Efficient Immunosensor Research Articles

Amine Functionalized Gadolinium Oxide Nanoparticles-Based Electrochemical Immunosensor for Cholera.

Herein, we report the synthesis and functionalization of gadolinium oxide nanoparticles (Gd2O3 NPs) to fabricate a highly efficient immunosensor for the detection of Vibrio cholera toxin (CT). Gd2O3 NPs were produced in a straightforward manner utilizing the microwave irradiation technique using a domestic microwave oven. X-ray diffraction, transmission electron microscopy, and spectroscopic techniques were used to characterize the structural and physical aspects of Gd2O3 NPs. The Gd2O3 NPs were then functionalized with 3-(Aminopropyl) triethoxysilane (APTES) and electrophoretically deposited onto an ITO-coated glass substrate. The anti-CT monoclonal antibodies were covalently attached to the APTES-Gd2O3/ITO electrode via EDC-NHS chemistry, followed by bovine serum albumin (BSA). For CT detection, electrochemical response experiments using BSA/anti-CT/APTES-Gd2O3/ITO immunoelectrodes were carried out (5-700 ng mL-1). The immunoelectrode demonstrated an outstanding electrochemical reaction against CT, with a sensitivity of 8.37 mA ng-1 mL cm-2 and a detection limit of 1.48 ng mL-1.

Electrochemical Immunosensor for Early Detection of β-Amyloid Alzheimer's Disease Biomarker Based on Aligned Carbon Nanotubes Gold Nanocomposites.

Beta-amyloid (βA) peptides accompanying the physiological change in brain induce Alzheimer's disease. In this work, a highly sensitive electrochemical (EC) immunosensor platform has been developed for the quantitative detection of βA peptides, using the gold nanoparticle functionalized chitosan-aligned carbon nanotube (CS-aCNT-Au) nanocomposites on glassy carbon electrodes (GCE). The immunosensor has been fabricated by immobilization of the anti-βA antibody upon CS-aCNT-Au/GCE. In the CS-aCNT nanocomposite, CS has high biocompatibility. Hydroxy and amine functionalities favor the antibody immobilization and prevent the leaching of nanocomposites of the modified electrode due to the adhesive environment. Moreover, aCNT offers high conductivity, stability, and a large surface area (the calculated effective surface area of the CS-aCNT/GCE is 8.594 × 10-2 cm2). However, the incorporation of AuNPs further enhances the conductivity of the CS-aCNT-Au nanocomposite based on differential pulse voltammetry (DPV) results, and also improves the effective surface area (9.735 × 10-2 cm2). The surface morphology and electrochemical studies of the nanocomposite, as well as its modifications by the anti-βA antibody and BSA, were carried out through field emission scanning electron microscope (FESEM), cyclic voltammetry (CV), and DPV. The quantitative immunosensing of the βA in phosphate-buffered saline (PBS) solution is accomplished via DPV, which reveals that the immunosensor has a high sensitivity of 157.60 µA pg-1 mL cm-2 and a broad detection range of 10.0 pg mL-1-100.0 µg mL-1, with a limit of detection (LOD) of 0.87 pg mL-1. Subsequently, we detected the spiked βA in diluted serum with a linear detection range of 10.0 pg mL-1-1.0 ng mL-1 and LOD of 0.95 pg mL-1. Moreover, a selectivity study exhibited a high affinity of immunosensors towards βA. Thus, we propose that this highly efficient immunosensor can potentially be applied for the point-of-care (POC) sensing of βA in clinical samples.

