Evaluation of Analytical Performances of Magnetic Force-Assisted Electrochemical Sandwich Immunoassay for the Quantification of Carcinoembryonic Antigen

Carcinoembryonic antigen (CEA) is a biomarker indicated in different cancers, targeted for quantitative analysis via immunoassay. Here we introduce a new technique called magnetic force-assisted electrochemical sandwich immunoassay (MESIA) for determination of CEA level in a drop of human serum using a fully automated point-of-care testing (POCT) device. The analytical performances of the assay are assessed based on precision, accuracy, limit of blank (LoB), limit of detection (LoD) and limit of quantitation (LoQ), linearity, Hook effect, interference, cross-reactivity, and method comparison following the guidelines of the Clinical Laboratory Standards Institute (CLSI). The LoD is 0.50 ng/ml. A linear relationship is shown in the range of 0.5–200 ng/ml. A high dose effect is not seen up to approximately 500,000 ng/ml. The recovery range is from 94.7 to 108.9%. The %CV of run-to-run and within-lab variations are less than 2.04 and 4.41% across the CEA concentrations, respectively, whereas reproducibility is 4.45–6.24%. Method comparison shows that the assay correlates well with the reference device (R2 = 0.9884). The assay demonstrates acceptable precision, accuracy, LoB, LoD and LoQ, hook effect, linearity, interference, cross-reactivity, and high correlation with its reference device. Thus, the system is suitable for the quantification of CEA in clinical practices with a POCT manner.

Stabile SERS Encoded Silver Silica Nanocomposites for Industrial Labeling – the Case of COVID-19 Diagnosis

Biosensors, especially those with a SERS readout, are required for an early and precise healthcare diagnosis. Unreproducible SERS platforms hampers the clinical translation of SERS. Here we report a synthetic procedure to obtain stabile, reproducible and robust highly-SERS performing nanocomposites for labelling. We control the NPs agglomeration and codification which results in an increased number of hot spots, thus exhibiting reproducible and superior Raman enhancement. We studied fundamental aspects affecting the plasmonic thiol bond resulting in pH exhibiting a determining role. We validated their biosensing performance by designing a SERS-based sandwich immunoassay against COVID-19. The limits of detection for the recombinant SARS-CoV-2 protein is below 0.01 ng/μL. We offered herein one nanostructure with robust and homogeneous SERS signal which can be potentially applied for biodiagnosis.

Anti-Metatype Antibody Screening, Sandwich Immunoassay Development, and Structural Insights for β-Lactams Based on Penicillin Binding Protein

Theoretically, sandwich immunoassay is more sensitive and has a wider working range than that of competitive format. However, it has been thought that small molecules cannot be detected by the sandwich format due to their limited size. In the present study, we proposed a novel strategy for achieving sandwich immunoassay of β-lactams with low molecular weights. Firstly, five β-lactam antibiotics were selected to bind with penicillin binding protein (PBP)2x* to form complexes. Then, monoclonal and polyclonal antibodies against PBP2x*-β-lactams complexes were produced by animal immunization. Subsequently, the optimal pairing antibodies were utilized to establish sandwich immunoassay for detection of 18 PBP2x*-β-lactam complexes. Among them, ceftriaxone could be detected at as low as 1.65 ng/mL with working range of 1–1000 ng/mL in milk. To reveal the detection mechanism, computational chemistry and molecular recognition study were carried out. The results showed that β-lactams with a large size and complex structures maybe conducive to induce conformational changes of PBP2x*, and then exhibit greater possibility of being detected by sandwich immunoassay after combination with PBP2x*. This study provides insights for subsequent investigations of anti-metatype antibody screening and sandwich immunoassay establishment for small-molecule detection.

Double-Antibody Sandwich Immunoassay and Plasmonic Coupling Synergistically Improved Long-Range SPR Biosensor with Low Detection Limit

A long-range surface plasmonic resonance (LR-SPR) biosensor modified with double-antibody sandwich immunoassay and plasmonic coupling is demonstrated for human-immunoglobulin G detection with a low limit of detection (LOD). The double-antibody sandwich immunoassay dramatically changes the average refractive index of the medium layer on the sensor surface. The near-field electron coupling between the localized surface plasmon and the long-range surface plasmon leads to a significant perturbation of the evanescent field. The large penetration depth and the long propagation distance of the long-range surface plasmonic waves facilitate the LR-SPR sensor in the detection of biological macromolecules. The unique light absorption characteristic of the nanocomposite material in the sensor provides the in situ self-compensation for the disturbance. Therefore, besides the inherent advantages of optical fiber sensors, the developed biosensor can realize the detection of biomolecules with high sensitivity, low LOD and high accuracy and reliability. Experimental results demonstrate that the LOD of the biosensor is as low as 0.11 μg/mL in the detection of the phosphate-buffered saline sample, and the spike-and-repetition rate is 105.56% in the detection of the real serum sample, which partly shows the practicability of the biosensor. This indicates that the LR-SPR biosensor provides better response compared with existing similar sensors and can be regarded as a valuable method for biochemical analysis and disease detection.

Oriented Antibody Covalent Immobilization for Label-Free Impedimetric Detection of C-Reactive Protein via Direct and Sandwich Immunoassays

The detection and monitoring of biological markers as disease indicators in a simple manner is a subject of international interest. In this work, we report two simple and sensitive label-free impedimetric immunoassays for the detection of C-reactive protein (CRP). The gold electrode modified with boronic acid–terminated self-assembled monolayers afforded oriented immobilization of capture glycosylated antibody (antihuman CRP monoclonal antibody, mAb). This antibody-modified surface was able to capture human CRP protein, and the impedance signal showed linear dependence with CRP concentration. We confirmed the immobilization of anti-CRP mAb using surface sensitive X-ray photoelectron spectroscopy (XPS) and electrochemical impedance. The oriented covalent immobilization of mAb was achieved using glycosylated Fc (fragment, crystallizable) region specific to boronic acid. The direct immunoassay exhibited a linear curve for concentration range up to 100 ng ml−1. The limit of detection (LoD) of 2.9 ng ml−1, limit of quantification (LoQ) of 9.66 ng ml−1, and sensitivity of 0.585 kΩ ng−1 ml cm−2 were obtained. The sandwich immunoassay was carried out by capturing polyclonal anti-CRP antibody (pAb) onto the CRP antigen immunoreaction. The impedance signal after pAb capture also showed linear dependence with CRP antigen concentration and acted as a CRP antigen detection signal amplifier. The detection of the CRP antigen using sandwich pAb immunoassay improved LoD to 1.2 ng ml−1, LoQ to 3.97 ng ml−1, and enhanced the sensitivity to 0.885 kΩ ng−1 ml cm−2. The real sample analysis, using newborn calf serum, showed excellent selectivity and % recovery for the human CRP ranging from 91.2 to 96.5%. The method was reproducible to 4.5% for direct immunoassay and 2.3% for sandwich immunoassay.