturization, multiplexing opportunities, and enhanced performance label-free biodetection in a wide field of applications. Between them, engineering tissues represent a novel, challenging, and prolific application field for nanostructured plasmonic biosensors considering the previously described benefits and the low levels of secreted biomarkers (≈pM–nM) to detect. Here, we present an integrated plasmonic nanocrystals-based biosensor using high throughput nanostructured polycarbonate substrates. Metallic film thickness and incident angle of light for reflectance measurements were optimized to enhance the detection of antibody–antigen biorecognition events using numerical simulations. We achieved an enhancement in biodetection up to 3× as the incident angle of light decreases, which can be related to shorter evanescent decay lengths. We achieved a high reproducibility between channels with a coefficient of variation below 2% in bulk refractive index measurements, demonstrating a high potential for multiplexed sensing. Finally, biosensing potential was demonstrated by the direct and label-free detection of interleukin-6 biomarker in undiluted cell culture media supernatants from bioengineered 3D skeletal muscle tissues stimulated with different concentrations of endotoxins achieving a limit of detection (LOD) of ≈ 0.03 ng/mL (1.4 pM).

Electrochemical Impedance Spectroscopy (EIS): Principles, Construction, and Biosensing Applications.

Electrochemical impedance spectroscopy (EIS) is a powerful technique used for the analysis of interfacial properties related to bio-recognition events occurring at the electrode surface, such as antibody–antigen recognition, substrate–enzyme interaction, or whole cell capturing. Thus, EIS could be exploited in several important biomedical diagnosis and environmental applications. However, the EIS is one of the most complex electrochemical methods, therefore, this review introduced the basic concepts and the theoretical background of the impedimetric technique along with the state of the art of the impedimetric biosensors and the impact of nanomaterials on the EIS performance. The use of nanomaterials such as nanoparticles, nanotubes, nanowires, and nanocomposites provided catalytic activity, enhanced sensing elements immobilization, promoted faster electron transfer, and increased reliability and accuracy of the reported EIS sensors. Thus, the EIS was used for the effective quantitative and qualitative detections of pathogens, DNA, cancer-associated biomarkers, etc. Through this review article, intensive literature review is provided to highlight the impact of nanomaterials on enhancing the analytical features of impedimetric biosensors.

Ultrasensitive Detection of SARS-CoV-2 Antibody by Graphene Field-Effect Transistors.

The fast spread of SARS-CoV-2 has severely threatened the public health. Establishing a sensitive method for SARS-CoV-2 detection is of great significance to contain the worldwide pandemic. Here, we develop a graphene field-effect transistor (g-FET) biosensor and realize ultrasensitive SARS-CoV-2 antibody detection with a limit of detection (LoD) down to 10-18 M (equivalent to 10-16 g mL-1) level. The g-FETs are modified with spike S1 proteins, and the SARS-CoV-2 antibody biorecognition events occur in the vicinity of the graphene surface, yielding an LoD of ∼150 antibodies in 100 μL full serum, which is the lowest LoD value of antibody detection. The diagnoses time is down to 2 min for detecting clinical serum samples. As such, the g-FETs leverage rapid and precise SARS-CoV-2 screening and also hold great promise in prevention and control of other epidemic outbreaks in the future.

Addressing the Theoretical and Experimental Aspects of Low-Dimensional-Materials-Based FET Immunosensors: A Review

Electrochemical immunosensors (EI) have been widely investigated in the last several years. Among them, immunosensors based on low-dimensional materials (LDM) stand out, as they could provide a substantial gain in fabricating point-of-care devices, paving the way for fast, precise, and sensitive diagnosis of numerous severe illnesses. The high surface area available in LDMs makes it possible to immobilize a high density of bioreceptors, improving the sensitivity in biorecognition events between antibodies and antigens. If on the one hand, many works present promising results in using LDMs as a sensing material in EIs, on the other hand, very few of them discuss the fundamental interactions involved at the interfaces. Understanding the fundamental Chemistry and Physics of the interactions between the surface of LDMs and the bioreceptors, and how the operating conditions and biorecognition events affect those interactions, is vital when proposing new devices. Here, we present a review of recent works on EIs, focusing on devices that use LDMs (1D and 2D) as the sensing substrate. To do so, we highlight both experimental and theoretical aspects, bringing to light the fundamental aspects of the main interactions occurring at the interfaces and the operating mechanisms in which the detections are based.

Liposomal Controlled Release Ag-Activated DNAzyme Cycle Amplification on a 2D Pyrene COF-Based Photocathode for α-Synuclein Immunosensing.