Copper doped terbium metal organic framework as emitter for sensitive electrochemiluminescence detection of CYFRA 21-1

Lanthanide metal organic frameworks (L-MOFs) are emerging as promising electrochemiluminescence (ECL) emitters for bioanalysis. This work proposed a copper doped terbium MOF as a luminescent tag for construction of a “signal-on” ECL immunosensing method. The Tb–Cu-PA MOF was prepared using Tb3+ and Cu2+ ions as metal linkers and m-phthalic acid as bridge ligand, and exhibited strong ECL emission with K2S2O8 as a coreactant. The immunosensor was prepared by immobilizing capture antibody on Pd nanoparticles modified Ni–Co layered double hydroxide (Pd-ZIF-67@LDH) nanoboxes, which showed strong electrocatalytic activity toward the reduction of S2O82− for amplifying the ECL signal. Upon the sandwich-typed immunoreactions, Tb–Cu-PA MOF labeled antibody was introduced onto the immunosensor for sensitive ECL detection of target protein. Using cytokeratin 19 fragment 21–1 (CYFRA21-1), a representative lung cancer biomarker, as target model, the ECL immunosensing method showed a linear range of 0.01–100 ng/mL and a detection limit of 2.6 pg/mL (S/N = 3). This immunosensing strategy highlighted the advances of using luminescent and electroactive MOFs in the developments of highly efficient immunosensors for bioanalysis.

Amine Functionalized Noble Metal: Metal Oxide Nanohybrid for Efficient Electrochemical Determination of 25-Hydroxy Vitamin-D3 in Human Serum

Quantification of 25-hydroxy vitamin-D3 (25-OHVD3) is important because its deficiency is related to numerous diseases, including osteoporosis, depression, diabetes, heart disease, certain autoimmune conditions, and even Covid-19. In the present study, noble metal-metal oxide nanohybrid based on L-cysteine functionalized gold decorated zirconia nanoparticles (Cys-Au@ZrO2 NPs) were synthesized with the objective of enhanced electrochemical behavior, stability, availability of functional group to covalently bind with biomolecules, and developing an efficient immunosensor for 25-OHVD3 detection. The formation of Au@ZrO2 NPs and further Cys functionalization was validated by various characterizations. Cys-Au@ZrO2 NPs were electrodeposited onto ITO substrate and further modified with antibodies specific to 25-OHVD3 (ab-25VD3) and bovine serum albumin (BSA) to build an immunosensing platform for 25-OHVD3 detection. The fabricated BSA/ab-25VD3/Cys-Au@ZrO2/ITO immunosensor demonstrated an improved sensitivity of 2.01 μA ng−1 ml cm−2, LOD of 3.54 ng ml−1 for the linear detection range of 1–50 ng ml−1 with regression constant of 0.98. Moreover, the analytical performance of the BSA/ab-25VD3/Cys-Au@ZrO2/ITO immunosensor in determining 25-OHVD3 in human serum samples collected from four healthy people yielded satisfactory results that were highly correlated with the conventional enzyme-linked immunosorbent assay (ELISA).

Non-invasive bioassay of Cytokeratin Fragment 21.1 (Cyfra 21.1) protein in human saliva samples using immunoreaction method: An efficient platform for early-stage diagnosis of oral cancer based on biomedicine

Oral cancer (OC) is considered as sixth most common cancer in the world. The challenge facing oral cancer is the lack of non-invasive, rapid, sensitive, accurate, and inexpensive screening and diagnosis methods. Given the increasing importance of prevention, prognosis, and early-stage diagnosis of cancer in improving of survival rate, the use of efficient diagnostic devices is essential. In this study, novel bioassay based on antigen and antibody immunocomplex was proposed for early stage diagnosis of OC. For the first time, an efficient immunosensor (Cys-GA-anti-Cyfra21.1-BSA-Cyfra21.1 antigen/AuE) was successfully designed and developed to the detection and determination of the Cyfra21.1 biomarker in unprocessed human saliva samples. The Au electrode was modified by Cysteamine (CysA) and Glutaraldehyde (GA) respectively via self-assembly as a substrate to immobilize the biological agents. The engineered immunosensor exhibit an excellent ability to detect and determine of Cyfra21.1 biomarker in low concentrations in unprocessed human saliva samples. Under the optimized operating conditions, the results demonstrate that the desired platform has a good sensitivity in the detecting of Cyfra21.1 with the low limit of quantitation (LLOQ) of 2.5 ng/mL, which this evaluation was performed at a wide linear range of 2.5−50 ng/mL. The use of the CysA-GA nano-hybrid as extraordinary stable substrate and extensive platform to place recognition elements was investigated using various electrochemical methods including cyclic voltammetry (CV) and square wave voltammetry (SWV). In this study, the engineered biosensor was used to non-invasive detection of Cyfra21.1 in unprocessed human saliva sample. Based on results, CysA-GA-anti-Cyfra21.1 antibody-BSA- Cyfra21.1 antigen/AuE with significantly high current intensity can provide appropriate, reliable, affordable, quick, and user-friendly diagnostic device to monitoring oral abnormality by detection and determination of Cyfra21.1 biomarker in human real sample. Above all, the easy to prepared designed immunosensor can be an extremely promising candidate to specific and favorable for a vast range of clinical diagnosis of OC in near future.

Ultrahigh sensitive enhanced-electrochemiluminescence detection of cancer biomarkers using silica NPs/graphene oxide: A comparative study

The increasing progress in using nano-biomaterials for medical purposes has opened new horizons toward researchers around the globe. To investigate the presence of these nanomaterials and the impacts they might have, a comparative enhanced-electrochemiluminescence immunosensing study has been designed. The effects of utilizing graphene oxide, silica, and gold nanoparticles in cancer diagnosis were evaluated during the quantification of two major cancer biomarkers (CEA and AFP) in different approaches. In other words, first and second approaches were designed to employ nanomaterials while third and fourth approaches were developed in absence of those. Accordingly, resulted LODs experienced dramatic amplification when nano-biomaterials were included in the immunosensor modification (for AFP: 1st and 3rd approaches: 1.36fg/ml in comparison with 0.39ng/ml, and for CEA: 2nd and 4th approaches: 1.90fg/ml versus 0.46ng/ml, respectively). Correspondingly, capability of nano-biomaterials for developing highly sensitive and more efficient immunosensors was validated through selectivity, stability, reproducibility, and feasibility examinations.

L-cysteine capped lanthanum hydroxide nanostructures for non-invasive detection of oral cancer biomarker

In this paper, we present the result of studies related to the in situ synthesis of amino acid (L-Cysteine) capped lanthanum hydroxide nanoparticles [Cys-La(OH)3 NPs] towards the fabrication of efficient immunosensor for non-invasive detection of oral cancer. The characterization of Cys-La(OH)3 NPs was carried out by different techniques including X-ray diffraction, scanning electron microscopy, transmission electron microscopy, fourier transform infrared spectroscopy and electrochemical techniques. These Cys-La(OH)3 NPs were electrophoretically deposited onto an indium-tin-oxide glass substrate and used for immobilization of anti-cytokeratin fragment-21-1 (anti-Cyfra-21-1) for the electrochemical detection of Cyfra-21-1. This immunosensor shows a broad detection range of 0.001–10.2ngmL−1, the low detection limit of 0.001ngmL−1, and high sensitivity of 12.044µA (ng per mL cm−2)−1 with a response time of 5min. This immunosensor was found to be more advanced in terms of high sensitivity and low detection limit as compared to previously reported biosensors and commercially available ELISA kit (Kinesis DX).

Bismuth oxide nanorods based immunosensor for mycotoxin detection

We report results of the studies relating to fabrication of an efficient immunosensor based on bismuth oxide nanorods (nBi2O3), electrophoretically deposited onto indium-tin-oxide (ITO) coated glass substrate. This immunosensor was fabricated by immobilization of anti-aflatoxin monoclonal antibodies (Ab-AFB1) and bovine serum albumin (BSA) for aflatoxin B1 detection. The structural and morphological studies of n-Bi2O3 have been carried out by XRD, UV–vis spectrophotometer; SEM, AFM and FTIR. It was found that the nBi2O3 provided improved sensing characteristics to the electrode interface in terms of electroactive surface area, diffusion coefficient, charge transfer rate constant and electron transfer kinetics. The results of electrochemical response studies of this BSA/Ab-AFB1/nBi2O3/ITO immunosensor revealed good linearity in the range of 1–70ngdL−1 with low detection limit of 8.715ng/dL, improved sensitivity of 1.132μA/(ng/dLcm−2), regression coefficient R2 of 0.918 and reproducibility of >11 times. The association constant for the BSA/Ab-AFB1/nBi2O3/ITO immunosensor was determined as 7.318ng/dL.