Ultrasensitive and accurate monitoring of ultralow-level biomarkers is imperiously needed in clinical diagnosis. So far, exploring high-performance photocathodes and developing new sensing strategies have remained central challenges in photoelectrochemical bioassays. Herein, a two-dimensional (2D) pyrene covalent organic framework (COF, PAF-130) is exemplified for the first time as a high-performance photocathode for precise immunosensing of α-synuclein (α-Syn) by integrating a DNAzyme-induced signal cycle amplification strategy with Ag nanoparticles (NPs)-mediated liposomal immunoassay. Through sequential immunobinding, lysis treatment, and acidolysis, numerous Ag+ ions are released, and then they activate the DNAzyme, which further recycles the cleavage of hairpin DNA (HDNA) on the photoelectrode and induces signal cycle amplification. As a result, an ultralow detection limit (3.6 fg/mL) and a wide linear range (10-5-103 ng/mL) are achieved, which surpass those of most methods reported so far. The proposed sensing approach can be readily extended to detect various biomarkers by substituting the biorecognition events, providing great promise for biomedical and related applications.

BIO bragg gratings on microfibers for label-free biosensing

Discovering nanoscale phenomena to sense biorecognition events introduces new perspectives to exploit nanoscience and nanotechnology for bioanalytical purposes. Here we present Bio Bragg Gratings (BBGs), a novel biosensing approach that consists of diffractive structures of protein bioreceptors patterned on the surface of optical waveguides, and tailored to transduce the magnitude of biorecognition assays into the intensity of single peaks in the reflection spectrum. This work addresses the design, fabrication, and optimization of this system by both theoretical and experimental studies to explore the fundamental physicochemical parameters involved. Functional biomolecular gratings are fabricated by microcontact printing on the surface of tapered optical microfibers, and their structural features were characterized. The transduction principle is experimentally demonstrated, and its quantitative bioanalytical prospects are assessed in a representative immunoassay, based on patterned protein probes and selective IgG targets, in label-free conditions. This biosensing system involves appealing perspectives to avoid unwanted signal contributions from non-specific binding, herein investigated in human serum samples. The work also proves how the optical response of the system can be easily tuned, and it provides insights into the relevance of this feature to conceive multiplexed BBG systems capable to perform multiple label-free biorecognition assays in a single device.

Interferometric multilayered nanomaterials for imaging unlabeled biorecognition events

Abstract There is an intense scientific activity on nanomaterials for biosensing, supported by their great potential to conceive powerful applications in chemistry, biology, and medicine. In addition to meet the required analytical parameters, the social impact of these advances is also highly influenced by cost, simplicity, and portability aspects, which are critical in label-free systems. This paper addresses the design, development, and experimental assessment of multilayered nanomaterials to transduce unlabeled biorecognition assays as both constructive and destructive interferences, by means of simple and effective imaging setups. The materials rely on superposed nanometric films of metals (gold and Ag-In-Sb-Te alloy) and a dielectric (zinc sulphide) deposited on a polymeric substrate. They are tailored to display maximal (constructive and destructive) interferences for immunoassays performed on their surface, and then fabricated by sputtering and characterized by focused ion beam scanning electron microscopy. Herein we also address the development of a simple optical setup that exploits standard DVD laser units to scan by imaging the reflected interferometric response of microarrayed immunoassays. The bioanalytical performance of the approach is experimentally assessed using a representative model immunoassay (BSA/anti-BSA) and a competitive immunoassay to quantify a low molecular weight drug (sulfasalazine), reaching detection limits of 460 and 11 ng mL−1 of unlabeled targets, respectively. This study also explores two alternative one-shot interferometric imaging approaches that provide insights into simpler and faster strategies for label-free biosensing.

Co Single-Atom Catalysts Boost Chemiluminescence.

Co single-atom catalysts (SACs) with good aqueous solubility and abundant labelling functional groups were prepared in Co/Fe bimetallic metal-organic frameworks by a facile solvothermal method without high-temperature calcination. In contrast to traditional chemiluminescence (CL) catalysts, Co SACs accelerated decomposition of H2 O2 to produce a large amount of singlet oxygen (1 O2 ) rather than superoxide (O2 .- ) and hydroxyl radical (OH. ). They were found to dramatically enhance the CL emission of the luminol-H2 O2 reaction by 1349 times, and, therefore, were employed as very sensitive signal probes for conducting CL immunoassay of cardiac troponin I. The detection limit of the target analyte was as low as 3.3 pg mL-1 . It is the first time that employment of SACs for boosting CL reactions has been validated. The Co SACs can also be employed to trace other biorecognition events with high sensitivity.