Correlation of Capture Efficiency with the Geometry, Transport, and Reaction Parameters in Heterogeneous Immunosensors.

Higher capture efficiency of biomarkers in heterogeneous immunosensors would enable early detection of diseases. Several strategies are used to improve the capture efficiency of these immunosensors including the geometry of the system along with the transport and reaction parameters. Having a prior knowledge of the behavior of the above parameters would facilitate the design of an efficient immunosensor. While the contributions of the transport and reaction parameters toward understanding of the mechanism involved in capture have been well studied in the literature, their effect in combination with the geometry of the sensors has not been explored until now. In this work, we have experimentally demonstrated that the capture efficiency of the antigen-antibody systems is inversely related to the size of the sensor patch. The experimental system was simulated in order to get an in-depth understanding of the mechanism behind the experimental observation. Further, the extent of heterogeneity in the system was analyzed using the Sips isotherm to obtain the heterogeneity index (α) and the reaction rate constant (K(D)) as fitted parameters for a sensor patch of 1.5 mm radius. The experimental kinetic data obtained for the same sensor patch matched reasonably with the simulation results by considering K(D) as the global affinity constant, which indicated that our system can be considered to be homogeneous. Our simulation results associated with the size dependency of the capture efficiency were in agreement with the trends obtained in our experimental observations where an inverse relation was observed owing to the fact that the mass-transfer limitation decreases with the decrease in the size of the sensor patch. The possible underlying mechanism associated with size dependency of capture efficiency was discussed based on the time-dependent radial variation of captured antigens obtained from our simulation results. A study on the parametric variation was further conducted for the nonmixed and mixed systems on the transport (Deff), reaction (K(D)), and geometric parameters (R). Two different correlations were established for the nonmixed and mixed systems between the capture efficiency (f) and a nondimensional number (t(D)/t(R)) consisting of the above-mentioned parameters. Such unified relations will be useful in designing heterogeneous immunosensors and can be extended to microfluidic immunosensors.

Visual and efficient immunosensor technique for advancing biomedical applications of quantum dots on Salmonella detection and isolation.

It is a great challenge in nanotechnology for fluorescent nanobioprobes to be applied to visually detect and directly isolate pathogens in situ. A novel and visual immunosensor technique for efficient detection and isolation of Salmonella was established here by applying fluorescent nanobioprobes on a specially-designed cellulose-based swab (a solid-phase enrichment system). The selective and chromogenic medium used on this swab can achieve the ultrasensitive amplification of target bacteria and form chromogenic colonies in situ based on a simple biochemical reaction. More importantly, because this swab can serve as an attachment site for the targeted pathogens to immobilize and immunologically capture nanobioprobes, our mAb-conjugated QD bioprobes were successfully applied on the solid-phase enrichment system to capture the fluorescence of targeted colonies under a designed excitation light instrument based on blue light-emitting diodes combined with stereomicroscopy or laser scanning confocal microscopy. Compared with the traditional methods using 4-7 days to isolate Salmonella from the bacterial mixture, this method took only 2 days to do this, and the process of initial screening and preliminary diagnosis can be completed in only one and a half days. Furthermore, the limit of detection can reach as low as 10(1) cells per mL Salmonella on the background of 10(5) cells per mL non-Salmonella (Escherichia coli, Proteus mirabilis or Citrobacter freundii, respectively) in experimental samples, and even in human anal ones. The visual and efficient immunosensor technique may be proved to be a favorable alternative for screening and isolating Salmonella in a large number of samples related to public health surveillance.

Biocompatible capped iron oxide nanoparticles for Vibrio cholerae detection

We report the studies relating to fabrication of an efficient immunosensor for Vibrio cholerae detection. Magnetite (iron oxide (Fe3O4)) nanoparticles (NPs) have been synthesized by the co-precipitation method and capped by citric acid (CA). These NPs were electrophoretically deposited onto indium-tin-oxide (ITO)-coated glass substrate and used for immobilization of monoclonal antibodies against Vibrio cholerae (Ab) and bovine serum albumin (BSA) for Vibrio cholerae detection using an electrochemical technique. The structural and morphological studies of Fe3O4 and CA-Fe3O4/ITO were characterized by x-ray diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, and dynamic light scattering (DLS) techniques. The average crystalline size of Fe3O4, CA-Fe3O4 nanoparticles obtained were about 29 ± 1 nm and 37 ± 1 nm, respectively. The hydrodynamic radius of the nanoparticles was found to be 77.35 nm (Fe3O4) and 189.51 nm (CA-Fe3O4) by DLS measurement. The results of electrochemical response studies of the fabricated BSA/Ab/CA-Fe2O3/ITO immunosensor exhibits a good detection range of 12.5–500 ng mL−1 with a low detection limit of 0.32 ng mL−1, sensitivity 0.03 Ω/ng ml−1 cm−2, and reproducibility more than 11 times.

Zeta potential and Reynolds number correlations for electrolytic solutions in microfluidic immunosensors

Efficient designs are critical toward obtaining optimal performances in microfluidic immunosensors. In this manuscript, we report a systematic study to characterize the flow characteristics of KCl solutions of different concentrations in trapezoidal Si microchannels with non-functionalized, silanized and antibody-immobilized surfaces. Streaming potentials and pressure drops were measured, and the respective zeta potentials were calculated from the Helmholtz–Smoluchowski equation. The effects of the type of microchannel surfaces, the concentrations and flow rates of the electrolyte concentrations were investigated. Attempts were made to understand the role of zeta potential on the flow characteristics of the solutions in the functionalized microchannels. To the best of our knowledge, such studies have not been reported in the literature. A highlight of the work is that correlations between the zeta potentials with the Reynolds number were found with ζRe0.5 appearing to be reasonably independent of Re for the cases studied here. The results for the antibody-immobilized surfaces could help in further investigations of the electro-viscous effects with phosphate buffer saline solutions containing antigens through the microfluidic immunosensors. We believe that this work advances the knowledge in the area and could be utilized to design efficient immunosensors.

Tunable surface free energies of functionalized molecular layers on Si surfaces for microfluidic immunosensor applications

Enhanced antigen–antibody interactions in microfluidic immunosensors can be effected by tailoring the surface free energies of the antibody immobilized surfaces to obtain the appropriate fluid–wall interactions. We report a systematic study to evaluate the surface free energies from contact angle measurements, using the LWAB method, of different antibody (anti-BSA, anti-PSA, and anti-CRP) surfaces, each immobilized separately on to non- and nanotextured Si surfaces via a stack of functionalized layers including aminosilanes of which three different types were used. The apolar surface free energy components were independent of the physical modification in the non-functionalized and the intermediate hydrolyzed surfaces where as they depended on the nature of the surface and the chemical modifications in the subsequent functionalized stages. Surface free energies of the different antibodies immobilized with the shorter chain length aminosilane (APTES) on non- and nanotextured surfaces were in the order of anti-BSA<anti-PSA<anti-CRP. A tunability of the surface free energy up to 9.6mJ/m2 was achieved which is reasonably significant when compared to the surface free energy window (Δγs=40mJ/m2) of biofunctionalized surfaces. This fundamental understanding of the surface energetics of the biofunctionalized surfaces can be utilized in modulating the surface properties to design efficient immunosensors.

Performance of Eudragit Coated Whispering Gallery Mode Resonator-Based Immunosensors

Whispering gallery mode resonators (WGMR) are an efficient tool for the realization of optical biosensors. A high Q factor preservation is a crucial requirement for good biosensor performances. In this work we present an Eudragit®L100 coated microspherical WGMR as an efficient immunosensor. The developed resonator was morphologically characterized using fluorescence microscopy. The functionalization process was tuned to preserve the high Q factor of the resonator. The protein binding assay was optically characterized in terms of specificity in buffer solution.